Electrochemical Synthesis of Metabolites, Degradants, Reference Materials ASMS 2018 San Diego, CA, USA

Outline Requirements Instrumentation Examples from Pharma - Synthesis of Degradants - Synthesis of Metabolites Conclusions

General Requirements for Synthesis - Substance (starting material) must be electroactive - Solubility must be known (aqueous, non-aqueous solvents, mixtures) - MS Voltammogram (I/V curve) must be measured to determine optimal oxidation/reduction potential(s) - Optimal electrode selection for Ox  GC, BDD for Red  TiBlue/TiGrey - Optimal pH conditions acidic, neutral or basic

Instrumentation 1 2 3 Electrodes 1) Reticulated Glassy Carbon (RGC) 1 2 3 Electrodes 1) Reticulated Glassy Carbon (RGC) 2) Magic Diamond™ (BDD) 3) Pt SynthesisCell™ ROXY™ Potentiostat 4

Electrochemical Synthesis of Degradants Fesoterodine (Pfizer) (1) and (2) are known oxidation products form long-term stability studies

I/V Curve Fesoterodine Scanning voltammogram Fesoterodine. Current (Icell) vs potential (Ecell) for a scan cycle (solid line) and corresponding first derivative (dotted line) Maximum Ecell = 830 mV. Conditions: E1 = 0 V; E2 = 1.5 V; scan rate: 20 mV/s; flow rate: 50 μL/min; 0.1 mg/mL in 5 mM aqueous ammonium acetate

SynthesisCell™ - 80 mL Bulk Cell t = 0 t = 15 min t = 30 min after synthesis experiment

Workflow - Electrochemical Synthesis 2400 mV 1. Potential based fractionation in vial(s) e.g., 2400 mV for 394 and 520 metabolite 2. Inject in HPLC (semiprep) 3. Fractionation/purification 4. ID by NMR

Synthesis of Oxidation Products Fesoterodine at 0V Reaction mixture after 2 hrs at 950 mV UV chromatograms at 224 nm.0.25 mg/mL fesoterodine fumarate solution in 50 mM aqueous ammonium acetate.

NMR Spectra of Oxidation Product 1 & 2 S. Torres et al. Org. Process Res. Dev., 2015, 19 (11),1596-1603

EC Synthesis of Metabolite M16 of Cipargamin (Malaria Treatment) In this study, a new oxidative metabolite, M16, was identified in all biological matrices albeit at low levels. All 19 recombinant human CYP enzymes were capable of catalyzing the hydroxylation of M23 to form M16 even though the extent of turnover was very low. Electrochemical synthesis using the ROXY EC system equipped with SynthesisCell(Antec Scientific) generated sufficient quantity of M16 to allow full structural characterisation for the 1st time. Group of Dr. Jürgen Kühnöl, Novartis Basel

Metabolic pathway Cipargamin

Electrochemical synthesis is especially useful for compounds that are poorly water soluble and that therefore not reactive in aqueous enzyme reactions. The proposed structure of M16 suggested benzylic oxidation, therefore electrochemical synthesis was used for producing sufficient material for identification by NMR. A boron doped diamond electrode under acidic conditions was used. Surprisingly the oxidation proceeded stereo selectively, as the hydroxyl radical attacked from the opposite site of the neighboring methyl group.

Conclusions Electrochemical Synthesis Fast and cost effective synthesis of mg quantities of REDOX products Complementing (wet)chemical and enzymatic synthesis Stereoselective synthesis? Sufficient reference material for structural elucidation, (MS and NMR), toxicity and bioactivity studies In both cases - Pfizer and Novartis - EC Synthesis was the only way to produce the degradants/metabolites in sufficient amounts for subsequent characterization. 16