Crystal structure and Hirshfeld surface analysis of pinaverium bromide dihydrate

Abstract

In the present study, we have discovered and identified a new crystalline form of pinaverium bromide, pinaverium bromide dihydrate (C₂₆H₄₁BrNO₄⋅Br⋅2H₂O), whose single crystals can be obtained by recrystallization from a mixture of water and acetonitrile at room temperature. The obtained crystals were characterized by X-ray single-crystal diffraction, and their crystal structure was also solved based on X-ray single-crystal diffraction data. The results show that the final pinaverium bromide dihydrate model contains an asymmetric unit of one pinaverium bromide (C₂₆H₄₁Br₂NO₄) molecule and two water molecules that combine with the bromine ion through O–H⋯O and O–H⋯Br hydrogen bonds. Then, the adjacent pinaverium bromide dihydrates are linked by O–H⋯O, O–H⋯Br, and C–H⋯O hydrogen bonds. On the other hand, the experimentally obtained X-ray powder diffraction pattern is in good agreement with the simulated diffraction pattern from their single-crystal data, confirming the correctness of the crystal structure. Hirshfeld surface analysis was employed to understand and visualize the packing patterns, indicating that the H⋯H interaction is the main acting force in the crystal stacking of pinaverium bromide dihydrate.

I. INTRODUCTION

Pinaverium bromide (4-[(2-bromo-4,5-dimethoxyphenyl)methyl]-4-[2-[2-(6,6-dimethyl-bicyclo[3,1,1]-hept-2-yl)ethoxy]ethyl]morpholinium bromide, C₂₆H₄₁Br₂NO₄) is a musculotropic spasmolytic drug virtually devoid of anticholinergic action (Christen, 1990; Annaházi et al., 2014). As a calcium antagonist, it inhibits the flux of calcium into intestinal smooth muscle cells. It exerts this effect by blocking the L-type voltage-dependent calcium channels (the most common type of voltage-operated calcium channel in the intestinal smooth muscle) at the level of the α1-subunit by blocking voltage-dependent calcium channels within the intestinal smooth muscle cells (Christen, 1990; Froguel et al., 1990; Dai et al., 2003; Bor et al., 2021). As a typical crystalline drug, so far the crystal structure of pinaverium bromide has not been reported, and only X-ray powder diffraction patterns of its polycrystalline forms were collected and reported (Mendoza et al., 2021).

Here, a new crystalline form, pinaverium bromide dihydrate (C₂₆H₄₁BrNO₄⋅Br⋅2H₂O) as shown in Figure 1, was discovered and its single crystals were also obtained by solution volatilization, and then its crystal structure was characterized and solved by X-ray single-crystal diffractometry. Furthermore, the intermolecular forces and stacking patterns of pinaverium bromide dihydrate were analyzed using the Hirshfeld surface analysis. Meanwhile, the polycrystalline products of pinaverium bromide were also prepared by grinding the single crystals and characterized using X-ray powder diffractometry. X-ray powder diffraction (XPRD) is the most effective characterization tool for identifying polycrystalline forms of drugs. The XPRD pattern can also be used to verify the accuracy of the single-crystal data.

Figure 1. The pinaverium bromide dihydrate.

II. EXPERIMENTAL

A. Preparation and testing of single crystals

The pinaverium bromide raw material was provided by Zhejiang Apeloa Medical Technology Co., Ltd (Jinhua, China). Analytical grade acetonitrile (ACN) was purchased from Hangzhou ShuangLin Chemical Reagent Co., Ltd (Hangzhou, China), and the purified water was obtained using a Millipore Milli-Q Purification System. All chemical reagents were used without further purification.

In a typical experiment, pinaverium bromide (0.2 g) and ACN/H₂O (1/1, v/v, 10 ml) were mixed in a beaker under stirring until the solution became clear and then covered with a PVC film with a few pores. After slowly evaporating at room temperature, colorless lumpy single crystals appeared in the solution. Subsequently, a suitable single crystal was selected for measurement by single-crystal methods and the determination of the crystal structure.

A suitable crystal (0.15 × 0.05 × 0.03 mm³) was selected and mounted on a glass fiber support on a Bruker APEX-II CCD diffractometer. The crystal was kept at a steady T = 296 K during data collection.

B. Refinement

Crystal data, data collection, and structure refinement details for pinaverium bromide dihydrate are summarized in Table I. All H atoms were placed geometrically and treated with riding constraints. The U _iso(H) values of all CH₂ and CH groups are fixed to 1.2 times the U _eq value of the attached C atom. CH₃ groups are idealized as freely rotating groups, and their U _iso(H) values are fixed to 1.5 times the U _eq value of the attached C atom, and for H atoms on water molecules, their U _iso(H) values are also fixed to 1.5 times the U _eq value of the attached oxygen atom.

TABLE I. Experimental details.

For the single-crystal structure: triclinic, $P\bar{1}$, Z = 2. Experiments were carried out at 296 K with Mo Kα

radiation using a Bruker APEX-II CCD diffractometer. Absorption was corrected for by multi-scan methods (SADABS; Bruker, 2016).

Computer programs: APEX2 (Bruker, 2016), SAINT (Bruker, 2016), OLEX2 (Dolomanov et al., 2009), SHELXT (Sheldrick, 2015a), SHELXL2014 (Sheldrick, 2015b).

In the determined crystal structure, the middle portion of pivibromium dihydrate exhibited disorder, and the refinement of the disordered group was made anisotropically. The disorder was modeled as two site occupancies with occupancy factors of 0.668(19) and 0.332(19) for the C15/O4 and C15A/O4A groups, respectively.

C. X-ray powder diffraction

The sample was carefully mounted on a flat plate to ensure uniform exposure to the X-ray source. X-ray powder diffraction patterns were collected using a D8 Advance diffractometer (Bruker, Germany), which is equipped with a Cu Kα ₁ X-ray (λ = 1.5406 Å). The measurements were conducted using a fixed slit of 0.6 mm and a solar slit of 2.5° to optimize the beam divergence and intensity. The X-ray tube was operated at a voltage of 40 kV and a current of 40 mA. A LYNXEYE- XE-T detector was employed to collect XRD data over a 2θ range from 5° to 40°, with a step size of 0.02° and a total of 1750 data points were recorded.

D. Hirshfeld surface analysis

Hirshfeld surface analysis and the related two-dimensional (2D) fingerprint plots were calculated using CrystalExplorer (Jayatilaka et al., 2006; Spackman et al., 2021) from the structure input file in the CIF format.

III. RESULTS AND DISCUSSION

A. Crystal and molecular structure

During recrystallization from a mixture of ACN and water, water molecules were incorporated into the lattice to form pinaverium bromide dihydrate with a stoichiometric ratio of 1:2. Table I shows the crystallographic data, data collection, and structural refinement details of pivibromium dihydrate, i.e., crystal system, space group, and cell metric.

The single-crystal X-ray diffraction data show that the pinaverium bromide dihydrate crystallizes in the triclinic space group P $\bar{1}$. An asymmetric unit includes one pinaverium bromide molecule and two water molecules, where as one pinaverium molecule contains a cation and an anion. The view of the asymmetric unit is shown in Figure 2. From the crystal stacking, the molecules are laminated along the a-axis, and the benzene ring C1–C6 is located in a planar position and interconnected with the morpholine group through the methylene group, which is basically in the same plane as the benzene ring, and the torsion angle between them is only 1.1(5)° for C4–C5–C6–C1. In addition, the torsion angle between the morpholine group and the methylene group is 90.9(4)° for C5–C6–C9–N1.

Figure 2. The asymmetric unit of pinaverium bromide dihydrate, with displacement ellipsoids drawn at the 40% probability level and H atoms represented by spheres of arbitrary size.

Hydrogen bonding plays a crucial role in the crystal formation process. Pinaverium bromide dihydrate exhibits a unique hydrogen bonding as well as molecular stacking pattern. As shown in Figure 3 (red for oxygen atoms, purple for nitrogen atoms, white for hydrogen atoms, gray for carbon atoms, and dark yellow for bromine atoms), the molecules can stack stably through intramolecular and intermolecular hydrogen bonding interactions. Firstly, pinaverium bromide and water molecules are hydrogen bonded by groups of O5–H5B⋯O6 hydrogen bonds as well as O6–H6⋯Br2, and the pinaverium bromide cations in adjacent molecules are connected through C10–H10B⋯O2^ⅰ and C11–H11B⋯O1^ⅰ, while Br^– is connected to a water molecule through O6–H6A⋯Br2ⁱⁱⁱ and O6–H6B⋯Br2 hydrogen bonds. At the same time, the cation also forms a C14–H14A⋯O5^ⅱ hydrogen bond with the nearest water molecule, which further stabilizes the crystal structure of pinaverium bromide dihydrate. Hydrogen bond geometry (Å, °) for pinaverium bromide dihydrate is listed in Table II. See supplementary material for the details.

Figure 3. The single-crystal cell of pinaverium bromide dihydrate is viewed along the a-axis direction. The blue line represents the hydrogen bonding. And viewed along the b-axis and c-axis direction are given in Figures 8–9 (supplementary material).

TABLE II. Hydrogen bond geometry (Å, °) for pinaverium bromide dihydrate

Symmetry code: (ⅰ) −x + 2, −y + 1, −z + 1; (ⅱ) x–1, y, z; (ⅲ) −x + 2, −y + 2, −z + 1.

B. X-ray powder diffraction analysis

After knowing the atomic coordinates in the cell, the experimental X-ray diffraction pattern of pinaverium bromide dihydrate was simulated using Mercury software (Macrae et al., 2019) and compared with the experimentally obtained XRPD pattern. Figure 4(a) shows good agreement between the experimental diffraction pattern and the simulated pattern from the single-crystal structure. It can be concluded that the crystal structure of the powder crystal samples is highly consistent with that of a single crystal and shows high purity and homogeneous structure. It is worth noting that some diffraction planes, especially those with h = 0 such as $\left({\matrix{ 0 \hfill & 1 \hfill & 1 \hfill \cr } } \right)$ and $\left({\matrix{ 0 \hfill & 3 \hfill & 2 \hfill \cr } } \right)$, show lower intensity compared to the fitted data, indicating the existence of preferred orientations.

Figure 4. (a) X-ray powder diffraction pattern of pinaverium bromide dihydrate and (b) Whole Pattern Fitting and Rietveld refinement results.

In order to obtain more accurate results, this paper used the Whole Pattern Fitting and Rietveld Refinement features of MDI Jade 9 software (MDI, 2019) to perform Rietveld refinement of the powder XRD pattern and then compares it with the simulated XRD data from the single crystals. As shown in Figure 4(b), the Whole Pattern Fitting and Rietveld refinement, including the background, specimen displacement, lattice constants, overall intensity scale factor, peak width, peak symmetry parameters, skewness factor, absorption correction, and atomic structure, was carried out successfully. In addition, it is clear that the crystals mainly grow along the $\left\langle {\matrix{ 1 \hfill & 0 \hfill & { 0 \rangle } \hfill \cr } } \right.$ axis; here, the orientation coefficient of the $\left({\matrix{ 1 \hfill & 0 \hfill & 0 \hfill \cr } } \right)$ diffraction plane is calculated to be 0.75. The residuals of the final fit, R = 13.63%, is less than 15%, which is an acceptable error range. The relevant data for refinement, including 2θ _obs, 2θ _cal, Δ2θ, d, I, height, area, and full width at half maximum (FWHM), are listed in Table III.

TABLE III. X-ray powder diffraction data of pinaverium bromide dihydrate before and after whole Pattern Fitting and Rietveld refinement.

C. Hirshfeld surface analysis

The stacking properties of crystals are explored by using the Hirshfeld surface analysis in the Crystalexplorer program. This method allows the identification and analysis of close contacts occurring between molecules in a crystal. Figures 5 displays the Hirshfeld surfaces of the pinaverium bromide dihydrate, d _i and d _e denote the distance from the surface to the nearest atom inside or outside to the surface (Sundareswaran and Karuppannan, 2020), respectively, and d _norm is the surface map that depicts the individual hydrogen bonding interactions on the d _norm surface, respectively. In addition, the strong hydrogen bonding contacts are depicted as large circular depressions (dark red) on the d _norm surface. Other contacts, i.e., those that are weaker and longer than the hydrogen bonds, are depicted as small dots and very lightly colored regions (Spackman and McKinnon, 2002).

Figure 5. Hirshfeld surface analysis of pinaverium bromide dihydrate.

Figure 5 represents the Hirshfeld surfaces of pinaverium bromide dihydrate mapped over d _norm, d _i, d _e, shape index, and curvedness. In the map, the larger red dots indicate stronger hydrogen bonding interactions between molecules. It can be seen from Figure 5 that the Br atoms in the pinaverium ammonium bromide dihydrate molecule and the O–H in the water molecule form strong (O–H⋯Br) interactions with the O–H and Br atoms in the neighboring molecules. The shape index represents the bumps (blue) and hollows (red) present in the system, and the curvedness shows that the structure contains almost flat surface areas (green) with sharp edges (blue).

Different molecular interactions have different two-dimensional fingerprints, and the two-dimensional fingerprints are very sensitive to the environment in which the molecules are located, so they can be used to reflect the modes of interaction between molecules within different crystalline forms and the contribution range of each mode of interaction to the overall action (Nikpour et al., 2009). The two-dimensional fingerprint plots of the Hirshfeld surface of pinaverium bromide dihydrate and the percentage contribution of various modes of action to Hirshfeld are shown in Figures 6 and 7.

Figure 6. The 2D fingerprint plots (atom–atom interactions) of the Hirshfeld surface of pinaverium bromide dihydrate.

Figure 7. Relative contributions of various intermolecular interactions to the Hirshfeld surface area in pinaverium bromide dihydrate.

It can be seen from Figures 6 and 7 that there are seven main intermolecular interactions of pinaverium bromide dihydrate, among which the H⋯H interactions between the methyl and methylene groups of adjacent molecules are distributed in the middle region of the fingerprint profile, which is the most dominant mode with the largest contribution of 59.4% to the Hirshfeld surface, followed by the intermolecular O⋯H and Br⋯H interactions, which contributed 11.4%. In fact, according to the d _norm diagram, Br⋯H interactions are mainly from the C–H⋯Br van der Waals forces between H and Br atoms and O–H⋯Br hydrogen bonding forces; meanwhile, O⋯H interactions can be attributed to the van der Waals forces between O and H atoms and the intermolecular C–H⋯O hydrogen bonding forces, indicating that hydrogen bonding plays a key role in the intermolecular interaction.

The corresponding interaction distances based on Hirshfeld analysis are calculated to be 2.897 Å (C5–H5⋯Br2), 2.869 Å (C10–H10A⋯Br2), 2.593 Å (O6–H6⋯Br2), 1.913 Å (O5–H5B⋯O6), and 2.597 Å (C14–H14C⋯O6). It is obvious that the interaction distance is related to the force strength of the hydrogen bond. The stronger the hydrogen bond is, the shorter the interaction distance is.

IV. CONCLUSION

In this paper, single crystals of pinaverium bromide dihydrate were prepared from the ACN/water system by slow volatilization and its crystal structure was determined by single-crystal X-ray diffraction analysis. The results show that the pinaverium bromide dihydrate molecules crystallize in the triclinic space group P $\bar{1}$, with one cell containing two pinaverium bromide dihydrate molecules arranged along the a-axis. The dihydrate molecules are connected by four intramolecular and four intermolecular hydrogen bonds when the crystals are stacked. Particularly, H⋯H, Br⋯H, and O⋯H interactions play an important role in the self-assembly process of stabilized pinaverium bromide dihydrate crystals. In addition, the XRPD pattern simulated from the single-crystal structure of pinaverium bromide dihydrate is in good agreement with the experimentally obtained powder pattern, implying the correctness of the crystal structure model solved from the single-crystal data.