2. EXPERIMENTAL

Cloxacillin sodium monohydrate was a commercial reagent, purchased from USP (Lot #R095J0) and was used as-received. 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 295K at beamline 11-BM (Lee et al., Reference Lee, Shu, Ramanathan, Preissner, Wang, Beno, Von Dreele, Ribaud, Kurtz, Antao, Jiao and Toby2008; Wang et al., Reference Wang, Toby, Lee, Ribaud, Antao, Kurtz, Ramanathan, Von Dreele and Beno2008) of the Advanced Photon Source at Argonne National Laboratory using a wavelength of 0.412 849 Å from 0.5 to 50° 2θ with a step size of 0.001° and a counting time of 0.1 s per step.

The pattern was indexed on a primitive orthorhombic unit cell with a = 7.999 00, b = 10.919 88, c = 25.540 42 Å, V = 2230.9 Å³, and Z = 4 using Jade 9.8 (MDI, 2018) and N-TREOR (Altomare et al., Reference Altomare, Cuocci, Giacovazzo, Moliterni, Rizzi, Corriero and Falcicchio2013). Both programs suggested the space group P2₁2₁2₁, which was confirmed by the successful solution and refinement of the structure. A reduced cell search in the Cambridge Structural Database (Groom et al., Reference Groom, Bruno, Lightfoot and Ward2016) with the chemistry C, H, Cl, N, O, S, and Na only yielded no hits. A reduced cell search with no chemistry yielded 120 hits, including BEBCAM (Blanpain et al., Reference Blanpain, Nagy, Laurent and Durant1980). A cloxacillin anion was built using Spartan ‘18 (Wavefunction, 2018) and converted into a .mol2 file using OpenBabel (O'Boyle et al., Reference O'Boyle, Banck, James, Morley, Vandermeersch and Hutchison2011). The structure was solved by Monte Carlo simulated annealing techniques using DASH (David et al., Reference David, Shankland, van de Streek, Pidcock, Motherwell and Cole2006), using a cloxacillin, Na, and O (from the water molecule) as fragments, and including Mogul Distribution Bias. The success rate was ~10%.

Rietveld refinement was carried out using GSAS-II (Toby and Von Dreele, Reference Toby and Von Dreele2013). Only the 1.7–22.0° portion of the pattern was included in the refinement (d _min = 1.082 Å). All non-H bond distances and angles were subjected to restraints based on a Mercury/Mogul Geometry Check (Bruno et al., Reference Bruno, Cole, Kessler, Luo, Motherwell, Purkis, Smith, Taylor, Cooper, Harris and Orpen2004; Sykes et al., Reference Sykes, McCabe, Allen, Battle, Bruno and Wood2011) of the molecule. The results were exported to a csv file. The Mogul average and standard deviation for each quantity were used as the restraint parameters and were incorporated using the new feature Restraints/Edit Restraints/Add MOGUL Restraints, which reads the bond distance and angle restraints from the csv file. The restraints contributed 1.7% to the final χ ². The hydrogen atoms were included in calculated positions, which were recalculated during the refinement using Materials Studio (Dassault, 2019). The positions of the active hydrogen atoms were deduced by analysis of potential hydrogen bonding patterns. The U _iso of the non-H atoms were grouped by chemical similarity. A common U _iso was refined for the β-lactam ring system, the methyl groups C12 and C16, the carboxylate group, the oxazole ring system plus the amide group, and the phenyl ring. The U _iso for each hydrogen atom was constrained to be 1.3× that of the heavy atom to which it is attached. Two sharp additional peaks revealed the presence of 0.3 wt% NaCl impurity. The background was modeled using a five-term shifted Chebyshev polynomial, and a five-term diffuse scattering function to model the scattering from the Kapton capillary and any amorphous component.

The final refinement of 123 variables using 20 333 observations and 82 restraints yielded the residuals R _wp = 0.0.07726 and GOF = 1.66. The largest peak (0.42 Å from C10) and hole (1.29 Å from O22) in the difference Fourier map were 0.12 and −0.12(3) eÅ⁻³. The Rietveld plot is included as Figure 2. The largest errors in the fit are in the shapes and positions of some of the low-angle peaks and may represent subtle changes in the specimen during the measurement.

Figure 2.

The Rietveld plot for the refinement of cloxacillin sodium monohydrate. The blue crosses represent the observed data points, and the green line is the calculated pattern. The cyan curve is the normalized error plot. The vertical scale has been multiplied by a factor of 8× for 2θ > 10.2°.

A density functional geometry optimization was carried out using CRYSTAL14 (Dovesi et al., Reference Dovesi, Orlando, Erba, Zicovich-Wilson, Civalleri, Casassa, Maschio, Ferrabone, De La Pierre, D-Arco, Noël, Causà and Kirtman2014). The basis sets for the H, C, N, and O atoms were those of Gatti et al. (Reference Gatti, Saunders and Roetti1994), and the basis sets for S, Cl, and Na were those of Peintinger et al. (Reference Peintinger, Vilela Oliveira and Bredow2013). The calculation was run on eight 2.1 GHz Xeon cores (each with 6 Gb RAM) of a 304-core Dell Linux cluster at IIT, using 8 k-points and the B3LYP functional, and took ~35 h.

3. RESULTS AND DISCUSSION

The refined atom coordinates of cloxacillin sodium monohydrate and the coordinates from the DFT optimization are reported in the CIFs attached as Supplementary Material. The root-mean-square Cartesian displacement of the non-hydrogen atoms in the Rietveld-refined and DFT-optimized structures is 0.113 Å (Figure 3), and the maximum displacement is 0.256 Å, at the methyl group C32. The excellent agreement between the refined and optimized structures provides evidence that the experimental structure is correct (van de Streek and Neumann, Reference van de Streek and Neumann2014). This discussion concentrates on the CRYSTAL-optimized structure. The asymmetric unit (with atom numbering) is illustrated in Figure 4, and the crystal structure is presented in Figure 5.

Figure 3.

Comparison of the Rietveld-refined (red) and CRYSTAL14-optimized (blue) structures of cloxacillin sodium monohydrate.

Figure 4.

The asymmetric unit of cloxacillin sodium monohydrate, with the atom numbering. The atoms are represented by 50% probability spheroids.

Figure 5.

The crystal structure of cloxacillin sodium monohydrate, viewed down the a-axis.

The crystal structure is characterized by corner-sharing chains of irregular NaO₅ polyhedra along the a-axis (Figure 5). The carboxylate group chelates to the Na, and O21 bridges two Na cations. The water molecule O48 coordinates to the Na, as well as the carbonyl O26. The Mulliken overlap populations (0.04–0.05 e) indicate that although the Na–O bonds are mostly ionic, they contain some covalent character. The crystal structure of cloxacillin sodium monohydrate is very similar to that of the fluorinated derivative BEBCAM, reflecting the similarity of the lattice parameters.

All of the bond distances, bond angles, and torsion angles in the cloxacillin anion fall within the normal ranges indicated by a Mercury Mogul Geometry check (Macrae et al., Reference Macrae, Bruno, Chisholm, Edington, McCabe, Pidcock, Rodriguez-Monge, Taylor, van de Streek and Wood2008). The bond valence sum of the Na cation is 1.14, so the cation is slightly “crowded”.

Quantum chemical geometry optimization of the cloxacillin anion (DFT/B3LYP/6-31G*/water) using Spartan ‘18 (Wavefunction, 2018) indicated that the observed conformation is 4.1 kcal mol⁻¹ higher in energy than the local minimum. The geometry differences lie mainly in the orientation of the β-lactam system with respect to the rest of the molecule. Molecular mechanics conformational analysis indicated that the minimum-energy molecular conformation is only 0.7 kcal mol⁻¹ lower in energy than the observed solid-state conformation. The differences lie mainly in the conformation of the β-lactam ring system and show that the observed conformation is close to a local minimum.

Analysis of the contributions to the total crystal energy using the Forcite module of Materials Studio (Dassault, 2019) suggests that angle distortion terms are dominant in the intramolecular deformation energy and that torsion terms are also significant, as might be expected for a fused ring system. The intermolecular energy is dominated by electrostatic attractions, which in this force-field-based analysis include cation coordination and hydrogen bonds. The hydrogen bonds are better analyzed using the results of the DFT calculation.

Hydrogen bonds are prominent in the crystal structure (Table I). The water molecule acts as a donor to the carboxylate oxygen O22 and the carbonyl oxygen O7. It is an acceptor in C–H⋯O hydrogen bonds from the methyl group C16 and the ring carbon C5. The energies of the O–H⋯O hydrogen bonds were calculated from the correlation of Rammohan and Kaduk (Reference Rammohan and Kaduk2018). The amide nitrogen N23 forms a strong intramolecular hydrogen bond to S8. Several C–H⋯O hydrogen bonds to ionized carboxylate and carbonyl oxygens also contribute to the crystal energy.

TABLE I.

Hydrogen bonds (CRYSTAL14) in cloxacillin sodium monohydrate. (The table is garbled in the PDF, but is OK here.)

a Intramolecular.

The volume enclosed by the Hirshfeld surface (Hirshfeld, Reference Hirshfeld1977; Turner, et al., Reference Turner, McKinnon, Wolff, Grimwood, Spackman, Jayatilaka and Spackman2017) is 546.06 Å³, 97.97% of 1/4 the unit cell volume. The molecules are thus not tightly packed. All of the significant close contacts (red in Figure 6) involve the hydrogen bonds. The volume/non-hydrogen atom is 17.4 Å³.

Figure 6.

The Hirshfeld surface of cloxacillin sodium monohydrate. 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.

The Bravais–Friedel–Donnay–Harker (Bravais, Reference Bravais1866; Donnay and Harker, Reference Donnay and Harker1937; Friedel, Reference Friedel1907) morphology suggests that we might expect platy morphology for cloxacillin sodium monohydrate, with {002} as the principal faces. No preferred orientation model was necessary, indicating that preferred orientation was not significant. The powder pattern of cloxacillin sodium monohydrate from this synchrotron data set has been submitted to ICDD for inclusion in the Powder Diffraction File™.