Crystal structures of tolfenamic acid polymorphic forms I and II with precise hydrogen-atom positions for nuclear magnetic resonance studies

The structures of tolfenamic acid polymorph forms I and II have been redetermined with improved precision of the hydrogen-atom positions by Hirshfeld atom refinement to provide improved data for solid- and solution-state nuclear magnetic resonance studies.

The structures of tolfenamic acid [TFA; 2-(3-chloro-2-methylanilino)benzoic acid, C 14 H 12 ClNO 2 ] polymorph forms I and II have been redetermined [compare Andersen et al. (1989). J. Chem. Soc., Perkin Trans. 2, pp. 1443-1447 with improved precision using high-resolution X-ray diffraction data and Hirshfield atom refinement in order to better define both hydrogen-atom locations and their associated bond lengths. Covalent bond lengths to hydrogen were found to be significantly longer throughout both structures, especially for the anilino H atom, which is involved in an important intramolecular N-HÁ Á ÁO hydrogen bond to the carboxylic acid group. This hydrogen bond is shown to clearly perturb the electron density around both oxygen atoms in the latter group. The extended structures of both polymorphs feature carboxylic acid inversion dimers. These structures provide an improved foundation for nuclear magnetic resonance studies in both solution and the solid state.

Chemical context
Tolfenamic acid (TFA; 2-[(3-chloro-2-methylphenyl)amino]benzoic acid; C 14 H 12 ClNO 2 ) is a non-steroidal anti-inflammatory drug (NSAID). It is frequently used as a model for crystallography studies because it displays interesting polymorphism, with eight forms identified to date (Andersen et al., 1989;Ló pez-Mejías et al., 2009;Case et al., 2018). Moreover, its small size and simple crystal structures permit timely computational calculations, which is advantageous for studies investigating its behaviour by nuclear magnetic resonance (NMR).
The two most common polymorphs of TFA (forms I and II) differ principally in the dihedral angles between the phenyl rings, giving different overall conformations and attendant packing arrangements (Andersen et al., 1989). A number of experimental and theoretical studies have been performed on TFA to explore the origin of this (see e.g. Ang et al., 2016;Du et al., 2015;Mattei & Li, 2012Mattei et al., 2013). Both form I and form II are easily prepared in sufficient amounts and purity under standard laboratory conditions to permit ISSN 2056-9890 solid-state NMR studies and they are readily distinguishable by their colour (form I, white; form II, yellow) (Andersen et al., 1989). TFA is soluble in a variety of crystallization solvents and is suitable for having its conformational behaviour precisely characterized by solution-state NMR methods (Blundell et al., 2013).
Our motivation for the present study was the desire to generate high resolution structures of both forms I and II in which the locations of the hydrogen atoms and their attendant bond lengths were precisely resolved. Accurate hydrogen positions are important for both solid-and solution-state NMR studies because 1 H is a chief nucleus for acquiring experimental data through and a variety of experimental observables depend sensitively on bond lengths to hydrogen (e.g. chemical shifts of hydrogen atoms involved in hydrogen bonding, see Siskos et al., 2017; residual dipolar couplings, see Lipsitz & Tjandra, 2004). Structures with precise hydrogen positions therefore provide more robust starting points for density functional theory (DFT) calculations of NMR observables in the solid-state and, in the solution state, a better mean-average geometry to found conformational analysis upon.
Accordingly, large single crystals (needles 0.5-1 mm in length) of forms I and II were grown that diffracted to d ' 0.48 Å (2 max = 95.5 , T = 100 K) and 0.53 Å (83.6 , 150 K), respectively with Mo K radiation. The structures of each form were solved and refined using Hirshfeld atom refinement, which determines the structural parameters from single crystal X-ray diffraction data by using an aspherical atom partitioning of ab initio quantum mechanical molecular electron densities (Capelli et al., 2014). Significantly for our purpose, the precision of the determined bond lengths and anisotropic displacement parameters for the hydrogen atoms calculated with Hirshfeld atom refinement with data of this resolution is comparable to that from neutron diffraction measurements (Fugel et al., 2018).

Structural commentary
The crystal structure determination of forms I and II was achieved (Fig. 1). Both forms are monoclinic, with form I in space group P2 1 /c and form II in P2 1 /n; both structures comprise Z 0 = 1 and Z = 4 and form an internal hydrogen bond between N7-H7 and O15 with very similar geometry (Tables  1 and 2); this internal hydrogen bond makes the aminobenzoic acid group adopt an essentially planar conformation. The chief difference between the two forms is the dihedral angle between the C1-C6 and C8-C13 phenyl rings, being 72.82 (4) for form I and 44.34 (3) for form II. This is also seen in the torsion angle C8-N7-C1-C6, with values of 74.34 (12) and À143.00 (6) for forms I and II, respectively (in the crystal an equal number of molecules have the opposite sign for these torsion angles). Additionally, the C8-N7-C1 angle also differs somewhat [form I 123.97 (7); form II 129.34 (5)  The molecular structures of TFA in form I and form II, with atom labelling. The internal N7-H7Á Á ÁO15 hydrogen bond is indicated with a dotted line. Displacement ellipsoids are drawn at the 50% probability level. Table 1 Hydrogen-bond geometry (Å , ) for form I. Symmetry codes: (i) Àx; Ày þ 1; Àz þ 1; (ii) x; Ày þ 3 2 ; z À 1 2 .

Table 2
Hydrogen-bond geometry (Å , ) for form II. the 3-chloro-2-methylphenyl ring and form II having one hydrogen atom orthogonal to it. The displacement parameters of the ellipsoids show that the methyl groups in both structures display greater motion relative to the rest of their structures.

Supramolecular features
The packing arrangement for both forms are shown in Fig. 2  Crystal structure of TFA form II, showing the hydrogen-bond network (red dotted lines) and the deformation of the electron density calculated by TONTO (Capelli et al., 2014). Negative electron density is shown with green surfaces and positive electron density is shown with red (threshold level À0.25, Res/Å ). Ellipsoids of carbon atoms are shown in grey, nitrogen blue, oxygen red, hydrogen white and chlorine green.

Figure 4
Detail of the hydrogen-bond network of TFA form I in two orientations, showing the hydrogen bonds (red dotted lines) and the deformation of the electron density calculated by TONTO (Capelli et al., 2014). The differing molecular orbitals for O15 and O16 are clearly visible. Negative electron density is shown with green surfaces and positive electron density is shown with red (threshold level À0.25, Res/Å ). Ellipsoids of carbon atoms are shown in grey, nitrogen blue, oxygen red and hydrogen white.
The structure determination has precisely resolved not only hydrogen-atom positions but also the shape and positions of the electron density corresponding to the molecular orbitals throughout the structure (Figs. 3 and 4). Very clear differences between the carboxylate oxygen atoms in the dimer hydrogenbonding motif are now apparent. Bond C14-O15 has more electron density than C14-O16, revealing its greater doublebond character. Oxygen atom O15 accordingly adopts an sp 2 geometry, with its two lone-pairs clearly located in the expected co-planar positions (i.e., at AE120 from the C15-O16 bond); one lone pair accepts the intramolecular hydrogen bond from H7 while the other receives an intermolecular hydrogen bond from H16. Atom O16 in contrast has a covalent bond to H16, for which electron density is clearly visible. Despite the apparently dominant double-bond character of C14-O15, O16 is interestingly not simply forming a purely single bond with C14 and adopting an sp 3 hydridization state: the typical positions of the two sp 3 molecular orbitals projecting away from and above and below the carboxylate plane have smeared into a single lobe of density with a significant amount of coplanar (i.e., sp 2 -like) electron density.
In short, the typical equivalence of the O atoms in the carboxylic acid has been significantly perturbed by the N7-H7Á Á ÁO15 intramolecular hydrogen bond. These electronic perturbations should be remembered when molecules containing carboxylic acid groups are being designed to interact with protein targets via hydrogen bonding.  Surov et al., 2015, Wittering et al., 2015. In all cases, the internal hydrogen-bond equvialent to N7-H7Á Á ÁO15 in the present structures is present and the hydroxyl hydrogen atom is attached to the corresponding O16 equivalent. The C8-N7-C1-C6 torsion angle ranges from 75.0 to 138.4 in the pure forms and from 76.1 to 156.9 in the cocrystals.

Database survey
The database structures with refcodes KAXXAI01 and KAXXAI correspond to the crystal structures of form I and form II redetermined here at higher resolution. The locations of the heavy atoms in these new structures do not differ significantly from those reported previously even though, as expected, some hydrogen-atom locations differ substantially (Fig. 5). H7 is in a significantly different position in both structures, materially affecting both its associated hydrogenbond geometries (compare Tables 1 and 2 with Table 3) and covalent bond lengths. The N-H bond length is some 23% longer for form I and 17% for form II, which would correspond to a considerable calculated difference in residual dipolar couplings by factors of 1.9 and 1.6 times smaller, respectively. The O-H bond length is slightly longer by 5% for form I and 6% for form II. Carbon-hydrogen bond lengths are also notably longer at 13 AE 3% (min. 6%, max. 17%) for form I and 10 AE 3% (min. 5%, max. 14%) for form II; C-C-H bond angles differ absolutely by 2.6 AE 1.8% (min. 0.0, max 5.7 ) for form I and 1.6 AE 1.3 (min. 0.2, max 5.2 ) for form II. This improved precision in hydrogen-atom placement provides a significant structural enhancement for subsequent solid-and solution-state NMR studies.   Table 3 Hydrogen-bond geometry (Å , ) for structures of forms I and II determined by Andersen et al. (1989).  Symmetry codes: (i) Àx, 1 À y, 1 À z; (ii) 2 À x, 1 À y, 1 À z.

Synthesis and crystallization
Tolfenamic acid was used as received from Sigma-Aldrich (Gillingham, UK). Large single crystals of form I (needles 0.5-3 mm in length) were grown by slow evaporation at room temperature: 3 mg of compound was dissolved in an initial volume of 200 ml of ethyl acetate and the mixture was allowed to evaporate to dryness over 24-36 h.
Large single crystals of form II (needles 0.3-1 mm in length) were obtained serendipitously from an attempted salt crystallization experiment, during which form II crystals suitable for single-crystal X-ray diffraction were isolated. A 1:1 molar ratio of TFA (20 mg of compound) and N-(2-hydroxyethyl)pyrrolidine were dissolved into approximately 5 ml ethyl acetate. The mixture was then left to slowly evaporate, during which large yellow needles formed. Single-crystal diffraction performed on multiple crystals indicated that these were pure form II. There was no evidence of form I within the product, nor of any inclusion of N-(2-hydroxyethyl)-pyrrolidine within the form II crystals. Clearly the presence of N-(2-hydroxyethyl)-pyrrolidine either inhibited the growth of form I crystals and/or promoted the growth of form II crystals; the mechanism for this has not been investigated.

Refinement
Crystal data, data collection and structure refinement details are summarized in Table 4.
Intensity data for form I and form II were collected using Mo K radiation at 100 and 150 K respectively using a Rigaku FR-X rotating anode diffractometer, equipped with an HyPix-6000HE detector and an Oxford Cryosystems nitrogen flow gas system. Data were measured and reduced using the CrysAlis PRO suite of programs. Absorption correction was performed using empirical methods implemented in the SCALE3 ABSPACK scaling algorithm (Blessing, 1995;Sheldrick, 1996). The crystal structures were solved and refined against all F 2 values using the SHELXL and OLEX2 suite of programs (Sheldrick, 2008(Sheldrick, , 2015bDolomanov et al., 2009). All atoms (including H atoms) were refined anisotropically.
Hirshfeld atom refinement was achieved using the recently implemented HARt option in OLEX2 (Fugel et al., 2018). It precisely estimates the atomic positions and deformation electron densities in crystal structures by deconvolution of accurate static electron density calculated by TONTO from the thermally smeared electron density calculated from an independent atomic model (IAM) obtained from the X-ray diffraction data (Capelli et al., 2014).
The wavefunctions of crystal structures of form I and form II were calculated using TONTO (Capelli et al., 2014), with the cc-pVTZ basis set (Dunning, 1989) and the Hartree-Fock method. Wavefunctions for each crystal structure were calculated with the crystal structure grown in order to account for the hydrogen bond formed between the carboxylate groups. Each model obtained was then refined against the single crystal X-ray diffraction data collected, using OLEX2 and the L-M method (Fugel et al., 2018).  For both structures, data collection: CrysAlis PRO (Rigaku OD, 2017); cell refinement: CrysAlis PRO (Rigaku OD, 2017); data reduction: CrysAlis PRO (Rigaku OD, 2017); program(s) used to solve structure: SHELXT (Sheldrick, 2015a); program(s) used to refine structure: olex2.refine (Bourhis et al., 2015); molecular graphics: OLEX2 (Dolomanov et al., 2009); software used to prepare material for publication: OLEX2 (Dolomanov et al., 2009).