Enantiopurity Determination of the Enantiomers of the Triple Reuptake Inhibitor Indatraline

Abstract

The present study describes the development of two approaches for the determination of the enantiopurity of both enantiomers of indatraline. Initially, a method was developed using different chiral solvating agents (CSAs) for diastereomeric discrimination regarding signal separation in (1)H nuclear magnetic resonance (NMR) spectroscopy, revealing MTPA as a promising choice for the differentiation of the indatraline enantiomers. This CSA was also tested for its ideal molar ratio, temperature, and solvent. Optimized conditions could be achieved that made determination of enantiopurity for (1R,3S)-indatraline up to 98.9% enantiomeric excess (ee) possible. To quantify even higher enantiopurities, a high-performance liquid chromatography (HPLC) method based on a modified β-cyclodextrine phase was established. The influence of buffer type, concentration, pH value, percentage and kind of organic modifier, temperature, injection volume as well as sample solvent on chromatographic parameters was investigated. Afterwards, the reliability of the established HPLC method was demonstrated by validation according to the ICH guideline Q2(R1) regarding specificity, accuracy, precision, linearity, and quantitation limit. The developed method proved to be strictly linear within a concentration range of 1.25-1000 μM for the (1R,3S)-enantiomer and 1.25-750 μM for its mirror image that enables a reliable determination of enantiopurities up to 99.75% ee for the (1R,3S)-enantiomer and up to 99.67% ee for the (1S,3R)-enantiomer.

MS Binding Assays for the Three Monoamine Transporters Using the Triple Reuptake Inhibitor (1R,3S)‐Indatraline as Native Marker

“…We intended to initially characterize the affinities of the available indatraline stereoisomers for hDAT, hNET, and hSERT (except for (1 R ,3 S )‐ 1 toward hNET, which was previously published)5b in saturation experiments using the MS Binding Assays recently published (Figure 2). 5b From the aforementioned study, samples of the enantiomers of indatraline with high enantiomeric purities of >99.75 % ee [(1 R ,3 S )‐ 1 ] and >99.67 % ee [(1 S ,3 R )‐ 1 ] (determined according to the published HPLC method)10 were available to us. Unfortunately, only the racemate of the cis ‐configured indatraline diastereomer was available, as attempts to separate the compound by crystallization after the formation of diastereomers using various acids (e.g., tartaric acid, mandelic acid, and dibenzoyl tartaric acid), an approach recently applied to indatraline, have so far been unsuccessful.…”

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

“…Since an MS binding assay using (1 R ,3 S )‐indatraline as a marker (Grimm et al, ) was analyzed on the same instrument, we wanted to avoid introducing high concentrations of indatraline into the MS, which could interfere with the (1 R ,3 S )‐indatraline MS Binding Assays. As an alternative to native indatraline, rac ‐[ 2 H 3 ]‐indatraline ( rac ‐[ 2 H 3 ]‐ 4 ) was previously synthesized in our group [according to the procedure published in the Supporting Information of Grimm et al, ; synthesis is described in detail in the Supporting Information of this publication] for an earlier project. When used as inhibitor for the determination of the nonspecific binding in the newly developed MS Binding Assays, rac ‐[ 2 H 3 ]‐indatraline ( rac ‐[ 2 H 3 ]‐ 4 ) did not interfere with the (1 R ,3 S )‐indatraline‐MS Binding Assays, allowing its use in this project.…”

“…[ 2 H 4 ]‐4‐(2‐benzhydryloxyethyl)‐1‐(4‐fluorobenzyl)piperidin‐3‐ol ( rac ‐[ 2 H 4 ]‐D‐84, rac ‐[ 2 H 4 ]‐ 1 ) was synthesized utilizing an H/D exchange reaction; the chemical purity was 99.2% (Allmendinger & Wanner, ; total synthesis is to be published soon). rac ‐[ 2 H 3 ]‐Indatraline ( rac ‐[ 2 H 3 ]‐ 4 ) HCl was synthesized according to Grimm et al (; for synthesis see Supporting Information).…”

Synthesis of [2H7]indatraline

“…For the preparation of [ 2 H 7 ]indatraline ( rac )‐ 2 , we decided to follow the synthetic route recently published by us for the preparation of the unlabelled compound that deviates from the original approach reported by Bøgesø only to a small extent. Analytical issues were the main reasons that this approach has been chosen.…”

“…2 In addition, it is widely used as a pharmacological tool compound in biological studies concerning the previously mentioned transporters. 3 The importance of indatraline (1), which is also commercially available, becomes, in addition, apparent from several studies on the asymmetric synthesis [4][5][6][7][8][9][10] and the determination of the enantiopurity 11 published very recently.…”