A Spectroscopic and Diffractometric Study of Polymorphism in Ethyl 3

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A Spectroscopic and Diffractometric Study of Polymorphism in Ethyl 3‑{3-[((2R)‑3-{[2-(2,3-dihydro‑1H‑inden-2-yl)-1,1dimethylethyl]amino}-2-hydroxypropyl)oxy]-4,5difluorophenyl}propanoate Hydrochloride Frederick G. Vogt,*,† Glenn R. Williams,†,# Matthew N. Johnson,‡,⊥ and Royston C. B. Copley§ †

Product Development, GlaxoSmithKline plc., 709 Swedeland Road, King of Prussia, PA, United States, 19406 Product Development, GlaxoSmithKline plc, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, U.K. § Molecular Discovery Research, GlaxoSmithKline plc., Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, U.K. ‡

S Supporting Information *

ABSTRACT: Two polymorphic forms of ethyl 3-{3-[((2R)3-{[2-(2,3-dihydro-1H-inden-2-yl)-1,1-dimethylethyl]amino}2-hydroxypropyl)oxy]-4,5-difluorophenyl} propanoate hydrochloride, an investigational pharmaceutical compound, are characterized using spectroscopic and diffractometric techniques. These polymorphic forms exhibit very similar spectra and diffraction patterns and present challenges for analytical and physical characterization techniques. Capillary powder Xray diffraction (PXRD) patterns for the two forms show minor but distinct differences. A single crystal X-ray diffraction structure for one of the forms was obtained. The unit cell of the other form was obtained by PXRD indexing. Detailed solid-state nuclear magnetic resonance (SSNMR) studies observing the 1H, 13C, 15N, 19F, and 35Cl nuclei are performed to characterize the subtle structural differences between the two forms. Molecular spectroscopic methods including infrared, Raman, UV−visible, and fluorescence spectroscopy are also applied. The combined results, particularly the results obtained from X-ray diffraction analysis, 13C, 15N, and 35Cl SSNMR, and fluorescence spectroscopy, are consistent with the more thermodynamically stable form having a structure that is an extended, perturbed superstructure of the less stable form.

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INTRODUCTION

Achieving understanding and control of polymorphic crystalline drug phases remains an important consideration in the development of pharmaceutical compounds.1−4 Lack of control over polymorphic form can lead to active pharmaceutical ingredient processes that yield solids with irreproducible bulk properties, dissolution performance, appearance, physical stability, and chemical stability.1−4 Analytical methods for studying polymorphic forms, obtaining insight into their structures, and ensuring the presence of a single reproducible phase are thus of great interest.3−5 In some cases, structural similarity between polymorphic forms can challenge the ability of analytical techniques to discriminate between forms and to interrogate their structural differences. This challenge is illustrated in the present study, which examines two polymorphic forms of a developmental drug compound, ethyl 3-{3-[((2R)-3-{[2-(2,3-dihydro-1H-inden-2-yl)-1,1dimethylethyl]amino}-2-hydroxypropyl)oxy]-4,5-difluorophenyl} propanoate hydrochloride (I).6 Compound I crystallizes in two polymorphic forms that exhibit similar spectroscopic and diffractometric properties. This similarity presents challenges for structural analysis of the polymorphs. In the present work, these polymorphs are studied © XXXX American Chemical Society

using a range of analytical techniques to examine subtle structural differences. One of the polymorphs was successfully characterized by single-crystal X-ray diffraction (SCXRD) analysis. Suitable single crystals of the other polymorph could not be grown for SCXRD analysis. Instead, the structural relationships between the forms are elucidated using powder Xray diffraction (PXRD), PXRD indexing analysis, and sensitive Received: August 12, 2013 Revised: October 6, 2013

A

dx.doi.org/10.1021/cg401232g | Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

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spectroscopic techniques including 1H, 13C, 15N, 19F, and 35Cl solid-state nuclear magnetic resonance (SSNMR), diffuse reflectance (DR) UV−visible spectroscopy, fluorescence spectroscopy, infrared (IR) spectroscopy, and Raman spectroscopy. The multinuclear SSNMR approach applied here has been found to be particularly useful in studies of pharmaceutical polymorphism.7 High static field strengths (B0) of 16.4 T were used for 35Cl SSNMR analysis and for 1H SSNMR analysis, the latter in conjunction with recently introduced homonuclear decoupling methods. The results are interpreted collectively and with reference to density functional theory (DFT) calculations to evaluate the structural differences between the two polymorphs and the relationship between spectroscopic observables and the stability of the forms.

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X-ray Diffraction. SCXRD analysis was performed on a single crystal of Form 1 grown by ethyl acetate and heptane vapor diffusion. Single-crystal diffraction experiments were performed using a Nonius KappaCCD diffractometer (Bruker AXS, Madison, WI, USA). A normal focus sealed tube Mo Kα radiation source (0.71073 Å) was employed. Further details of the data collection, structure solution and refinement are summarized in Table 1, and additional details can be

Table 1. Summary of the SCXRD Analysis of Form 1 moiety formula empirical formula formula weight temperature (K) wavelength (Å) crystal size crystal habit crystal system space group unit cell dimensions a, α b, β c, γ volume formula units per cell (Z) formula units in the asymmetric unit (Z′) calculated density (g/cm3) absorption coefficient (μ) (mm−1) F000 theta range (°) index ranges

EXPERIMENTAL AND COMPUTATIONAL METHODS

Preparation of Materials and Optical Microscopy. Compound I was synthesized by previously reported methods.6 Form 1 was prepared by antisolvent addition of tert-butylmethylether to the final step of the reaction mixture (consisting primarily of toluene and dioxane) and can also be prepared by addition of a concentrated (60 mg/mL) acetone solution of I to an excess of either cyclohexane or water. Form 2 was prepared by competitive ripening of Form 1 using phase mixtures of Forms 1 and 2, after Form 2 was first obtained during larger scale unseeded preparations of Form 1. Competitive ripening experiments were performed by charging 1:1 mixtures of the two forms in toluene and other solvents and ripening for six weeks. These experiments established that Forms 1 and 2 have a monotropic relationship and that Form 2 is the more thermodynamically stable form. Optical microscopy was performed with brightfield illumination and polarized light using an Optiphot POL microscope (Nikon Metrology, Inc., Brighton, MI, USA). Samples were dispersed in silicone oil. Thermal Analysis. Differential scanning calorimetry (DSC) experiments were performed using a Q1000 calorimeter (TA Instruments, New Castle, DE, USA). The analysis was performed on preweighed samples with masses of approximately 2−3 mg. Samples were placed in aluminum pans that were sealed by applying hand pressure. The temperature was ramped from 25 to 300 °C at a rate of 10 °C/min. Dry nitrogen was used as a purge gas with a flow rate of 20 mL/min. The calorimeter was calibrated for enthalpy and temperature using indium. Vibrational Spectroscopy. Infrared (IR) spectra were obtained using a Spectrum One Fourier transform (FT) spectrometer equipped with a deuterated triglycine sulfate detector (Perkin-Elmer, Waltham, MA, USA). A total of 32 scans were averaged at a resolution of 1 cm−1 for each of the spectra, requiring approximately 20 min per spectrum. Spectra were obtained using a single-bounce attenuated total reflectance (ATR) accessory with a diamond window. Samples were covered with a glass slide and pressed against the ATR window for analysis. DR IR spectra were obtained using a Vertex 70 spectrometer (Bruker Optics, Billerica, MA, USA) and an Auto Seagull variable angle DR accessory set to a 50° angle (Harrick Scientific Products, Pleasantville, NY, USA). A cryogenic mercury cadium telluride detector was used (Infrared Associates, Stuart, FL, USA). DR IR spectra were obtained using 512 scans with 1 cm−1 resolution, and CsI powder was used as a background. Raman spectra were obtained using a MultiRAM FT Raman spectrometer equipped with a cooled germanium diode detector (Bruker Optics, Billerica, MA, USA). Laser excitation utilized a 1 W, 1.064 μm diode-pumped neodymium-doped yttrium aluminum garnet (Nd:YAG) laser that was attenuated to an output power of 0.6 W (Klastech GmbH, Dortmund, Germany). The 180° scattered signal was collected using a resolution of 1 cm−1 and 2048 scans. Samples were placed in 5 mm outer diameter borosilicate glass tubes for Raman analysis.

measured reflections independent reflections R(int) coverage of independent reflections (%) absorption correction max/min transmission data/restraints/parameters goodness of fit on F2 final R indices for I > 2σ(I) data final R indices for all data absolute structure parameter largest diff peak and hole (e·Å−3)

[C27H36F2NO4]+Cl− C27H36ClF2NO4 512.02 150(2) 0.71073 0.40 × 0.06 × 0.06 mm colorless needle triclinic P1 7.9028(13) Å, 61.675(14)° 13.740(3) Å, 86.564(13)° 13.9223(16) Å, 84.041(17)° 1323.5(4) Å3 2 2 1.285 0.191 544 3.96−25.00 −9 ≤ h ≤ 9 −16 ≤ k ≤ 16 −16 ≤ l ≤ 16 29679 9019 0.0720 99.3 semiempirical from equivalents 0.989 and 0.928 9019/640/753 1.008 R1 = 0.0551 wR2 = 0.1051 R1 = 0.0952 wR2 = 0.1195 0.00(8) 0.322 and −0.247

found in the Supporting Information and in the crystallographic information file (CIF) deposited in the Cambridge Structural Database (see Supporting Information).8 The structure was solved using direct methods and refined using full-matrix least-squares procedures that minimized the function Σw(Fo2 − Fc2)2. The SHELXTL software package (version 6.10, Windows NT, Bruker AXS, Madison, WI, USA) was used for solution and refinement. Coordinates and anisotropic atomic displacement parameters were refined for the non-hydrogen atoms, with geometric and atomic displacement restraints being applied to several disordered sections found to be present in the two independent cations. The disorders were modeled using two alternative sites for each affected atom using refined site occupancy factors. Hydrogen atom positions on heteroatoms were also refined with distance restraints, although the hydrogen on O17 could not be located. Hydrogen atoms bonded to carbons were placed in idealized positions and allowed to ride on attached atoms. The isotropic atomic displacement parameters for all hydrogen atoms were used as an B



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appropriate multiple of the atomic displacement parameters of the bonded atom. PXRD patterns were obtained with an X’Pert Pro diffractometer using Debye−Scherrer transmission geometry with a focusing mirror and an X’Celerator real time multistrip detector (PANalytical B.V., Almelo, The Netherlands). Samples were loaded into 0.91 mm (inner diameter) polyimide capillaries and analyzed at ambient temperature and humidity. Cu Kα radiation (with wavelengths of 1.54056 and 1.54439 Å) was employed with generator voltage and current settings of 45 kV and 40 mA. Samples were scanned in continuous mode from 3° to 60° with a 2θ step size of 0.0167°. The sample was rotated in a capillary spinner at a rate of >1000 rpm. The diffractometer incident beam path was equipped with a focusing mirror, 0.02 rad Soller slit, and a 0.5° fixed divergence slit, while the diffracted beam path was equipped with a programmable antiscatter slit (fixed at 0.25°) and a 0.02 rad Soller slit. Pawley refinement of the PXRD data was performed using the Reflex module within the Materials Studio version 6.0 software package (Accelrys, San Diego, CA, USA).9 Indexing of the Form 2 PXRD pattern was accomplished using the DICVOL package as implemented in Materials Studio.10 The X-cell package in Materials Studio was also employed to ensure exhaustive search results were obtained.11 Solid-State NMR Spectroscopy. SSNMR spectra were obtained using two spectrometers, an Avance I system with a 1H frequency of 400.13 and a B0 of 9.4 T, and an Avance III system with a 1H frequency of 700.13 MHz and a B0 of 16.4 T (Bruker Biospin, Billerica, MA, USA). Experiments were performed using several magic-angle spinning (MAS) probes as described below with zirconia rotors spinning at different rates (νr). 1H−13C, 1H−15N, and 1H−19F crosspolarization (CP) transfers were performed at matching conditions of approximately 40−80 kHz using a linear ramp on the 1H radiofrequency (RF) channel.12 13C CP spectra were obtained with a fivepulse total sideband suppression (CP-TOSS) sequence, 1944 scans, and a 10 s relaxation delay using a 4 mm Bruker HFX MAS probe.13 19 F spectra were obtained using the same probe and a conventional CP-MAS pulse sequence. 15N spectra were also obtained using a CPMAS pulse sequence with a 7 mm Bruker HX MAS probe using 16384 scans and a 5 s relaxation delay. 13C, 15N, and 19F CP experiments utilized a 2 ms contact time and SPINAL-64 heteronuclear 1H decoupling performed at an RF power of approximately 105 kHz for 19 F and 13C experiments and 70 kHz for 15N experiments.14,15 Edited 13 C spectra were measured using 3888 scans and a 10 s relaxation delay using dipolar dephasing by interrupting the 1H decoupling during the TOSS period and three subsequent rotor periods that were formed with a shifted echo.13,16 1H−19F−13C double CP (DCP) experiments were obtained using a TOSS sequence added to a conventional DCP pulse sequence.17,18 1H−13C CP-HETCOR experiments were performed using frequency-switched Lee−Goldburg (FSLG) decoupling of 1H nuclei and a ramped 1H CP pulse.19 1H and 35 Cl spectra were obtained using a Bruker 2.5 mm HX MAS probe with an X-channel tunable to 35Cl, 14N, and 17O frequencies using single-pulse direct polarization MAS (DP-MAS) experiments. The 35 Cl DPMAS spectra were obtained using a π/2 pulse width of 2 μs. 1 H homonuclear decoupling during the free-induction decay period was performed by means of the “decoupling using mind boggling optimization” (DUMBO) scheme using the windowed eDUMBO-122 variant of this sequence.20,21 The 1H T1 relaxation times of Forms 1 and 2 were measured using a saturation recovery pulse sequence by direct 1H observation. 1H spectra were externally referenced to the tetramethylsilane (TMS) scale using a sample of crystalline L-alanine with the methyl peak set to 1.6 ppm. 13C spectra were referenced to the TMS scale using an external standard of solid hexamethylbenzene at 17.36 ppm.22 15N spectra were externally referenced to the nitromethane scale using a solid sample of NH4Cl at −341.0 ppm.23 19 F spectra were referenced to CFCl3 by use of frequency ratios relative to the 13C reference.24 35Cl spectra were externally referenced to crystalline NaCl at 0 kHz. UV−visible Spectroscopy. UV−visible spectra were obtained using a Varian Cary 50 spectrometer (Agilent, Palo Alto, CA, USA).

For solid samples, diffuse reflectance (DR) spectra were obtained using a Barrelino diffuse reflectance probe (Harrick Scientific Products, Pleasantville, NY, USA). DR UV−visible spectra were collected on neat powders over the region of 200−1100 nm using a scan rate of 60 nm/min. Measurements were also performed on diluted samples prepared by physical blending with magnesium oxide powder as discussed below. Transmission UV−visible spectra were obtained in solution using quartz cuvettes with a 1 cm path length. The instrument was calibrated for wavelength accuracy using four maxima in the spectrum of holmium oxide (in the range 279.4−637.5 nm) and calibrated for photometric accuracy using four maxima in the spectrum of K2Cr2O7. Diffuse reflectance calibration for 0% and 100% reflectance was performed with Spectralon standards (Labsphere, North Sutton, NH, USA). Fluorescence Spectroscopy. Fluorescence spectra were recorded using a Fluorolog 3 spectrometer equipped with double-grating Czeny-Turner monochromators and reflective optics (Horiba Jobin Yvon, Edison, NJ, USA). The excitation source was a 450 W Xe lamp, the signal detector was a photomultiplier tube, and the reference detector was a stabilized Si photodiode. Diffraction gratings were ruled at 1200 tr/mm and blazed at 330 and 500 nm for the excitation and emission monochromators, respectively. A slit width of 1−5 nm was used for both monochromators. Monochromator dispersion was 2.1 nm/mm. Detection times of 1−5 s were used. Samples were analyzed in front-facing mode at an angle of 30° using a black-anodized aluminum solid powder holder with a quartz cover plate. The excitation monochromator was calibrated using the lamp emission line at 467 nm, and the emission monochromator was calibrated using 350 nm excitation of the Raman line in water at 397 nm. All spectra shown are the result of dividing the detector response by the reference diode response. Spectra were analyzed using FluorEssence version 2.0 software (Horiba Jobin Yvon, Edison, NJ, USA). Computational Methods. Periodic boundary condition DFT calculations of NMR parameters were performed using Materials Studio version 6.0 (Accelrys, San Diego, CA, USA). The Form 1 crystal structure contains disordered atoms as discussed below. The disorder was approximated for the purpose of calculating NMR parameters by separating the disordered components into two crystal structures referred to as the “major” component (containing the disordered regions with atomic occupancies near to 0.6) and the “minor” component (containing the disordered regions with occupancies near 0.4). The DMol3 DFT package in Materials Studio was used to optimize the hydrogen atom positions in both structures.25,26 The hydrogen position optimization was performed using the HCTH/407 generalized gradient approximation (GGA) density functional (referred to as the HCTH functional) with a double numerical basis set that included polarization functions on all atoms (referred to as the DNP basis set).27,28 The Brillouin zone integration for the optimization used a 2 × 1 × 1 k-space. The hydrogen attached to O17 was not located in the crystal structure of Form 1. This position was added to the structure in several alternative orientations and energy-minimized, and the lowest energy geometry found was used for calculations. Additional CIFs were generated for each of the two optimized structures (see Supporting Information). The CASTEP package in Materials Studio was used for electric field gradient (EFG) and chemical shielding calculations.29 Chemical shielding calculations were performed using the gauge-including projector augmented wave (GIPAW) method.30 The GIPAW calculations were performed using the PBE functional, a plane wave cutoff energy of 500 eV, on-the-fly generated pseudopotentials, and the Γ-point.30,31 The GIPAWcalculated isotropic chemical shielding is denoted by σiso. Additional details about the principal components of the chemical shielding tensor are given in the Supporting Information. The quadrupolar coupling constant is defined as CQ = (eQVzz)/h, where Vzz refers to the largest absolute component of the EFG tensor ordered according to the convention |Vzz| ≥ |Vyy| ≥ |Vxx|. The dimensionless quadrupolar asymmetry parameter (ηQ) is given by ηQ = (Vxx − Vyy)/Vzz. C



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RESULTS AND DISCUSSION PXRD and SCXRD Analysis. The structural similarity between Forms 1 and 2 is highlighted by their capillary PXRD patterns, which are compared in Figure 1a. The patterns were

means of the Pawley procedure using the experimental PXRD patterns of both Forms 1 and 2 as input (see Supporting Information). The pattern from Form 1 refined well, with no significant unmodeled features, a weighted-profile residual value (Rwp) of 3.76%, and a nonweighted residual value (Rp) of 8.37%. In contrast, the best Rwp that could be obtained for a Pawley refinement of the SCXRD unit cell against the Form 2 experimental PXRD pattern was 22.47%, and many major features of the pattern were unmodeled. The most prominent of the unmodeled features occurred at 2θ angles of 11.5°, 12.1°, 15.0°, 16.7°, and 18.2°. Despite the similarity of their PXRD patterns, Pawley refinement was able to readily distinguish the form for which the single crystal structure was obtained. The unit cell from the Pawley refinement of Form 1 expanded to a volume of 1338.5 Å3. This expansion was consistent with a unit cell of Form 1 that was determined by raising the temperature of the single crystal to ambient temperature (approximately 295 K) and measuring the positions of 26 reflections, which resulted in a unit cell with a cell volume of 1340.3(6) Å3. A comparison of the SCXRD unit cells at 150 K and ambient temperature showed that the caxis of the Form 1 cell contracted by about 0.7% and the b-axis contracted by 0.1%, while the a-axis dimension increased by 0.6% when the crystal was warmed by 145 K (see Supporting Information). A notable increase of 2.5% in the value of the angle α occurred upon warming of the crystal. The overall changes in cell angles and dimensions between 150 and ambient temperature lead to a small increase in cell volume of 1.3%. Views of the Form 1 crystal structure showing atomic displacement ellipsoids are depicted in Figure 2. The asymmetric unit contains two independent cations of I (Z′ = 2), referred to as cation A (Figure 2a) and cation B (Figure 2b),

Figure 1. Capillary transmission PXRD patterns of the two polymorphs of I obtained at ambient conditions showing the similar patterns obtained for the polymorphs. The patterns are compared in (a), with an expanded view shown in (b). The patterns have been baseline corrected to allow for easier comparison.

scanned from 3° to 60° 2θ, but no significant reflections were observed after 45°. Over the most useful range of 10−30°, the patterns exhibit a very similar appearance with many of the Bragg reflections overlapped or indistinguishable, as seen in Figure 1b. The strongest reflection in each pattern appears at a maximum of 7.21° in Form 1 and at 7.17° in Form 2, values which differ by an amount within the peak width of these reflections. The d-spacing values associated with this reflection are 12.26 Å in Form 1 and 12.33 Å in Form 2. Despite their similarity and peak overlap, peaks specific for each of the polymorphs can be observed and used to confirm that Form 1 is not detected in Form 2 and vice versa. In Form 1, the most specific reflections occur at 13.22°, 13.81°, 15.14°, 16.33°, 16.54°, 18.06°, and 27.81°. In Form 2, the most specific reflections appear at 14.18°, 15.01°, 16.73°, 17.51°, 18.19°, 22.87°, 25.43°, and 27.04°. A crystal structure with a final R1 of 5.51% was obtained by SCXRD for a crystal grown by vapor diffusion using ethyl acetate and heptane. The results of the structure determination are summarized in Table 1 and in the Supporting Information.8 To ensure that the single crystal used to obtain this structure was representative of one of the polymorphs of interest here, the unit cell obtained from the SCXRD structure was refined by

Figure 2. (a) A view of cation A from the Form 1 crystal structure, showing the numbering scheme employed. Both components of the indanyl disorder are shown. The hydroxyl hydrogen atom was not located. (b) A view of cation B and the two chloride anions from the Form 1 crystal structure, showing the numbering scheme employed. Atoms from all of the disordered components are shown. For both cations, anisotropic atomic displacement ellipsoids for the nonhydrogen atoms are shown at the 50% probability level. Hydrogen atoms are displayed with an arbitrarily small radius. The numbering scheme for cation B can be related to cation A by adding 40 to the atomic number in cation A. D



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together with two independent chloride counterions. The numbering scheme for cation B is obtained by adding 40 to the equivalent atom number in cation A. The SCXRD study unambiguously proved that C16 and C56 have the (R)configuration. The indanyl group of cation A, the linker between the protonation site and the indanyl group of cation B, and the ester side-chain of cation B were all disordered over two positions. In each case, the atomic site occupancy factor for atoms in the major components of the various disordered regions refined to a value of approximately 0.6 (see Supporting Information). The minor components of the disordered regions in the Form 1 crystal structure refined to approximately 0.4. The hydrogen atom of the cation A hydroxyl group could not be located and was not included in the final refinement model, although it was added later to the models for DFT optimization and NMR parameter predictions. The conformations of the two independent cations are essentially inverted variations of one another, as shown in Figure 3a. The only major exception to

Table 2. Comparison of the Unit Cell and Space Group Obtained for Form 2 by Indexing of the PXRD Pattern with the Unit Cell and Space Group Obtained by SCXRD for Form 1a parameter crystal system space group a (Å) b (Å) c (Å) α (°) β (°) γ (°) volume (Å3) formula units per cell (Z) formula units in the asymmetric unit (Z′) calculated density (g/cm3) a

Form 1 SCXRD

Form 2 PXRD indexing solution

triclinic P1 7.947(3) 13.726(3) 13.826(5) 63.23(3) 86.79(3) 84.51(2) 1340.3(6) 2 2

monoclinic P21 47.2361(12) 4.8044(2) 14.6132(4) 90 89.2094(19) 90 5386.7 8 4

1.269

1.263

All measurements were performed at ambient temperature.

pattern of Form 2 are shown in Figure 4 after Pawley refinement, which achieved a final Rwp of 2.37%. The

Figure 3. (a) Least-squares fit for all the non-hydrogen atoms, except O17, of cation A from the Form 1 crystal structure (colored by atom type) with the corresponding atoms of cation B (inverted, colored magenta, RMS = 0.168 Å). (b) A view of cations A and B, together with two chloride anions, showing the dimeric unit formed by the hydrogen bonding interactions (dashed lines) in Form 1. For clarity, only one component of the various disorders is shown. Figure 4. Pawley refinement of the indexed unit cell of Form 2 (calculated pattern) against the experimental capillary PXRD pattern. The cell was indexed to a monoclinic cell in the P21 space group. The Pawley refinement yielded an Rwp of 2.37% and an Rp of 1.77%.

this, disregarding the disordered regions, is at the chiral center. Here, inversion is not possible owing to the cations of I each being a single enantiomer. Cation A and cation B and the chloride anions form a dimeric unit about a pseudoinversion center, as shown in Figure 3b. The two independent cations are linked by hydrogen bonds that are mediated through the chloride ions. No other classical hydrogen bonds are associated with the crystal packing. A single crystal of Form 2 could not be obtained during this study. The PXRD pattern of Form 2 was therefore indexed to determine unit cell parameters for comparison with Form 1. The best result from PXRD indexing analysis of the Form 2 pattern was obtained for a monoclinic cell with space group P21, with unit cell metrics given in Table 2. The results of fitting this indexing solution to the experimental capillary PXRD

monoclinic cell solution was located after an extensive search using the DICVOL package, and the reported cell was found reliably using a large number of peaks, in this case 30, in the indexing calculations. The P21 space group was selected because I is a single enantiomer. Attempts to use fewer peaks led to alternative indexing solutions that did not fit as well to the experimental data using Pawley refinement. Many of the other possible cells located for Form 2 were triclinic and exhibited dimensions similar to those of the monoclinic cell but usually with a c-axis dimension that was 1/2 of that for the monoclinic cell. This cell requires a Z′ value of 4 to achieve a E



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Figure 5. (a) Comparison of the 13C CP-TOSS spectra of Forms 1 and 2. (b) 13C CP-TOSS spectra of the forms acquired using dipolar dephasing for a duration of 4 τr. Assignments are shown using the numbering scheme for the first molecule in the asymmetric unit of Form 1 for clarity (with disordered carbon positions referred to by their basic numbering shown in Scheme I). Spectra were obtained at 9.4 T and 273 K with νr set to 8 kHz.

realistic density. As discussed in detail in later sections, the SSNMR results support the assertion that Form 2 has a Z′ of 4, but the results are subject to ambiguity caused by structural disorder. Because of the inability to decisively determine Z′ for Form 2 by SSNMR, these triclinic cells could not be completely ruled out, but there was also no evidence that would lead to a preferred selection of one of these cells. A figure-of-merit was also estimated for each unit cell obtained from the indexing searches, and the monoclinic cell found for Form 2 yielded the highest figure-of-merit.32,33

The unit cell parameters of the polymorphs are compared in Table 2. Although there is no obvious similarity in cell constants, the volume of the Form 2 cell obtained from the Pawley refinement in Figure 4 is approximately four times that of the Form 1 cell, and the a axis of Form 2 is considerably lengthened. When combined with the similar diffraction patterns observed for the forms, this suggests that Form 2 may be an extended superstructure relative to Form 1.34,35 Although not a true superlattice exhibiting periodic or aperiodic noncrystallographic symmetry,36 this designation (or the F



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alternative designation of “quasisuperlattice”) has been applied to a polymorph with a unit cell that extends that of another polymorph.34,35 In this type of structure, the molecules may be slightly perturbed in the extended volume in comparison to the original cell (e.g., with respect to conformation, packing, or hydrogen bonding), and disorder and higher Z′ values may be observed.34,35,37 Forms that are related in this manner can exhibit similar PXRD patterns.34,35 Thermal Analysis and Optical Microscopy. DSC analysis of Form 1 showed a single melting endotherm with an onset of 121.8 °C, a peak at 123.2 °C, and an enthalpy of 61.6 J/g (see Supporting Information). Form 2 exhibited a single melting endotherm with an onset of 122.9 °C, a peak at 124.3 °C, and an enthalpy of 65.3 J/g. Both DSC analyses show decomposition of compound I beginning at 210 °C. No evidence of solvation or other thermal events prior to melting and decomposition was observed in batches of both Forms 1 and 2 by DSC. The higher melting polymorph (Form 2) thus has a higher heat of fusion, and following the heat of fusion rule reported by Burger and Ramburger, this indicates that the forms are monotropically related with Form 2 being the more thermodynamically stable form.3,38 This finding is consistent with the monotropic relationship between the forms established independently by competitive ripening experiments, where Form 2 was also found to be the more thermodynamically stable form. Because the Form 2 cell shows nearly the same density as Form 1, as seen in Table 2, the density rule that the thermodynamically stable polymorph should exhibit a higher density could not be used to further confirm this finding.38 DSC was not useful as an analysis technique to distinguish the polymorphs. Polarized light microscopy of both polymorphs showed birefringence that is indicative of crystallinity (see Supporting Information). The morphology of Form 1 was characterized as small columnar-shaped particles and large fan-shaped radial clusters. The morphology of Form 2 was characterized as large and small columnar and rod-shaped particles. Aggregation was observed for both polymorphs. 13 C SSNMR Analysis. With the crystal structure of Form 1 in hand and a likely unit cell determined for Form 2, detailed spectroscopic characterization was performed to explore the structural relationships between the two polymorphs. First, to help design the SSNMR experimental approach, the 1H T1 relaxation times for Forms 1 and 2 were measured by saturation recovery experiments in which the 1H isotope is directly observed. With νr set to 8 kHz, a temperature of 273 K, and a B0 of 9.4 T, the values of 1H T1 were determined to be 1.28 s for Form 1 and 1.25 s for Form 2. With νr set to 30 kHz, a temperature of 293 K, and at the highest B0 used in this study of 16.4 T, the values of 1H T1 were determined to be 2.18 s for Form 1 and 2.39 s for Form 2. These short relaxation times allow for a wide variety of CP-based SSNMR experiments to be conducted on the polymorphs without excessive experiment times, although the relaxation times are too similar for resolution of the two forms in mixtures using heteronucleardetected 1H T1 experiments for assessment of phase purity.39 13 C SSNMR spectra obtained with the CP-TOSS pulse sequence are shown in Figure 5a. A longer than necessary 10 s relaxation delay was used to acquire the spectra to detect the presence of potential phase impurities that might have a longer 1 H T1 relaxation time, such as another polymorph besides those examined here (which was not observed). The 13C spectrum of Form 1 is highly complex and is reflective of the Z′ = 2 nature

of the crystal structure of this phase, but with additional splitting from disorder. The spectrum of Form 2 is similar to that of Form 1 but shows greater complexity through the presence of additional signals at 21.9, 22.6, 33.7, 52.8, 53.8, 60.0, 63.4, 69.3, 107.9, 126.0, and 174.3 ppm. Form 1 shows at least two resolved resonances at 124.3 and 143.1 ppm that are not observed in Form 2. A minor amount of Form 2 may be present in the Form 1 sample as evidenced by weak resonances at approximately 22 and 53 ppm. (Form 2 could be ripened to achieve phase purity, whereas no similar process was available for Form 1.) The 13C assignments shown in Figure 5a for both polymorphs have been made using general 13C chemical shift trends, the edited 13C spectra discussed below, and the GIPAW calculations performed using a model which incorporates all of the major components of the disorders and a second model which incorporates all of the minor components of the disorders (see Supporting Information). The assignments are generally analogous between the polymorphs. For simplicity, the assignments in Figure 5a refer to atomic positions in I and represent the first molecule in the asymmetric unit of Form 1. Two 13C resonances in Form 2 at 52.8 and 53.8 ppm are noteworthy. These signals are assigned to either C10 or C15, both of which may be affected by changes in their surroundings because of their proximity to the flexible chain (from C10 to O19) connecting the fluoroaryl and indanyl portions of Compound I. This chain is more likely to be susceptible to a significant conformational change than the atoms surrounding C2 and/or C5, hence the tentative assignment of these resonances to C10 or C15. Significant disorder was observed in this chain in the crystal structure of Form 1. The GIPAW calculations indicated that the C10 and C15 positions (as well as the corresponding and C50 and C55 positions) each a covered a range of about 10 ppm in this spectral region. In the 2D 1H−13C CP-HETCOR spectrum of Form 2, the carbon positions at 52.8 and 53.8 ppm are observed to correlate with overlapped aliphatic 1H positions at 1.7 and 2.6 ppm at a short mixing time of 500 μs (see Supporting Information). This result does not provide further insight into the assignment of C10 or C15. Two additional resonances observed in the 13C CP-TOSS spectrum of Form 2 at 22.4 and 21.9 ppm are also notable because of their uniqueness compared to Form 1. These resonances are assigned to C12 and C13 (and their counterparts C52 and C53). These methyl positions were also refined as two disordered positions with partial occupancies in the Form 1 SCXRD structure, and the appearance of a new set of signals for these carbons may indicate that they are subject to greater disorder in Form 2. Overall, the extra 13C resonances seen for Form 2 are suggestive of the presence of additional different C10−O19 chain conformations beyond the to conformations already present in Form 1. This offers evidence that the Form 2 polymorph includes additional conformers of I involving the C10−O19 chain relative to Form 1. The carbonyl 13C resonances assigned to the C30 and C70 sites in Forms 1 and 2 offer information about hydrogen bonding differences between the two forms. Lower frequency 13 C carbonyl resonances (corresponding to more deshielded nuclei) are generally associated with shorter hydrogen bonds in cases where the carbonyl oxygen acts as an acceptor.40 In Form 1, a single carbonyl 13C resonance is observed at 173.7 ppm, and no hydrogen bonds are observed to either the O31 or O71 acceptor positions (see Supporting Information for a summary G



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very similar packing motifs. 2D 1H−13C CP-HETCOR spectra of the two forms are also compared in the Supporting Information and also showed no indication of packing differences between the forms.19 15 N SSNMR Analysis. 15N CP-MAS spectra of the polymorphs are shown in Figure 6. The structure of I contains

table of hydrogen bonding). A weak, nonclassical interaction is observed between C33 and O71 in the major component of the disorder with a donor−acceptor (D···A) distance of 3.38 Å and an O71···H33A distance of 2.45 Å. The corresponding interaction between C73 and O31 is also observed with a D···A distance of 3.36 Å and an O31···H73B distance of 2.70 Å. The O31 position is involved in a still weaker interaction with C53 at a D···A distance of 3.53 Å and an O31···H53C distance of about 2.62 Å. Disorder is observed in the region around C53 and could affect these weak interactions, which are quoted for the major component. The O71 position engages in an even longer range interaction with C12 with a D···A distance of 3.80 Å and an O71···H12B distance of 2.83 Å that is similarly affected by disorder. In spite of all of the differences in the interactions involving C30 and C70, only a single 13C carbonyl resonance is observed in Form 1. In Form 2, the major 13C carbonyl resonance is also observed at 173.7 ppm but is joined by a second resonance at 174.3 ppm with an intensity that is approximately half of the major resonance (see Supporting Information for an expanded plot). Although other structural effects could be responsible for the resonance at 174.3 ppm, its appearance may be indicative of a slightly stronger hydrogen bond involving one or both of these nonclassical interactions or possibly another interaction involving the O31 or O71 acceptors, and if so would suggest that Form 2 contains more energetically favorable hydrogen bonding to these acceptors than Form 1. In Figure 5b, edited 13C CP-TOSS spectra obtained with dipolar dephasing are plotted. These spectra show only methyl and quaternary carbon resonances and are useful in confirming the assignments in Figure 5a and in the assessment of Z′ because they are simplified compared to conventional CPTOSS spectra.7 Quaternary aryl carbons that appear in these spectra are often sensitive to conformational changes involving pendant groups in higher Z′ structures.7 In the spectrum of Form 1, the Z′ = 2 condition found by SCXRD is not easily observed by the usual doubling of resonances, reflecting the similarity in conformation for the two molecules in the asymmetric unit (as shown in Figure 3a). A split resonance is only clearly observed for one of the methyl positions at 27.7 and 26.8 ppm in the dipolar dephasing spectrum. The remainder of the dipolar dephasing spectrum of Form 1 shows only minor broadening effects arising from the Z′ = 2 structural feature or from disorder. Additional splitting is observed in the spectrum of Form 2 shown in Figure 5b, indicating that Form 2 has greater structural complexity than Form 1. For example, at least three resonances are observed for the C11 position, and up to six resonances are observed for the two methyl positions (C12 and C13). Some of these vary significantly in intensity, suggesting disorder models with variable occupancies are present in Form 2. 13 C CP-TOSS and 1H−19F−13C DCP-TOSS spectra were obtained using 19F π-pulse heteronuclear decoupling in addition to 1H decoupling (see Supporting Information).17,18 The 13C CP-TOSS spectra obtained using 19F π-pulse heteronuclear decoupling showed no significant gain in resolution except for the C21 and C22 positions directly attached to fluorine, where heteronuclear J-coupling was removed by decoupling. 1 H−19F−13C DCP-TOSS spectra of both polymorphs were also obtained to observe packing or other structural differences between the polymorphs caused by differences in fluorine− carbon distances.17,18 The 1H−19F−13C DCP-TOSS spectra were very similar, which suggests that the polymorphs exhibit

Figure 6. 15N CP-MAS (νr = 5 kHz) spectra of Forms 1 and 2. Although the spectra arise from the single amine site in I (N14 and N54), they each show extensive splitting (see text). Spectra were obtained at 9.4 T and 298 K.

a single nitrogen atom (N14), and the complex 15N spectra obtained for each form arise solely from this position. Both spectra show resonances at similar 15N chemical shifts, with the most intense resonance in each spectrum at −309.9 ppm (Form 1) and −309.4 ppm (Form 2), but the spectrum of Form 2 shows an additional splitting affecting the strongest resonance at −308.7 ppm. Both spectra exhibit a resonance at −307.1 ppm that is more intense in Form 2. Both spectra also contain more shielded resonances at −314.1 ppm in Form 1 and −312.4 ppm in Form 2, with the latter resonance again more intense in Form 2. Although subtle and tentative, this effect is suggestive of more favorable hydrogen bonding in Form 2 because amine nitrogen hydrogen bond donors (including salts) are known to deshield upon formation of shorter hydrogen bonds.41 While the 15N CP-MAS spectra of the polymorphs are too complex to analyze in more detail given the observed signal-to-noise, the results indicate that the N14 sites in Form 2 occupy a greater range of environments, consistent with the presence of additional molecules in the asymmetric unit or disorder in a superstructure. The 15N spectrum of Form 1 is affected by disorder in the structure along the C10−O19 chain, as it shows at least three resolved resonances instead of the two resonances that would be expected from the Z′ = 2 crystal structure. 19 F SSNMR Analysis. 19F CP-MAS spectra of Forms 1 and 2 are compared in Figure 7a. Expanded regions of the centerbands are shown with assignments in Figure 7b. Assignments have been made using the results of the GIPAW calculation for Form 1, which predicted F26 and F66 to be 35 ppm more shielded than F27 and F67 using the average of the values predicted for these positions (see Supporting Information). Both forms exhibit splitting involving the F27 and F67 H



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positions. This is consistent with the observed splitting in Figure 7b for the F27 and F67 positions and the reduced splitting for the F26 and F66 positions and allows for the assignment shown for the F27 and F67 positions. Finally, the similarity of the 19F chemical shifts for the two forms is likely indicative of similar intermolecular arrangements involving the indanyl and difluoroaryl rings in the two polymorphs, suggesting similar packing motifs for the polymorphs. 1 H SSNMR Analysis. 1H SSNMR spectra of the two polymorphs were obtained at a high magnetic field strength of 16.4 T (700.13 MHz), using fast MAS (νr = 30 kHz), and with 1 H homonuclear dipolar decoupling to maximize spectral resolution given the broadening effects of 1H homonuclear dipolar coupling. In Figure 8, 1H spectra obtained using DP-

Figure 7. (a) 19F CP-MAS spectra (νr = 14 kHz) of Forms 1 and 2. Spinning sidebands are marked with asterisks. (b) Expanded region of the CP-MAS spectra showing only the centerband resonances. Spectra were obtained at 9.4 T and 273 K.

positions that is consistent with an even Z′, suggesting two dominant molecular environments for the fluoroaryl rings in both forms (and supportive of the Z′ = 2 cell determined for Form 1 and the Z′ = 4 cell found by PXRD indexing for Form 2). The fluorine positions do not appear to be strongly affected by the disorder that impacts the N14 position and many of the carbon sites in Form 1. The similarity of the 19F CP-MAS spectra of the polymorphs is relatively unusual given that aryl fluoride chemical shifts are normally very sensitive to polymorphism because of aromatic π-stacking changes that often occur between forms.7 In the crystal structure of Form 1, the difluoroaryl ring is engaged in a distorted edge-on interaction with the indanyl ring, a change which would strongly affect 19F shielding. The F27 and F67 positions are positioned such that they should be affected more strongly by π-stacking than F26 and F66. Slight differences in the fluorine positions in the two molecules in the Form 1 asymmetric unit thus lead to a 3-fold greater difference in the GIPAW-calculated σiso values for the F27 and F67 positions relative to the difference in calculated σiso values for the F26 and F66

Figure 8. 1H DP-MAS spectra of Forms 1 and 2 shown in comparison to DUMBO spectra of the same samples. Assignments are shown using the numbering scheme for the first molecule in the asymmetric unit and the higher occupancy (major) component of the disordered regions, with the exception of the hydrogen bonded H57 signal (which is significantly more deshielded than its counterpart, H17, in the GIPAW calculations). Spectra were obtained at 16.4 T and 283 K with νr = 30 kHz.

MAS and with DUMBO homonuclear decoupling are compared for the polymorphs. Individual 1H resonances are resolved for aromatic and aliphatic positions. The 1H positions in Form 1 can be partially assigned as shown in Figure 8 with the assistance of the GIPAW calculation (see Supporting Information) but are too complex for detailed interpretation I



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MHz and an ηQ of 0.79. In the second model, containing the minor components of the disordered regions, the parameters calculated for Cl81 changed to a CQ of 5.48 MHz and an ηQ of 0.46, while those for Cl82 changed to a CQ of 4.01 MHz and an ηQ of 0.74. 35Cl σiso values were also highly sensitive to disorder. Cl81 was calculated to be 16 ppm more shielded than Cl82 in the major component of the disorder, but Cl82 was found to be 35 ppm more shielded than Cl81 in the minor component. Given that the degree of multisite disorder present in Form 2 was even more complex than Form 1, additional fitting the 35Cl spectra of either polymorph to extract chemical shift or quadrupolar parameters was not attempted. This precludes analysis of hydrogen bonding trends to the chlorine anions in both structures using the reported trend that CQ decreases with an increasing number of hydrogen bond donors.43 Although detailed fitting was not possible, a key observation can be made from the 35Cl DP-MAS spectra of the two polymorphs because Form 2 contains an additional set of signals at a higher frequency in comparison to Form 1, which arise from additional chlorine sites with significantly different CQ, ηQ, and/or δiso values. The additional signals seen in the 35 Cl DP-MAS spectrum of Form 2 are consistent with the additional signals observed in the 13C and 15N spectra of Form 2 and suggest the presence of additional nonequivalent chloride sites in the superstructure. Even without the availability of fitted 35 Cl EFG and NMR parameters, the 35Cl spectra offer useful structural information similar to that gained from the 13C spectra. Vibrational Spectroscopy. Vibrational spectroscopic measurements were performed on the polymorphs to gain further insights into the structural differences and particularly differences in hydrogen bonding. IR spectra obtained with ATR sampling are shown in Figure 10a. The spectra were obtained with 1 cm−1 spectral resolution to highlight the subtle differences expected for the polymorphs. The IR spectra generally lack analytical specificity for the forms but still contain useful structural information. A minor but noticeable difference in the C30−O31 stretching vibration can be observed in Figure 10b. This band appears at 1727.7 cm−1 in Form 1 and at 1726.8 cm−1 in Form 2 and exhibits a minor but reproducible shift in peak maximum. Since carbonyl hydrogen bond acceptors are known to be sensitive to hydrogen bond geometry, even in the weak CH...O interactions probed here, this could be indicative of a minor hydrogen bonding difference between the polymorphs.46−48 A frequency shift of an IR band to lower wavenumbers is generally associated with an ester carbonyl oxygen atom (here O31 and/or O71) accepting a shorter hydrogen bond.46−48 The trend observed in Figure 10b is suggestive of a slightly shorter and stronger hydrogen bond to at least a fraction of the O31 and/or O71 sites in Form 2, consistent with its thermodynamic stability.38 This trend is also consistent with the observation of a second deshielded resonance assigned to C30 and/or C70 in the 13C SSNMR spectrum of Form 2. The region of the IR spectrum between 3700 and 3100 cm−1 is also useful for analysis of hydrogen bonding, in the present case through potential observation of frequency differences between the polymorphs for NH and OH vibrational stretching modes. The NH stretching vibrations in I are expected to be a series of broad bands in the 3000 to 2700 cm−1 region as is typically found for secondary amine salts.49 Inspection of the ATR IR spectra of the polymorphs in Figure 10a showed no evidence of measurable differences in this region, precluding

with the exception of the deshielded resonances assigned to the protons attached to N14 and N54. The DUMBO spectra of Forms 1 and 2 offer sufficient resolution to obtain a chemical shift for the most deshielded 1H peaks, which are assigned to the hydrogen-bonding H14A and H14B sites and their counterparts in the second molecule in the asymmetric unit. The 1H resonances in Form 2 show a more pronounced maximum at 9.7 ppm and are slightly shifted to higher frequency relative to the group of resonances assigned to these hydrogen bonded positions in Form 1, which are centered at about 9.0 ppm. More deshielded 1H positions are associated with shorter hydrogen bonds.42 This result suggests that the N14 and N54 donors are able to form a shorter hydrogen bond with the chlorine acceptors in at least some of the molecules in Form 2. 35 Cl SSNMR Analysis. 35Cl SSNMR is increasingly applied in the analysis of hydrochloride salts of pharmaceutical compounds.43−45 The 35Cl DP-MAS spectra obtained at 16.4 T of the two polymorphs are shown in Figure 9. The spectra

Figure 9. 35Cl DP-MAS spectra of Forms 1 and 2. The frequency axis is referenced with respect to crystalline NaCl at 0 kHz. The spectra show signals from Cl81 and Cl82, with Form 2 exhibiting additional signals between 0 and 4 kHz (see text). Spectra were obtained at 16.4 T and 283 K with νr = 30 kHz.

exhibit the appearance of complex, overlapping second-order quadrupolar patterns arising from multiple chlorine sites. Unsatisfactory results were obtained from attempts to fit the 35 Cl DP-MAS spectrum of Form 1 to a two-site model based upon the two nonequivalent chloride anions in the Z′ = 2 structure using parameters for CQ, ηQ, isotropic chemical shift (δiso), and line broadening, most likely because of the influence of disorder in the crystal structure. The complexity observed for the 35Cl spectra of both forms may be related to the complexity of the 15N spectra shown in Figure 6 because both spectra may be strongly affected by the distribution of the electrostatic interactions involving these sites. DFT calculations of 35Cl NMR parameters indicate that the chlorine positions experience a wide range of environments in the Form 1 structure (see Supporting Information). The EFG parameters at the 35Cl nuclei were found to be sensitive to disorder by comparing results calculated for the major and minor components of the disorder. In the first model used here, which incorporates all of the major components of the disordered regions, the Cl81 site was calculated to have a CQ of 5.06 MHz and an ηQ of 0.68, while Cl82 yielded a CQ of 4.50 J



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On the basis of these observations and known trends for OH stretching vibrations in O−H···Cl interactions,50 the two maxima in the spectrum of Form 1 are assigned to O17− H17 (3345 cm−1) and O57−H57 (3300 cm−1) stretching vibrations. The observation of similar bands in Form 2 suggests a similar environment, but the band assigned to the more weakly interacting O17−H17 vibration increases in intensity. This suggests a less energetically favorable environment for this hydrogen bonding interaction in Form 2 relative to Form 1, in contrast to the other observations that showed more favorable hydrogen bonding in Form 2. Closer inspection of the ATR IR spectra of the polymorphs in Figure 10a showed an additional weak band at about 3555 cm−1 in both forms. The weak intensity results from the limited penetration depth of the ATR evanescent wave at this high frequency and made it difficult to measure the peak maxima. DR IR spectroscopy was used to more clearly observe these bands, which are shown in Figure 10c. This band appears at 3556.4 cm−1 in Form 1 and shifts slightly to 3554.7 cm−1 in Form 2. The position of this band is consistent with very weakly interacting or noninteracting hydroxyl groups.49,50 The presence of this band in both forms may be indicative of a second disordered component for H17 and/or H57 that is not engaged in hydrogen bonding. Although Figure 10c is scaled to show both bands at similar intensity, this band was significantly more intense in Form 1 than in Form 2 (see Supporting Information). This suggests that Form 1 contains a greater population of this weakly interacting component and could contribute to its destabilization relative to Form 2. The combined spectroscopic data, including the shifts observed in the 1H resonances assigned to the protons attached to N14, the additional deshielded resonance seen in the 13C signals assigned to C30 and/or C70, the peak shifts and intensities in the 15N spectra, and the IR frequency shifts and band intensities observed for the C30−O31 and hydroxyl vibrations, are together indicative of a slightly more favorable hydrogen bonding stabilization in Form 2 relative to Form 1. The relative intensities of the broad maxima at 3345 and 3300 cm−1 are the only observed spectral feature in any of the SSNMR or vibrational spectra presented here that favors a stronger hydrogen bonding environment in Form 1. Since Form 2 is the more thermodynamically stable form, the overall trends agree with the rule proposed by Burger and Ramburger based on IR studies of a range of polymorphic systems for which thermodynamic stability was established.38 This rule posits that the polymorph showing the shorter hydrogen bonds will generally be the more stable form. Although the analysis of Burger and Ramburger was confined to IR spectroscopic observables, SSNMR spectral trends are useful for the same purpose. Raman spectra of the two forms are shown in Figure 11a. A laser wavelength of 1.064 μm was used to avoid strong fluorescence from these phases (see below). The spectra show minor differences, particularly in the 600 to 550 cm−1 and 300 to 250 cm−1 regions, as seen in Figures 11b,c. Because of their low frequency, these bands are primarily associated with phonon modes that differ slightly between the two crystal structures. The differences in these bands are consistent with a slight perturbation of the Form 1 and 2 structures relative to each other. As observed in the IR spectra, the band assigned to the C30−O31 stretching vibration shifts from 1728.5 cm−1 in Form 1 to 1727.5 cm−1 in Form 2. No additional structural

Figure 10. (a) DATR IR spectra of Forms 1 and 2, showing the full spectral region. (b) Expansion of the carbonyl band region of the ATR IR spectra, showing a subtle difference between the polymorphs. (c) Expansion of the hydroxyl band region of the DR IR spectra, showing differences between the polymorphs.

analysis of hydrogen bonding involving the N14 donor by this method. The hydroxyl bands associated with O17−H17 and O57−H57 vibrations are generally expected to produce strong and broad bands in the range of 3700 to 3200 cm−1, with bands from hydroxyl groups not engaged in hydrogen bonding expected at about 3600 cm−1.49 In Figure 10a, two broad maxima at 3345 and 3300 cm−1 are observed, with the band at 3345 cm−1 exhibiting more intensity in Form 2. In the crystal structure of Form 1, the O57 atom in the second cation in the asymmetric unit donates a hydrogen bond with an O57···Cl82 D···A distance of 3.10 Å (see Supporting Information). In Form 1, the H17 position could not be located and the corresponding O17···Cl82 D···A distance is longer at 3.15 Å. K



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respectively, also showing strong absorbance below 230 nm and a shoulder at 216.2 nm with a molar absorption coefficient of 18800 AU L mol−1 cm−1 (see Supporting Information). DR UV−visible spectra were first acquired on neat solids to detect weaker bands (see Supporting Information), although the use of neat solids obscures features in the most absorptive region of the spectra. No weak bands with absorption maxima in the 300−400 nm range were observed for either polymorph. When 5% w/w of each form is diluted into an MgO blend prior to analysis, the resulting UV−visible spectra of both forms show strong absorption bands with two maxima at 265 and 272 nm, in agreement with the bands observed in acetonitrile solution (see Supporting Information). Both polymorphs exhibit no significant absorption above 400 nm, consistent with their white to off-white appearance. No evidence of splitting from excitonic states was observed in the spectra.53 Although Form 2 shows enhanced absorption for the bands at 265 and 272 nm, the UV−visible spectra are almost indistinguishable, consistent with the structural similarity observed by other techniques. Solid-state fluorescence spectroscopy has not been widely employed in studies of solid-state phenomena, although it has been shown to provide useful structural information in other polymorphic systems.54−57 In acetonitrile solution at a concentration of 0.01 mg/mL, weak emission at 405 nm was detected for I using excitation at 270 nm (see Supporting Information). This excitation wavelength was chosen from the midpoint of the UV−visible absorption bands in this range in this solvent (see above). In aqueous solution at a similar concentration, a slightly stronger emission maximum was observed at 415 nm. Solid-state fluorescence emission spectra of the two polymorphs obtained using excitation at 270 nm show strong emission at 375 to 380 nm as seen in Figure 12a. The same excitation wavelength was used as in the solutionphase experiments because the DR UV−visible absorption bands also occur in this range. Although the emission spectra of the polymorphs are similar, a small but reproducible shift in the maximum emission wavelength is seen from 380 nm in Form 1 to 375 nm in Form 2. The excitation spectra of the polymorphs were detected via emission at 380 nm and are shown in Figure 12b. These spectra show a more distinctive pattern for the two forms. Because the xenon lamp used in the fluorescence spectrometer does not excite efficiently at wavelengths of 250 nm or less, the electronic absorbances observed below this wavelength in the DR UV−visible spectra (see Supporting Information) were suppressed in the excitation spectra, allowing for more selective observation of the electronic absorbances in the 260−350 nm region as shown in Figure 12b. The excitation spectrum of Form 2 contains additional maxima or shoulders at 278 and 322 nm in comparison to the similarly obtained spectrum of Form 1. The presence of additional bands likely arises from the added electronic transitions of structurally distinct molecules or the appearance of excitonic effects, consistent with the observation of extra signals in Form 2 as previously noted in the 13C, 15N, and 35Cl SSNMR results. The 3D total luminescence (or excitation−emission) spectra of each of the polymorphs were also obtained (see Supporting Information) and suggested that synchronous scanning of the monochromators might offer better specificity for the two forms and allow observation of other structural features.58−61 Different offsets between the monochromators were examined for Forms 1 and 2, and the best results with respective to specificity for the polymorphs were obtained using offsets of

Figure 11. (a) Raman spectra of Forms 1 and 2 obtained using laser irradiation at 1.064 μm. Expansions of the spectra are shown in (b) and (c), highlighting the minor differences between the polymorphs.

information could be gleaned from the Raman spectra of the polymorphs. UV−Visible and Fluorescence Spectroscopy. Solid state DR UV−visible spectra can in some cases distinguish between polymorphic forms, particularly when they differ in color or appearance, and are also useful because they allow for assessment of the optical properties of the solids.51,52 In acetonitrile solution at a concentration of 0.25 mg/mL, the UV−visible spectrum of I shows weak absorbances at 272.5 and 266.0 nm with molar absorption coefficients of 1920 and 2050 AU L mol−1 cm−1 (absorbance units L mol−1 cm−1), L



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Figure 12. (a) Fluorescence emission spectra of Forms 1 and 2 obtained using an excitation wavelength of 270 nm. (b) Fluorescence excitation spectra of the two forms detected via emission at 380 nm. (c) Synchronous fluorescence spectra of Forms 1 and 2 obtained with a 50 nm offset. (d) Synchronous fluorescence spectra of Forms 1 and 2 obtained with a 100 nm offset. For all spectra, the detector response divided by the reference diode response is plotted.

+50 and +100 nm, as shown in Figure 12c,d.58,59 These spectra represent higher resolution “slices” through the 3D total luminescence spectra. In these spectra, Form 2 contains features than generally appear to be broader or more complex than those in Form 1, in agreement with the previously noted trend toward greater complexity in Form 2.

seen in the DR UV spectra. Form 2 was observed to have stronger hydrogen bonds by 1H SSNMR, 13C SSNMR, and IR spectroscopy, consistent with the experimental determination that it is the thermodynamically stable form. The spectroscopic data are particularly valuable in the event that a crystal of Form 2 could be grown in the future, since significant efforts may be needed to obtain the structure of Form 2 given its complexity and the spectroscopic data may help resolve ambiguities. This study also provided an opportunity to assess the analytical specificity of a suite of techniques in the analysis of two polymorphic forms that are difficult to distinguish. In pharmaceutical development, techniques are needed for specific detection and potentially quantitative analysis of polymorphs in batches of active pharmaceutical ingredient or in formulated drug products. The methods found in this study to provide the best specificity for these two closely related polymorphs were capillary PXRD and 13C SSNMR, and to a more limited extent, fluorescence excitation spectroscopy. PXRD would be especially useful if the method is optimized for several key reflections, although time-consuming high resolution scans would be needed. 13C SSNMR has the drawback of long acquisition times, particularly in formulations where the drug is significantly diluted. 19F SSNMR provides much more sensitivity, but the degree of spectral overlap would likely require more complex data analysis such as multivariate methods. Fluorescence excitation spectra are also sensitive but may suffer from interference from autofluorescence of many excipients used in formulations. IR and Raman spectroscopy are unlikely to be of much use in the present system because of their very limited specificity. The results presented here illustrate the analytical challenges still faced in characterizing a complex polymorphic pharmaceutical system.

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CONCLUSIONS The structural features of two similar polymorphic forms of I were investigated using spectroscopic and diffractometric methods. SCXRD was invaluable in obtaining the structure of Form 1, which allowed for inferences to be drawn about Form 2. Despite the structural similarity between the forms and the unavailability of a crystal structure for Form 2, a number of structural features could be observed and related to the overall structure and thermodynamic stability of the two polymorphs. The powder pattern of Form 2 was indexed, and the most likely cell was found to be approximately four times the volume of that determined for Form 1 by SCXRD. This suggests that Form 2 might be described as an approximate superstructure of the Form 1 structure, with similar packing and molecular structures of I, but with greater disorder (particularly involving the C10−O19 chain) and other slightly perturbed structural features. A similar effect has been reported for other organic molecular polymorphs.34,35 Despite the complexity of the Form 2 structure, a number of structural features were identified using a suite of solid-state spectroscopic methods. In several of the spectra, additional signals were observed that are consistent with additional molecules in a larger unit cell. These additional signals were observed in the 13C, 15N, and 35Cl SSNMR spectra and also in the fluorescence excitation spectra, which revealed UV absorbances in the region masked by larger absorbances M



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(12) Metz, G.; Wu, X.; Smith, S. O. Ramped-amplitude crosspolarization in magic-angle spinning NMR. J. Magn. Reson. A 1994, 110, 219−227. (13) Antzutkin, O. N. Sideband manipulation in magic-angle spinning NMR. Prog NMR Spectros. 1999, 35, 203−266. (14) Fung, B. M.; Khitrin, A. K.; Ermolaev, K. An improved broadband decoupling sequence for liquid crystals and powders. J. Magn. Reson. 2000, 142, 97−101. (15) Hodgkinson, P. Heteronuclear decoupling in the NMR of solids. Prog. NMR Spectros. 2005, 46, 197−222. (16) Opella, S. J.; Frey, M. H. Selection of non-protonated carbon resonances in solid-state NMR. J. Am. Chem. Soc. 1979, 101, 5854− 5856. (17) Hagaman, E. W.; Burns, J. H. The determination of local structure in organofluorides using fluorine-19 carbon-13 dipolar coupling. Fuel 1993, 72, 1239−1243. (18) Vogt, F. G.; Katrincic, L. M.; Long, S. T.; Mueller, R. L.; Carlton, R. A.; Sun, Y. T.; Johnson, M. N.; Copley, R. C. B.; Light, M. E. Enantiotropically-related polymorphs of {4-(4-chloro-3-fluorophenyl)-2-[4-(methyloxy)phenyl]-1,3-thiazol-5-yl} acetic acid. Crystal structures and multinuclear solid-state NMR. J. Pharm. Sci. 2008, 97, 4756−4782. (19) van Rossum, B. J.; Förster, H.; de Groot, H. J. M. High-field and high-speed CP-MAS 13C NMR heteronuclear dipolar-correlation spectroscopy of solids with frequency-switched Lee-Goldburg homonuclear decoupling. J. Magn. Reson. 1997, 124, 516−519. (20) Elena, B.; de Paëpe, G.; Emsley, L. Direct spectral optimization of proton-proton homonuclear dipolar decoupling in solid-state NMR. Chem. Phys. Lett. 2004, 398, 532−538. (21) Madhu, P. K. High-resolution solid-state NMR spectroscopy of protons with homonuclear dipolar decoupling schemes under magicangle spinning. Solid State NMR 2009, 35, 2−11. (22) Earl, W. L.; Vanderhart, D. L. Measurement of 13C chemical shifts in solids. J. Magn. Reson. 1982, 48, 35−54. (23) Ratcliffe, C. I.; Ripmeester, J. A.; Tse, J. S. Nitrogen-15 NMR chemical shifts in solid ammonium salts. Chem. Phys. Lett. 1983, 99, 177−180. (24) Harris, R. K.; Becker, E. D.; Cabral de Menezes, S. M.; Goodfellow, R.; Granger, P. NMR nomenclature. Nuclear spin properties and conventions for chemical shifts (IUPAC recommendations 2001). Pure Appl. Chem. 2001, 73, 1795−1818. (25) Delley, B. An all-electron numerical method for solving the local density functional for polyatomic molecules. J. Chem. Phys. 1990, 92, 508−517. (26) Delley, B. From molecules to solids with the DMol3 approach. J. Chem. Phys. 2000, 113, 7756−7764. (27) Boese, A. D.; Handy, N. C. A new parametrization of exchangecorrelation generalized gradient approximation functionals. J. Chem. Phys. 2001, 114, 5497−5503. (28) Flurchick, K. M. DFT functionals and molecular geometries. Chem. Phys. Lett. 2006, 421, 540−543. (29) Clark, S. J.; Segall, M. D.; Pickard, C. J.; Hasnip, P. J.; Probert, M. J.; Refson, K.; Payne, M. C. First principles methods using CASTEP. Z. Kristallogr. 2005, 220, 567−570. (30) Profeta, M.; Mauri, F.; Pickard, C. J. Accurate first principles prediction of 17O NMR parameters in SiO2: Assignment of the zeolite ferrierite spectrum. J. Am. Chem. Soc. 2003, 125, 541−548. (31) Perdew, J. P.; Burke, K.; Ernzerhof, M. Generalized gradient approximation made simple. Phys. Rev. Lett. 1996, 77, 3865−3868. (32) Markvardsen, A. J.; David, W. I. F.; Johnson, J. C.; Shankland, K. A probabilistic approach to space-group determination from powder diffraction data. Acta Crystallogr. 2000, A57, 47−54. (33) Smith, G. S.; Snyder, R. L. F(N): A criterion for rating powder diffraction patterns and evaluating the reliability of powder indexing. J. Appl. Crystallogr. 1979, 12, 60−65. (34) Rheingold, A. L.; Figueroa, J. S.; Dybowski, C.; Beckmann, P. A. Superlattices, polymorphs and solid-state NMR spin-lattice relaxation (T1) measurements of 2,6-di-tert-butylnaphthalene. Chem. Commun. 2000, 651−652.

ASSOCIATED CONTENT

S Supporting Information *

Additional spectral data, crystallographic data, results of DFT calculations, and other characterization results. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Present address: Morgan, Lewis & Bockius, L.L.P., 1701 Market Street, Philadelphia, PA, 19103-2921, USA. E-mail: [email protected]. Present Addresses #

(G.R.W.) Rigaku Corporation, 9009 New Trails Drive, The Woodlands, TX, 77381, USA. ⊥ (M.N.J.) Kuecept Ltd., 16/17 Station Close, Potters Bar, Hertfordshire, EN6 1TL, UK. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Ms. Ann Diederich (GlaxoSmithKline) is acknowledged for assistance in preparation of samples, Ms. Maria Barnett (GlaxoSmithKline) is thanked for assistance with characterization studies, and Dr. Mark Strohmeier (Vertex Pharmaceuticals) is thanked for helpful discussions. Prof. William Clegg and Dr. Neil R. Brooks (Newcastle University) are thanked for assistance with X-ray diffraction studies.

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REFERENCES

(1) Yu, L. Pharmaceutical quality by design: product and process development, understanding and control. Pharm. Res. 2008, 25, 781− 791. (2) Lee, S. L.; Raw, A. S.; Yu, L. Significance of drug substance physiochemical properties in regulatory Quality by Design. In Drugs and the Pharmaceutical Sciences, Vol. 178 (Preformulation in Solid Dosage Form Development); Informa: Zug, Switzerland, 2008; pp 571−583. (3) Bernstein, J. Polymorphism in Molecular Crystals; Oxford University Press: New York, 2002. (4) Byrn, S. R.; Pfeiffer, R. R.; Stowell, J. G. Solid-State Chemistry of Drugs; SSCI: West Lafayette, 1999. (5) Threllfall, T. L. Analysis of organic polymorphs: A review. Analyst 1995, 120, 2435−2460. (6) Marquis, R. W.; Casillas, L. N.; Ramanjulu, J. M.; Callahan, J. F. Preparation of phenylalkanoic acids as calcilytic compounds. U. S. Patent 7,514,473, July 4, 2009. (7) Vogt, F. G. Solid-state nuclear magnetic resonance of polymorphic materials, in The Encyclopedia of Analytical Chemistry, Meyers, R. A.; Ed., John Wiley & Sons, Inc.: New York (2011), pp 1− 49. (8) The crystal structure data for Form 1 has been deposited with the Cambridge Crystallographic Data Center under deposition number CCDC 965940. This material can be obtained free of charge via www. ccdc.cam.ac.uk/data_request/cif, by emailing [email protected], or by contacting the Cambridge Crystallographic Data Center, 12, Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033. (9) Pawley, G. S. Unit-cell refinement from powder diffraction scans. J. Appl. Crystallogr. 1981, 14, 357−361. (10) Boultif, A.; Louër, D. Indexing of powder diffraction patterns for low-symmetry lattices by the successive dichotomy method. J. Appl. Crystallogr. 1991, 24, 987−993. (11) Neumann, M. X-Cell - a novel indexing algorithm for routine tasks and difficult cases. J. Appl. Crystallogr. 2003, 36, 356−365. N



Article

(56) Brittain, H. G. Fluorescence studies of the dehydration of cefadroxil monohydrate. J. Pharm. Sci. 2007, 96, 2757−2764. (57) Brittain, H. G. Fluorescence studies of the transformation of carbamazepine anhydrate Form III to its dihydrate phase. J. Pharm. Sci. 2004, 93, 375−383. (58) Lloyd, J. B. F. Synchronized excitation of fluorescence emission spectra. Nature 1971, 231, 64−65. (59) Rubio, S.; Gomez-Hens, A.; Valcarcel, M. Analytical applications of synchronous fluorescence spectroscopy. Talanta 1986, 33, 633− 640. (60) Karim, M. M.; Jeon, C. W.; Lee, H. S.; Alam, S. M.; Lee, S. H.; Choi, J. H.; Jin, S. O.; Das, A. K. Simultaneous determination of acetylsalicylic acid and caffeine in pharmaceutical formulation by first derivative synchronous fluorimetric method. J. Fluoresc. 2006, 16, 713−721. (61) Vilchez, J. L.; Navalon, A.; Rohand, J.; Avidad, R.; CapitanVallvey, L. F. Simultaneous determination of benomyl and morestan residues in waters by synchronous solid-phase spectrofluorimetry. J. Fluoresc. 1995, 5, 225−229.

(35) Beckmann, P. A.; Burbank, K. S.; Clemo, K. M.; Slonaker, E. N.; Averill, K.; Dybowski, C.; Figueroa, J. S.; Glatfelter, A.; Koch, S.; Liable-Sands, L. M.; Rheingold, A. L. 1H NMR spin-lattice relaxation, 13 C magic-angle spinning NMR spectroscopy, differential scanning calorimetry, and X-ray diffraction of two polymorphs of 2,6-di-tertbutylnaphthalene. J. Chem. Phys. 2006, 113, 1958−1965. (36) Yamamoto, A. Determination of composite crystal structures and superspace groups. Acta Crystallogr. 1993, A49, 831−846. (37) Steed, J. W. Should solid-state molecular packing have to obey the rules of crystallographic symmetry? CrystEngComm 2003, 5, 169− 179. (38) Burger, A.; Ramberger, R. On the polymorphism of pharmaceuticals and other molecular crystals. I. Theory of thermodynamic rules. Mikrochem. Acta 1979, 2, 259−272. (39) Zumbulyadis, N.; Antalek, B.; Windig, W.; Scaringe, R. P.; Lanzafame, A. M.; Blanton, T.; Helber, M. Elucidation of polymorph mixtures using solid-state 13C CP/MAS NMR spectroscopy and direct exponential curve resolution algorithm. J. Am. Chem. Soc. 1999, 121, 11554−11557. (40) Stothers, J. B. Carbon-13 NMR Spectroscopy; New York: Academic Press, 1972; p 299. (41) Duthaler, R. O.; Roberts, J. D. Nitrogen-15 nuclear magnetic resonance spectroscopy. Solvent effects on the 15N chemical shifts of saturated amines and their hydrochlorides. J. Magn. Reson. 1979, 34, 129−139. (42) Jeffrey, G. A.; Yeon, Y. The correlation between hydrogen-bond lengths and proton chemical shifts in crystals. Acta Crystallogr. 1986, B42, 410−413. (43) Bryce, D. L.; Gee, M.; Wasylishen, R. E. High-field chlorine NMR spectroscopy of solid organic hydrochloride salts: A sensitive probe of hydrogen bonding environment. J. Phys. Chem. A 2001, 105, 10413−10421. (44) Hamaed, H.; Pawlowski, J. M.; Cooper, B. F. T.; Fu, R.; Eichhorn, S. H.; Schurko, R. W. Application of solid-state 35Cl NMR to the structural characterization of hydrochloride pharmaceuticals and their polymorphs. J. Am. Chem. Soc. 2008, 130, 11056−11065. (45) Vogt, F. G.; Williams, G. R.; Copley, R. C. B. Solid-state NMR analysis of a boron-containing pharmaceutical compound. J. Pharm. Sci. 2013, 102, 3705−3716. (46) Bellamy, L. J.; Pace, R. J. The origins of group frequency shifts. Part III. Spectrochim. Acta 1963, 19, 1831−1839. (47) Bellamy, L. J. Advances in Infrared Group Frequencies; Meuthen & Co.: New York, 1968; pp 173−174. (48) Novak, A. Hydrogen bonding in solids: correlation of spectroscopic and crystallographic data. In Structure and Bonding (Berlin); Springer-Verlag: New York, 1974; Vol. 18, pp 177−216. (49) Silverstein, R. M.; Webster, F. X. Spectrometric Identification of Organic Compounds, 6th ed; John Wiley and Sons: New York, 1998; pp 88−89 and 103. (50) Jeffrey, G. A. An Introduction to Hydrogen Bonding; Oxford University Press: New York, 1997; pp 220−225. (51) Brittain, H. G. UV/Vis Reflectance Spectroscopy. In Drugs and the Pharmaceutical Sciences; Taylor & Francis: New York, 2006; Vol. 160, pp 121−149. (52) Zhou, L.; Vogt, F. G.; Overstreet, P. A.; Dougherty, J. T.; Clawson, J. S.; Kord, A. S. A systematic method development strategy for quantitative color measurement in drug substances, starting materials, and synthetic intermediates. J. Pharm. Innov. 2011, 6, 217−231. (53) Agranovich, V. M. Excitations in Organic Solids; Oxford University Press: New York, 2009. (54) Brittain, H. G.; Elder, B. J.; Ibester, P. K.; Salerno, A. H. Solidstate fluorescence studies of some polymorphs of diflunisal. Pharm. Res. 2005, 22, 999−1006. (55) Brittain, H. G. Photoluminescence of pharmaceutical materials in the solid state. Fluorescence studies of various solvated and desolvated solvatomorphs of erythromycin A. Rev. Fluoresc. 2007, 4, 379−392. O


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