An Updated Structure of Oxybutynin Hydrochloride

Abstract

Oxybutynin (Ditropan), a widely distributed muscarinic antagonist for treating the overactive bladder, has been awaiting a definitive crystal structure for nearly 50 years due to the sample and technique limitations. Past reports used powder X-ray diffraction (PCRD) to shed light on the possible packing of the molecule however a 3D structure remained elusive. Here we used Microcrystal Electron Diffraction (MicroED) to successfully unveil the 3D structure of oxybutynin hydrochloride. We identify several inconsistencies between the reported PXRD analyses and the experimental structure. Using the improved model, molecular docking was applied to investigate the binding mechanism between M₃ muscarinic receptor (M₃R) and (R)-oxybutynin, revealing essential contacts/residues and conformational changes within the protein pocket. A possible universal conformation was proposed for M₃R antagonists, which is valuable for future drug development and optimization. This study underscores the immense potential of MicroED as a complementary technique for elucidating the unknown pharmaceutical crystal structures, as well as for the protein-drug interactions.

Figure 1

Chemical (A) and MicroED structures (B) of (R)-oxybutynin hydrochloride 1R and (S)-oxybutynin hydrochloride 1S. 2F_o-F_c density map was shown in blue mesh. (C) Overlay of MicroED and literature reported PXRD structure of 1R showing the missing O atom error and X-ray induced photoreduction in C≡C bond in the PXRD structure. MicroED structure was colored in grey, PXRD structure was colored in orange.

Figure 2

(A) Packing diagram of oxybutynin hydrochloride 1, viewed along b and c axes; (B) Hydrogen bonding interactions in 1, viewed along b axis; (C) Π-stacking interactions in 1 (less than 5 Å). 1R was colored in blue, 1S was colored in violet. Hydrogen bonding and π-stacking interactions were represented by the dashed lines in orange. Cl⁻ anions were highlighted in spacefill style. Extra Cl⁻ anions were omitted in Figures 2B and 2C for clarification.

Figure 3

(A) Overlay of protein-drug interaction diagram of complexes between M₃R and four antagonists; (B) Topside view of M₃R/1R complex structure predicted by molecular docking; (C) Topside view of M₃R/2 complex structure predicted by molecular docking; (D) Topside view of M₃R/3 complex structure determined by X-ray diffraction (PDB entry: 4U15); (E) Topside view of M₃R/4 complex structure predicted by molecular docking. Hydrogen bonding interactions were colored by the dashed line in greencyan, and salt bridges were colored by the dashed line in light magenta. π-stacking, π-cation and hydrophobic interactions were omitted for clarification (see details in Tables S3–S6, Supporting Information). The fusion parts of T4 lysozyme were omitted for clarification. Compounds were symbolled as 1-4: (R)-Oxybutynin 1R, (R)-4-Dimethylamino-2-butynyl-2-cyclohexyl-2-hydroxy-2-phenylacetate hydrochloride 2, Tiotropium 3, Trospium 4.