Unexpected scaffold rearrangement product of pirenzepine found in commercial samples

Abstract

Pharmacovigilance aims at a better understanding of the molecular events triggered by medications to prevent adverse effects, which despite significant advances in our analytical repertoire plague the use of drugs until today. In this study, we find that clinically prescribed and commercially available pirenzepine may not be the correct compound. Pirenzepine can undergo an unexpected scaffold rearrangement from the pharmaceutical active ingredient (API) to a previously uncharacterized benzimidazole. The rearrangement occurs under highly acidic conditions, which were believed to favour the dihydrochloride formation of pirenzepine. The rearranged products of pirenzepine and the structurally related telenzepine have significantly decreased affinity for the muscarinic acetylcholine receptor, the pharmacological target of these compounds. Fortunately, in situ rearrangement after oral application is no safety issue, as we show that reaction kinetics in gastric acid prevent rearrangement. The research community should consider appropriate measures to perform reliable receiving inspections in the commercial supply of well described and frequently used chemicals, in particular if experiments yield unexpected results.

Figure 1

Overview of synthetic routes of pirenzepine dihydrochloride. Emphasis is given on the synthetic conditions for the formation of the dihydrochloride.

Figure 2

Crystal structure of 4 (left) and 5 (right), drawn with 50% displacement ellipsoid. The bond precision for C–C single bonds is 0.0017 Å (4) and 0.0039 Å (5), respectively (Supplementary Tables S5–S8). Solvent omitted for clarity.

Figure 3

A: ¹H-NMR spectra of 4 (top, sample #2) and 2 (bottom, sample #4) in D₂O at 400 MHz. B: UV-HPLC traces of 4 (top, sample #2) and 2 (bottom, sample #4).

Figure 4

A: Kinetic data of the rearrangement of 2 in 6 M HCl at an initial concentration of 50 µg/mL as determined by HPLC. Error bars indicate the standard deviation of n = 3 measurements. Left: The kinetic profile of the rearrangement shows the conversion of 2 to 4 as function of time at different temperatures. The conversion is calculated normalized to the maximum area under the curve of 4. The rearrangement follows a pseudo first order kinetic with half lifes of 25 ± 2 min (75 °C), 117 ± 16 min (55 °C) and 830 ± 30 min (37 °C). Right: Arrhenius plot illustrating a linear relationship between inverse temperature and logarithm of the rate constants. The activation energy of the reaction was delineated from the slope: 83.2 ± 1.9 kJ/mol. B: Reaction scheme showing the proposed reactive intermediate. Oxygens in the carboxylic acid group are indistinguisable, but for reasons of clarity only one of them is coloured like the hydrolyzing water.

Figure 5

Acid catalyzed rearrangement of telenzepine.

Figure 6

Competition binding curves using 0.2 nM [³H]NMS on mAChR M1 membranes for compounds 2, 4, 5 and telenzepine dihydrochloride. Filter bound decay rate is normalized to 1 for binding in absence of competitor and to 0 for nonspecific binding. Error bars indicate the standard deviation of n = 3 measurements. The table shows K_i-values (nM) as determined by a competitive radioligand binding assay using [N-methyl-³H]scopolamine methyl chloride ([³H]NMS) on CHO cell membranes containing human mAChR receptors. All obtained binding displacement curves showed the expected sigmoidal shape. * Binding was carried out for 90 min at 37 °C, using membranes from rat heart tissue (M2) and transfected A9Lcells (M1 and M3) and in NG108-15 cells (M4).

Figure 7

Graphical illustration of the investigated scientific literature about pirenzepine between January 2000 and February 2021 showing the literature research workflow (left) and the specified sources (right).

Figure 8

Isomerization of 2 using HCl in [¹⁸O]H₂O. The UV-HPLC chromatogram and HRMS spectra of collected fractions after 60 min at 75 °C are shown: [¹⁸O]4 (t_R = 2.9 min, expected mass: 354.1816 m/z), 2 (t_R = 8.3 min, expected mass: 352.1768 m/z).