Molecular Conformation and Crystallization: The Case of

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Molecular Conformation and Crystallization: The Case of Ethenzamide Published as part of a Crystal Growth and Design virtual special issue of selected papers presented at the 10th International Workshop on the Crystal Growth of Organic Materials (CGOM10) Kevin R. Back,† Roger J. Davey,*,† Tudor Grecu,‡ Christopher A. Hunter,‡ and Lynne S. Taylor§ †

SCEAS, University of Manchester, Oxford Road, Manchester M13 9PL, U.K. Centre for Chemical Biology, Krebs Institute for Biomolecular Science, Department of Chemistry, University of Sheffield, Sheffield S3 7HF, U.K. § Department of Industrial and Physical Pharmacy, School of Pharmacy, Purdue University, West Lafayette, Indiana 47907, United States ‡

S Supporting Information *

ABSTRACT: Ethenzamide is a small, conformationally labile pharmaceutical molecule which crystallizes in an unusual conformation relative to both its co-crystals and to similar molecules found in the Cambridge Structural Database. Relative to its co-crystals, large ethenzamide crystals are challenging to grow, a property which may be linked to the crystal structure conformation. We have explored this link through computational and spectroscopic techniques, studying the conformational properties of the individual molecules in vacuo and in solution. Structures built from alternative conformations have been generated theoretically, but crystallizations under a wide range of conditions have resulted in no new polymorph, though single crystal structures for the known form and two new co-crystals have been solved.

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INTRODUCTION The importance of molecular conformation in crystal structures has long been recognized.1−9 Links between conformation in the solid state and solution have also been the subject of investigation10−13 for pharmaceutical materials, as analysis of the known solid state conformation can aid in the hunt for the difficult to determine solution phase conformation.13 It is only recently, however, that the effect of solution conformation on the process of crystal growth has begun to be studied.14−16 In crystal structures the existence of high energy molecular conformers can be explained in terms of ‘crystal forces’,4 where an improvement in intermolecular interactions compensates for an energetically unfavorable conformation. In solution, the population of these high energy conformers is likely to be low, as the solute molecule environment is dominated by solvation. This suggests that the observed conformer must appear during crystal growth. It has been suggested14 on the one hand that lattice interactions at the crystal−solution interface may assist in this process, while on the other that molecules with conformations which differ significantly from the conformation found in the crystal structure are rejected at the crystal surface. In both cases, crystals containing conformations which are high in energy would be predicted to grow more slowly, due to the energetic and time penalty of rearrangement or because of the low relative concentration of the required conformer in solution. © XXXX American Chemical Society

Ethenzamide, or 2-ethoxybenzamide, is a small pharmaceutical molecule, with amide and ether groups positioned ortho to each other on a benzene ring (Figure 1). It has a single known

Figure 1. The molecular structure of ethenzamide.

crystal form, the structure of which has been previously determined through X-ray powder diffraction techniques17  no single crystal structure has been found in the Cambridge Structural Database (CSD).18 On the other hand a number of single crystal structures of co-crystals of ethenzamide are in the CSD (including polymorphic co-crystals19−22 multicomponent co-crystals23 and solvates of co-crystals21), suggesting that crystallization of the pure material may be problematic. In the single component crystal ethenzamide adopts a conformation Received: August 28, 2012 Revised: November 5, 2012

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dx.doi.org/10.1021/cg301244x | Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

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recorded after each addition. Changes in chemical shift of the ethenzamide signals were fitted to a dimerization isotherm using purpose written software. FTIR Analysis. Spectra were measured on a Thermo Nicolet iS10 FTIR spectrometer, with a resolution of 4 cm−1, using a Golden Gate ATR stage for solid samples, and an Omni Transmission stage with a liquid cell consisting of two KBr discs separated by a changeable spacer for liquid spectra. Solutions of ethenzamide (Aldrich, 97%) were prepared in dichloromethane (BDH, 99.8%). Single Crystal Structures. An ethenzamide single crystal was grown by cooling a 95 mg mL−1 solution in acetonitrile from 60 to 0 °C at 0.1° min−1. The ethenzamide−2,6-dihydroxybenzoic acid co-crystal was grown by cooling a solution containing approximately 300 mg of ethenzamide and 200 mg of dihydroxybenzoic acid in 5 mL of acetonitrile from 60 to 10 °C over 12 h. The solution was held at this temperature for 2 days prior to isolating the crystals. The ethenzamide−3,5-dichlorobenzoic acid co-crystal was grown by dissolving 102 mg of ethenzamide and 116 mg of dichlorobenzoic acid in approximately 5 mL of tetrahydrofuran, and allowing this to evaporate to dryness in a vial covered with pinholed foil. Structures were determined at 100 K (233 K for the 3,5dichlorobenzoic acid co-crystal) on an Oxford Xcaliber 2 diffractometer, using Mo Kα radiation with a graphite monochromator. The data were collected and processed using the CrysAlisPro software, and the structures were solved using the OLEX226 program as an interface together with the SHELXS and SHELXL programs,27 in order to solve and refine the structure respectively. Heavy atoms were refined anisotropically. Hydrogen atoms were placed in geometric positions and refined as riding atoms with the exception of those bound to nitrogen and oxygen which were refined freely. In the case of the room temperature structure for ethenzamide, all hydrogens were refined as riding atoms. Polymorph Screen. A search for other polymorphs of ethenzamide was undertaken in a series of evaporative, room temperature crystallizations at approximately 2 mL scale using 18 different solvents. Three of these were repeated at 70 °C. Cooling crystallizations in nine different solvents were carried out, typically under rapid cooling conditions to try to promote the appearance of a lower energy conformer. A series of additives was tried, including known and potential co-formers, as well as other additives chosen to try to inhibit the ladder formation seen in the single component crystal (Figure 7a), such as an additive that has been shown to act as a motif capper for benzamide.28 Details of the solvents (combination of a selection of hydrogen bonding and non-hydrogen bonding solvents) and additives tested can be found in the Supporting Information. Nonsolution methods were also tested. Liquid ethenzamide was crash cooled in liquid nitrogen and vapor growth was carried out by allowing liquid ethenzamide to evaporate under a glass Petri dish. All crystals were characterized by pXRD, using a Rigaku Miniflex. Samples were typically scanned between 2 and 40° 2θ at a rate of 4.5° per minute, with a step size of 0.03°.

in which the amide group is out of the plane of the benzene ring, with the oxygen of the amide positioned close to the ether group (Figure 2a). This arrangement is in conflict with Etter’s

Figure 2. Conformations of the ethenzamide molecule in (a) pure ethenzamide (determined at 100 K) and (b) ethenzamide-3,5dichlorobenzoic acid co-crystal (structure determined at 233 K).

second rule,24 as for this molecule there is a possible intramolecular hydrogen bond between an amide proton and the ether oxygen, which if satisfied would form a six-membered ring. This intramolecular hydrogen bond (Figure 2b), not seen in the single component crystal, is however found in all the known co-crystal structures. Ethenzamide thus appears to exhibit an interesting phenomenon in which as a pure material it has an unexpected conformation in comparison to that in its co-crystals and may be more difficult to crystallize. This suggests that it may be an ideal candidate for this study, in which we attempt to understand this behavior through an examination of the crystallization and conformational properties of this material. Our approach combines existing data from the CSD, conformational calculations, and polymorph prediction together with experiments designed to probe the state of ethenzamide in solutions and to search for other polymorphs of this material.

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EXPERIMENTAL SECTION

NMR Analysis. All 1H NMR titrations were carried out using a Bruker Avance III 400 spectrometer at 298 K. 1 H NMR Titration Experiments. In carbon tetrachloride (CCl4): A 5 mL sample of ethenzamide dissolved in carbon tetrachloride was prepared at a known concentration (1 mM). A 0.6 mL fraction of this solution was used to record a 1H NMR spectrum using a capillary of deuterium oxide as a lock signal. The ethenzamide stock solution was used to prepare a 3 mL solution of tri-n-butyl phosphine oxide at known concentration (3−500 mM), so that the concentration of ethenzamide remained constant throughout the titration. Aliquots of tri-n-butyl phosphine oxide solution were successively added to the NMR tube containing the ethenzamide solution and a 1H NMR spectrum was recorded after each addition. Changes in chemical shift of the ethenzamide signals were fitted to a 1:1 binding isotherm using purpose written software.25 In deuterated chloroform (CDCl3): A 5 mL sample of ethenzamide dissolved in deuterated chloroform was prepared at a known concentration (50−100 mM). A 0.6 mL fraction of this solution was used to record a 1H NMR spectrum. The ethenzamide stock solution was used to prepare a 3 mL solution of tri-n-butyl phosphine oxide at known concentration (1.0−1.5 M), so that the concentration of ethenzamide remained constant throughout the titration. Aliquots of tri-n-butyl phosphine oxide solution were successively added to the NMR tube containing the ethenzamide solution and a 1H NMR spectrum was recorded after each addition. Changes in chemical shift of the ethenzamide signals were fitted to a 1:1 binding isotherm using purpose written software. 1 H NMR Dilution Experiment. A 5 mL sample of ethenzamide dissolved in deuterated chloroform (CDCl3) was prepared at a concentration of 594.5 mM as a stock solution. A 0.6 mL fraction of this solution was used to record a 1H NMR spectrum. Aliquots of pure CDCl3 were successively added to the 0.6 mL fraction of ethenzamide, the tube was shaken thoroughly and a 1H NMR spectrum was

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RESULTS Crystal Structures. The crystal structure of ethenzamide has only previously been determined from powder data,17 which is likely to be due to the difficulty in growing large crystals of the material. Ethenzamide typically crystallizes as thin (typically 10−50 μm thick) needles. A cooling crystallization of ethenzamide in acetonitrile (as described in the methods section for the single crystal structures) resulted in a few crystals that were large enough to diffract, though these crystals still have dimensions in the micrometer range, as shown in Figure 3. Two determinations of the crystal structure were carried out, at 293 K and 100 K  the parameters for these are shown in Table 1, where a good agreement with the structure solved from powder diffraction data is also seen. Figure 2a shows the conformation of the ethenzamide molecule observed in the 100 K structure. B



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Table 2 compares the conformations present in the crystal structures of ethenzamide and its co-crystals, by comparing the Table 2. Torsion Angles of the Ether (TOR1) and Amide (TOR2) Groups of Ethenzamide in the Known Crystal Structures Containing the Molecule CSD Refcode or Deposition Number

co-former(s) none (single component) gentisic acid and acetic acid23 gentisic acid

Figure 3. Microscope image of ethenzamide crystals obtained from a cooling crystallization in acetonitrile.

Table 1. Comparison of Crystal Structure Parameters between Powder and Single Crystal Determinations CSD reference space group temperature of determination (K) cell lengths (Å) a b c angle (°) β cell volume (Å3) R-factor (%) ether torsion angle (TOR1, °) amide torsion angle (TOR2, °) a

DUKXAJ17 (Powder)

CCDC 891072

P21/n 283−303

19

TOR2 (deg)

CCDC 891073

6.80

129.67

NURFOW

3.55a

−9.14a

CCDC 891074

5.55b −1.01b c −6.68 9.48c d 7.63 −9.85d −3.85 1.22 −12.26 0.2 −1.67 −6.33 8.58 −1.67 −10.75 −2.32 −10.79 −14.62 −8.05 −7.06 −8.47 −6.53 −8.67 −6.88 −7.55 −4.01 7.37 6.47 7.49 6.64 7.25 6.11 −1.1 3.05 2.99 −3.07 −4.94 −6.17 planar, no structure available 1.86 3.37

CCDC 891075

−4.83

QULLUF

saccharin20

CCDC 891073

3,5-dinitrobenzoic acid21

P21/n 288−298

P21/n 100

14.2141 12.0446 5.05009 96.5811 858.894 6.32a 5.44

5.0512 12.027 14.251 96.774 859.716 13.77 6.92

5.0339 11.8782 14.1229 96.590 838.881 5.04 6.80

with acetone21 dioxane21 diethyl ether21 toluene21 acetonitrile21 ethyl acetate21 para-xylene21 mesitylene21 ethylmalonic acid22

129.36

129.58

129.67

thiourea29 2,6-dihydroxybenzoic acid 3,5-dichlorobenzoic acid

Rwp for the refined structure determined from powder data.

TOR1 (deg)

QULLUF01 QULLUF02 VUHFIO VUHFIO01 WUZHOP WUZHOP01 WUZJAD WUZJEH WUZJIL WUZJOR WUZJUX WUZKAE WUZKEI WUZKIM VAKTOS VAKTOS01 KITWOA

−1.69

a

Ethenzamide molecule bonded to gentisic acid. bEthenzamide molecule bonded to acetic acid. cBridging ethenzamide molecule. d Ethenzamide molecule bonded to single gentisic acid.

During this study, two further co-crystals of ethenzamide were found, with 2,6-dihydroxybenzoic acid (CCDC 891074) and with 3,5-dichlorobenzoic acid (CCDC 891075). In both of these new co-crystal forms ethenzamide exhibited the intramolecular hydrogen bond seen in all previously determined cocrystal structures. In the case of an uncontrolled evaporative crystallization from methanol of the 3,5-dinitrobenzoic acid cocrystal a very large crystal resulted, as seen in Figure 4. It is interesting to note that, as suspected, the appearance of large crystals of the co-crystals of ethenzamide was significantly more common than for the pure component.

torsion angles of the ether and amide groups (defined in Figure 5). No co-crystal torsion is more than 15° away from 0°,

Figure 5. Structure used as basis for Cambridge Structural Database searches, with definition of the ether (TOR1) and amide (TOR2) torsion angles considered in this paper.

whereas the angle of the amide group in ethenzamide is 130°. It is also noted that in every co-crystal structure, the carbonyl is hydrogen bonded to either OH or NH, satisfying Etter’s first rule that all good proton donors and acceptors are used in hydrogen bonding. These data confirm the unusual conformation adopted by ethenzamide in its crystal structure, as all 21 solved co-crystal

Figure 4. A photographic image of an ethenzamide-3,5-dinitrobenzoic acid co-crystal. C



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and the amide is at an angle of 35° to the ring, but the amide nitrogen is close to the ether oxygen. This appears the more favorable conformation, as it prevents any oxygen−oxygen repulsion while possibly allowing some intramolecular hydrogen bonding interactions. Ab Initio Calculations. Density functional theory (DFT) based ab initio calculations31 (B3LYP/6-31++G**) were used to generate a relaxed potential energy surface for ethenzamide. The amide and ether torsions defined earlier were varied in 5° steps, and at each point the molecule was allowed to relax (though the ring carbon and hydrogen atoms were fixed in position). The potential energy surface can be seen in Figure 8

structures exhibit the expected intramolecular hydrogen bond and thus an almost planar conformation. To investigate this apparent anomaly further, searches were carried out in the Cambridge Structural Database to explore the existence of the ethenzamide conformation in other, similar compounds. Cambridge Structural Database Search. The CSD was searched for ethenzamide-like molecules (see Figure 5), excluding those which, due to a second group ortho to the amide and/or ring systems linking the amide or ether groups to other parts of the ethenzamide fragment, may have restricted conformations. The torsion angles of the ether and amide groups in the structures remaining are shown plotted against each other in Figure 6. The distribution of torsion angles

Figure 8. Relaxed potential energy surface for ethenzamide, measured in kJ mol−1 above the minimum energy conformation found. The cross denotes the crystal structure conformation of ethenzamide.

Figure 6. Torsion angles of amide and ether groups in ethenzamide like molecules in the CSD. The ethenzamide structure (DUKXAJ) is denoted by the star.

and shows that the lowest energy vacuum conformation is predicted to be planar, with an intramolecular hydrogen bond between the amide protons and the ether oxygen, as seen in the co-crystals of ethenzamide. The conformation seen in the single component crystal is calculated to be over 20 kJ mol−1 higher in energy than this planar conformation, though there is no other potential barrier to overcome in moving between the two conformers. Full optimizations (excluding the amide and ether torsion angles only) were carried out at the planar geometry and at the geometry seen in the single component crystal, and this confirmed the energy difference as 22.5 kJ mol−1. OPiX Structure Generation. In order to explore the possibility of a second polymorph of ethenzamide utilizing the planar conformation, the program PROM from Gavezzotti’s OPiX32 suite was used to generate structures. Taking the nearplanar conformation seen in one of the co-crystals (NURFOW), theoretical ethenzamide crystal structures were generated using this program, in all the space groups permitted (C2/c, P1,̅ P21, P21/c, P212121, and Pbca). Each set of generated structures was minimized (though the conformation was held rigid), and any replicates were removed in an iterative process using the Minop and Sorter programs in the OPiX suite. An energy/density plot of the structures obtained is shown in Figure 9 in which the minimized experimental structure is also included for comparison. The lowest energy structure (−133.9 kJ mol−1) was found in the space group Pbca and is made up of R228 dimers of ethenzamide, in which individual molecules are slightly offset as shown in Figure 10. The offset leads to an N−H···OC angle of 153°, which is close to the mean angle of 158° observed by Taylor et al.33 for these bond

confirms that the amide group of the pure component crystal of ethenzamide has an unusual torsion angle compared to known structures of similar molecules, which are distributed around an amide torsion angle of 0°. There are four other structures that have unusually high and negative amide torsion angles, whose refcodes, from left to right at the bottom of Figure 6, are RIRXUM, SUQGIV, RUFJEI and DUMRAF. In contrast to ethenzamide, all four have bulky groups attached to the amide nitrogen. An additional feature of the ethenzamide conformation is the close proximity of the oxygen atoms, which is related to the angle of the amide (see Figure 2a). If the angle of the amide is required in order to build up the ladder network of hydrogen bonding, then the structure of methenzamide (RECQIA) makes an interesting comparison (see Figure 7). In the methenzamide crystal structure, a similar ladder motif exists

Figure 7. Ladder motifs in (a) ethenzamide and (b) methenzamide.30 D



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energy. However, the computational methods used to calculate the conformational energy and the structure energy are different, and so the result of this summation can only be considered as an indication that there may be other polymorphs of ethenzamide with the more stable molecular conformation. It is interesting to note that all of the predicted structures with lattice energies below −130 kJ mol−1 are made up of R228 dimers of ethenzamide, and that none of them exhibit the extended ladder motif seen in the observed ethenzamide structure (see Figure 7a). Because of the planar conformation of the amide group the ether group would be expected to sterically hinder the formation of this ladder. This highlights a possible explanation for the appearance of the high energy conformation in the known crystal structure  the ladder motif may not be able to form using the planar, low energy conformation due to this steric interference. The cost of assuming the high energy conformation would be paid back at least in part by the ability to form these chains of hydrogen bonded dimers. An additional factor favoring the appearance of the high energy conformation is that it would be consistent with Etter’s first rule through the use of the stronger H-bond acceptor, the carbonyl. As noted above, methenzamide forms a similar but staggered ladder motif30 (see Figure 7b)  but in this case, in contrast to ethenzamide, the NH2 group of the amide and not the carbonyl oxygen is closest to the ether oxygen. From the potential energy surface generated, if ethenzamide assumed methenzamide-like torsion angles (ether torsion −5° and amide torsion 50°), then the energy of this conformer is calculated to be 10.7 kJ mol−1 above the minimum energy conformation, a considerably more favorable geometry than that seen in the known crystal structure. This conformation was examined further by rotating the amide group of ethenzamide to a methenzamide-like angle while keeping the other elements of the structure of ethenzamide identical, and minimizing the obtained structure in OPiX. The energy of this structure was determined to be −150.0 kJ mol−1, very close to the minimized energy of the observed ethenzamide structure (−153.5 kJ mol−1). When the relative conformational energy obtained from DFT is considered (10.7 vs 22.5 kJ mol−1 above the minimum), the methenzamide-like structure would appear to be 8.3 kJ mol−1 more stable, though again the summation is only an indicator. From these theoretical results, it would be predicted that a structure containing a methenzamide-like conformation of ethenzamide would both be feasible and easier to form, as the conformer is lower in energy than that in the observed structure of ethenzamide, and thus more likely to appear in solution. All of these results confirm that the conformation of ethenzamide in its known structure is unusual and suggest that a second polymorph may exist. Polymorph Screen. None of the crystallizations resulted in a new pure component crystal form. This is of particular interest when the vapor phase crystallization is considered. The ab initio predictions for conformer energy were for molecules in a vacuum, and the presence of solvent is very likely to change the relative energies of conformers. Crystallization from the vapor phase is thus the condition closest to that modeled in the simulations, and it is noteworthy that the crystals grown from this phase contained the usual high energy twisted conformer. If the ab initio predictions are accurate, the predominant conformer in the vapor phase would be the planar conformer, and yet despite this, crystals containing the twisted conformer were grown from this phase. This suggests that molecules may

Figure 9. Density−energy plot of OPiX generated structures for a planar ethenzamide molecule, with the minimized experimental (DUKXAJ) structure included for comparison.

Figure 10. Minimum energy structure predicted by OPiX, highlighting the ethenzamide dimer and showing the unit cell.

types. In contrast to the three intermolecular hydrogen bonds of the known structure, this prediction has one intra- and two intermolecular hydrogen bonds per molecule of ethenzamide. The DUKXAJ ethenzamide structure was also minimized and evaluated using OPiX, and has a lattice energy of −153.5 kJ mol−1. Clearly the known structure has a considerably lower energy than the predicted structure. However, OPiX does not take into account any intramolecular energy, treating each molecule as a rigid unit. If the difference in energy between the two conformers (evaluated using DFT) is added to the OPiX energy (evaluated using the UNI forcefield) of the known structure, then the known structure has a total energy of −131.0 kJ mol−1, slightly above the energy of the predicted structure. If these values are accurate, they suggest there is a potential second polymorph of ethenzamide which is lower in E



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well be able to rearrange into the correct conformer on contact with the crystal surface, in spite of the energetic cost of this process. Solution State Analysis. While the minimum energy vacuum phase conformation of ethenzamide appears to be planar, the solution phase conformation may well be different owing to the interactions of solute and solvent. In order to determine the solution phase conformation and in particular test for the presence of an intramolecular hydrogen bond, a combination of spectroscopic techniques was used. Nuclear Magnetic Resonance. The conformational properties of ethenzamide in solution were investigated using 1H NMR titrations with tri-n-butyl phosphine oxide in deuterated chloroform and in carbon tetrachloride. Typical titration results are shown in Figure 11 for addition of tri-n-butyl phosphine

Figure 12. Changes in 1H NMR chemical shift of the signals due to H1 (filled circles) and H2 (open circles) in CCl4 at 298 K. The lines of best fit to a 1:1 binding isotherm are shown.

contrast, H1 forms strong intermolecular interactions with trin-butyl phosphine oxide. Dilution experiments with ethenzamide showed no changes in 1H NMR chemical shift indicating that self-association is negligible at the concentrations used for the titration experiments. However above 58 mM, there are some changes in 1H NMR chemical shift with concentration (Figure 16). The signal due to the non-intramolecularly hydrogen bonded amide proton (H1) increases in chemical shift, indicating that it is participating in intermolecular hydrogen bonding, which must be with the carbonyl oxygens of other ethenzamide molecules. This would appear to be consistent with the results from the infrared analysis discussed below which also indicate that both the carbonyl and the free amino hydrogen are available for intermolecular hydrogen bonding.

Figure 11. Downfield region of 400 MHz 1H NMR spectra recorded for titration of tri-n-butyl phosphine oxide into a solution of ethenzamide in CDCl3 at 298 K. Similar spectra were obtained in CCl4.

oxide to ethenzamide. There are large changes in chemical shift indicative of complexation. Tri-n-butyl phosphine oxide is a very strong H-bond acceptor and will form H-bonds with any available polar protons on ethenzamide. H-bond interactions are characterized by large positive changes in chemical shift. The data shown in Figure 11 suggest that one of the two amide protons (H2) is involved in an intramolecular H-bond in ethenzamide in the free state, because this signal is shifted by +1.7 ppm compared with H1. When tri-n-butyl phosphine oxide is added, the signal due to H1 increases in chemical shift indicative of formation of an intermolecular H-bond between the amide proton and the tri-n-butyl phosphine oxide. In contrast, the signal due to H2 shows a decrease in chemical shift on addition of tri-n-butyl phosphine oxide, which implies that this amide proton does not form a new intermolecular Hbond with the tri-n-butyl phosphine oxide. The stability of the H-bonded complex is too weak to allow accurate measurement of an association constant in CDCl3, but the complex is much more stable in CCl4, a less competitive solvent. The data for H1 and H2 fit well to the same 1:1 binding isotherm giving an association constant of 9 ± 1 M−1 (Figure 12). This means that the changes in chemical shift illustrated in Figure 11 are a consequence of a single binding event, and the tri-n-butyl phosphine oxide does not interact at multiple sites on ethenzamide. In other words, the upfield changes in chemical shift observed for H2−H6 are a consequence of formation of an intermolecular H-bond between H1 and the tri-n-butyl phosphine oxide. These experiments clearly demonstrate that H2 is involved in an intramolecular H-bond with the alkoxy substituent in solution, and this intramolecular interaction competes with intermolecular interactions rendering the H-bond interaction with tri-nbutyl phosphine oxide too weak to detect in solution. In

Figure 13. Downfield region of 400 MHz 1H NMR spectra recorded for dilution of a solution of ethenzamide in CDCl3 at 298 K, going from 58 mM (top trace) to 600 mM (bottom trace). The signal that moves significantly is due to proton H1.

Infrared Spectroscopy. FTIR measurements of dichloromethane solutions of ethenzamide at different concentrations were used to study the behavior of the amine and carbonyl groups in the molecule. The NH stretch region of low concentration solutions of ethenzamide is shown in Figure 14. Two major peaks can be seen at 3507 cm−1 and 3387 cm−1. These peaks could be the antisymmetric and symmetric stretches if the two protons experience the same environment, or they could be individual stretches of two inequivalent NH bonds, perhaps involved in hydrogen bonding interactions. The results from the NMR indicate the latter is the most likely interpretation. As the ethenzamide concentration is increased, toward saturation in dichloromethane, a new peak appears in the NH stretching region, as shown in Figure 15. The height of this new, central peak (3456 cm−1) increases with respect to both outer peaks as concentration increases, suggesting that it could F



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from 28 mM to 602 mM in dichloromethane. Interestingly, although it is at 1670 cm−1, indicative of H-bonding, it shows no shift with concentration. Changing solvents to chloroform and methanol shows this stretch at 1663 cm−1 and 1661 cm−1 respectively, 7 cm−1 and 9 cm−1 lower than in dichloromethane, indicating that the carbonyl group is available for solvation, probably through H-bonding. This was further confirmed by addition of a powerful hydrogen bond donor, 4-nitrophenol, to a solution of ethenzamide (30 mM) in dichloromethane which resulted in a new carbonyl stretching peak at 1648 cm−1. Overall these results suggest that the carbonyl is hydrogen bonded at all concentrations, either because the amide dimer is present throughout the range or because exchanging solvent for solute has little impact on the extent of interaction. Given the NMR results it seems that the latter is more likely and leads to the conclusion that in all solutions the solvent interaction with the carbonyl is competitive with the formation of an amide Hbond with a second ethenzamide molecule.

Figure 14. The NH stretch region of the infrared spectra of ethenzamide in dichloromethane, in a 200 μm cell, at concentrations of 6.3, 13.8, 18.5, 26.5, and 30.1 mM.

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CONCLUSIONS Ethenzamide has an unusual conformation in its crystal structure, not only when compared to the co-crystals it forms, of which there are many, but also when compared to the wider family of similar compounds within the Cambridge Structural Database. The conformation does not obey Etter’s rules regarding intramolecular hydrogen bonding and additionally places two oxygens in unfavorable proximity. Interestingly, methenzamide adopts a very similar structure, but utilizing a conformation in which the oxygen and amide groups are swapped, thus avoiding this repulsion. Ab initio studies have shown that the conformer in the ethenzamide crystal structure is over 20 kJ mol−1 higher in energy than the global minimum, and that this global minimum is very similar to the conformer observed in the co-crystal structures. Theoretical structure generation using this planar geometry has predicted the existence of a polymorph, similar in energy if not lower than the known ethenzamide crystal structure. Additionally, a further polymorph has been predicted with a methenzamide-like conformation, which again is calculated to be lower in energy than the known structure. However, a thorough polymorph screen has not resulted in the discovery of any new structures, suggesting that the ethenzamide structure is at the least strongly kinetically favored, or more likely that it is the stable crystal form despite containing a high energy conformation. If the ethenzamide structure is considered in terms of explaining the structural factors which compensate for the conformational cost,6 then it is clear that the network of hydrogen bonding linking each ethenzamide molecule to three others is important. This network cannot be built out of planar ethenzamide molecules due to the steric interactions, and so the twist in the amide is necessary. However, it could assume a methenzamide-like conformation and still fulfill the same hydrogen bonding pattern, and yet does not. Spectroscopic analyses of solutions of ethenzamide have indicated that in deuterated chloroform and carbon tetrachloride, the solution conformer exhibits an intramolecular hydrogen bond, as predicted from the ab initio calculations, and that at high concentrations there may be a change in solution speciation from fully solvated molecules to higher order aggregates. In the light of the spectroscopic data and the failure to find a second polymorph even from the vapor phase, it seems most likely that crystallization from solutions involves

Figure 15. The NH stretch region of the infrared spectra of ethenzamide in dichloromethane, in a 25 μm cell, at concentrations of 28, 146, 292, 453, and 602 mM.

relate to an intermolecularly hydrogen bonded dimer or higher aggregate of ethenzamide. This is consistent with the NMR result and suggests the possibility of dimerization via the carbonyl of other solute molecules. This is explored further in Figure 16 which shows the position of the carbonyl stretch as the concentration increases

Figure 16. The CO stretch region of the infrared spectra of ethenzamide in dichloromethane, in a 25 μm cell, at concentrations of 28, 146, 292, 453, and 602 mM. G



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(19) Aitipamula, S.; Chow, P. S.; Tan, R. B. H. CrystEngComm 2009, 11, 1823−1827. (20) Aitipamula, S.; Chow, P. S.; Tan, R. B. H. CrystEngComm 2009, 11, 889−895. (21) Aitipamula, S.; Chow, P. S.; Tan, R. B. H. Cryst. Growth Des. 2010, 10, 2229−2238. (22) Aitipamula, S.; Chow, P. S.; Tan, R. B. H. CrystEngComm 2010, 12, 3691−3697. (23) Aitipamula, S.; Chow, P. S.; Tan, R. B. H. Acta Crystallogr., Sect. E 2010, 66, 1045−1046. (24) Etter, M. C. Acc. Chem. Res. 1990, 23, 120−126. (25) Chiarella, R. A.; Gillon, A. L.; Burton, R. C.; Davey, R. J.; Sadiq, G.; Auffret, A.; Cioffi, M.; Hunter, C. A. Faraday Discuss. 2007, 136, 179−193. (26) Dolomanov, O. V.; Bourhis, L. J.; Gildea, R. J.; Howard, J. A. K.; Puschmann, H. J. Appl. Crystallogr. 2009, 42, 339−341. (27) Sheldrick, G. Acta Crystallogr., Sect. A 2008, 64, 112−122. (28) Blagden, N.; Song, M.; Davey, R. J.; Seton, L.; Seaton, C. C. Cryst. Growth Des. 2005, 5, 467−471. (29) Moribe, K.; Tsuchiya, M.; Tozuka, Y.; Yamaguchi, K.; Oguchi, T.; Yamamoto, K. Chem. Pharm. Bull. 2004, 52, 524−529. (30) Moribe, K.; Tsuchiya, M.; Tozuka, Y.; Yamaguchi, K.; Oguchi, T.; Yamamoto, K. J. Incl. Phenom. Macrocycl. Chem. 2006, 54, 9−16. (31) Frisch, M. J. et al. Gaussian 03; Gaussian Inc.: Wallingford, CT, 2004. (32) Gavezzotti, A. OPiX; University of Milano: Italy, 2003. (33) Taylor, R.; Kennard, O.; Versichel, W. Acta Crystallogr., Sect. B 1984, 40, 280−288.

ethenzamide molecules rearranging on contact with the crystal surface, rather than the crystal growing from a very low concentration of the high energy conformer. The time for this rearrangement to occur together with its high energy cost means that not all molecules do rearrange on contact, resulting in a high number of rejected growth units at the crystal surface which may well result in the poor growth. This study highlights our lack of understanding of the role of conformation in determining the outcome of a crystallization process, even in such a simple molecule as ethenzamide. The structure of ethenzamide is built from a high energy conformer, and we suspect that a second polymorph of this material may one day be found. This would allow for a direct comparison of crystallization properties, in order understand more fully the effects of solution conformation on crystal growth.

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ASSOCIATED CONTENT

S Supporting Information *

Further details of the polymorph screen and cif files for ethenzamide and cocrystals. This material is available free of charge via the Internet at http://pubs.acs.org

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS K.R.B. and R.J.D. would like to acknowledge Sally Price, Geoff Dent and Colin Seaton for helpful conversations, the EPSRC and GlaxoSmithKline for KRB’s CASE award which funded this research, and AstraZeneca for funding LST’s study visit to the University of Manchester.

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REFERENCES

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