I. INTRODUCTION
Pimecrolimus (trade name Elidel) is used to treat atopic dermatitis (eczema) and other inflammatory skin diseases. It is usually prescribed in the form of a topical skin cream. Pimecrolimus binds to the receptor macrophilin-12 (FKBP-12) forming a complex that blocks the calcium-dependent signal transduction cascade mediated by calcineurin. Pimecrolimus weakens mast cell response, thus reducing inflammation on the region of skin where it is applied. Pimecrolimus is typically prescribed after a patient has not positively responded to other treatments. A small number of pimecrolimus users have reportedly developed skin cancer, but it is not known if this is the explicit result of pimecrolimus usage. More research is needed to determine whether long-term pimecrolimus administration increases the risk of any type of cancer. The IUPAC name (CAS Registry Number 137071-32-0) is (1R,9S,12S,13R,14S,17R,18E,21S,23S,24R,25S,27R)-12-[(E)-1-[(1R,3R,4S)-4-chloro-3-methoxycyclohexyl]prop-1-en-2-yl]-17-ethyl-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.04,9]octacos-18-ene-2,3,10,16-tetrone. A two-dimensional molecular diagram is shown in Figure 1.
Figure 1. The molecular structure of pimecrolimus. Carbon is presented in dark green, hydrogen in white, nitrogen in blue, oxygen in red, and chloride in light green.
A star-quality powder pattern without atomic coordinates of pimecrolimus, based on a Le Bail fit to the synchrotron data of this study, is contained in the Powder Diffraction File (Gates-Rector and Blanton, Reference Gates-Rector and Blanton2019) as entry 00-066-1619. Pimecrolimus was reported in amorphous form in European Patent Application EP0427680A1 (Baumann, Reference Baumann1990; Sandoz). Amorphous pimecrolimus was also claimed in U.S. Patent 7,589,100B2 (Kovacsne-Mezei et al., Reference Kovacsne-Mezei, Racz, Aronhime, Gyollai, Molnar and Pinchasov2009; Teva), and a pattern of the amorphous phase was presented. Crystalline pimecrolimus was reported in U.S. Patent 6,423,722B2 (Dosenbach et al., Reference Dosenbach, Grassberger, Hartmann, Horvath, Mutz, Penn, Pfeiffer and Wieckhusen2002; Novartis). Powder patterns for both Form A (a hydrate) and Form B (anhydrous) were provided.
This work was carried out as part of a project (Kaduk et al., Reference Kaduk, Crowder, Zhong, Fawcett and Suchomel2014) to determine the crystal structures of large-volume commercial pharmaceuticals and includes high-quality powder diffraction data for these pharmaceuticals in the Powder Diffraction File.
II. EXPERIMENTAL
Pimecrolimus was a commercial reagent, purchased from Carbosynth (Batch #FP270461501), 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 295 K 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 0.414157 Å from 0.5 to 50° 2θ with a step size of 0.001° and a counting time of 0.1 s step−1.
The pattern was indexed on a monoclinic unit cell with a = 15.329, b = 13.309, c = 10.952 Å, β = 96.2°, V = 2215.3 Å3, and Z = 2 using Jade 9.5 (MDI, 2014). Analysis of the systematic absences using EXPO2014 (Altomare et al., Reference Altomare, Cuocci, Giacovazzo, Moliterni, Rizzi, Corriero and Falcicchio2013) suggested the space group P2 1. A reduced cell search in the Cambridge Structural Database (Groom et al., Reference Groom, Bruno, Lightfoot and Ward2016) yielded eight hits, but no pimecrolimus structures.
A pimecrolimus molecule was built using Spartan '18 (Wavefunction, 2018), saved as a .mol2 file, and converted to .fh and .mop files using OpenBabel (O'Boyle et al., Reference O'Boyle, Banck, James, Morley, Vandermeersch and Hutchison2011). Many attempts to solve the structure by Monte Carlo-simulated annealing techniques using several different programs yielded plausible, but ultimately unsatisfactory, structure models. The pimecrolimus molecule was downloaded as Structure2D_CID_6509979.sdf from PubChem. The 2D model was converted to 3D using Spartan, and the local minimum energy conformation was computed. The conformations of the two models (PubChem and manually built) were very different. Attempts to solve the structure using the 6509979 model also yielded plausible, but unsatisfactory (wR in the mid-teens) models. Close examination of the models used by FOX (Favre-Nicolin and Černý, Reference Favre-Nicolin and Černý2002), DASH (David et al., Reference David, Shankland, van de Streek, Pidcock, Motherwell and Cole2006), and EXPO2014-simulated annealing revealed an unexpectedly small number of torsional degrees of freedom. The C59–C60 bond in the large ring was broken, and the structure solutions attempted with more torsional degrees of freedom. In two of the 100 solutions from DASH, the ends of the broken ring were close together. The bond was manually remade using Materials Studio (Dassault Systèmes, 2018), and the structure was optimized using the Forcite module. Refinement was begun from this model.
Rietveld refinement was carried out using GSAS-II (Toby and Von Dreele, Reference Toby and Von Dreele2013). Only the 1.2–20.0° portion of the pattern was included in the refinement (d min = 1.192 Å). The y-coordinate of C2 was fixed to define the origin. 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 10.3% to the final χ 2. The hydrogen atoms were included in calculated positions, which were recalculated during the refinement using Materials Studio (Dassault Systèmes, 2018). 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. The U iso for each hydrogen atom was constrained to be 1.3× that of the heavy atom to which it is attached. The background was modeled using a four-term shifted Chebyshev polynomial, with two peaks at 1.42 (sharp) and 5.31° (broad) 2θ to model the scattering from the Kapton capillary.
The final refinement (begun from the result of the DFT calculation) of 196 variables using 18 802 observations and 143 restraints yielded the residuals Rwp = 0.1192 and GOF = 1.68. The largest peak (0.19 Å from Cl16) and hole (1.48 Å from C60) in the difference Fourier map were 0.60 and −0.58(10) éÅ−3. The Rietveld plot is included in Figure 2. The largest errors in the fit are in the positions of some of the low-angle peaks and probably represent the changes in the specimen during the measurement.
Figure 2. The Rietveld plot for the refinement of pimecrolimus. 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 20× for 2θ >10.6°.
A density functional geometry optimization (fixed experimental unit cell) was carried out using VASP (Kresse and Furthmüller, Reference Kresse and Furthmüller1996) through the MedeA graphical interface (Materials Design, 2016). The calculation was carried out on 16 2.4 GHz processors (each with 4 Gb RAM) of a 64-processor HP Proliant DL580 Generation 7 Linux cluster at North Central College. The calculation 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 × 1 × 2 mesh, and took ~81 h. A single-point density functional calculation on the VASP-optimized structure 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 set for Cl was that 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 eight k-points and the B3LYP functional, and took ~34 h.
III. RESULTS AND DISCUSSION
This synchrotron powder pattern of pimecrolimus matches that reported for Form B by Dosenbach et al. (Reference Dosenbach, Grassberger, Hartmann, Horvath, Mutz, Penn, Pfeiffer and Wieckhusen2002; Novartis) well enough to conclude that they correspond to the same material (Figure 3). The refined atom coordinates of pimecrolimus and the coordinates from the DFT optimization have been deposited with ICDD. The root-mean-square (rms) Cartesian displacement of the non-hydrogen atoms in the Rietveld-refined and DFT-optimized structures is 0.354 Å (Figure 4), and the maximum displacement is 1.076 Å, at C59. This difference represents a difference in conformation of this part of the macrocycle. The agreement between the refined and optimized structures is at the upper end of the expected range for correct structures (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 5, and the crystal structure is presented in Figure 6.
Figure 3. Comparison of the synchrotron pattern of pimecrolimus to the pattern of Form B reported by Dosenbach et al. (Reference Dosenbach, Grassberger, Hartmann, Horvath, Mutz, Penn, Pfeiffer and Wieckhusen2002; Novartis). The published pattern was digitized using UN-SCAN-IT (Silk Scientific, 2013) and scaled to the synchrotron wavelength of 0.414157 Å using MDI JADE Pro (MDI, 2019).
Figure 4. Comparison of the Rietveld-refined (red) and VASP-optimized (blue) structures of pimecrolimus. The rms Cartesian displacement is 0.354 Å.
Figure 5. The asymmetric unit of pimecrolimus Form B, with the atom numbering. The atoms are represented by 50% probability spheroids.
Figure 6. The crystal structure of pimecrolimus Form B, viewed down the c-axis.
The crystal of this large complex molecule is difficult to visualize in a static image (Figure 6). Although there are an intramolecular six-ring hydrogen bond and some larger chain and ring patterns, the structure is dominated by van der Waals interactions.
All of the bond distances and most of the bond angles and torsion angles in pimecrolimus 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 O31–C29–C32 angle of 109.5° [average = 106.9 (11)°; Z-score = 3.1] is flagged as unusual. This lies at the edge of a narrow distribution of similar angles. The C29–O31–C118–C104 torsion angle lies in the tail of a narrow planar distribution. The C58–C56–C47–C45 and C56–C58–C59–C60 torsion angles lie in the tails of broad distributions. The O100–C99–C85–C86, O79–C85–C99–C101, and C86–C85–C99–C101 torsion angles lie in the tails of distributions of only a few similar torsion angles. The O40–C38–C32–C29 and C42–C38–C32–C34 torsion angles are truly unusual.
Quantum chemical geometry optimization of the pimecrolimus molecule (DFT/B3LYP/6-31G*/water) using Spartan '18 (Wavefunction, 2018) indicated that the observed solid-state conformation is 8.6 kcal mol−1 higher in energy than the local minimum. The local minimum is much more similar to the DFT-optimized structure at C59 than to the Rietveld-refined structure (Figure 7) but differs significantly in other places in the molecule. The minimum energy conformation of an isolated pimecrolimus molecule is very different than that observed in the solid state (Figure 8). The differences show that, although weak, the intermolecular interactions are important in determining the solid-state conformation.
Figure 7. Comparison of the Rietveld-refined (red), DFT-optimized solid-state (blue), and DFT-optimized local minimum isolated molecule (green) structures of pimecrolimus.
Figure 8. Comparison of the DFT-optimized solid-state structure of pimecrolimus (blue) and the global minimum energy conformation of an isolated molecule (orange).
Analysis of the contributions to the total crystal energy using the Forcite module of Materials Studio (Dassault Systèmes, 2018) suggests that bond, angle, and torsion distortion terms contribute to the intramolecular deformation energy, as might be expected in a macrocyclic fused-ring system. The intermolecular energy is dominated by van der Waals and electrostatic repulsions, which in this force-field-based analysis include hydrogen bonds. The hydrogen bonds are better analyzed using the results of the DFT calculation.
There are only two classical hydrogen bonds (Table I). The hydroxyl group O97–H98 acts as a donor in an intermolecular hydrogen bond to the ketone oxygen O46. The hydroxyl group O40–H41 acts a donor in an intramolecular hydrogen bond to the same ketone O46. The energies of these O–H⋯O hydrogen bonds were calculated from the Mulliken overlap populations using the correlation of Rammohan and Kaduk (Reference Rammohan and Kaduk2018).
TABLE I. Hydrogen bonds (CRYSTAL14) in pimecrolimus.
a Intramolecular.
The volume enclosed by the Hirshfeld surface (Figure 9; Hirshfeld, Reference Hirshfeld1977; Turner et al., Reference Turner, McKinnon, Wolff, Grimwood, Spackman, Jayatilaka and Spackman2017) is 1097.53 Å3, 99.03% of half of the unit cell volume. The molecules are thus more loosely packed than usual. Most of the significant close contacts (red in Figure 9) involve the hydrogen bonds, and most intermolecular contacts are larger than the sums of van der Waals radii. The volume/non-hydrogen atom is rather large at 19.8 Å3.
Figure 9. The Hirshfeld surface of pimecrolimus. 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; Friedel, Reference Friedel1907; Donnay and Harker, Reference Donnay and Harker1937) morphology suggests that we might expect blocky morphology for pimecrolimus, with {100} as major faces. A fourth-order spherical harmonic model for preferred orientation was incorporated into the refinement. The texture index was 1.021, indicating that preferred orientation was slight in this rotated capillary specimen. The powder pattern of pimecrolimus from a Le Bail fit to this synchrotron data set is included in the Powder Diffraction File as entry 00-066-1619.
IV. DEPOSITED DATA
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 info@icdd.com.
ACKNOWLEDGEMENTS
The use of the Advanced Photon Source at Argonne National Laboratory was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-06CH11357. This work was partially supported by the International Centre for Diffraction Data. We thank Lynn Ribaud and Saul Lapidus for their assistance in the data collection and Andrey Rogachev for the use of computing resources at IIT.
CONFLICTS OF INTEREST
The authors have no conflicts of interest to declare.
