I. INTRODUCTION
Tofacitinib citrate, marketed under the brand names Xeljanz® and others, is used to treat rheumatoid and psoriatic arthritis. Tofacitinib works by blocking the body's production of enzymes called Janus kinases (JAKs), specifically JAK1 and JAK3. Tofacitinib citrate is also prescribed to treat ulcerative colitis. The IUPAC name (CAS Registry number 540737-29-9) is 3-[(3R,4R)-4-methyl-3-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]piperidin-1-yl]-3-oxopropanenitrile 2-hydroxypropane-1,2,3-tricarboxylic acid. The molecular structure of tofacitinib citrate is illustrated in Figure 1. The literature indicates that it is a 1:1 salt, so is better named tofacitinib dihydrogen citrate.
Figure 1. The molecular structure of tofacitinib dihydrogen citrate.
Tofacitinib citrate was claimed in International Patent Applications WO 01/42246 A2 (Blumenkopf et al., Reference Blumenkopf, Flanagan and Munchhof2001) and WO 02/096909 A1 (Wilcox et al., Reference Wilcox, Koecher, Vries, Flanagan and Munchhof2002), but no powder diffraction data were provided. A powder pattern is included in European Patent EP 1451192 B1 (Flanagan and Li, Reference Flanagan and Li2002). A similar powder pattern is reported in International Patent Application WO 2015/051738 A1 (Lei et al., Reference Lei, Peng and Guo2015). Many other crystalline and amorphous salts of tofacitinib are claimed in the patent literature.
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 include high-quality powder diffraction data for these pharmaceuticals in the Powder Diffraction File (Gates-Rector and Blanton, Reference Gates-Rector and Blanton2019).
II. EXPERIMENTAL
The sample was a commercial reagent, purchased from Sigma (Lot #0000068911), 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 0.457899 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 primitive orthorhombic unit cell with a = 5.91175, b = 12.93239, c = 30.43566 Å, V = 2326.9 Å3, and Z = 4 using N-TREOR (Altomare et al., Reference Altomare, Cuocci, Giacovazzo, Moliterni, Rizzi, Corriero and Falcicchio2013). The suggested space group was P212121 (#19), which was confirmed by 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) yielded 23 hits, but no structures for tofacitinib derivatives.
A neutral tofacitinib molecule was built using Spartan ‘18 (Wavefunction, 2020), saved as a .mol2 file, and the chirality was checked to ensure that it was correct. It was converted into a Fenske-Hall Z-matrix using OpenBabel (O'Boyle et al., Reference O'Boyle, Banck, James, Morley, Vandermeersch and Hutchison2011). The structure was solved by Monte Carlo simulated annealing (parallel tempering) techniques as implemented in FOX (Favre-Nicolin and Černý, Reference Favre-Nicolin and Černý2002), using a tofacitinib and a citrate (without the acidic and hydroxyl hydrogen atoms) as fragments. One of the 34 runs yielded a cost factor much lower than the others, and was used to begin refinement.
Once the structure was solved, the task of locating the “interesting” hydrogen atoms remained. The relative orientation of the cation and the anion (Figure 2) made the process easier. As noted in Rammohan and Kaduk (Reference Rammohan and Kaduk2018), the central carboxylic acid of citric acid almost always ionizes first. The negatively charged C49–O58–O59 carboxylate group meant that N35 was protonated in preference to other potential N atoms in the tofacitinib molecule. Thus, the terminal carboxyl groups of the citrate were protonated. Examining the O⋯O distances revealed that O56⋯O59 (negative carboxylate) = 2.667 Å, and O55⋯O21 (carbonyl) = 2.658 Å. O54 and O57 were not involved in any short O⋯O distances, so the acid protons were located on O56 and O55. Similar analysis of the O⋯O distances around the hydroxyl group O60 indicated that it formed an intramolecular hydrogen bond to the negatively charged O58. Approximate H positions were placed 0.85 Å from the bonded O on these O⋯O vectors, and later optimized by the density functional theory (DFT) calculation.
Figure 2. The close contacts (indicated by cyan and red dashed lines) between the tofacitinib cation and the citrate anion, contacts which permitted deduction of the “additional” protons in the cation and the anion.
Rietveld refinement was carried out using GSAS-II (Toby and Von Dreele, Reference Toby and Von Dreele2013). Only the 1.5–27.0° portion of the pattern was included in the refinement (d min = 0.981 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 model. The Mogul average and standard deviation for each quantity were used as the restraint parameters. The restraints contributed 6.6% to the final χ 2. The hydrogen atoms were included in calculated positions, which were recalculated during the refinement using Materials Studio (Dassault, 2020). The U iso of the heavy atoms were grouped by chemical similarity. The U iso of the hydrogen atoms were constrained to be 1.3× that of the heavy atoms to which they are attached. The background was modeled using a 6-term shifted Chebyshev polynomial, along with a peak at 5.85° to model the scattering from the Kapton capillary and any amorphous component of the sample. The peak profiles were described using the generalized microstrain model, and a secnd-order spherical harmonic preferred orientation model was included.
The final refinement of 136 variables using 25 541 observations and 89 restraints yielded the residuals R wp = 0.0701 and GOF = 1.39. The largest peak (0.72 Å from O21) and hole (0.70 Å from O57) in the difference Fourier map were 0.29 and −0.28(7) eÅ−3, respectively. The largest errors in the fit (Figure 3) are in the shape of the lowest-angle peak. A weak unindexed peak at 2.29° indicates that an unidentified trace impurity is present, consistent with the stated purity of >98% in the Sigma catalog.
Figure 3. The Rietveld plot for the refinement of tofacitinib dihydrogen citrate. 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 is multiplied by a factor of 10 for 2θ > 10.0°.
A density functional geometry optimization (fixed experimental unit cell) was carried 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). 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 ~96 h.
III. RESULTS AND DISCUSSION
The synchrotron powder pattern from this study of tofacitinib dihydrogen citrate matches that reported by Flanagan and Li (Reference Flanagan and Li2002) well enough to conclude that they represent the same material (Figure 4). The refined atom coordinates of tofacitinib dihydrogen citrate and the coordinates from the DFT optimization are reported in the CIFs deposited with ICDD.
Figure 4. Comparison of the synchrotron pattern (black) of tofacitinib citrate to the experimental pattern (green) reported by Flanagan and Li (Reference Flanagan and Li2002). The published pattern was digitized using UN-SCAN-IT (Silk Scientific, 2013) and scaled to the synchrotron wavelength of 0.457899 Å using MDI JADE Pro (MDI, 2020).
The root-mean-square (rms) Cartesian displacement of the non-hydrogen atoms in the Rietveld-refined and DFT-optimized cation structures of the cation is 0.071 Å (Figure 5), and the rms displacement for the citrate anion is 0.052 Å (Figure 6), and is well within the normal range for correct structures (van de Streek and Neumann, Reference van de Streek and Neumann2014). This discussion concentrates on the DFT-optimized structure. The asymmetric unit (with atom numbering) is illustrated in Figure 7, and the crystal structure is presented in Figure 8.
Figure 5. Comparison of the Rietveld-refined (red) and DFT (blue) structures of the tofacitinib cation in tofacitinib dihydrogen citrate.
Figure 6. Comparison of the Rietveld-refined (red) and DFT (blue) structures of the dihydrogen citrate anion in tofacitinib dihydrogen citrate.
Figure 7. The asymmetric unit of tofacitinib dihydrogen citrate, with the atom numbering. The atoms are represented by 50% probability spheroids.
Figure 8. The crystal structure of tofacitinib dihydrogen citrate, viewed down the a-axis.
The crystal structure (Figure 8) consists of corrugated layers perpendicular to the c-axis. Within the layers, cation⋯anion and anion⋯anion hydrogen bonds link the fragments into a two-dimensional network parallel to the ab-plane. Between the layers, there are only van der Waals contacts.
Almost all of the bond distances, angles, and torsion angles fall within the normal ranges indicated by a Mercury/Mogul Geometry check (Macrae et al., Reference Macrae, Sovago, Cottrell, Galek, McCabe, Pidcock, Platings, Shields, Stevens, Towler and Wood2020). Only the C33–N32–C31 angle of 121.4 (average = 112.1(25)°, Z-score = 3.6) is flagged as unusual. This angle in the fused ring system lies in a minor population of the distribution.
Quantum chemical geometry optimization of the tofacitinib cation (DFT/B3LYP/6-31G*/water) using Spartan ‘18 (Wavefunction, 2020) indicated that the observed solid-state conformation is 4.1 kcal mol−1 higher in energy than the local minimum (Figure 9); the rms Cartesian displacement is 0.354 Å. The global minimum-energy conformation (MMFF, molecular mechanics force field) is lower in energy by 7.6 kcal mol−1 (Figure 10). The rms Cartesian displacement is 1.794 Å, indicating that intermolecular interactions are important in determining the observed solid-state conformation. The citrate anion is in the trans,trans conformation (about the C45–C46 and C46–C47 bonds), one of the two low-energy conformations of a citrate (Rammohan and Kaduk, Reference Rammohan and Kaduk2018). The O58–C49–C46–O60 torsion angle is 3.4 degrees indicating that the central portion of the citrate is in the normal planar conformation.
Figure 9. Comparison of the DFT-optimized solid-state conformation of the tofacitinib cation (blue) to the local minimum conformation (green). The rms cartesian displacement is 0.354 Å.
Figure 10. Comparison of the local minimum conformation (green) to the global minimum-energy conformation of an isolated tofacitinib cation (orange). The rms cartesian displacement is 1.794 Å.
Analysis of the contributions to the total lattice energy using the Forcite module of Materials Studio (Dassault, 2020) suggests that angle deformation terms are the dominant contributions to the intramolecular deformation energy, as expected for a molecule which contains a fused ring system. The intermolecular energy is dominated by electrostatic attractions, which in this force-field-based analysis include hydrogen bonds. The hydrogen bonds are better analyzed using the results of the DFT calculation.
As expected, hydrogen bonds are important in the crystal structure (Table I). The carboxylic acid O56–H64 forms a strong charge-assisted hydrogen bond to the ionized carboxylate O59. The other carboxylic acid O55–H63 acts as a donor to the carbonyl group O21. The citrate hydroxy group O60–H61 forms an intramolecular charge-assisted hydrogen bond to the ionized carboxylate O58. The two protonated nitrogen atoms N35–H62 and N38–H39 act as donors to the ionized carboxylate O59 and O58. These hydrogen bonds form a ring with the graph set symbol R2,2(8) (Etter, Reference Etter1990; Bernstein et al., Reference Bernstein, Davis, Shimoni and Chang1995; Shields et al., Reference Shields, Raithby, Allen and Motherwell2000). The energies of the O–H⋯O hydrogen bonds were calculated using the correlation of Rammohan and Kaduk (Reference Rammohan and Kaduk2018), and the energies of the N–H⋯O hydrogen bonds according to the correlation of Wheatley and Kaduk (Reference Wheatley and Kaduk2019). Several C–H⋯O and C–H⋯N hydrogen bonds also contribute to the lattice energy.
Table I. Hydrogen bonds (CRYSTAL14) in tofacitinib dihydrogen citrate.
* indicates intramolecular.
The volume enclosed by the Hirshfeld surface (Figure 11; Hirshfeld, Reference Hirshfeld1977; Turner et al., Reference Turner, McKinnon, Wolff, Grimwood, Spackman, Jayatilaka and Spackman2017) is 571.98 Å3, 98.34% of one-fourth the unit cell volume. The molecules are, thus, not tightly packed. All of the significant-close contacts (red in Figure 11) involve the hydrogen bonds. The volume/non-hydrogen atom is 16.1 Å3.
Figure 11. The Hirshfeld surface of tofacitinib dihydrogen citrate. 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 elongated morphology for tofacitinib dihydrogen citrate, with <100> as the principal axes. A second-order spherical harmonic model for preferred orientation was incorporated into the refinement. The texture index was only 1.000(0), indicating that preferred orientation was not present in this rotated capillary specimen. The powder pattern of tofacitinib dihydrogen citrate from this synchrotron data set has been submitted to ICDD for inclusion in the Powder Diffraction File.
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
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.
