Dopamine-β-hydroxylase (bovine IC50 = 25.1 ± 0.6 nM; human IC50 = 18.3 ± 0.6 nM) [1]
(R)-Nepicastat HCl targets dopamine-β-hydroxylase (DBH), the enzyme responsible for converting dopamine to norepinephrine. It is a potent and selective inhibitor with IC50 values of 25.1 nM and 18.3 nM for bovine and human DBH, respectively. By inhibiting DBH, it reduces the biosynthesis of norepinephrine, a key neurotransmitter in the sympathetic nervous system. Its selectivity for DBH over other enzymes and receptors makes it a valuable tool for studying the role of DBH in catecholamine signaling.

(R)-Nepicastat produces concentration-dependent inhibition of bovine and human dopamine-β-hydroxylase activity in vitro.

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In vitro enzyme inhibition: (R)-Nepicastat HCl [RS-25560-198 HCl] produced concentration-dependent inhibition of bovine dopamine-β-hydroxylase with an IC50 of 25.1 ± 0.6 nM and human dopamine-β-hydroxylase with an IC50 of 18.3 ± 0.6 nM, showing approximately 2-3 fold lower potency compared to the S-enantiomer (nepicastat) [1].
In vitro, (R)-Nepicastat HCl is a potent and selective DBH inhibitor. It produces concentration-dependent inhibition of bovine and human DBH activity. Its IC50 values for bovine and human DBH are 25.1 nM and 18.3 nM, respectively. It exhibits negligible affinity for twelve other enzymes and thirteen neurotransmitter receptors, confirming its selectivity. These in vitro studies confirm its mechanism as a DBH inhibitor and its potential for studying catecholamine biosynthesis.

(R)-Nepicastat (30 mg/kg, p.o.) reduces noradrenaline content, dopamine content and dopamine/noradrenaline ratio in mesenteric artery and left ventricle of spontaneously hypertensive rats (SHRs).

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In vivo catecholamine modulation in spontaneously hypertensive rats (SHRs): When administered orally at 30 mg/kg three consecutive times 12 h apart, (R)-Nepicastat HCl [RS-25560-198 HCl] significantly altered tissue noradrenaline and dopamine content. In the mesenteric artery, it reduced noradrenaline to 8.91 ± 0.9 μg/g (control 10.4 ± 1.03) and increased dopamine to 0.67 ± 0.09 μg/g (control 0.25 ± 0.02), resulting in dopamine/noradrenaline ratio of 0.08 ± 0.01 (control 0.03 ± 0.00). In the left ventricle, noradrenaline was 1.16 ± 0.08 μg/g (control 1.30 ± 0.06), dopamine 0.07 ± 0.00 μg/g (control 0.02 ± 0.00), ratio 0.06 ± 0.01 (control 0.02 ± 0.00). In the cerebral cortex, noradrenaline was 0.88 ± 0.02 μg/g (control 0.76 ± 0.03), dopamine 0.18 ± 0.00 μg/g (control 0.14 ± 0.01), ratio 0.20 ± 0.01 (control 0.19 ± 0.01). Compared to nepicastat, RS-25560-198 produced significantly smaller changes in noradrenaline and dopamine content in the mesenteric artery and left ventricle [1].
In vivo, (R)-Nepicastat HCl is an orally active inhibitor of DBH. It crosses the blood-brain barrier, allowing it to inhibit DBH in the central nervous system. By inhibiting DBH, it reduces norepinephrine levels and increases dopamine levels, which can have various physiological and behavioral effects. It has been studied in preclinical models for its potential therapeutic applications in conditions such as heart failure, post-traumatic stress disorder, and cocaine addiction.

Bovine and human dopamine-β-hydroxylase activity was assayed by measuring the conversion of tyramine to octopamine. Bovine adrenal enzyme was obtained commercially, while human enzyme was purified from neuroblastoma cell line SK-NSH culture medium. The assay was performed at pH 5.2 and 32°C in a medium containing sodium acetate, fumarate, CuSO4, catalase, tyramine, and ascorbate. Enzyme was added to the reaction mixture, followed by substrate mixture containing catalase, tyramine, and ascorbate to initiate the reaction. Samples were incubated with or without test compound at 37°C for 30-40 min. The reaction was quenched with a stop solution containing EDTA and 3-hydroxytyramine (internal standard). Octopamine was analyzed by reverse-phase HPLC with UV detection at 280 nm. The HPLC run used a LiChroCART RP-18 column with isocratic elution using acetic acid, 1-heptane sulphonic acid, tetrabutyl ammonium phosphate, and methanol at a flow rate of 1 ml/min. Remaining activity percentage was calculated based on controls, corrected with internal standards, and fitted to a non-linear four-parameter concentration-response curve [1].
Additionally, the affinity of (R)-Nepicastat HCl [RS-25560-198 HCl] for twelve other enzymes (including tyrosine hydroxylase, acetyl CoA synthetase, acyl CoA-cholesterol acyl transferase, Ca2+/calmodulin-protein kinase II, cyclo-oxygenase-I, HMG-CoA reductase, neutral endopeptidase, nitric oxide synthase, phosphodiesterase III, phospholipase A2, and protein kinase C) and thirteen neurotransmitter receptors (α1A, α1B, α2A, α2B, β1, β2 adrenoceptors, M1 muscarinic, D1, D2 dopamine, μ opioid, 5-HT1A, 5-HT2A, 5-HT2C serotonin receptors) was evaluated using established radioligand binding or enzymatic assays. The compound had negligible affinity (IC50 or Ki > 10 μM) for all these targets [1].
Non-cellular enzyme assays for (R)-Nepicastat HCl typically involve measuring its inhibition of DBH activity. These assays use purified bovine or human DBH enzyme and a substrate, such as dopamine or tyramine, to measure the rate of enzymatic reaction in the presence of varying concentrations of the compound. The compound's ability to inhibit DBH is assessed by determining the half-maximal inhibitory concentration (IC50). Such assays are essential for characterizing its potency and selectivity as a DBH inhibitor.

In vitro cell-based assays for (R)-Nepicastat HCl are conducted using neuronal cell cultures or cell lines expressing DBH to assess its effects on catecholamine biosynthesis. Cells are treated with the compound, and changes in dopamine and norepinephrine levels are measured using HPLC or ELISA. These experiments are crucial for confirming its mechanism of action at the cellular level and for studying its effects on catecholamine signaling.

Dissolved in distilled water; 30 mg/kg; p.o. Animal Models Spontaneously hypertensive rats (SHRs).

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Spontaneously hypertensive rats (SHRs): Male SHRs (15-16 weeks old) were randomly assigned to receive either vehicle (distilled water) or (R)-Nepicastat HCl [RS-25560-198 HCl] at 30 mg/kg (free base equivalent) orally via gavage needle, three consecutive times, twelve hours apart. Six hours after the third dose, rats were anaesthetized with halothane, decapitated, and tissues (cerebral cortex, mesenteric artery, left ventricle) were rapidly harvested, weighed, placed in ice-cold perchloric acid, frozen in liquid nitrogen, and stored at -70°C until analysis. Noradrenaline and dopamine concentrations were quantified by HPLC with electrochemical detection after tissue homogenization and centrifugation [1].
In vivo animal studies for (R)-Nepicastat HCl are typically conducted in rodent models to evaluate its effects on catecholamine levels and behavior. The compound is administered orally or intraperitoneally, and brain and plasma levels of dopamine and norepinephrine are measured. Behavioral tests, such as the elevated plus maze or forced swim test, can be used to assess its effects on anxiety and depression-like behaviors. These studies are essential for validating its in vivo efficacy and for understanding its pharmacological profile.

(R)-Nepicastat HCl has a molecular weight of 331.81 g/mol. It is soluble in DMSO at 50 mg/mL. The compound should be stored as a powder at -20°C for up to 3 years or in solution at -80°C for up to 6 months. It is an orally active compound that crosses the blood-brain barrier. Its pharmacokinetic properties have been studied in preclinical models.

Comprehensive toxicological data for (R)-Nepicastat HCl are limited, as it is primarily a research compound. It is intended for laboratory use only and is not for human consumption. As with all research chemicals, appropriate safety precautions should be taken when handling (R)-Nepicastat HCl. Its safety profile in vivo has not been extensively characterized.

Br J Pharmacol.1997 Aug;121(8):1803-9.

(R)-Nepicastat HCl [RS-25560-198 HCl] is the R-enantiomer of nepicastat (RS-25560-197). It is a less potent inhibitor of dopamine-β-hydroxylase compared to the S-enantiomer, exhibiting stereospecificity in its inhibitory activity. The compound was tested alongside nepicastat in comparative studies of catecholamine modulation [1].
(R)-Nepicastat HCl (RS-25560-198 HCl) is a potent and selective inhibitor of dopamine-β-hydroxylase (DBH) with IC50 values of 25.1 nM and 18.3 nM for bovine and human DBH, respectively. It exhibits negligible affinity for other enzymes and receptors. The compound is used in research to study the role of DBH in catecholamine biosynthesis and related neurological disorders. (R)-Nepicastat HCl is not an approved drug and is available as a research compound.

C14H15F2N3S.HCL

331.81

331.072

195881-94-8

54536287

Typically exists as solid at room temperature

4.514

2

4

2

20

429

1

Cl.NCC1=CNC(=S)N1[C@@H]1CCC2=C(C=C(C=C2C1)F)F

YZZVIKDAOTXDEB-SNVBAGLBSA-N

InChI=1S/C14H15F2N3S/c15-9-3-8-4-10(1-2-12(8)13(16)5-9)19-11(6-17)7-18-14(19)20/h3,5,7,10H,1-2,4,6,17H2,(H,18,20)/t10-/m1/s1

4-(aminomethyl)-3-[(2R)-5,7-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl]-1H-imidazole-2-thione

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)