Solid-State NMR Analysis of Organic Cocrystals and Complexes Frederick G. Vogt,*,† Jacalyn S. Clawson,† Mark Strohmeier,† Andrew J. Edwards,‡ Tran N. Pham,‡ and Simon A. Watson‡ Chemical DeVelopment, GlaxoSmithKline plc., 709 Swedeland Road, King of Prussia, PennsylVania 19406, and Chemical DeVelopment, GlaxoSmithKline plc., Gunnels Wood Road, SteVenage, Hertfordshire SG1 2NY, U.K.
CRYSTAL GROWTH & DESIGN 2009 VOL. 9, NO. 2 921–937
ReceiVed July 1, 2008; ReVised Manuscript ReceiVed October 19, 2008
ABSTRACT: Solid-state NMR (SSNMR) is capable of providing detailed structural information about organic and pharmaceutical cocrystals and complexes. SSNMR nondestructively analyzes small amounts of powdered material and generally yields data with higher information content than vibrational spectroscopy and powder X-ray diffraction methods. These advantages can be utilized in the analysis of pharmaceutical cocrystals, which are often initially produced using solvent drop grinding techniques that do not lend themselves to single crystal growth for X-ray diffraction studies. In this work, several molecular complexes and cocrystals are examined to understand the capabilities of the SSNMR techniques, particularly their ability to prove or disprove molecular association and observe structural features such as hydrogen bonding. Dipolar correlation experiments between spin pairs such as 1H-1H, 1 H-13C, and 19F-13C are applied to study hydrogen bonding, intermolecular contacts, and spin diffusion to link individual molecules together in a crystal structure and quickly prove molecular association. Analysis of the principal components of chemical shift tensors is also utilized where relevant, as these are more sensitive to structural effects than the isotropic chemical shift alone. In addition, 1H T1 relaxation measurements are also demonstrated as a means to prove phase separation of components. On the basis of these results, a general experimental approach to cocrystal analysis by SSNMR is suggested. Introduction Pharmaceutical cocrystals and complexes, which may be formed between an active ingredient (API) and one or more small organic molecules, are a key element of the growing field of crystal engineering.1,2 Typically, cocrystals and complexes are held together by strong directed interactions such as hydrogen bonds. In small-molecule pharmaceutical science, cocrystals and complexes offer an alternative approach to the delivery of active ingredients with the desired physical properties. In comparison to traditional solid forms such as crystalline polymorphs, salts, hydrates, solvates, or amorphous forms, delivering an active substance via a cocrystal can access a wider design space because of the large number of potential cocrystal formers, although the list is restricted to compounds with acceptable safety profiles, and is further restricted by the need to match the synthons of the API and the cocrystal former. In some cases, cocrystals have enabled custom development of a crystalline phase with tailored properties including solubility,3,4 bioavailablity,5 and chemical stability.6 In other cases, cocrystals have been found to have little or no impact upon solubility.7 In the pharmaceutical industry, the development of pharmaceuticals as cocrystals for eventual marketing faces several barriers to widespread acceptance, foremost of which is the need to develop large-scale processes capable of delivering cocrystals in high yields. Although recent work has shown that an understanding of the solubility relationships between the desired cocrystal and its components can allow for successful process scale-up, much work remains to be done to demonstrate the ability to make these phases with the batch scale and quality expected of marketed pharmaceuticals.8 The present work addresses another key barrier to cocrystal acceptance, namely, the analytical characterization of hits obtained from cocrystal screening. In general, limited analytical resources are available * To whom correspondence should be addressed. E-mail: Fred.G.Vogt@ gsk.com. † Chemical Development, GlaxoSmithKline plc., King of Prussia, PA. ‡ Chemical Development, GlaxoSmithKline plc., U.K.
early in the drug development process when the solid form is being chosen. The most definitive analytical data for cocrystal and complex identification, single crystal X-ray diffraction (SCXRD), may not be available in time to make development decisions. This is partially related to the most common method presently used to discover cocrystals, the solvent drop grinding (SDG) method, which does not lend itself to the production of single crystals.9 SDG avoids the potential problems of generating cocrystals from solution crystallization, as explained by ternary phase diagrams, and is believed to maximize the chance of success in finding pharmaceutical cocrystals.10 Vibrational and powder X-ray diffraction (PXRD) methods are commonly used to compare SDG-produced samples with spectra and patterns of the known input phases. The appearance of a new polymorph or solvate during the SDG process can confound the interpretation. In the absence of SCXRD data and when the cocrystal involves simple molecules, vibrational spectroscopy can be used to confirm an interaction between the molecules based on trends in carbonyl, hydroxyl, or other relevant vibrational frequencies.11 However, with the larger molecules commonly found in pharmaceutical development, a more discriminating technique would be helpful in verifying that a cocrystal has actually formed. Solid-state nuclear magnetic resonance spectroscopy (SSNMR) offers an analytical solution to cocrystal analysis; it is nondestructive, can study small amounts of powders, has nuclear-site specificity, is sensitive to hydrogen bonding and local conformational changes, and can evaluate mixed-phase samples and solvates.12 SSNMR allows access to a variety of one- and two-dimensional (1D and 2D) experiments which target different nuclei and can probe internuclear connectivity. To date, SSNMR has been used sparingly in the analysis of cocrystals, and only a limited set of experimental approaches have been illustrated in the literature.4,13,14 In the present work, we demonstrate the applicability of multinuclear 1D and 2D SSNMR techniques to the study of organic cocrystals and complexes with a range of NMR properties (e.g., accessible isotopes, chemical shifts and relax-
10.1021/cg8007014 CCC: $40.75 2009 American Chemical Society Published on Web 12/10/2008
922 Crystal Growth & Design, Vol. 9, No. 2, 2009
ation times). Here we use the terms “cocrystal” to refer to systems made from materials that are solids at room temperature and “complex” to refer to systems made from at least one liquid at room temperature.2 The SSNMR techniques demonstrated in this work are considered to be robust in that they can be readily applied to typical samples within reasonable data acquisition time periods. The examples studied include cocrystals of palmitic acid nicotinamide,15 piroxicam saccharin,7 fluoxetine hydrochloride succinic acid,16 and triphenylphosphine oxide (TPPO) 6-chloro-2-pyridone,17 all of which have been fully characterized by SCXRD. Studies of ternary complexes with a host framework of caffeine and succinic acid and different guest compounds are also described.9 Another host-guest complex examined is cholic acid 4′-fluoropropiophenone, which is a member of a group of cholic acid and cholic acid derivatives that form inclusion and clathrate-type complexes.18-21 Examples of relatively long and short organic hydrogen bonds are also included, namely, the complex of 4-picoline and pentachlorophenol and the cocrystal carbamazepine nicotinamide.22,23 SSNMR data supporting cocrystal formation in an “unknown” system involving nicotinamide and {4-(4-chloro-3-flurophenyl)2-[4(methyloxy)phenyl]-1,3-thiazol-5-yl} acetic acid is also presented. Because empirical trends in chemical shifts are interpreted as part of this work, computational methods are used in some cases to examine detailed aspects of chemical shifts and verify results. However, these calculations serve only in a supporting role; the experimental data with empirical knowledge of chemical shifts drive the analyses, allowing application to systems without the SCXRD structure needed for most computations. The present study is focused on nonionized cocrystals and only briefly treats the determination of ionization state with SSNMR, which can in practice be simultaneously addressed through closely related studies, particularly those that investigate chemical shifts and proton multiplicitity.24-28 The goal of this work is to demonstrate a general SSNMR approach to cocrystal analysis for a range of systems produced by SDG and direct crystallization, while also showing that it is possible to obtain detailed information rapidly from small amounts of powders even in the presence of substantial phase impurities. Experimental and Computational Methods Preparation of Materials. Materials were purchased from SigmaAldrich (St. Louis, MO, USA) unless otherwise noted and were used without further purification. PXRD patterns of each cocrystal and complex were obtained and compared with the pattern simulated from the respective crystal structures or a reported reference pattern; unless otherwise noted, the simulated and experimental PXRD patterns matched after refinement of unit cell, line shape, and preferred orientation parameters. Plots that compare simulated and experimental PXRD patterns for the systems discussed here are presented in the Supporting Information to demonstrate that the bulk powders used in SSNMR analyses are the same phases studied in earlier reports. Where applicable, the Cambridge Structural Database (CSD) reference code (refcode) for the crystal structure is given below for each example. Throughout this work, the numbering schemes reported in the CSD crystal structures have been used where possible in the SSNMR results, except where it deviates from other established numbering schemes. Hydrogen numbering, however, has been simplified in some cases to share the numbering of the attached heavy atom, except where explicitly noted on a chemical structure. Palmitic acid nicotinamide cocrystals (CSD refcode JEMDIP)15 were produced by grinding an equimolar mixture of the components (total mass of ∼300 mg) and two drops of acetone with a 1-cm stainless steel ball for 20 min in a Restch MM2000 mixer at a frequency of 30 Hz. A sample was also produced by recrystallization of an equimolar solution (containing 166 mg of palmitic acid, 79 mg of nicotinamide, and 4 mL of acetone) as described previously15 and was found to be indistinguishable by PXRD and SSNMR from the SDG sample; that
Vogt et al. is, both processes produced phase-pure palmitic acid nicotinamide cocrystals. The sample produced by SDG was used in the majority of the work described here. Caffeine succinic acid cocrystals forming inclusion complexes with dioxane and 1,4-difluorobenzene were prepared by SDG following published procedures.9 A 1:1 molar ratio of caffeine and succinic acid with a total mass of ∼300 mg was placed in a stainless steel mixing jar with five drops of the liquids (dioxane or 1,4-difluorobenzene) and with two additional drops of acetonitrile. The mixture was ground using a 1-cm stainless steel ball for 20 min at a frequency of 30 Hz. The PXRD patterns of these preparations matched those previously published including the presence of phase impurities in the 1,4-difluorobenzene complex (see Supporting Information).9 Piroxicam saccharin (CSD refcode YANNEH) was prepared by dissolving an equimolar mixture of the two components in 300 mL of a 1:1 mixture of chloroform and methanol.7 The mixture was gently heated until a clear yellow solution was obtained. The solution was allowed to evaporate and dark yellow crystals were recovered, filtered, and dried. A yield of approximately 2 g of piroxicam saccharin was obtained using 1.07 g of saccharin and 1.88 g of piroxicam. Fluoxetine HCl succinic acid (CSD refcode RAJFEO) was prepared by the procedure outlined by Childs et al.16 Briefly, about 400 mg of fluoxetine HCl (USP, Rockville, MD, USA) and 70 mg of succinic acid was dissolved in 4.5 mL of acetonitrile with gentle warming, and allowed to stand for several days until crystals appeared. The crystals were filtered and dried, and a yield of 60% was obtained. TPPO 6-chloro-2-hydroxypyridone was prepared by recrystallization of 1 g of 6-chloro-2-hydroxypyridone and 2.15 g of TPPO (Avacado Chemicals, UK) from a 400 mL solution of a 1:1 mixture of CH2Cl2 and hexanes.17 The solution was left to evaporate for five days, and crystals that formed were filtered and dried, with a yield of about 80%. The crystal structure of this cocrystal is not included in the CSD, and was recreated from published atomic coordinates.17 Cholic acid 4′-fluoropropiophenone (CSD refcode POZQAW) was prepared using SDG by placing approximately 400 mg of cholic acid, eight drops of 4′-fluoropropiophenone, and two drops of methanol in a stainless steel mixing jar with a single 1-cm ball and grinding for 20 min at 30 Hz. The samples were air-dried for 1 h after grinding. Previous literature reports have prepared this complex via recrystallization of cholic acid from 4′-fluoropropiophenone, which was repeated during the course of this work and found to produce the same phase made by SDG.18 A complex of 4-picoline and pentachlorophenol (CSD refcode GADGUN05) was obtained on a 1 g scale by evaporation of 4-picoline and pentachlorophenol from CHCl3.22 Initially, a phase impurity was detected by PXRD and SSNMR when an equimolar amount (∼0.01 mol) of the two compounds was evaporated from 100 mL of CHCl3. To reduce this phase impurity, a 5-fold excess of the volatile liquid 4-picoline was then utilized with a smaller amount of CHCl3 (25 mL) and seeds from the first batch were added; the solids were isolated by filtration while a large volume of 4-picoline still remained. The solids were then vacuum-dried to remove residual 4-picoline and solvent. Excepting this phase impurity, the PXRD patterns of the 4-picoline pentachlorophenol complex matched the pattern predicted from the crystal structure (see Supporting Information). Carbamazepine nicotinamide (CSD refcode UNEZES) was prepared by evaporation of a solution of 2.6 g carbamazepine and 2.2 g of nicotinamide from 100 mL of a 1:1 mixture of dimethylsulfoxide and methanol.23 A large molar excess of nicotinamide was used to enhance supersaturation.8 The solution was left to evaporate at ambient conditions for several days; solids that crystallized were filtered and dried. SDG experiments involving equimolar mixtures of carbamazepine and nicotinamide produced a different polymorph of this cocrystal that is not discussed here. NMR Spectroscopy. SSNMR experiments were performed on a Bruker Avance 400 triple-resonance spectrometer operating at a 1H frequency of 399.87 MHz and a Bruker Avance II+ 500 tripleresonance spectrometer operating at a 1H frequency of 500.13 MHz. Both systems were equipped with variable temperature control including a BCU-05 chiller. Experiments were performed at 273 K unless otherwise noted to minimize the effects of frictional heating upon the samples. 1H SSNMR experiments were performed using Bruker 2.5mm double- and triple-resonance probes spinning at an MAS rate (υr) of 35 kHz (except where otherwise noted). 1H experiments used the following typical parameters: a 2.5-µs excitation pulse, 60 to 120-s
Solid-State NMR Analysis of Organic Cocrystals relaxation delays, and between 8 and 32 acquired transients. 1H referencing was achieved by addition of minor amounts of tetramethylsilane (TMS) to solid powders and repeating the 1H analysis at a spinning rate of 25 kHz, referencing the TMS to 0.0 ppm. However, because of frictional heating effects, the accuracy of 1H referencing in this work is no better than 0.1 ppm; hence, all values are reported to the nearest 0.1 ppm, although additional significant digits are available given the spectral resolution. 13C and 19F SSNMR spectra were obtained with Bruker 4-mm triple resonance MAS probes tuned to 1H, 19F, and 13 C frequencies. Cross-polarization (CP) transfers were performed at power levels of 55-80 kHz; the power level was ramped linearly during the contact time over a depth of 15 to 20 kHz on the 1H channel to enhance CP efficiency.29 13C CP spectra were obtained at υr ) 8 kHz with a five-pulse total sideband suppression (CP-TOSS) sequence.30 1 H heteronuclear decoupling was performed at an RF power of 105 kHz using the SPINAL-64 pulse sequence and 19F decoupling was performed at 60 kHz using a series of π pulses (one per rotor period).31,32 13C direct-polarization (DP) spectra were obtained using a single π/2 pulse with 1H decoupling. Proton spin-lattice relaxation times (T1) were determined via 13C detection using a saturation-recovery version of the five-pulse CP-TOSS experiment including 100 closely spaced π/2 saturation pulses prior to the recovery period.33 This pulse sequence obtains similar results to 1H T1 experiments that have been previously reported,34 but offers advantages in speed and sideband suppression for dealing with more complex pharmaceutical systems. Edited 13C spectra containing only quaternary and methyl signals were obtained via dipolar dephasing (DD) during the TOSS period and three subsequent rotor periods using a shifted echo pulse sequence.30,35 13C spectra were referenced to tetramethylsilane (TMS) using an external reference sample of hexamethylbenzene.36 19F MAS spectra were also obtained using both CP and DP methods. 19F spectra were referenced to CFCl3 via calculation from the experimental 13C value with the unified scale method.37 15N and 31P experiments (both CP and direct polarization) were performed using Bruker 7-mm double resonance MAS probes. 15N spectral editing was achieved with a spin-echo version of the DD experiment including CP (CP-DD). 15N chemical shifts were referenced to nitromethane using NH4Cl as a secondary reference standard; 31P chemical shifts were referenced to 85% H3PO4 using the unified scale method.37 Besides those mentioned above, a variety of more advanced 1D and 2D SSNMR experiments were applied in this work as discussed in detail in the following sections. Experimental details for these experiments are given here. 1D homonuclear-decoupled 1H spectra were obtained using 4-mm probes with the windowed phase-modulated LeeGoldburg pulse sequence (w-PMLG).38 2D rotor-synchronized 1H double-quantum broadband back-to-back (DQ-BABA) MAS experiments were performed with 2.5-mm probes at υr ) 35 kHz using two rotor periods of double-quantum excitation and two rotor periods of reconversion.39 2D CP heteronuclear correlation (CP-HETCOR) experiments between 1H and 13C nuclei were obtained using 4-mm probes at υr ) 12.5 kHz with frequency-switched Lee-Goldburg (FSLG) homonuclear decoupling at 105 kHz.40 Ramp CP transfers were used for HETCOR experiments with durations ranging from 400 µs to 2 ms, and additionally Lee-Goldburg CP (LGCP) periods were used instead to prevent relay dipolar transfers as discussed below.41 When FSLG or w-PMLG-5 was used for homonuclear decoupling in either 1D or 2D experiments, the sample was restricted to the center of the 4-mm rotors to maximize RF homogeneity. 1H-19F-13C DCP-TOSS experiments, also performed using 4-mm probes, used a slightly modified version of a previously reported sequence that is described in detail elsewhere.42,43 A 1 ms contact time was used for the first (1H-19F) CP period, and the second (19F-13C) contact time was varied from 1 to 5 ms as described in the following sections. The principal components of the 13C chemical shift tensor (CST) were measured with the CPFIREMAT experiment.44 Slow spinning sideband patterns were extracted from the 2D data sets using TIGER processing.44 The principal components of the CST were extracted from the individual sideband patterns by nonlinear least-squares fitting with software that uses a banded matrix approach to compute sideband amplitudes.45 The convention used for the principal components is δ11 > δ22 > δ33. Fitting of 31P spectra was accomplished using Bruker Topspin 2.0 software (Bruker Biospin, Billerica, MA, USA). 1 H solution-state NMR experiments were performed using a Bruker Avance 400 spectrometer operating at 400.13 MHz and equipped with
Crystal Growth & Design, Vol. 9, No. 2, 2009 923 a Bruker QNI 5 mm probe. Spectra were obtained in DMSO-d6 solution to check the purity and stoichiometry of cocrystals and complexes where necessary. Computational Methods. Crystal structures were retrieved from the CSD Version 5.28 (November 2006) using the Conquest software package (Version 1.8)46 or were recreated from published coordinates. Density functional theory (DFT) calculations were used to predict NMR chemical shielding. Prior to NMR calculations, the hydrogen atom positions in the crystal structures were energy-minimized using the DMol3 module in Materials Studio versions 4.0 and 4.2 (Accelrys, San Diego, CA, USA), with an energy convergence of 2.0 × 10-5 Hartree, a gradient convergence of 4.0 × 10-3 Å and a displacement convergence of 5.0 × 10-3 Å.47,48 Pseudopotentials were not used and a atom-centered basis function cutoff of 3.6 Å was applied. The HCTH/ 407 generalized gradient approximation (GGA) functional was employed using a double numerical basis set with polarization functions on all atoms, referred to as a DNP basis set.47-49 Brillioun-zone integrations utilized different k-point sets that were adjusted for the unit cell of the particular crystal structure. Chemical shielding calculations were performed on gas-phase clusters extracted from the optimized solid-state structures using the Gaussian 03 software package with the gauge-including atomic orbitals method, the B3LYP functional and the 6-311+G(2d,p) or 6-311+G(3df,3pd) basis sets.50,51 The embedded ion method (EIM) was used to supplement several of the gas-phase cluster calculations to provide for electrostatic field effects as described below.52 For EIM calculations, optimization of the hydrogen positions was performed directly in Gaussian 03 and the D95**++ basis set was used with the B3LYP functional.53 Calculated 13C chemical shielding values were linearized and translated into chemical shifts; shieldings for other nuclei (1H and 31P) were not linearized and are reported as relative values.
Results Palmitic Acid Nicotinamide. The simple binary cocrystal of palmitic acid and nicotinamide serves to introduce many of the features of SSNMR approach that will be of relevance in more complex cocrystals and complexes. This cocrystal (Structure I) is shown with a numbering scheme adapted from the published SCXRD structure (CSD refcode JEMDIP).15
The structure features a simple hydrogen bonding motif involving the carboxylic acid donor (H1) and a pyridine acceptor (N1) with a reverse interaction between the H33 hydrogen and the carbonyl (O1) acceptor; this arrangement occurs with a high probability when a carboxylic acid donor and nitrogen base acceptor are present in a crystal.54 The 1D 1H and 13C SSNMR spectra of I and the input materials are shown in Figure 1. These spectra fulfill the first basic criterion for cocrystal formation, also used for vibrational spectroscopy and PXRD analyses, in that the spectra of the cocrystal do not match the spectra of the known phases of the input materials. (All of the systems presented in this work meet this basic criterion. Previously unknown crystalline forms of cocrystal components have been discovered during cocrystal screens, so this possibility must be taken into consideration.55-57) A 1H-13C CP-HETCOR spec-
924 Crystal Growth & Design, Vol. 9, No. 2, 2009
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Figure 1. (a) 13C CP-TOSS spectra of palmitic acid nicotinamide made by SDG (I), and the input phases of palmitic acid and nicotinamide, obtained at 11.7 T and 273 K, showing the distinctive spectra obtained. (b) 1H MAS spectra (υr ) 35 kHz) of the same three samples at the same field and temperature.
trum of I obtained with a 500 µs ramp-CP contact time is shown in Figure 2a with detailed spectral assignments, made in reference to known solution-state shift trends for these compounds and with the aid of an 13C dipolar-dephasing spectrum (not shown). If the structure of I is considered to be unknown for the purpose of this discussion, the spectrum in Figure 2a shows several features that together confirm cocrystal formation. The first feature of interest is the 1H chemical shift of the hydrogen bonding proton (H1), which is observed at 13.7 ppm and indicates that a hydrogen bond is present in the sample.58 The 1H chemical shift of the H1 site is greatly deshielded relative to the most deshielded site observed in a sample of nicotinamide (8.0 ppm) and slightly deshielded relative to the H1 signal obtained from palmitic acid (13.4 ppm). This is explained by the SCXRD structure, where the donor-acceptor (D · · · A) distance between O2 and N1 is 2.70 Å, and is a typical example of a cocrystal containing moderate-strength hydrogen bonds with D · · · A distances in the range of 2.5-3.2 Å as defined by Jeffrey.59 Nicotinamide lacks deshielded protons in its 1H spectrum as expected from the longer hydrogen bonds of 2.96 and 3.08 Å reported in its crystal structure (CSD refcode NICOAM01).60 Palmitic acid forms a structure containing a carboxylic acid dimer with D · · · A distance of 2.62 Å (CSD refcode YEFWEM),61 similar to the hydrogen bond in the JEMDIP structure and consistent with the similar 1H shifts
Figure 2. (a) The 1H-13C CP-HETCOR spectrum (υr ) 14 kHz) of palmitic acid nicotinamide (I), with the 1H MAS (υr ) 35 kHz) and 13 C CP-TOSS (υr ) 8 kHz) spectra shown as projections. (b) The 1H DQ-BABA (υr ) 35 kHz) spectrum of I. The dashed line represents the skew line for single and double-quantum frequencies. All spectra were obtained at 11.7 T and 273 K.
observed for the hydrogen bonding protons in the two structures. The 1H chemical shifts for I were confirmed by B3LYP/6311+G(3df,3pd) calculations on a molecular cluster containing one molecule of palmitic acid hydrogen-bonded to one molecule of nicotinamide. The H1 position was calculated to be 4.7 ppm more deshielded than that of the H33 position, in agreement with the 4.7 ppm difference observed experimentally. Since the API and cocrystal formers can also demonstrate hydrogen bonding among themselves, more than a deshielded proton signal is necessary to confirm cocrystal formation. Additional evidence is readily provided in the 1H-13C CPHETCOR spectrum in Figure 2a through correlations between nuclei of the different molecules. Correlations in the 1H-13C
Solid-State NMR Analysis of Organic Cocrystals
CP-HETCOR experiment may arise from intra- or intermolecular close contacts between nuclei within a shared crystal structure. For example, the carboxylic acid carbon (C1) shows correlations to the H33 and H36 aromatic protons of nicotinamide. Correlations may also occur because of spin diffusion processes among the tightly packed 1H spins in organic crystals (also known as relay transfers); these effects are also noted in Figure 2a.62,63 For example, the aliphatic protons show correlations to the C17 and C21 sites of nicotinamide. Although not as useful for structural analysis, spin diffusion effects can also prove association between 1H and 13C nuclei that are more distant (beyond 5 Å) and provide further evidence that the two components of the cocrystal system are located in the same crystal structure or phase. When a longer CP mixing period is used (typically 800 µs to 2 ms) greater contributions from spin diffusion are often observed, while experiments performed at shorter contact times (100-500 µs) or with LGCP offer more insight into direct dipolar interactions.40,41 The effects of spin diffusion on the 1H-13C CP-HETCOR spectrum are shown for I for contact times between 500 µs and 2 ms in the Supporting Information. Dipolar correlation techniques, combined with the nuclear site resolution of SSNMR, give it a distinct advantage in structural studies over techniques such as vibrational spectroscopy that can only distinguish functional groups to a limited extent. The 1H-13C CP-HETCOR results thus provide convincing evidence that nicotinamide and palmitic acid are associated on a molecular level, and have formed a cocrystal. Further evidence of cocrystal formation is provided by the 1 H DQ-BABA spectrum shown in Figure 2b. The 1H DQ-BABA experiment exploits homonuclear dipolar coupling by exciting DQ coherences between the 1H nuclei.39 Protons within ∼3.0 Å of each other will generally show a SQ-DQ correlation with the ∼57 µs mixing times used here.39 DQ-based experiments have an advantage over other 2D 1H homonuclear correlation experiments in that diagonal peaks are observed only when a site is near to magnetically equivalent site; cross peaks still show correlations between chemically unique protons. This experiment is performed at a high spinning rate to maximize 1H resolution while limiting potential spin diffusion effects. Figure 2b shows correlations between H1 and H33/H36, quickly proving the association between the two molecules in this cocrystal. The H1-H33 and H1-H36 internuclear distances in the JEMDIP structure are 2.7 and 2.8 Å, respectively.15 The 1H DQ-BABA spectrum also reports on other aspects of the structure; for example, the lack of a correlation between H1 and itself can be taken as evidence that a carboxylic acid dimer or catamer is not present as these motifs force hydrogen atoms into close proximity;54 this ability of the DQ-BABA experiment to detect the dimer motif has also been demonstrated for other systems,43 and would be useful in studies of unknown cocrystals. While both the 1H DQ-BABA and 1H-13C CP-HETCOR spectra provide clear evidence for association between the cocrystal components, the 1H DQ-BABA spectrum of I was acquired in less than one-sixth of the time and used only 10 mg of material (because it is run using a 2.5-mm MAS probe). However, the 1H DQ-BABA experiment suffers from limited 1 H resolution, which reduces its utility in many cases. Successful application of the 1H DQ-BABA experiment to cocrystal analysis requires at least two protons with distinctive chemical shifts. Fortunately, the hydrogen bonds in cocrystal systems often lead to proton chemical shifts that meet this requirement. Because of spin diffusion, a single 1H T1 (spin-lattice) relaxation time is generally observed for protons in the same phase, as long as homonuclear dipolar interactions between
Crystal Growth & Design, Vol. 9, No. 2, 2009 925 Table 1.
13
C-Detected 1H T1 Values for Palmitic Acid Nicotinamidea
position
T1 (s)
T1 (s)
T1 (s)
T1 (s)
T1 (s)
C1 (175.7 ppm) C22 (173.0 ppm) C17 (154.7 ppm) C21 (147.7 ppm) C19 (136.1 ppm) C18 (128.7 ppm) C20 (126.1 ppm) chain (33-37 ppm) C3/C15 (26.0 ppm) C16 (15.3 ppm) average 99.7% CIb
15.8 15.7 15.4 15.5 15.2 15.9 15.1 15.6 15.3 15.5 15.5 0.8
15.9 15.6 15.7 15.8 15.7 15.9 15.5 15.8 15.4 15.6 15.7 0.5
16.5 15.4 15.9 16.2 15.9 15.8 15.9 16.1 15.8 16.1 16.0 0.9
15.9 15.6 15.8 16.3 15.4 16.3 15.4 15.7 15.4 15.6 15.7 1.0
16.8 16.4 16.5 16.5 15.7 16.4 15.7 16.6 15.9 16.0 16.3 1.2
average 99.7% (s) CIa (s) 16.2 15.7 15.8 16.1 15.6 16.1 15.5 16.0 15.6 15.8 15.8c
1.4 1.2 1.3 1.2 0.8 0.8 0.9 1.2 0.8 0.9 1.2c
a Five replicates are shown along with averages for replicate measurements and statistics across the integrable peaks in the spectra. b 99.7% confidence intervals; value is reported as three times the standard deviation. c For all values reported in this table across all peaks.
protons are strong, and the MAS spinning rate is moderate (e.g., 8 to 15 kHz).34 This is the case for virtually all organic cocrystals of interest, and 1H T1 measurements are thus a useful tool to identify phase mixtures in cocrystal analysis. The pseudo-2D 13 C-detected 1H T1 saturation recovery experiment takes advantage of the high resolution in the 13C dimension to obtain 1 H T1 relaxation times of each component.34 The critical step in this experiment is the need to assign peaks to each component and establish that those peaks have a statistically different 1H T1 value. The results of five separate 13C-detected 1H T1 saturation recovery experiments on I, each requiring 18 h of acquisition time, are presented in Table 1. The results illustrate the error that can be expected from the experiment and nonlinear fitting for a sample with a typical 1H T1 relaxation time and a 13 C signal-to-noise ratio of approximately 200:1. The values reported for the five replicates indicate that the largest 99.7% confidence interval (CI), obtained as (3 times the standard deviation, is (1.4 s for the peak at 175.7 ppm. The magnitude of these intervals is largely related to the signal-to-noise of the peaks in the 13C CP spectrum. The error in fitting, which was of lesser importance in this case, can be improved if desired by selecting optimal recovery delays for the experiment based on initial coarse estimates of T1.64 Table 1 shows that the error across peaks in an individual spectrum is similar to that obtained from replicates, so that multiple peaks within a single 13Cdetected 1H T1 saturation recovery experiment can be used to estimate a CI. Thus, a simplified approach may be used for cocrystal analysis: the 1H T1 values for 13C resonances assigned to each molecule are compared, and if any peak falls outside the CI established by the other molecule, the experiment is considered to have detected phase separation. If needed, other approaches can be used to test whether a given set of peaks has a different T1 than another given set of peaks, or to transform the exponential decay into a frequency representation for more detailed analysis.34,65 Cocrystal 1H T1 times may vary based on the purity of input materials, the level and range of defects occurring upon milling or SDG, and the resultant particle size and surface area. For example, an acetone-recrystallized sample of I produced from the same lots of input materials used to prepare the SDG sample had a 1H T1 value of 18.2 ( 1.8 s measured across all peaks in a single experiment. Since variations in 1H T1 times can occur from sample to sample, they cannot be interpreted as proof of cocrystal formation. In light of this behavior and also the possibility that different phases may have statistically indistinguishable 1H T1 times, the results of a 13C-detected 1H T1
926 Crystal Growth & Design, Vol. 9, No. 2, 2009
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experiment can only be taken as evidence of phase separation, and not of cocrystal formation. The experiment can only show that two sets of T1 values are statistically indistinguishable, unlike dipolar connectivity experiments that can directly prove association. However, although failed cocrystal preparations are not the focus here, the 13C-detected 1H T1 experiment is invaluable in practice for its ability to prove that two phases are not associated, which is difficult to do using dipolar connectivity experiments. The results of 13C-detected 1H T1 experiments will be briefly mentioned for many of the cocrystals and complexes presented here to highlight the T1 values and typical errors obtained. The SSNMR experiments discussed so far each have benefits and limitations that are readily seen in their application to a simple cocrystal. In the case of I, sufficient evidence was found in the SSNMR data to conclude that this material was a cocrystal even if a SCXRD structure was not available. With more complicated crystal systems, spectral overlap can lead to less plentiful evidence of molecular association. However, using the basic SSNMR experiments shown here as a set along with additional experiments involving heteronuclei increases the likelihood that that their complementary nature will offer some key piece of evidence for cocrystal formation, as will be illustrated now for systems of increasing structural complexity. Caffeine Succinic Acid 1,4-Dioxane. Ternary cocrystals and complexes represent more complicated systems with even greater flexibility in possible intermolecular interactions. An example is caffeine succcinic acid 1,4-dioxane (Structure II), which is a member of a class of host-guest complexes made from a caffeine succinic acid molecular scaffold.9 The numbering scheme given reflects the crystallographic equivalence of many of the atomic positions.
The crystal structure of II contains two crystallographically unique succinic acid half-molecules, one containing atoms H6, C9, and C10 and the other containing H4, C11, and C12.9 One succinic acid molecule donates hydrogen bonds to caffeine (N1 · · · H6-O6) while accepting hydrogen bonds from the second molecule of succinic acid (O5 · · · H4-O4). The succinic acid molecule containing H6, C9, and C10 acts as a “bridge” between two caffeine molecules along the a-axis of the unit cell, while the succinic acid molecule containing H4, C11, and C12 forms a second bridge between succinic acid molecules along the c-axis. The succinic acid and caffeine molecules thus form a host structure along the ac-plane, in which dioxane molecules reside in a channel-like fashion.9 A single 13C-detected 1H T1 relaxation experiment determined the T1 of all peaks in a sample of II to be 3.1 ( 0.2 s with no individual peak or set of peaks falling outside of the 99.7% CI established by peaks assigned to each molecule. The 1H-13C CP-HETCOR spectrum of II is shown in Figure 3a, with the 1D 13C CP-TOSS and 1H MAS spectra as projections. In this case, the presence of 1,4-dioxane signals in the 13C CP-TOSS spectrum provides immediate evidence that it has joined in a
Figure 3. (a) The 1H-13C CP-HETCOR spectrum (υr ) 14 kHz) of caffeine succinic acid 1,4-dioxane (II), shown with the 13C CP-TOSS spectrum (υr ) 8 kHz) and the 1H MAS spectrum (υr ) 35 kHz) as projections. In (b), the 1H DQ-BABA spectrum (υr ) 35 kHz) of II is shown with a dashed skew line and the DQ signal (F1 dimension) from the aliphatic groups off-scale. All spectra were obtained at 11.7 T and 273 K.
solid complex, as this compound is a liquid when phaseseparated and would otherwise not give rise to signal. The 1 H-13C CP-HETCOR spectrum shows a number of correlations between the resonances of the caffeine and succinic acid molecules, some of which may be caused by spin diffusion (those marked with distances greater than 3.0 Å distances). However, no correlation is observed between the dioxane carbon signal and any characteristic protons of the caffeine or succinic acid. This result is partially explained by the crystal structure of this system, which does not show short contacts (
