Determination of the enantiomeric purity of the selective dopamine transporter inhibitor (+)‐R,R‐4‐(2‐benzhydryloxyethyl)‐1‐(4‐fluorobenzyl)piperidin‐3‐ol

Abstract

(+)-R,R-D-84 ((+)-R,R-4-(2-benzhydryloxyethyl)-1-(4-fluorobenzyl)piperidin-3-ol) is a promising pharmacological tool for the dopamine transporter (DAT), due to its high affinity and selectivity for this target. In this study, an analytical method to ascertain the enantiomeric purity of this compound was established. For this purpose, a high-performance liquid chromatographic (HPLC) method, based on a cellulose derived chiral stationary phase (CSP) was developed. The method was characterized concerning its specificity, linearity, and range. It was shown that the method is suitable to determine an enantiomeric excess of up to 99.8%. With only a few adjustments, this analytical CSP-HPLC method is also well suited to separate (+)-R,R-D-84 from its enantiomer in a semipreparative scale.

Development and validation of an LC‐ESI‐MS/MS method for the quantification of D‐84, reboxetine and citalopram for their use in MS Binding Assays addressing the monoamine transporters hDAT, hSERT and hNET

“…For the sake of simplicity, the racemic mixture of ( R , R )‐D‐84 and ( S , S )‐D‐83 [the enantiomer of ( R , R )‐D‐84] is referred to as rac ‐D‐84 throughout this work. The enantiomer ( R , R )‐D‐84 [( R , R )‐ 1 ] was obtained after semipreparative enantioseparation of rac ‐D‐84 (Neiens, Höfner, & Wanner, ) in a chemical purity of 97.0%. rac ‐Reboxetine ( rac ‐ 2 ) was extracted from Edronax tablets (4 mg, Pfizer Europe, Kent, UK) as a racemate which was further used to perform a semipreparative CSP‐HPLC to obtain ( S , S )‐reboxetine in a chemical purity of 97.1% [( S , S )‐ 2 , see Supporting Information].…”

“…and (S,S)-D-83 [the enantiomer of (R,R)-D-84] is referred to as rac-D-84 throughout this work. The enantiomer (R,R)-D-84 [(R,R)-1] was obtained after semipreparative enantioseparation of rac-D-84 (Neiens, Höfner, & Wanner, 2017) To elute the bound marker in the MS Binding Assays (see Section 2.10), the 100 pM internal standard solution was diluted with acetonitrile (9 parts 100 pM internal standard solution +1 part acetonitrile) to obtain a solution containing all three internal standards at a concentration of 90 pM (elution solution). All solutions were prepared in micro tubes (1.5 mL, polypropylene, Sarstedt, Nürmbrecht, Germany) and…”

Simultaneous Multiple MS Binding Assays for the Dopamine, Norepinephrine, and Serotonin Transporters

“…Chemicals LC-MS grade acetonitrile and water were obtained from VWR Prolabo (Darmstadt, Germany), water for incubation buffers and washing buffers was prepared by distillation of demineralized water (reverse osmosis) and filtration through a0 .45 mmf ilter.A mmonium formate was bought in mass spectrometry grade from Fluka (Taufkirchen, Germany). [29,42] and racemic reboxetine (rac-2)w as extracted from Edronax tablets (4 mg, Pfizer Europe, Kent, UK) which was further used to perform as emipreparative CSP-HPLC to obtain (S,S)reboxetine ((S,S)-2). [27] Enantiopurities of !…”

“…99.8 % ee for (R,R)-D-84 ((R,R)-1)a nd 99.3 % ee for (S,S)-reboxetine ((S,S)-2)w ere determined by CSP-HPLC. [27,42] [43,44] -is referred to as rac-[ 2 H 4 ]-D-84 throughout this work;s ynthesis of rac-[ 2 H 4 ]-D-84 is to be published soon) and racemic [ 2 H 3 ]-indatraline (rac-[ 2 H 3 ]-4)w ere synthesized in-house. [27] Polyethylenimine (PEI) solution (50 %i nH 2 O) was obtained from Fluka, sodium chloride from Bernd Kraft (Duisburg, Germany), potassium chloride and dimethylacetamide (DMA) from Sigma-Aldrich (Taufkirchen, Germany), HEPES and roti-quant (5 concentrated) for determination of total protein in membrane preparations from Roth (Karlsruhe, Germany).…”

“…( S )‐citalopram (( S )‐ 3 ) oxalate was bought from TCI (Eschborn, Germany), racemic [ 2 H 6 ]‐citalopram ( rac ‐[ 2 H 6 ]‐ 3 ) oxalate from Alsachim (Illkirch, France), racemic [ 2 H 5 ]‐reboxetine ( rac ‐[ 2 H 5 ]‐ 2 ) mesylate was obtained from TRC (North York, ON, Canada), 3‐α‐bis‐(4‐fluorophenyl)methoxytropane hydrochloride from Tocris (Wiesbaden‐Nordenstadt, Germany), hydrochlorides of desipramine and ( S )‐fluoxetine from Sigma–Aldrich (Taufkirchen, Germany), rac ‐bupropione from Alfa Aesar (Karlsruhe, Germany), rac ‐nisoxetine from Biotrend (Cologne, Germany), and sertraline hydrochloride from abcr GmbH (Karlsruhe, Germany), each in the highest commercially available quality. ( R , R )‐4‐(2‐Benzhydryloxyethyl)‐1‐(4‐fluorobenzyl)piperidin‐3‐ol (( R , R )‐D‐84, ( R , R )‐ 1 ) was synthesized and purified in‐house and racemic reboxetine ( rac ‐ 2 ) was extracted from Edronax tablets (4 mg, Pfizer Europe, Kent, UK) which was further used to perform a semipreparative CSP‐HPLC to obtain ( S , S )‐reboxetine (( S , S )‐ 2 ) . Enantiopurities of ≥99.8 % ee for ( R , R )‐D‐84 (( R , R )‐ 1 ) and 99.3 % ee for ( S , S )‐reboxetine (( S , S )‐ 2 ) were determined by CSP‐HPLC .…”