Solid-State NMR Analysis of a Complex ... - American Chemical Society

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Solid-State NMR Analysis of a Complex Crystalline Phase of Ronacaleret Hydrochloride Frederick G. Vogt,*,†,# Glenn R. Williams,†,⊥ Mark Strohmeier,†,∥ Matthew N. Johnson,‡,○ and Royston C. B. Copley§ †

Product Development, GlaxoSmithKline plc. 709 Swedeland Road, King of Prussia, Pennsylvania 19406, United States Product Development, GlaxoSmithKline plc., Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, United Kingdom § Analytical Chemistry, GlaxoSmithKline plc., Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, United Kingdom ‡

S Supporting Information *

ABSTRACT: A crystalline phase of the pharmaceutical compound ronacaleret hydrochloride is studied by solid-state nuclear magnetic resonance (SSNMR) spectroscopy and single-crystal X-ray diffraction. The crystal structure is determined to contain two independent cationic molecules and chloride anions in the asymmetric unit, which combine with the covalent structure of the molecule to yield complex SSNMR spectra. Experimental approaches based on dipolar correlation, chemical shift tensor analysis, and quadrupolar interaction analysis are employed to obtain detailed information about this phase. Density functional theory (DFT) calculations are used to predict chemical shielding and electric field gradient (EFG) parameters for comparison with experiment. 1H SSNMR experiments performed at 16.4 T using magicangle spinning (MAS) and homonuclear dipolar decoupling provide information about hydrogen bonding and molecular connectivity that can be related to the crystal structure. 19F and 13C assignments for the Z′ = 2 structure are obtained using DFT calculations, 19F homonuclear dipolar correlation, and 13C−19F heteronuclear dipolar correlation experiments. 35Cl MAS experiments at 16.4 T observe two chlorine sites that are assigned using calculated chemical shielding and EFG parameters. SSNMR dipolar correlation experiments are used to extract 1H−13C, 1H−15N, 1H−19F, 13C−19F, and 1H−35Cl through-space connectivity information for many positions of interest. The results allow for the evaluation of the performance of a suite of SSNMR experiments and computational approaches as applied to a complex but typical pharmaceutical solid phase.

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INTRODUCTION

validated using structures where an SCXRD structure is available for use in calculations and for structural parameters. In this work, a detailed analysis using complementary 1D and 2D SSNMR methods is conducted on a novel crystalline form of ronacaleret hydrochloride, which is a calcium-sensing receptor antagonist under development for the treatment of osteoporosis.13 The chemical structure of ronacaleret HCl and the numbering scheme used here is shown in Scheme 1. The molecular weight of ronacaleret free base is 447.52 Da, which is typical of a modern pharmaceutical compound.14,15 The crystal structure of the novel phase of interest of ronacaleret HCl, designated form 1, was determined by SCXRD and is reported here. Form 1 is found to have two molecules in its asymmetric unit (Z′ = 2), a common occurrence in organic molecular crystals,16 which combines with the chemical structure of ronacaleret to lead to moderately complex SSNMR spectra relative to many small molecule

Control of the solid phase of an active pharmaceutical ingredient is a critical component of modern drug development.1 The analytical and physical characterization of polymorphism and other solid-state phenomena is an essential aspect of this control.2,3 Solid-state NMR spectroscopy (SSNMR) has proven to be an invaluable technique for the structural characterization of complex pharmaceutical solids.4−9 However, SSNMR is still most commonly applied to pharmaceutical solids in the form of 1D experiments that observe the 13C nucleus.4−9 When 2D methods are applied to pharmaceutical solids, the published examples often focus on the performance of particular 2D SSNMR pulse sequences.10−12 The capabilities of a range of multinuclear 1D and 2D SSNMR experiments operating in concert are not usually explored for the complex pharmaceutical crystalline phases often encountered in drug development. In particular, fully interpreted 2D correlation spectra involving as many accessible NMR nuclei as possible may be useful in studies of active pharmaceutical ingredient phases for which a single crystal Xray diffraction (SCXRD) structure is not available, if suitably © 2014 American Chemical Society

Received: May 22, 2014 Revised: August 11, 2014 Published: August 11, 2014 10266

dx.doi.org/10.1021/jp505061j | J. Phys. Chem. B 2014, 118, 10266−10284

The Journal of Physical Chemistry B

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Scheme 1

calorimetry (DSC) (see Supporting Information). DSC analysis was performed using a Q2000 instrument with a heating rate of 10 °C/min (TA Instruments, Inc., New Castle, DE). A sample with a mass of 3 mg was heated in a closed aluminum pan with a lid that was not crimped. Nitrogen with a flow rate of 20 mL/ min was used as a purge gas. X-ray Diffraction. The SCXRD study was performed at 150 K on a single crystal grown by the slow evaporation of an aqueous acetone solution of I. Experiments were performed using a Nonius KappaCCD diffractometer (Bruker AXS, Madison, WI). A normal focus sealed tube generating Mo Kα radiation (0.71073 Å) was employed. Further details of the data collection, structure solution, and refinement are summarized in Table 1, and a refinement summary together with full

pharmaceutical compounds studied by 1D and 2D SSNMR to date.4−12 In this study, the capabilities of 2D SSNMR experiments exploiting the 1H, 13C, 15N, 19F, and 35Cl nuclei are explored using form 1. Dipolar, chemical shift, and quadrupolar interactions are utilized and related to density functional theory (DFT) calculations where applicable. The extent of resonance assignment possible in form 1 is explored using complementary SSNMR methods, but without recourse to less sensitive natural-abundance 2D 13C−13C correlation SSNMR techniques or isotopic labeling.10,17 The approach taken here begins with the assessment of the simpler 19F, 15N, and 1H spectra of form 1 before proceeding to the more complex 13C spectral analysis. Homonuclear and heteronuclear correlation experiments among 1H, 13C, 15N, and 19F nuclei, including 2D dipolar correlation experiments based on 19F−19F dipolarassisted rotational resonance (DARR) and 1H−19F−13C double cross-polarization heteronuclear correlation (DCP-HETCOR), are used to assist with assignments. The 13C assignments are further improved by the results of DFT calculations. The 35Cl SSNMR spectrum of form 1 is also analyzed in detail. 35Cl SSNMR has seen increasing applications to pharmaceutical analysis.18−21 DFT methods are also used to calculate 35Cl chemical shielding and electric field gradient (EFG) parameters. Different NMR computational methods are explored along with the use of two versions of the form 1 structure as input, namely a “low temperature” (LT) structure obtained by hydrogen position optimization of the SCXRD structure, and a “room temperature” (RT) structure obtained by Rietveld refinement of a capillary powder X-ray diffraction (PXRD) pattern and optimization of the unit cell and all atom positions. Finally, a novel application of a 1H−35Cl cross-polarization heteronuclear correlation (CP-HETCOR) experiment is shown to be useful in the assignment of 1H environments near to chloride anions. Throughout the analysis, the SSNMR results are related to conformational and intermolecular effects observed in the crystal structure of form 1.

Table 1. Summary of the SCXRD Analysis of Form 1 of Ronacaleret HCl moiety formula empirical formula formula weight temperature wavelength 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 absorption coefficient (μ) F000 θ range index ranges

measured reflections independent reflections R(int) coverage of independent reflections absorption correction maximum and minimum transmission data/restraints/parameters goodness of fit on F2 final R indices for I > 2σ(I), 6872 data final R indices for all data

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absolute structure parameter largest difference peak and hole

EXPERIMENTAL AND COMPUTATIONAL METHODS Preparation of Materials and Thermal Analysis. Ronacaleret HCl, 3-{3-[((2R)-3-{[2-(2,3-dihydro-1H-inden-2yl)-1,1-dimethylethyl]amino}-2-hydroxypropyl)oxy]-4,5difluorophenyl}propanoic acid hydrochloride salt (I), was prepared using previously described methods.22 Briefly, form 1 is prepared by crystallization from water and acetone solutions with concentrated HCl. Form 1 was observed to have a melting point of 171.4 °C by differential scanning

[C25H32F2NO4]+Cl− C25H32ClF2NO4 483.97 150(2) K 0.71073 Å 0.40 × 0.30 × 0.15 mm colorless block monoclinic P21 13.6490(13) Å, 90° 7.6848(14) Å, 96.889(18)° 23.486(7) Å, 90° 2445.7(9) Å3 4 2 1.314 Mg/m3 0.202 mm−1 1024 5.11 to 25.00° −16 ≤ h ≤ 16 −9 ≤ k ≤ 9 −27 ≤ l ≤ 27 33 318 8482 0.0407 98.8% numerical 0.979 and 0.862 8482/9/625 0.963 R1 = 0.0423 wR2 = 0.0974 R1 = 0.0609 wR2 = 0.1090 0.06(7) 0.605 and −0.278 e Å−3

crystallographic tables can be found in the Supporting Information. The crystallographic information file (CIF) is available in the Supporting Information and has been deposited with the Cambridge Crystallographic Data Centre.23 PXRD data were obtained at ambient temperature and humidity using an X’Pert Pro diffractometer equipped with an X’Celerator real time multi-strip (RTMS) detector (PANalyt10267



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scans were acquired with a 0.5 s delay for a total DP-MAS spectral acquisition time of 18 h. 13C and 19F SSNMR spectra were obtained at 125.76 and 470.53 MHz (B0 = 11.7 T), respectively, with 4 mm triple-resonance HFX MAS probes tuned to 1H, 19F, and 13C frequencies or HXY MAS probes in double-resonance mode and tuned to 1H and 13C frequencies. 15 N spectra were collected at 50.68 MHz (B0 = 11.7 T) using a 4 mm triple-resonance HFX MAS probe and at 40.53 MHz (B0 = 9.4 T) using a 7 mm double-resonance HX MAS probe. Cross-polarization (CP) and double CP (DCP) transfers were performed at power levels of 40−80 kHz with the radiofrequency (RF) field strength ramped down linearly during the contact time over a 50% range on the channel from which polarization was transferred.30 For 35Cl CP experiments, a weaker 35Cl spin lock RF field, as described below, was used to improve efficiency.31 13C CP spectra were obtained at νr = 8 kHz with a five-pulse total sideband suppression (CP-TOSS) sequence.32 1H heteronuclear decoupling was performed at an RF field strength of 105 kHz using the SPINAL-64 pulse sequence and 19F decoupling made use of a series of π pulses timed such that one pulse occurs during each rotor period (τr).33 Edited 13C spectra containing only quaternary and methyl signals were obtained using dipolar dephasing during the TOSS sequence and three subsequent echo periods (for a total of 4 τr) using a shifted echo pulse sequence; the use of the TOSS sequence forms echoes from rephasing and requires a longer dephasing duration to suppress rigid methylene and methine carbons.32,34 15N CP-MAS spectra were obtained using a basic CP-MAS pulse sequence. 19F spectra were obtained using both CP-MAS and DP-MAS methods with 4 mm and 2.5 mm HFX probes, respectively. Proton spin−lattice relaxation times (1H T1) were determined via 1H saturation recovery experiments with a 100-pulse saturation comb. Form 1 has a relatively short 1H T1 relaxation time of about 1.5 to 2.5 s across the range of static field strengths employed in this work, and relaxation delays in the range of 3 to 10 s were used with 1 H excitation and CP experiments. 13 C spectra were referenced to tetramethylsilane (TMS) using an external reference sample of hexamethylbenzene.35 19F spectra were referenced to CFCl3 by calculation from the experimental 13C references with the unified scale method.36 15 N spectra were referenced to nitromethane using an external ammonium chloride standard. 37 1 H spectra were also referenced with the unified scale method and checked by addition of a small amount of liquid TMS to samples. 35Cl spectra were referenced to solid NaCl at 0.0 ppm and fitted isotropic chemical shift values (δiso) were mathematically corrected to use dilute aqueous NaCl as a reference via eq 1:18

ical B.V., Almelo, The Netherlands). The experiment was performed using Debye−Scherrer transmission geometry. The sample was loaded into a 0.91 mm (inner diameter) polyimide capillary. Cu Kα radiation (with wavelengths of 1.54056 and 1.54439 Å) was used with a generator voltage and current of 45 kV and 40 mA, respectively. The sample was rotated in a capillary spinner at a rate of >1000 rpm and was scanned in continuous mode from 4 to 80° θ with a 2θ step size of 0.0167°. The incident beam path was configured with a focusing mirror, 0.02 rad Soller slit, and a 0.5° fixed divergence slit. The diffracted beam path included a programmable antiscatter slit (fixed at 0.25°) and a 0.02 rad Soller slit. PXRD data were refined and analyzed using the Pawley refinement24 and Rietveld refinement25 procedures in the Materials Studio Reflex software package, version 6.0 (Accelrys, San Diego, CA). The Pawley fit of the PXRD pattern of form 1 against the cell determined by SCXRD yielded a weighted profile residual value (Rwp) of 1.85% and a nonweighted profile residual value (Rp) of 1.40% (see Supporting Information). Rietveld refinement results are discussed in conjunction with DFT calculation results below. Both Pawley and Rietveld refinements used a polynomial background, pseudo-Voigt peak shape profiles, a Debye−Scherrer line shift correction, and a Finger−Cox−Jephcoat asymmetry correction.26 Solution-State NMR Spectroscopy. Solution-state NMR experiments were performed using a Bruker Avance 400 spectrometer operating at a 1H frequency of 400.13 MHz and equipped with an inverse QNI probe tunable to 1H, 13C, and 19 F frequencies (Bruker Biospin, Billerica, MA). Solution-state NMR assignments were made using standard 1D and 2D NMR experiments, including heteronuclear single-quantum coherence (HSQC) and heteronuclear multiple-bond coherence (HMBC) experiments.27 Solid-State NMR Spectroscopy. SSNMR experiments were performed using Bruker Avance I, Avance II+, and Avance III triple-resonance spectrometers operating at 1H frequencies of 399.87 MHz (with a static field strength B0 of 9.4 T), 500.13 MHz (11.7 T), and 700.13 MHz (16.4 T), respectively (Bruker Biospin, Billerica, MA). The 400 and 500 MHz spectrometers are wide-bore (89 mm) systems, while the 700 MHz spectrometer is a narrow-bore (54 mm) system. All systems are equipped with gas chillers for sample temperature control. 1 H SSNMR experiments were performed at 700.13 MHz using a 2.5 mm double-resonance magic-angle spinning (MAS) probe and at 500.13 MHz using a 2.5 mm triple-resonance HFX MAS probe. In both cases, a spinning rate (νr) of 30 to 35 kHz was used. 1H spectra were obtained using direct-polarization MAS (DP-MAS) experiments with 2.5−3 μs excitation pulses and between 16 and 64 acquired transients. 1H spectra were also obtained at 700 MHz with homonuclear decoupling using the eDUMBO-122 variant of the “decoupling using mind boggling optimization” (DUMBO) scheme.28,29 Both windowed 1D 1H experiments and 2D 1H−1H spin diffusion experiments with a windowless F1 dimension and windowed F2 dimension (where F1 is the indirectly detected 1H frequency dimension and F2 is the directly detected dimension) were performed using DUMBO.28,29 DUMBO spectra were referenced relative to the DP-MAS spectrum and linearly scaled to account for partial averaging of the chemical shift interaction by the sequence. 35Cl DP-MAS spectra were acquired without 1H decoupling at a frequency of 68.60 MHz (B0 = 16.4 T) using the 2.5 mm double-resonance MAS probe spinning at νr = 30 kHz. The central transition π/2 pulse width for 35Cl was 2 μs, and 128 K

δiso(NaCl at infinite dilution) = δiso(NaCl(s)) − 45.37 ppm)

(1) 1

2D rotor-synchronized H double-quantum broadband backto-back (DQ-BABA) MAS experiments were performed with a 2.5 mm probe at νr = 35 kHz using double-quantum excitation and reconversion periods of 2 τr.38 2D CP-HETCOR experiments between 1H and 13C, 15N, or 19F nuclei were acquired using a 4 mm probe at νr = 10 kHz with frequencyswitched Lee−Goldburg (FSLG) homonuclear decoupling at an RF field strength of 105 kHz.39 The CP-HETCOR experiments were performed using similar 2D parameters, with each 1H−13C experiment requiring 9 h, each 1H−19F experiment requiring 2 h, and each 1H−15N experiment 10268



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Figure 1. (a) View of the two cations and two anions in the asymmetric unit of the ronacaleret HCl form 1 crystal structure, showing the numbering scheme employed. Anisotropic atomic displacement thermal ellipsoids for the non-hydrogen atoms are shown at the 50% probability level. Hydrogen atoms are displayed with an arbitrarily small radius. A pseudoinversion center exists between the two independent cations. (b) Least-squares fit for all non-hydrogen atoms of cation A of ronacaleret HCl form 1 (colored by atom type) with the corresponding atoms of an inverted cation B (colored magenta, RMS error = 0.223 Å). (c) Illustration of the hydrogen bonding network (dashed lines) observed in the crystal structure of ronacaleret HCl form 1. Cl3* and Cl7* (where * refers to a letter) are symmetry equivalents of Cl33 and Cl73, respectively. Oxygen sites ending in a letter are also symmetry equivalents.

requiring about 2 days of acquisition time. 2D 1H−35Cl CPHETCOR experiments were acquired using the 2.5 mm double-resonance probe at 16.4 T with νr = 25 kHz, again using similar parameters, with the exception of a reduced spin lock RF field strength of 18 kHz used on the 35Cl channel (measured as an effective central transition nutation rate) and a correspondingly lower 1H CP field strength.31 Each 1H−35Cl CP-HETCOR spectrum required 1.9 days to obtain using 32 t1 increments, 1024 scans per increment, and a 5 s relaxation delay. Ramp CP transfers were used for all CP-HETCOR experiments with durations ranging from 25 μs to 2 ms.39 The 2D 1H−19F−13C DCP-HETCOR pulse sequence is given in the Supporting Information. This experiment was performed using a 1H−19F contact time of 2 ms and a 19F−13C contact time of 6

ms, and was obtained after a total acquisition time of 35 h using 24 time-domain increments in the F1 dimension. With 4 mm probes, the sample was restricted to the center of the rotor to maximize RF homogeneity for FSLG-based experiments. HETCOR analyses were performed with the spectrum recorded with F1 > 0 Hz to avoid quadrature images and artifacts at 0 Hz in the 1H spectrum (F1 = 0 Hz). The 19F CPDARR experiment was performed at νr = 10 kHz using the pulse sequence given in the Supporting Information, mixing periods given in the text, and a DARR RF field strength of 10 kHz.40 Chemical shift tensors (CSTs) were measured using a five-pulse 2D CP-PASS (phase adjusted spinning sidebands) pulse sequence.32 CSTs and quadrupolar parameters were fitted using Bruker Topspin version 3.0 (Bruker Biospin, Billerica, 10269



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10 torsion angles involving heavy atoms. The chloride ions were each allowed three translational degrees of freedom. A maximum angle change of 10° and a maximum distance change of 10% were enforced. A total of 37 degrees of freedom were thus refined, along with the unit cell parameters and the same polynomial background, peak shape profiles, line shift correction and asymmetry correction parameters used with the Pawley refinement. No preferred orientation corrections were used in the Rietveld refinement. An Rwp of 3.32% and an Rp of 2.56% were obtained. The resulting structure was subjected to an unconstrained DFT geometry optimization at the HCTH/DNP level of theory in which all atoms were allowed to move independently to minimize energy while the unit cell remained fixed. The Rietveld refinement was repeated to obtain a final Rwp of 3.29% and an Rp of 2.52%, and the resulting structure was used as the RT structure and is provided as a CIF in the Supporting Information.

MA) and the Herzfeld−Berger method implemented in the HBA software package.41,42 The convention δ11 ≥ δ22 ≥ δ33 is used for the principal components of the experimental CST, where δiso = (δ11 + δ22 + δ33)/3. Computational Methods. The DMol3 DFT package in Materials Studio version 6.0 (Accelrys, San Diego, CA), which uses the periodic boundary condition (PBC) approach, was used to optimize the hydrogen atom positions in the crystal structure using a fixed unit cell and fixed heavy atom positions from the SCXRD determination.43,44 Hydrogen atom positions were optimized with the HCTH/407 generalized gradient approximation (GGA) density functional (referred to here as the HCTH functional) and a double numerical basis set with polarization functions on all atoms (the DNP basis set).45,46 A 2 × 3 × 1 k-space was used for the Brillouin zone integration. The structure obtained using HCTH/DNP optimization of the hydrogen positions is referred as the LT structure (a CIF for this structure is available in the Supporting Information). The DMol3 package was also used for EFG calculations as described below.47 The CASTEP PBC package in Materials Studio was used for both EFG and chemical shielding calculations, with the latter performed using the gauge-including projector augmented wave (GIPAW) method.48,49 GIPAW calculations were performed using the PBE functional with a plane wave cutoff energy of 500 eV, on-the-fly generated pseudopotentials, and a 1 × 2 × 1 kspace.50 The principal components of the chemical shielding tensor are ordered as |σzz − σiso| ≥ |σxx − σiso| ≥ |σyy − σiso|, where σiso represents the isotropic chemical shielding. The chemical shielding anisotropy (σaniso) is defined as σaniso = σzz − (σxx + σyy)/2 and reported in ppm. The dimensionless chemical shielding asymmetry parameter (ηcs) is defined as ηcs = (σxx − σyy)/(σaniso − σzz). The Gaussian 09W software package (Gaussian, Inc., Wallingford, CT) was used for EFG and chemical shielding calculations with restricted Hartree−Fock (RHF) theory and DFT using the B3LYP functional.51−53 Molecular clusters were used for these calculations as shown in the Supporting Information. Chemical shielding calculations were performed with the gauge-independent atomic orbital (GIAO) method and Gaussian basis sets.53 A locally dense basis set approach was employed for individual atoms, with a 6-311+G(2d,p) basis set on Cl33/Cl73 and a 3-21G basis set on all other atoms.54 The EFG tensor values produced by DMol3, CASTEP, and Gaussian 09W were converted from the atomic units reported by each package into MHz by multiplication of the calculated EFG tensor components by the quadrupolar moment (−0.0817 barn for 35Cl) and then by eq/ℏ (approximately 234.956). The convention used for ordering EFG tensor components is |Vzz| ≥ |Vyy| ≥ |Vxx|. The quadrupolar coupling constant is defined as CQ = (eQVzz)/h. The dimensionless quadrupolar asymmetry parameter (ηQ) is defined as ηQ = (Vxx − Vyy)/Vzz. An RT structure was produced for use with the aforementioned NMR chemical shielding and EFG calculations. To produce this structure, the SCXRD structure was subjected to Rietveld refinement using the room temperature capillary PXRD pattern (see Supporting Information).25 In the Rietveld refinement, the first ronacaleret cation was allowed 6 translational and rotational degrees of freedom, while the second cation was allowed 5 degrees of freedom because the diffraction pattern in this case is invariant under some translations of the atoms in the asymmetric unit. The refinement of each of the two ronacaleret molecules included

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RESULTS AND DISCUSSION Crystal Structure of Form 1. Key experimental and structural parameters from the SCXRD analysis of ronacaleret HCl form 1 are given in Table 1. The final R1 [I > 2σ (I)] for the form 1 crystal structure was 4.23%. The structure contains two ronacaleret cations and two chloride anions in the asymmetric unit (Z′ = 2), as shown in Figure 1a. The two ronacaleret cations are referred to as cation A and cation B. The numbering scheme in cation B may be obtained by adding 40 to the atom number of the equivalent atom in cation A. The chloride anions also follow this scheme and are referred to Cl33 and Cl73. The absolute stereochemistry of ronacaleret was unambiguously determined on the basis of anomalous dispersion effects, with the chiral center found to have an (R)-configuration in both independent cations. The crystal structure of form 1 can be described as pseudocentrosymmetric and the two independent cations have conformations that are essentially inverted variations of one another. This relationship can be illustrated by fitting the non-hydrogen atoms of cation A to an inverted cation B, as shown in Figure 1b, although it should be noted that the latter inverted species does not actually exist in the structure and is presented for illustrative purposes only. Figure 1b illustrates that the only exception to the conformational inversion of the two independent cations is in the region of the chiral center, where true inversion is not possible owing to I being a single enantiomer. However, conformational variations around the chiral center allow for a similar overall position for the hydroxyl group in both molecules. Several other features of the crystal structure are noteworthy. In the following discussion, cation A is described with corresponding findings for cation B being presented in square brackets. The non-hydrogen atoms of the side-chain containing the carboxylic acid group are approximately coplanar, giving an root-mean-square (RMS) error of 0.040 Å [0.023 Å]. The C2− C1−C23-C24 torsion angle, describing the relationship of the side-chain relative to the attached phenyl ring, is 70.8(4)° [-62.7(4)°]. The chain linking the two ring systems is not fully extended and for cation A is a mix of anti and gauche conformations. For cation B, the drive to maintain a similar position for the hydroxyl group leads to one torsion angle that is nearly eclipsed. The torsion angles through the linker, from C4−C5−O7−C8 to C12−C13−C14−C15, are −176.7(3)° [164.6(4)°], 158.9(3)° [−168.9(3)°], 60.2(3)° [20.1(5)°], −174.3(2)° [168.9(3)°], 157.7(2)° [148.3(3)°], 56.9(4)° 10270



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[−72.3(4)°], −173.6(3)° [171.2(3)°] and 172.2(3)° [−173.0(3)°]. The five-membered ring has an envelope conformation, with C14 [C54] lying 0.422(4) Å [0.401(5) Å] from a plane defined by the other non-hydrogen atoms of the fused ring system. This plane has a normal that is inclined at 78.10(10)° [79.53(11)°] to the normal of the phenyl ring. There are eight classical hydrogen bonds associated with the form 1 crystal structure. Two of these link the independent cations to form a dimeric unit using the carboxylic acid groups, as depicted in Figure 1c. The side-chains containing the carboxylic acid groups are coincident with the (1 0 1) family of lattice planes. The remaining hydrogen bonds link the cations to the anions, with each chloride ion accepting three interactions from one independent cation or its symmetry equivalent. These hydrogen bonds link ronacaleret cations and chloride anions in the direction of the crystallographic b-axis. Detailed hydrogen bonding metrics are given in the Supporting Information. The unit cell constants were redetermined at 295 K for the form 1 crystal used in this study and are given in the Supporting Information. The unit cell expands relatively evenly with increasing temperature, with an overall volume change of approximately 2.5%. The unit cell from the structure determined at 150 K was refined as previously discussed using the Pawley method against the capillary PXRD pattern of form 1 (see Supporting Information).24 The difference between the refined and experimental patterns showed no significant unmodeled features, confirming that the single crystal was representative of the bulk form 1 powder used in the SSNMR studies. 1 H SSNMR Analysis. In Figure 2, the 1H DP-MAS spectrum of form 1 at νr = 35 kHz and the 1H spectrum obtained using DUMBO homonuclear decoupling at νr = 30 kHz are compared. The strong 1H homonuclear dipolar coupling is further suppressed by use of DUMBO decoupling, which improves the resolution. Given the highly crystalline nature of the form 1 batch used in this study (as observed by PXRD), the remaining broadening in the DUMBO spectrum may be caused by anisotropic bulk magnetic susceptibility (ABMS) arising from the presence of aromatic rings in the structure and their relative alignment in the crystal.55 This effect is often encountered in pharmaceutical materials containing aromatic or heteroaromatic rings.4−9 The resolution in the 1H DUMBO spectrum may also be limited by interference caused by similarities between the cycle time of the decoupling sequence and the rotor period.56 However, the DUMBO conditions used here were found to be effective in achieving higher resolution in other crystalline substances studied in our laboratory. Although resolution is improved, DUMBO introduces spectral artifacts from RF-rotor-resonance lines.57−59 Because of this, 1H 1D and 2D experiments based on DUMBO and conventional MAS-only techniques were both used in the present work. Although the resolution in the 1H DUMBO spectrum of form 1 is limited, a number of useful spectral assignments are still possible. These are informed by the solution-state 1H NMR shifts for ronacaleret in d6-DMSO solution and the GIPAW calculations performed on the form 1 crystal structure (see Supporting Information). In Figure 2, the carboxylic acid protons (H27 and H67) are assigned to the most deshielded 1 H peak at 13.0 ppm, as these protons participate in dimeric hydrogen bonding interactions in the form 1 structure with a donor−acceptor (D···A) distance of 2.69 Å. The observed 1H

Figure 2. 1H DP-MAS (νr = 35 kHz) and 1H 1D DUMBO (νr = 30 kHz) spectra of form 1. Additional resolution available from DUMBO decoupling (using the eDUMBO-122 variant of this sequence) allows for observation of the H11A/B and H51A/B positions as well as other positions of interest. Spectra were obtained at 16.4 T and 283 K.

chemical shift of 13.0 ppm is consistent with this type of hydrogen bonding interaction.60 The H27 and H67 protons in the Z′ = 2 structure could not be distinguished in the 1H DUMBO spectrum, although their corresponding carboxylate carbons (C25 and C65) can be resolved in the 13C SSNMR spectra described later in this work. The inability to resolve 1H chemical shifts for H27 and H67 is consistent with the σiso values calculated with the GIPAW method, which predicted the 1 H shift of these sites to be within 0.07 and 1.37 ppm of each other using the LT and RT structures, respectively. The H11A/ B and H51A/B positions, attached to N11 and N51, are partially resolved in the DP-MAS spectrum and are assigned in the spectrum in Figure 2 based on 1H−15N CP-HETCOR results discussed in detail below. The 1H DUMBO spectrum in Figure 2 also reveals the presence of highly shielded proton resonances at about 0.6 ppm, which are often associated with aromatic π-stacking interactions that cause shifts to higher frequency for 1H and 19F nuclei e.g. in close proximity to the region directly above the plane of an aromatic ring.61−63 Both GIPAW calculations predicted that H23B and H63B are highly shielded relative to the other sites in form 1. Inspection of the form 1 crystal structure reveals that the H23B position is 3.23 Å away from the centroid of the C57−C61 ring and the H63B position is 3.06 Å away from the centroid of the C16−C21 ring (although the true distances to the rings are shorter because of the artificially short distances that arise in X-ray diffraction studies between hydrogens and their attached carbons).60 The experimental observation of 1H resonances consistent with highly shielded H23B and H63B positions suggests that the GIPAW calculation accurately replicates the shielding effect of π-stacking in form 1. 10271



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2D 1H−1H spin diffusion experiments obtained with DUMBO decoupling in both dimensions were employed to detect interactions between resolved proton sites, as shown in Figure 3.58 Correlations between aliphatic and aromatic protons are observed at a mixing time of 200 μs but are not sufficiently resolved to draw any structural conclusions. More useful correlations are observed between the H11A/B and H51A/B protons at a mixing time of 400 μs, with evidence of the correlation first appearing at a mixing time of 200 μs. The 1 H assignments shown in Figure 3 for these positions are established by the results of a 1H−15N CP-HETCOR spectrum discussed below. Since the H11A/B and H51A/B protons have 1 H−1H distances of at least 10 Å in the crystal structure, this correlation likely arises from slow but favorable spin diffusion build-up assisted by partial spectral overlap between the resonances assigned to H11A/B on cation A and H51A/B on cation B. Because of complete 1H spectral overlap between the protons attached to the same nitrogen, the short-range correlations between e.g. H11A and H11B are not resolved. Weak correlations to aliphatic and aromatic protons involving the H11A/B and H51A/B protons as well the H27 and H67 protons were observed with a mixing time of 400 μs (not shown). The low intensity of these correlations results from reduced spin diffusion that is likely affected by the lack of chemical shift overlap and the detailed multispin transfer aspects of the spin diffusion network in the form 1 crystal structure. Examination of the GIPAW results for the LT and RT structures for support for the 1H assignments yielded 1H σiso values that were only in partial agreement, suggesting that the calculation is insufficiently accurate to fully reproduce the observed trend or that any remaining differences between the RT structure and the actual structure may affect the calculated 1 H shifts of the NH2 protons. Although extensive spectral overlap in the 1D 1H spectra and the 2D 1H−13C CPHETCOR spectra discussed below precluded detailed estimates of errors in 1H σiso values relative to δiso values for form 1, typical GIPAW 1 H isotropic shielding calculations in pharmaceutical solids yield average absolute errors on the order of about 0.3 to 0.4 ppm.20 1 H spectra were also obtained using a DQ-BABA pulse sequence using MAS (νr = 35 kHz) without dipolar decoupling, which yielded better results because of reduced artifacts relative to DQ experiments performed with DUMBO decoupling.38,58,59 The 1H DQ-BABA spectrum of form 1 is shown in Figure 4. The spectrum highlights the dimeric short-contact 1 H−1H interaction often seen in carboxylic acids via a correlation at a DQ frequency of 26.2 ppm.63 This correlation arises because of the 2.6 Å distance between H27 and H67. An additional resolved correlation is observed at a DQ frequency of 15.0 ppm and involves short-range contacts to H24A/B and H64A/B. The remainder of the 1H DQ-BABA spectrum shows evidence of correlations between aromatic and aliphatic positions but is too overlapped to assign. Other positions of interest for the evaluation of hydrogen bonding effects, most notably the H30 and H70 hydroxyl protons that are hydrogen-bonded to chlorine anions, are not resolved in the 1H spectra. Using the GIPAW results, the σiso values for H30 (26.2 ppm) and H70 (25.6 ppm) are calculated to be about 8.7 and 8.1 ppm more shielded than H27 and H67, suggesting an average δiso for the hydroxyl groups of about 4.5 ppm. This region is obscured in the 1H spectra in Figures 2, 3,

Figure 3. 1H 1D DUMBO spin diffusion spectra (νr = 30 kHz) of form 1 obtained with three different spin diffusion mixing periods. The 1D DUMBO spectrum is plotted along the F1 (vertical) and F2 (horizontal) axes. Distances shown are taken from the form 1 crystal structure. Spectra were obtained at 16.4 T and 283 K.

and 4, but can be observed using the 1H−35Cl CP-HETCOR spectra discussed below. 10272



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Figure 4. 1H DQ-BABA spectrum of form 1, obtained at νr = 35 kHz, showing 1H−1H dipolar interactions. The DP-MAS spectrum (νr = 35 kHz) is plotted along the F2 axis. A skyline column projection is plotted along the F1 axis. Distances shown are taken from the form 1 crystal structure. Spectra were obtained at 11.7 T and 283 K. 19

F SSNMR Analysis. The 19F SSNMR spectrum of form 1 is expected to show up to four distinct resonances arising from the two fluorine atoms in each molecule in the asymmetric unit. 19 F spectra obtained at different spinning rates are shown in Figure 5, and four resonances with different isotropic shifts are observed. All spectra in Figure 5 were obtained with CP-MAS except for the spectrum at νr = 32 kHz, which was obtained using a DP-MAS experiment because of reduced CP efficiency at this high spinning rate. The 19F spectrum of form 1 consists of two sets of isotropic resonances centered at chemical shifts of −135 and −165 ppm. The 19F lineshapes are affected by rotational resonance (R2) when νr is set to 14 kHz and 8 kHz because of the near-overlap of sidebands for the orthosubstituted fluorines, which exhibit strong dipolar coupling, leading to partial reintroduction of 19F−19F homonuclear dipolar coupling (see Figure 5 insets).64 The ortho-substituted fluorines in ronacaleret have an internuclear distance of approximately 2.7 Å and thus a dipolar coupling magnitude of about 5.5 kHz. The R2 condition is avoided when νr is set to 32 kHz and is largely avoided at 10 kHz (with minor effects on the F29/F69 resonances still observed). Assignment of the pairs of resonances to F28/F68 and F29/ F69 can be made by comparison with the GIPAW calculations and solution-state 19F NMR assignments (see Supporting Information). The pairs of signals obtained at approximately −135 and −165 ppm thus correspond to F28/F68 and F29/ F69, respectively. Assignment of the individual signals appearing at −133.9, −135.9, −164.2, and −165.0 ppm to cations A and B is challenging and requires extensive analysis presented in detail in the following sections. Direct assignment using the GIPAW calculation is not feasible, as calculations of 19 F σiso values are generally believed to be accurate only to within a few ppm, and the experimental difference between each pair of resonances is within this accuracy.65 The GIPAW

Figure 5. 19F DP-MAS spectrum (νr = 32 kHz) of form 1 compared with CP-MAS spectra obtained at νr = 14, 10, and 8 kHz. The insets show expansions of the centerbands in each spectrum. Assigned fluorine positions are shown over the centerbands of the DP-MAS spectrum. Centerbands in the CP-MAS spectra are denoted with arrows. The CP-MAS spectrum at νr = 14 kHz most clearly shows the effects of the R2 condition, while the spectrum at νr = 10 kHz was the best achievable combination of a slower spinning rate and the avoidance of the R2 condition (although minor R2 effects are still observed). All spectra were obtained at 11.7 T and 273 K.

calculations performed on the LT and RT structures illustrate the difficulties encountered, as the calculation performed on the LT structure predicts that F28 is more shielded than F68 by 0.11 ppm while the calculation performed on the RT structure predicts F28 to be more shielded than F68 by 2.06 ppm. The 19F CSTs for each of the fluorine sites were measured to assess whether they could offer a means of making assignments for each cation. For fitting, the sideband manifold of a 19F DPMAS spectrum performed with νr = 10 kHz was used to avoid the aforementioned R2 condition as much as possible (see Supporting Information). As shown in the Supporting Information, when experimental CSTs were linearized relative to their calculated counterparts from both GIPAW calculations, 10273



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the linearization was not significantly affected when the relative assignments of the F28/F68 and F29/F69 positions are reversed. This indicates that CST analysis cannot assist in the assignments. Other approaches using 19 F as the sole heteronucleus, such as 1H−19F CP-HETCOR experiments, also did not offer any confirmation of the 19F assignments (see Supporting Information). The 19F assignments shown in Figure 5 are the result of cointerpretation with the 13C spectra using additional correlation experiments described in later sections. The fluorine groups in ronacaleret are ortho-substituted relative to each other with an internuclear distance of approximately 2.7 Å, allowing a 19F dipolar correlation experiment to be used to associate F28 with F29 in cation A and F68 with F69 in cation B in the Z′ = 2 asymmetric unit. This provides an important link between the assignments made from the GIPAW calculations in later sections and the observed internuclear proximity. The 19F CP-DARR experiment was chosen for this task because it is an efficient method for detection of dipolar interactions between fluorine sites.66 The 19 F CP-DARR experiment was performed with νr = 10 kHz to allow for efficient CP while also avoiding the R2 condition to maximize resolution. During the mixing period, the 1H RF field of 10 kHz broadens the R2 condition through heteronuclear 1 H−19F dipolar coupling with nearby hydrogens, thus reintroducing the 19F homonuclear dipolar interaction. The range of spin diffusion in this experiment is potentially extensive because of the long 19F T1. The results of 19F CPDARR experiments on form 1 (each requiring 45 min of acquisition time) are shown in Figure 6. The spectrum obtained with a short mixing period of 2.5 ms (25 τr) shows immediate correlations between the more deshielded F28/F68 resonance and the more shielded F29/F69 resonance and vice versa, indicating these are present in the same molecule. Increasing the mixing period to 50 ms (500 τr) shows longer range spin diffusion between the two molecules in the asymmetric unit, which if the crystal structure was unknown would indicate that these peaks arise from a single crystalline phase.66 Full mixing is observed at 200 ms (2000 τr). As the use of 19F CP-DARR experiments in this role is relatively unexplored, a confirmatory 19F DQ-BABA experiment at νr = 32 kHz was also performed using direct excitation of 19F and with 1H decoupling active throughout the experiment (see Supporting Information). In cases involving complex fluorinated pharmaceuticals, the resolution gained in the DQ dimension might be an additional factor in favor of use of this experiment. However, for the present application, the 19F CP-DARR experiment was found to be an efficient approach to link the fluorine resonances present in each molecule in the asymmetric unit in form 1. 15 N SSNMR Analysis. Form 1 was analyzed using both 1D 15 N CP-MAS experiments and 1H−15N CP-HETCOR experiments, the latter to detect dipolar interactions that could aid in spectral assignment. The 1H−15N CP-HETCOR experiment is rarely applied to pharmaceutical compounds because of its limited sensitivity and long acquisition times, but it is feasible for phases with a short 1H T1, protonated nitrogen sites, and relatively narrow lineshapes.67 In Figure 7, the results of two 1 H−15N CP-HETCOR experiments on form 1 are shown. A 15 N CP-MAS spectrum of form 1 obtained with νr = 10 kHz is plotted along the F2 axis in Figure 7 and consists of two resonances at −307.0 and −310.2 ppm as expected from the Z′ = 2 crystal structure. The GIPAW chemical shielding

Figure 6. Expanded regions of the 1H−19F CP-DARR spectra (νr = 10 kHz) of form 1 obtained with three mixing periods: (top) 2.5 ms or 25 τr, (middle) 50 ms or 500 τr, and (bottom) 200 ms or 2000 τr. The expansions show the regions of the spectra assigned to spinning sidebands (denoted “ssb”). Expansions of the CP-MAS spectra (νr = 10 kHz) are plotted along the F1 and F2 axes. The F1 dimension was rotor-synchronized. Spectra were obtained at 11.7 T and 273 K. 10274



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Contact times of 200 and 500 μs were used in the 1H−15N CP-HETCOR experiments to highlight directly attached protons and longer range interactions, respectively. The higher frequency nitrogen site (N51) correlates to the higher frequency 1H signal in Figure 7a using a contact time of 200 μs. These correlations are assigned to protons that are directly attached to the NH2 groups, and the 1H shielding trend generally agrees with the correlations seen in the 1H−15N CPHETCOR spectrum in Figure 7a. This assignment of H51A/B to the higher and H11A/B to the lower frequency region between 8 to 10 ppm is consistent with the 1H−1H spin diffusion experiment shown in Figure 3 and discussed above. In Figure 7b, a 1H−15N CP-HETCOR experiment obtained using a 500 μs contact time is shown, where longer range 1H−15N correlations enhanced by 1H−1H spin diffusion are observed between both nitrogen sites and aliphatic proton environments at about 3 ppm. These 1H sites cannot be distinguished in the spectra but likely include protons attached to C10/C50, C31/ C71, and C32/C72. However, these correlations offer no further insight into structural differences between cations A and B. 13 C SSNMR Analysis. The 13C CP-TOSS spectrum of form 1, shown in Figure 8, exhibits the complexity expected from the SCXRD results and illustrates the challenges faced in assigning resonances to individual positions and molecules in the asymmetric unit. The solution-state 13C NMR assignments in DMSO-d6 for ronacaleret HCl (see Supporting Information) were combined with the results of the GIPAW calculations to provide initial assignments for the 13C SSNMR spectra shown in Figure 8. The characteristic splitting typically observed for a crystal structure with Z′ = 2 is visible in several regions of the spectra, although many resonances show no sign of this characteristic splitting. The similarity of the molecular conformations and intermolecular interactions for the two molecules in the asymmetric unit, as seen in Figure 1, is the likely cause of this effect. Only the carbons corresponding to cation A are noted in Figure 8 for simplicity; the individual 13C resonances assigned to cations A and B are discussed below. 19F heteronuclear decoupling was used to remove J-coupling effects in the CP-TOSS spectrum to maximize resolution for many of the overlapped 13C resonances. To help assess whether molecular motion affected the 13C spectra, several spectra were obtained with 19F dipolar decoupling over a temperature range of 100 K (see Supporting Information). No effects were observed that would be indicative of molecular motion by the variable temperature experiments. Given the highly crystalline nature of the sample, the remaining broadening of the 13C spectrum after 19F decoupling (with a line width of ∼45 Hz) is thus likely caused by ABMS effects, which are also likely responsible for the similar line width observed in the 19F and 15N spectra and also provide a source of line broadening in the 1H spectra reported here.55 ABMS broadening and spectral overlap hinders the use of experiments such as the CP-INADEQUATE experiment, which is highly useful for pharmaceutical materials exhibiting sharp 13C resonances but is limited in practice by low sensitivity and long acquisition periods (e.g., >1 week) for many compounds of interest.10,17 Thus, unlike the 19F CP-DARR experiment, there is no convenient 13C−13C correlation experiment in which the form 1 13C resonances can be linked to individual molecules in the asymmetric unit. Evidence of molecular motion in form 1 was detected using a spectrum obtained with 1H dipolar dephasing as shown in Figure 8,

Figure 7. 1H−15N CP-HETCOR spectra of form 1, obtained with νr = 10 kHz, showing 1H−15N dipolar interactions. In part a, a contact time of 200 μs was used, to show short-range dipolar interactions. In part b, a contact time of 500 μs shows longer-range interactions in form 1. Distances shown are taken from the form 1 crystal structure. The 15N CP-MAS (νr = 10 kHz) spectrum and the 1H DP-MAS spectrum (νr = 35 kHz) are plotted along the F2 and F1 axes, respectively, for both 2D spectra. All spectra were obtained at 11.7 T and 273 K.

calculations using the LT and RT structures as input predict N51 to be 3.41 and 1.84 ppm more shielded than N11, respectively. The experimental chemical shift difference between the two nitrogen sites of 3.2 ppm agrees with the trend in both calculations, which was used to tentatively assign the 15N resonances in Figure 7. A CP-MAS spectrum was also taken with νr set to 1.5 kHz, and no spinning sidebands were observed, indicating that the nitrogen sites possess a small chemical shift anisotropy. This result is in agreement with the GIPAW calculations, where σaniso for both 15N sites was predicted to be less than 43 ppm. 10275



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Figure 8. 13C CP-TOSS spectrum of form 1 of ronacaleret HCl (I) compared with the 13C CP-TOSS spectrum obtained with dipolar dephasing for a duration of 4 τr. The 13C CP-TOSS spectrum was obtained with additional 19F π-pulse heteronuclear decoupling, which primarily affects the C3 and C4 positions. The 13C CP-TOSS spectrum obtained with dipolar dephasing used 1H decoupling only. All spectra were obtained at 11.7 T and 273 K with νr = 8 kHz. For simplicity, the assignments shown refer to atomic positions in only the first molecule in the asymmetric unit.

which is also useful for confirming assignments. The 13C dipolar dephasing spectrum normally retains signals from quaternary and methyl carbons, the latter because of rapid molecular motion. However, the dipolar dephasing spectrum in Figure 8 also retains the signals from the C22 and C62 methylene carbons, indicating that these positions in the indanyl ring system undergo rapid motion not reflected in the SCXRD structure or in the variable temperature 13C CP-TOSS spectra. The motion involving C22 and C62 and their accompanying hydrogens, which are related to each other by the aforementioned pseudocentrosymmetry, may result from an envelope-type motional model common in five-membered rings. It is interesting to note that the chemically equivalent C15 and C55 methylene carbons do not show this effect, which may be the result of the intramolecular and intermolecular environments around C22 and C62, including conformation and dispersive forces. Future investigation using molecular dynamics methods may provide insight into this effect. The complex 13C spectrum of form 1 was studied and interpreted in greater detail using complementary 2D SSNMR methods. A 2D 1H−19F−13C DCP-HETCOR experiment was performed to link the fluorine sites with carbon sites via both intra- and intermolecular 19F−13C dipolar coupling, with results shown in Figure 9. This spectrum is useful for assigning the 13C spectrum and relating its assignment to the 19F spectrum and to the individual molecules in the asymmetric unit.66,68 A 2D 1 H−13C CP-HETCOR experiment was also performed to similarly correlate proton and carbon sites, as shown in Figure 10, and in the present work was cointerpreted with the 1 H−19F−13C DCP-HETCOR experiment.39 In Figure 9, four expanded regions of the 1 H− 19 F− 13 C DCP-HETCOR spectrum of form 1 are shown. In Figure 9a, long-range 19 F−13C correlations are observed between the C25 and C65 positions and the F29 and F69 positions. The distances noted

in Figure 9a are taken from the form 1 crystal structure and are among the longest-range correlations detected in this experiment. These correlations allow for the first tentative assignment of F29 and F69 to their respective molecules in the asymmetric unit. In Figure 9(a), the resonance assignments shown for C25 and C65 are in agreement with the trend predicted by the GIPAW calculation, although the shift/shielding difference between the two positions is too small for the calculation to be definitive (see Supporting Information). However, the assignment of the C25 and C65 chemical shifts is also supported by a well-established trend for hydrogen bonding to carbonyl acceptors.69,70 The O66 acceptor is likely engaged in a slightly shorter hydrogen bond than the O26 acceptor as measured by O···O distance in the crystal structure, although the differences in distance are just within the standard uncertainties (see Supporting Information). The trend is consistent with the attachment of the O66 acceptor to the more deshielded C65 nucleus, which was itself consistent with the GIPAW calculation using both the LT and RT structures as input. The 1H−13C CP-HETCOR experiment detected correlations between C25 and C65 and their nearby hydrogen-bonded protons (H27 and H67), as shown in Figure 10a, but was unable to distinguish between the two positions on the basis of 1 H chemical shift. This is consistent with the GIPAW calculations, as noted above, which predicted the 1H shift difference of these sites to be within the resolution of the 1H dimension. The principal components of the 13C CSTs for the C25 and C65 positions were determined via a 2D CP-PASS spectrum (see Supporting Information). Although slightly greater differences are observed between the principal components than between the δiso values alone, the differences in the components are too subtle to offer any support for the C25 and C65 resonance assignment using the DFT calculations. The δiso values observed by the 13C CP-TOSS 10276



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Figure 9. Expansions of the 1H−19F−13C DCP-HETCOR spectrum of form 1 (νr = 10 kHz) obtained using a 2 ms 1H−19F contact time and a 6 ms 19 F−13C contact time for 2D dipolar correlation between 19F and 13C spins. The 13C CP-TOSS spectrum (νr = 8 kHz) with 1H and 19F decoupling is plotted along the F2 axis. The 19F CP-MAS spectrum (νr = 10 kHz) is plotted along the F1 axis. The 19F axes are expanded to show regions of the spectra assigned to spinning sidebands (denoted “ssb”). Spectra were obtained at 11.7 T and 273 K. Distances shown are taken from the form 1 crystal structure and the shortest distance between the fluorine−carbon pair is shown in the case of multiple contacts. The intensity of the contour plots in (a) and (c) has been increased 4-fold relative to the other plots to show detail. Annotations are not shown where overlap prevented confident assignment.

spectrum and the correlations seen in the 1H−13C−19F DCPHETCOR spectrum thus offered the greatest specificity for the slightly different hydrogen bonding environments of the carboxylic acid groups, and support a tentative assignment of the resonances of cations A and B as shown. Further assignments can be made using other regions of the 1 H− 13 C− 19 F DCP-HETCOR spectrum. In Figure 9b, correlations between the fluorine positions and aromatic carbon resonances are observed that enable the assignment of

many aromatic 13C resonances for each of the molecules in the asymmetric unit. While a number of correlations are overlapped, at least 16 resolved correlations can be assigned clearly as shown. This type of information is not available from the aromatic region of the 1H−13C CP-HETCOR spectrum in Figure 10a. The region of the 1H−13C−19F DCP-HETCOR spectrum shown in Figure 9c provides information about C9, C49, C12, and C52. The portion of the 13C dimension including C8 and 10277



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The C9 and C49 positions are readily assigned by their correlations to H11A/B and H51A/B in the 1H−13C CPHETCOR spectrum in Figure 10(b), which is consistent with the previous assignment of these 1H positions from the 1 H−15N CP-HETCOR spectrum. C8 and C48 are assigned to the remaining resonances. These correlations allow for confirmation of the assignment of the molecules in the asymmetric unit by comparing GIPAW-calculated 13C resonances to those obtained experimentally from the spectrum in Figure 9c, as discussed below. The assignments of the resonances between 80 and 60 ppm were further verified by comparing principal components of the 13C CSTs measured by CP-PASS with the GIPAW calculation and then reversing the assignments of the sites for cations A and B, which led to reduced agreement (see Supporting Information). The remaining informative region of the 1H−13C−19F DCPHETCOR spectrum is shown in Figure 9d. As with the other regions, 19F−13C dipolar correlations are observed in the 3.5 to 5 Å range that enhance the ability to assign the 13C spectrum, particularly with respect to cations A and B. Several weak correlations are observed in this region that are difficult to explain based on direct dipolar contacts because of long distances of >5 Å, such as apparent correlations from F29 to C31 and/or C32. These correlations may arise because of the influence of strong 19F−19F dipolar coupling during the 19 F−13C contact time period. The overall agreement between the 13C σiso values from the GIPAW calculations of 13C chemical shielding and the experimental δiso values as assigned in Figures 8, 9, and 10 was assessed after linearization (see Supporting Information). The LT and RT structures yielded RMS errors of 1.89 and 1.66 ppm, respectively, with the latter structure thus showing better agreement. The largest outliers from each linearization were C22 and C62, which is explained by the influence of molecular motion involving these sites (as seen in the dipolar dephasing spectrum in Figure 8) on the δiso values through averaging of one or more components of the CSTs, which is not accounted for in the GIPAW calculations. Overall, interpretation of the 2D 1 H−19F−13C DCP-HETCOR spectrum of form 1 provided 13C assignments consistent with the GIPAW calculations as well as 19 F assignments for cations A and B by correlating each fluorine position to carbon sites for which additional DFT-predicted chemical shielding results were available. To further confirm the 19F and 13C assignments of cations A and B made above in relation to the GIPAW calculation on the LT and RT structures, the trend in calculated differences in σiso for corresponding carbons in cations A and B was compared to the assigned 13C δiso trend for positions where an observation could be made (see Supporting Information). For example, with the RT structure, δiso,C6 − δiso,C46 yielded −1.97 ppm, while σiso,C46 − σiso,C6 yielded −3.39 ppm, showing an agreement between the predicted and observed trends for these carbons in cations A and B. For the RT structure, using the assignments given above, nine observable carbon resonances agreed with the calculated trend, three observable carbon resonances (C10, C22, and C24) did not agree with the calculated trend, and a predicted trend for C32 was not experimentally observed. A similar result was obtained for the LT structure. 35 Cl SSNMR Analysis. In addition to the spin 1/2 nuclei already examined in detail, the chloride anions in the ronacaleret HCl form 1 structure were studied using 35Cl SSNMR. The 35Cl nucleus is a quadrupolar nucleus with a spin

Figure 10. 1H−13C CP-HETCOR spectrum of form 1, obtained with νr = 10 kHz, showing 1H−13C dipolar interactions. The spectrum was obtained without 19F decoupling. A contact time of 500 μs was used. In part a, the lower frequency 13C spectral region is shown, with the higher frequency region shown in part b. Noteworthy correlations that could be confidently interpreted are marked on the spectra (see text). Distances shown are taken from the form 1 crystal structure. The 13C CP-TOSS spectrum obtained with 1H and 19F decoupling is plotted along the F2 axis (νr = 8 kHz). The 1H DP-MAS spectrum (νr = 35 kHz) is plotted along the F1 axis. All spectra were obtained at 11.7 T and 273 K.

C48 is not shown because it did not contain useful correlations and was dominated by spinning sidebands from C3 and C43. An additional 1H−19F−13C DCP-HETCOR spectrum (not shown) was obtained with νr = 8 kHz to verify that no useful correlations could be detected involving C8 and C48. The 13C spectral region between 80 and 60 ppm was assigned by starting with the prior knowledge of the assignment of C12 and C52 from the dipolar dephasing spectrum shown in Figure 8. 10278



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transition line shape. The experimental spectrum shown in Figure 11a is referenced with respect to crystalline NaCl at 0 kHz and 0.0 ppm.18 The fidelity of the line shape observed by DP-MAS was verified using a spin−echo MAS experiment (see Supporting Information). The 35Cl DP-MAS spectrum of form 1 was fitted to a model consisting of two equally weighted central transition lineshapes as anticipated from the two nonequivalent chlorine sites in the crystal structure (Cl33 and Cl73). The results of the fitting are shown in Figure 11a and reported in Table 2. The fit used exponential line broadening factors of 400 Hz and included the quadrupolar coupling constant CQ and asymmetry parameter ηQ along with a δiso value for each 35Cl site. Anisotropic chemical shift parameters were not included, as these are known to be small for 35Cl in hydrochloride salts (which is further supported by the calculations described below).18,19 The δiso values given in Figure 11(a) have been converted using eq 1 and are referenced to dilute aqueous NaCl(aq).18 These δiso values, which differ by 12.8 ppm, are distinctive for the individual chlorine sites. Using the fitted δiso values and quadrupolar parameters, the two experimentally observed chlorine sites were assigned to the two chlorine atoms in the asymmetric unit of form 1. A series of calculations were performed using the PBC methods in DMol3 and CASTEP and a molecular cluster in Gaussian 09 (see Supporting Information and Table 2). The σiso values for the chlorine sites can be predicted by the Gaussian 09 and CASTEP programs, but not by the DMol3 program. All chemical shielding calculations predicted the same trend in shielding for Cl33 and Cl73. The CASTEP results predicted Cl33 to be less shielded than Cl73 by 20.38 and 29.88 ppm using the LT and RT structures, respectively. The molecular cluster calculation using Gaussian 09 yielded a similar 17.06 ppm difference using the LT structure. This suggests that the 35 Cl site with a δiso value of 64.7 ppm shown in Figure 11a corresponds to the more shielded Cl73 position. This shielding trend forms the basis of the 35Cl assignments given here. The CASTEP chemical shielding calculations also indicate that the anisotropic chemical shifts for 35Cl are likely small, with both sites showing σaniso values of less than 100 ppm, justifying their exclusion from fitting and in agreement with previous work.18−20 The EFG results in Table 2 were compared with calculated results from the DMol3, CASTEP, and Gaussian 09 packages. Calculation of EFG parameters is a useful tool to assist in the assignment of 35Cl spectra as well as the spectra of other quadrupolar nuclei.19,72−74 The accurate prediction of ηQ for 35 Cl has been reported to be particularly challenging.73 Given the larger size of the form 1 crystal structure relative to crystal structures used in previously reported 35Cl studies, and the wide range of computational methods used in other reports, the calculation of 35Cl EFG parameters in this work was extended to include different computational approaches and levels of theory for comparison with the experimental 35Cl parameters.19,72−77 The results in Table 2 that were predicted using different methods in DMol3 and CASTEP with the LT structure as input generally overestimate the magnitude of CQ in comparison to the experimental results, and also do not agree with the relative order of magnitudes of CQ for the two chlorine sites (i.e., the larger CQ was observed for Cl73 instead of Cl33, as expected). In contrast, the PBC-based calculations of ηQ generally agree with the experimental results using the assignments obtained from the 35Cl chemical shifts, with the

of 3/2 that offers direct structural insight into the chlorine environment. High-field 35Cl SSNMR is particularly useful for the analysis of organic hydrochloride salts because of their lower CQ magnitudes (typically

Solid-State NMR Analysis of a Complex ... - American Chemical Society

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