An Updated Structure of Oxybutynin Hydrochloride

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.

and microprobe beam size 11 under the parallel beam condition (45.2% C2 intensity).A 70 µm C2 aperture and a 50 µm selected area (SA) aperture were used to result in an approximately 1.4 µm width beam area (0.0098 e -1 /Å 2 /s). 3 Typical data collection used a constant rotation rate of 2° per second over an angular wedge of 130° from -65° to +65°, with 0.5s exposure time per frame, resulting a total dose of 0.65 e -/Å 2 for each dataset.

MicroED data processing.
MicroED data was saved in mrc format and converted to smv format using the mrc2smv software (https://cryoem.ucla.edu/microed). 2 The converted frames were indexed and integrated by XDS. 4,5One dataset with the highest resolution (0.87 Å) was scaled using XSCALE to achieve 83.9% completeness. 5Intensities were converted to SHELX hkl format using XDSCONV 5 and ab initio solved by SHELXT. 6The structure was refined by SHELXL 7 using Shelxle 8 as a graphical interference to yield the final MicroED structure (Table S1).

Molecular Docking
The ligand structures from MicroED structure 1R and X-ray structure 2 (CSD entry: MBCHPA) 9 and 4 (CSD entry: IPILUQ) 10 were extracted.The amine hydrogens were removed based on pKa value. 11All the water and Cl -anions were removed.The ligand structures were imported to AutoDock Tools 1.5.7, 12 making all active torsion bonds rotatable (Figure S4).The protein structure was downloaded from PDB (Protein Data Bank, https://www.rcsb.org/)entry 4U15. 13gands, ions, water were removed using Pymol 2.5.5. 14The resulting protein structure was modified by adding hydrogen atoms and charges as computed by AutoDock Tools 1.5.7. 12 The protein cavity was analyzed by CB-Dock2 webtool 15 at the center of x,y,z = (17, 85, 56), which is consistent with the orthosteric binding site observed in PDB structure 4U15. 13A grid box measuring 18.75 Å × 18.75 Å × 18.75 Å with 0.375 Å spacing was positioned.During the docking in AutoDock Vina 1.1.2, 16,17the ligands were set to be flexible (Figure S4), while the protein was treated with rigid model.The docked complex with minimum binding energy was analyzed in Protein-Ligand Interaction Profiler (PLIP) web tool and Pymol 2.5.5 (Figure 3 and Tables S3-S6). 14,18

Figure S2
Overlay of PXRD patterns of oxybutynin hydrochloride 1 and the literature reported oxybutynin hydrochloride hemihydrate. 19The PXRD patterns were back-calculated using the MicroED and PXRD structures in Mercury software 20 (wavelength was set as 0.457667Å with 0.001° 2 step to be consistent with literature 19 ), colored in green and orange, respectively.

Figure S1
Figure S1 Crystal appearance and diffraction pattern under the TEM.(A) Image of oxybutynin hydrochloride 1 under the imaging mode (SA 3400×).The diffraction beam area was highlighted in dashed red circles; (B) Diffraction pattern of oxybutynin hydrochloride 1 under diffraction mode (741 mm).

Figure
Figure S3 (A) Packing diagram of 1R layers; (B) Packing diagram of 1S layers.1R was colored in blue, 1S was colored in violet.Cl -anions were omitted for clarification.The dashed lines in orange indicated the different positions 1R and 1S occupied in the unit cell.

Figure S4
Figure S4The conformation changes of 1R from drug formulation state to biologically active state.1-9are colored in green which dramatically influence the whole structure; 1-3 are colored in red that have less impact on the overall structure.The major changes were marked with blue arrows.
c Angles were measured by the angles between planes of two aromatic rings.

Table S6
Protein-ligand interactions in M3R/4 complex predicted by molecular docking (Å, °).Notes: a Ligand group was represented by abbreviation: bicyclic ring (BR), carbonyl group (Carb), phenyl group (Ph).b Distances were measured between centroids determined as the center of aromatic rings.