Determination of the metabolic pathways of different Rycals in in vitro samples for doping control purposes

Code: 241C07MT

With the ongoing development of new drugs, the addition of newly developed substances to the World Anti-Doping Agency's Prohibited List has been and continues to be a critical part of anti-doping research. Also, the knowledge of the metabolism of such new compounds is an important tool to identify suspicious samples in routine doping controls. In recent years, ryanodine channel complex stabilizers (Rycals) have received growing attention in the anti doping community due to their potential performance-enhancing effects, by stabilizing the calcium channels which are located in the skeletal muscle. To date, the compounds S107, JTV-519, ARM036 and ARM210 have been investigated for such properties and hence are potential doping agents. While the metabolic behavior of S107 has been investigated, little is known on the metabolism of JTV 519, ARM036 and ARM210. Additionally, the ARM 036 and ARM 210 are either of limited availability or not commercially available.

In this project, we describe the synthesis of ARM 036 and ARM 210 using a multi step approach. After synthesis, the Rycals S107, JTV 519, ARM 036 and ARM 210 are metabolized in vitro using human liver preparations. The samples are analyzed by means of liquid chromatography (LC) coupled with to a high resolution mass spectrometer (HRMS) to identify possible transformation products. These possible metabolites are then analyzed by tandem mass spectrometry (MS/MS) to gain further information on their possible structures. Suitable metabolites are subjected to chemical synthesis in order to confirm their proposed structures. Precursor compounds as well as identified metabolites are potential targets for doping control analysis and are therefore of particular interest for anti doping research