Crystal structure of ractopamine hydrochloride, C 18 H 24 NO 3 Cl

The crystal structure of ractopamine hydrochloride has been solved and re ﬁ ned using synchrotron X-ray powder diffraction data, and optimized using density functional theory techniques. Ractopamine hydrochloride crystallizes in space group Pbca (#61) with a = 38.5871(49), b = 10.7691(3), c = 8.4003(2) Å, V = 3490.75(41) Å 3 , and Z = 8. The ractopamine cation contains two chiral centers, and the sample consists of a mixture of the S,S/R,R/S,R and R,S forms. Models for the two diaste-reomers S,S and S,R were re ﬁ ned, and yielded equivalent residuals, but the S,R form is signi ﬁ cantly lower in energy. The crystal structure consists of layers of molecules parallel to the bc -plane. In each structure one of the H atoms on the protonated N atom acts as a donor in a strong discrete N – H ⋯ Cl hydrogen bond. Hydroxyl groups act as donors in O – H ⋯ Cl and O – H ⋯ O hydrogen bonds. Both the classical and C – H ⋯ Cl and C – H ⋯ O hydrogen bonds differ between the forms, helping to explain the large microstrain observed for the sample. The powder pattern has been submitted to ICDD® for inclusion in the Powder Diffraction File ™ (PDF®). ©


I. INTRODUCTION
Ractopamine is an animal feed additive used to promote leanness and increase food conversion efficiency in farmed animals.It is the active ingredient in products marketed in the US as Paylean for swine, Optaflexx for cattle, and Topmax for turkeys.It was developed by Elanco Animal Health, a division of Eli Lilly and Company.As of 2014, the use of ractopamine was banned in 160 countries (Pacelle, 2014), including the European Union, China, and Russia, while 27 other countries, such as Japan, the United States, South Korea, and New Zealand have deemed meat from livestock fed with ractopamine to be safe for human consumption.The systematic name (CAS Registry Number 90274-24-1) is 4-(1-hydroxy-2-((4-(4-hydroxyphenyl)butan-2-yl)amino)ethyl)phenol hydrochloride.A two-dimensional molecular diagram of ractopamine as downloaded from PubChem (Kim et al., 2023) is shown in Figure 1.
Ractopamine has two chiral centers, one the so-called "OH-site" and the other the "Me-site" (Gunnar et al., 2009).Commercial ractopamine hydrochloride is a mixture of all four isomers.The R,R (OH,Me) isomer is known to be the most potent (Ricke et al., 1999;Mills et al., 2003aMills et al., , 2003b)).The hydrochloride salt of the R,R isomer is known as butopamine hydrochloride (Gunnar et al., 2009).R,R-ractopamine is not chirally stable, but epimerizes over a period of days.In contrast, R,R/S,R-ractopamine is an isomerically stable mixture (Gunnar et al., 2009).We are unaware of any published X-ray powder diffraction data for ractopamine hydrochloride.
This work was carried out as part of a project (Kaduk et al., 2014) to determine the crystal structures of large-volume commercial pharmaceuticals, and include high-quality powder diffraction data for them in the Powder Diffraction File (Gates-Rector and Blanton, 2019).

II. EXPERIMENTAL
Ractopamine hydrochloride was a commercial reagent, purchased from USP (Batch R066T0), and was used asreceived.The white powder was packed into a 1.5-mm diameter Kapton capillary, and rotated during the measurement at ∼50 Hz.The powder pattern was measured at 295 K at a beam line 11-BM (Antao et al., 2008;Lee et al., 2008;Wang et al., 2008) of the Advanced Photon Source at Argonne National Laboratory using a wavelength of 0.458208(2) Å from 0.5°to50°2θ with a step size of 0.001°and a counting time of 0.1 s/step.The high-resolution powder diffraction data were collected using twelve silicon crystal analyzers that allow for high angular resolution, high precision, and accurate peak positions.A mixture of silicon (NIST SRM 640c) and alumina (NIST SRM 676a) standards (ratio Al 2 O 3 :Si = 2:1 by weight) was used to calibrate the instrument and refine the monochromatic wavelength used in the experiment.
The synchrotron diffraction peaks are observed to be broad, and do not extend to high angles (Figure 2).After a number of unsuccessful attempts using several programs, the pattern was indexed using DICVOL06 (Louër and Boultif, 2007) as incorporated into EXPO2014 (Altomare et al., 2013) on a high-quality primitive orthorhombic unit cell with a = 38.49578,b = 10.75221,c = 8.39423 Å, V = 3474.49Å 3 , and Z = 8.A reduced cell search in the Cambridge Structural Database (Groom et al., 2016) with the chemistry C, H, Cl, N, and O only yielded six hits, but no ractopamine derivatives.
Several programs indicated different space groups, but both JANA2006 (Petricek et al., 2014) andDASH (David et al., 2006) suggested that Pbca was most probable.This suggestion was confirmed by a successful solution and refinement of the structure.
The ractopamine molecule (S,S) was downloaded from PubChem (Kim et al., 2023) as Conformer3D_CID_56052.sdf.It was converted to a *.mol2 file using Mercury (Macrae et al., 2020), and to a Fenske-Hall Z-matrix using OpenBabel (O'Boyle et al., 2011).The crystal structure was solved using Figure 1.The structure of the neutral ractopamine molecule, as downloaded from PubChem (Kim et al., 2023), showing the S,S configuration.Image generated using Mercury (Macrae et al., 2020).
Figure 2. The synchrotron powder pattern of ractopamine hydrochloride, measured at 11-BM at APS using a wavelength of 0.458208 Å. Image generated using JADE Pro (MDI, 2023).
Monte Carlo simulated annealing techniques as implemented in DASH (David et al., 2006), with the S,S cation and a Cl atom as fragments.
The neutral molecule was protonated at the N atom, and converted to the S,R configuration using Materials Studio (Dassault Systèmes, 2022).The structure was solved independently using this cation and a Cl atom with EXPO2014 (Altomare et al., 2013).
NMR analysis of ractopamine HCl was performed using a 400-MHz Bruker Avance spectrometer equipped with a multinuclear probe with the pharmaceutical in d 6 DMSO (d 6 DMSO was stored over flame dried 3 Å molecular sieves).As shown in Figure 3, the proton NMR spectra indicated that the molecule was protonated at the secondary amine of ractopamine.Interestingly, one of the resonances assigned to the N-H proton is the result of two closely overlapping broad singlets, which are likely the result of the two sets of diastereomers present in the sample.
Rietveld refinements of both structures (S,S and S,R) were carried out using GSAS-II (Toby and Von Dreele, 2013).A third refinement, with a 50/50 mixture of S,S and R,S molecules, yielded much higher residuals (R wp ∼0.14) and will not be discussed further.Only the 1.0°-25.0°(S,S)and 1.0°-22.0°(S,R)portions of the diffraction patterns were included in the refinements (d min = 1.058 and 1.201 Å, respectively).All non-H bond distances and angles were subjected to restraints, based on a Mercury/Mogul Geometry check (Bruno et al., 2004;Sykes et al., 2011).The Mogul average and standard deviation for each quantity were used as the restraint parameters.Planar restraints were also applied to the phenyl rings.The restraints contributed 6.9 and 3.6% to the final χ 2 .The hydrogen atoms were included in calculated positions, which were recalculated during the refinement using Materials Studio (Dassault Systèmes, 2022).The U iso of the heavy atoms were grouped by chemical similarity.The U iso for the H atoms were fixed at 1.3× the U iso of the heavy atoms to which they are attached.A fourth-order spherical harmonics model for preferred orientation was included in the refinements; the refined texture indices were 1.289 and 1.297.The peak profiles were described using the generalized microstrain model (Stephens, 1999).The background was modeled using  a six-term shifted Chebyshev polynomial, and a peak at 6.50°2 θ to model the scattering from the Kapton capillary and any amorphous component.
The final refinements yielded the residuals reported in Table I.The largest errors in the final difference plots (Figures 4 and 5) are small, and reflect fitting ordered models to a disordered system.
The structures of S,S and S,R ractopamine hydrochloride were optimized (fixed experimental unit cell) with density functional theory techniques using VASP (Kresse and Furthmüller, 1996) through the MedeA graphical interface (Materials Design, 2016).The calculations were carried out on 16 2.4 GHz processors (each with 4-Gb RAM) of a 64processor HP Proliant DL580 Generation 7 Linux cluster at North Central College.The calculations used the GGA-PBE functional, a plane wave cutoff energy of 400.0 eV, and a k-point spacing of 0.5 Å −1 leading to a 1 × 2 × 2 mesh, and took ∼35 (S,S) and 48 (S,R) days.Rietveld refinements using the fixed VASP-optimized structures yielded much higher residuals, with R wp around 32%. Single-point density  functional theory calculations (fixed experimental cell) and population analysis were carried out using CRYSTAL23 (Erba et al., 2023).The basis sets for the H, C, N, and O atoms in the calculation were those of Gatti et al. (1994), and that for Cl was that of Peintinger et al. (2013).The calculations were run on a 3.5 GHz PC using eight k-points and the B3LYP functional, and took ∼3.4 (S,S) and 3.5 (S,R) hr.

III. RESULTS AND DISCUSSION
Two different ordered models (S,S and S,R) for ractopamine hydrochloride have been refined, for a sample which consists of a mixture of S,S/R,R/S,R and R,S forms.Powder diffraction cannot distinguish the two racemic pairs, so refinement of two diastereomers was sufficient.In Pbca, each structural model contains both members of an enantiomeric pair.The asymmetric units (with atom numbering) are illustrated in Figures 6 and 7.The displacement coefficients in the centers of the molecules are large, reflecting the disorder.
Refinements of the two models yielded comparable residuals, but the S,R model is significantly lower in energy.The S, S model also resulted in an unreasonably short cation-anion distance.The S,R model is thus to be preferred for this disordered system.
The root-mean-square (rms) Cartesian displacement of the refined S,S and S,R molecules is 0.770 Å (Figure 8).The rms displacement for the VASP-optimized molecules is 1.079 Å (Figure 9).The overall shapes of the molecules are similar, making it non-unreasonable that they could be accommodated in the same lattice.The average microstrains for the two models are 17,479 (S,S) and 16,917 (S,R) ppm, reflecting the imperfect nature of the crystals.
The rms displacement between the Rietveld-refined and VASP-optimized S,S cations is 0.637 Å (Figure 10), and the displacement between the S,R cations is 0.668 Å (Figure 11).The agreements are outside the normal range for correct structures (van de Streek and Neumann, 2014), but this is to be expected from refining ordered models of a  disordered structure.Overlays of the refined and optimized structures of the S,S and S,R models are presented in Figures 12 and 13.A more-standard picture of the optimized S,R structure is included as Figure 14.The remaining discussion will concentrate on the VASP-optimized structures.
The crystal structure consists of layers of molecules parallel to the bc-plane (Figure 14).The mean planes of the phenyl rings in the S,S molecule are (5,2,7) and ( 7 Quantum chemical geometry optimizations of the isolated cations (DFT/B3LYP/6-31G*/water) using Spartan '18 (Wavefunction, 2020) indicated that the S,R cation is  1.2 kcal/mol lower in energy than the S,S cation, but this difference lies within the expected error range of such calculations, so the two molecules should be considered equivalent in energy.The global minimum-energy conformations (MMFF force field) of both the S,S and S,R cations are much more compact (fold on themselves), showing that intermolecular interactions are important in determining the observed solid-state conformations.
Analysis of the contributions to the total crystal energy of the structure using the Forcite module of Materials Studio (Dassault Systèmes, 2022) suggests that bond, angle, and torsion distortion terms dominate the intramolecular deformation energy.The intermolecular energy is dominated by electrostatic attractions, which in this force field analysis also include hydrogen bonds.The hydrogen bonds are better analyzed using the results of the DFT calculation.
Hydrogen bonds are prominent in both the S,S and S,R structures (Tables II and III).As expected, in each structure one of the H atoms on the protonated N atom acts as a donor in a strong discrete N-H⋯Cl hydrogen bond.In the S,R structure this H also forms a weak intramolecular N-H⋯O hydrogen bond, and the other H atom on the N also forms an intramolecular N-H⋯O hydrogen bond.In each structure, hydroxyl groups act as donors in O-H⋯Cl and O-H⋯O hydrogen bonds.The energies of the O-H⋯O hydrogen bonds were calculated using the correlation of Rammohan and Kaduk (2018), the energies of the N-H⋯O hydrogen bonds were calculated using the correlation of Wheatley and Kaduk (2019), and the energies of the O-H⋯Cl hydrogen bonds were calculated using a correlation implicit in Kaduk (2002).In the S,S structure the classical hydrogen bonds link the molecules into chains parallel to the a-axis, while in the S,R structure, they link the molecules into corrugated chains parallel to the b-axis.There are several C-H⋯Cl and C-H⋯O hydrogen bonds, which vary between the two structures.The volumes enclosed by the Hirshfeld surface of the ractopamine hydrochloride asymmetric unit (Figures 15 and 16, Hirshfeld, 1977;Spackman et al., 2021) are 428.59 and 428.02Å 3 , 98.1 and 98.2% of the unit cell volume.The packing density is thus fairly typical.The only significant close contacts (red in Figures 15 and 16) involve the hydrogen bonds.The volume/non-hydrogen atom is larger than normal, at 19.0 Å 3 , reflecting the presence of the large chloride anion.
The Bravais-Friedel-Donnay-Harker (Bravais, 1866;Friedel, 1907;Donnay and Harker, 1937) morphology suggests that we might expect platy morphology for ractopamine hydrochloride, with {100} as the major faces.Afourth-order spherical harmonics model for preferred orientation was included in the refinements; the refined texture indices were 1.289 and 1.297, indicating that preferred orientation was significant in this rotated capillary specimen.

IV. DEPOSITED DATA
The powder pattern of ractopamine hydrochloride from this synchrotron data set has been submitted to ICDD for inclusion in the Powder Diffraction File.The Crystallographic Information Framework (CIF) files containing the results of the Rietveld refinement (including the raw    data) and the DFT geometry optimization were deposited with the ICDD.The data can be requested at pdj@icdd.com.

Figure 3 .
Figure 3.The 1 H NMR spectrum of ractopamine HCl in d 6 DMSO.

Figure 4 .
Figure 4.The Rietveld plot for the refinement of S,S-ractopamine hydrochloride.The blue crosses represent the observed data points, and the green line is the calculated pattern.The cyan curve is the normalized error plot, and the red line is the background curve.The vertical scale has been multiplied by a factor of 10× for 2θ > 9.0°.

Figure 5 .
Figure 5.The Rietveld plot for the refinement of S,R-ractopamine hydrochloride.The blue crosses represent the observed data points, and the green line is the calculated pattern.The cyan curve is the normalized error plot, and the red line is the background curve.The vertical scale has been multiplied by a factor of 10× for 2θ > 9.0°.

Figure 6 .
Figure 6.The asymmetric unit of S,S-ractopamine hydrochloride, with the atom numbering.The atoms are represented by 50% probability spheroids.Image generated using Mercury (Macrae et al., 2020).

Figure 7 .
Figure7.The asymmetric unit of S,R-ractopamine hydrochloride, with the atom numbering.The atoms are represented by 50% probability spheroids.Image generated using Mercury(Macrae et al., 2020).
,8,7), while those in the S,R molecule are (1,1,−1) and (−5,−3,4); the orientations of the rings thus differ in the two structures.The Mercury Aromatics Analyser indicates only weak interactions (>4.9 Å) in the two structures.N-H⋯Cl hydrogen bonds link the cations and anions.Almost all of the bond distances and bond angles fall within the normal ranges indicated by a Mercury Mogul Geometry check (Macrae et al., 2020).Only the C6-C5-C4 angle of 117.8°(average = 113.6(14)°;Z-score = 3.0) in the S,S molecule is flagged as unusual.Torsion angles in S,S involving rotation about the C4-N44 and C5-C6 bonds lie in minor populations of trimodal gauche/trans distributions, and are flagged as unusual.Torsion angles involving rotation about the C7-C10 bond are approximately 0/180°, on the tails of a broad distribution centered about 90°.

Figure 12 .
Figure12.Overlap of the refined S,S, and S,R structures of ractopamine hydrochloride, viewed down the c-axis.Image generated using Mercury(Macrae et al., 2020).

Figure 13 .
Figure 13.Overlap of the VASP-optimized S,S, and S,R structures of ractopamine hydrochloride, viewed down the c-axis.Image generated using Mercury (Macrae et al., 2020).

Figure 14 .
Figure 14.The crystal structure of ractopamine hydrochloride, viewed down the b-axis.Image generated using Diamond (Crystal Impact, 2022).

Figure 16 .
Figure 16.The Hirshfeld surface of S,R-ractopamine hydrochloride.Intermolecular contacts longer than the sums of the van der Waals radii are colored blue, and contacts shorter than the sums of the radii are colored red.Contacts equal to the sums of radii are white.Image generated using CrystalExplorer(Spackman et al., 2021).

Figure 15 .
Figure 15.The Hirshfeld surface of S,S-ractopamine hydrochloride.Intermolecular contacts longer than the sums of the van der Waals radii are colored blue, and contacts shorter than the sums of the radii are colored red.Contacts equal to the sums of radii are white.Image generated using CrystalExplorer(Spackman et al., 2021).

TABLE I .
Refinement residuals for ractopamine hydrochloride