Variable Temperature Studies of a Polymorphic System Comprising

CRYSTAL GROWTH & DESIGN 2004 VOL. 4, NO. 3 431-439

Articles Variable Temperature Studies of a Polymorphic System Comprising Two Pairs of Enantiotropically Related Forms: [S,S]-Ethambutol Dihydrochloride Janice M. Rubin-Preminger,† Joel Bernstein,*,† Robin K. Harris,‡ Ivana Radosavljevic Evans,‡ and Phuong Y. Ghi‡ Department of Chemistry, Ben-Gurion University of the Negev, P.O. Box 635, Beer-Sheva, Israel, 84105, and Chemistry Department, University of Durham, South Road, Durham, DH1 3LE, UK Received October 26, 2003

W This paper contains enhanced objects available on the Internet at http://pubs.acs.org/crystal. ABSTRACT: Of the four known polymorphs of [S,S]-ethambutol dihydrochloride, two transform in an enantiotropic single-crystal-to-single-crystal phase transformation from Form II to Form I on heating. The crystal structures of these two forms have been solved and compared. Because the other two forms are metastable and are only obtained from the melt, single-crystal data could not be obtained on them. However, all four forms have been characterized, and their relative thermodynamic relationships have been investigated by variable-temperature solid-state 13C NMR, variable-temperature powder X-ray diffraction studies, differential scanning calorimetry, and optical microscopy. Introduction Diastereomers, like other conformational isomers, may have completely different physiological effects in vivo, and, therefore, should often be treated as completely different substances.1 The three diastereomers of 2,2′-(ethylenediimino)-di-1-butanol dihydrochloride (ethambutol dihydrochloride) (Scheme 1) are a typical example of this; the S,S-form is therapeutically active, while the R,S-form is 16 times less effective and the R,Rdiastereomer is completely inactive against Mycobacterium tuberculosis. This phenomenon has been studied since the therapeutic properties of ethambutol dihydrochloride (EB2HCl) were first discovered.2 The mode of action of this drug, like many antituberculosis drugs, is not completely understood.3 It is possible that EB2HCl forms a chelate with copper, and the ability of this chelate or of the EB2HCl itself to fit a specific enzyme receptor may account for the structural and stereoisomeric selectivity of the antimycobacterial activity of these diamines.4 It is also believed that EB2HCl can increase the effectiveness of other antibacterial drugs such as spermidine, mycolic acids, and arabinogalactin, which through biosynthetic inhibition specifically alter the Mycobacterial cell wall.3 * To whom correspondence should be addressed. E-mail: yoel@ bgumail.bgu.ac.il. † Ben-Gurion University of the Negev. ‡ University of Durham.

Scheme 1

[S,S]-Ethambutol dihydrochloride (SS-EB2HCl) was first prepared by Wilkinson et al. in 1961.2 RR-EB2HCl (mp ) 200.5-201.5 °C) was originally obtained in the same way as SS-EB2HCl but from (-)-2-amino-1-butanol in place of (+)-2-amino-1-butanol. The low solubility of RS-EB2HCl (mp ) 203.5-204.6 °C) in a number of solvents allowed for easy separation of it from SS-EB2HCl and RR-EB2HCl, since it is formed as a byproduct in the production of both SS-EB2HCl and RR-EB2HCl.2 Several studies of SS-EB2HCl have been previously carried out by Brancone and Ferrari (1966),5 by Ferrari and Grabar (1971),6 most thoroughly by KuhnertBrandsta¨tter and Moser (1979),7 and briefly by Gamberini et al. (1994).1 However, all of these studies are incomplete such that much confusion remains about the exact number of polymorphs that exist and the energetic relationships between the various polymorphic forms. Therefore, we have attempted to clarify the confusion surrounding this material. Our studies reported here have revealed the presence of four polymorphic forms of which only one is stable at

10.1021/cg0341959 CCC: $27.50 © 2004 American Chemical Society Published on Web 04/10/2004

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Crystal Growth & Design, Vol. 4, No. 3, 2004 Scheme 2

room temperature (Form II is the form that is used in the commercial drug formulations). The relationships between the polymorphic forms are summarized in Scheme 2. All the phase transformations shown, except that of Form IV to Form II, are reversible.8 Two of the polymorphic forms of SS-EB2HCl are known to exist with a reversible phase transformation occurring between them at approximately 74 °C.7 The phase transformation is single-crystal-to-single-crystal, developing rapidly as a front moving through the crystal. The single-crystal structure of only the room temperature form (Form II) has been previously reported.9 The nature of the phase transformation from Form II to Form I allowed us to solve the crystal structures of both forms from the same crystal; in this way we have completed a study similar to that which Sørensen and Simonsen (1989)10 carried out on R,SEB2HCl, one of the other diastereomers of this material. Form III is obtained on recrystallization from the melt and converts to Form IV at approximately 36 °C, on cooling. Form IV exists at room temperature but converts to Form II on standing under ambient conditions. Therefore, it is probably best to think of the material in terms of two pairs of polymorphic forms: Forms II and I, which exist and interconvert reversibly below the melting point; and Forms IV and III, which exist and convert from one form to the other only once recrystallization from the melt has taken place. It should be noted that once Form III has been obtained, subsequent melting and recrystallization cycles only produce Forms IV and III (regardless of the form of the original solid), but Form II can be reobtained by recrystallizing the material from solution, as well as from Form IV on standing. This indicates that Form II is the thermodynamically preferred form. All four forms have been characterized, and their relative thermodynamic relationships have been investigated by variable temperature solid-state 13C NMR, variable temperature powder X-ray diffraction studies, differential scanning calorimetry (DSC), and optical microscopy. Experimental Procedures [S,S]-Ethambutol dihydrochloride was purchased from SigmaAldrich and used without further purification. All DSC measurements were performed using Rheometric Scientific Plus V v5.42 software on a Polymer Laboratories PL-DSC differential scanning calorimeter. The reported mea-

Rubin-Preminger et al. surements were all run with a heating rate of 10 °C/min and an uncontrolled cooling rate, in sealed Al-pans. 13 C NMR solid-state spectra were acquired by cross polarization from protons on a Chemagnetics CMX200 spectrometer, operating at 50.33 MHz. The samples were spun at ca. 5 kHz in a 4-mm zirconia rotor at the magic angle (54.7°), using an HX probe double-tuned to 13C and 1H. A π/2 pulse of duration 3 µs was used with a contact time of 4 ms. 13C spectra were recorded with a flip-back pulse and a recycle delay of 5 s. Between 256 and 1024 transients were collected (accumulation times between 21 and 85 min), and each transient contained 1024 data points. A 10-Hz exponential function was applied before Fourier-transformation to 8192 data points. The sample was referenced to adamantane (38.4 δ for the highfrequency peak), and chemical shifts are quoted with respect to the signal for tetramethylsilane. The magic angle was checked by the resonance of 79Br in KBr. In all cases, at least half an hour was allowed to elapse between measurements at different temperatures to allow the temperature to equilibrate. 13 C NMR solution-state spectra were acquired in D2O on a Bruker DPX500 spectrometer, operating at 50.32 MHz. The sample was studied at 300 K in a 5-mm QNP probe doubletuned to 13C and 1H. The chemical shifts, given in ppm, are quoted with respect to the tetramethylsilane resonance. FT-IR microscopy was performed on a Bruker spectrometer, using an Equinox 55 connected to an IRScope II using Opus software. Only the room-temperature FT-IR spectrum of SSEB2HCl has been obtained. It agrees well with that of Amber et al.,17 and that presented by Kuhnert-Brandsta¨tter and Moser7 for their Form II modification. All powder X-ray diffraction data were collected using a Bruker D8 Advance powder diffractometer with Cu KR1 radiation, equipped with a germanium (111) monochromator and an mBraun linear position-sensitive detector. An Anton Paar HTK 1200 furnace was used in the variable temperature study. All temperatures should be considered as ( 2 °C. All data were collected in the flat plate mode. Samples were prepared by sieving a small amount of material onto an amorphous fused silica disk. Data were collected in the 2θ range between 5 and 50°, with a step size of 0.0144° and a counting time of 3 s per step, resulting in a total collection time of just over 2.5 h per scan. Between data collections, heating and cooling were performed at a rate of 10°/min. Suitable crystals were grown by vapor diffusion of methanol into an ethanol solution (both solvents being of commercial grade). All the single-crystal data were collected on a Bruker SMART 6000K diffractometer using Mo KR-radiation with a graphite monochromator. An Oxford Cryosystem 600 series open flow nitrogen cryosystem was used to raise the temperature to 80 °C for the data collection of Form I. All the atoms (including the hydrogen atoms) were located from the difference maps. The data were reduced by SAINT,11 solved using SHELXS,12 and then refined with SHELXL13 in SHELXTL.14

Discussion Thermal Methods of Analysis. In 1966, Brancone and Ferrari5 ran a series of differential thermal analysis (DTA) measurements on the various diastereomers of EB2HCl at 20 °C/min. On heating the sample, they observed endotherms in SS-EB2HCl at 75 and 200 °C. The first endotherm was reversible and repeatable, in that the sample could be cooled and reheated without affecting the presence or size of this endotherm, if the melting point (the second endotherm) was not reached. They accounted for this as a crystalline phase transition. Ferrari and Grabar6 continued the work started with Brancone,5 reporting both the heating and cooling cycles for the material. The DTA thermogram of SS-EB2HCl displayed two endotherms at 75 and 200 °C on heating, and the cooling cycle (starting from above the melting point) displayed three exotherms at 175 °C, 165 °C, and

[S,S]-Ethambutol Dihydrochloride

Figure 1. Pressure/temperature phase diagram of the four modifications of [S,S]-ethambutol dihydrochloride from Kuhnert-Brandsta¨tter and Moser (1979).9 Note that S (schmelze) indicates the melt, Fp is the melting point, and Up indicates a phase transition.

the largest at 150 °C. However, these authors did not speculate on the nature of the exotherms. On reheating of the sample, following the first cooling cycle with a second heating cycle, an additional endotherm at 44 °C was present, but that of the phase transition at 75 °C was absent.6 This is indicative of the presence of at least one previously unknown phase. DSC was carried out on SS-EB2HCl in 1979 by Kuhnert-Brandsta¨tter and Moser.7 They reported the existence of four polymorphic forms. Their Form II undergoes a reversible phase transition to Form I on heating to 72 °C, with a slight hysteresis on cooling, i.e., at 70 °C. They claimed that once recrystallization from the melt occurred Form I was obtained again. However, they did indicate that Form I prior to melting and Form

Crystal Growth & Design, Vol. 4, No. 3, 2004 433

I obtained after the melt were not “absolutely identical”. On cooling, a double peak was seen at 40 °C; this corresponds to the single peak seen by Ferrari and Grabar6 at 44 °C. They interpreted this as Form I converting to Form III at 42 °C with an additional polymorphic transformation to Form IV at 39 °C, on cooling. On heating of the sample, these temperatures shifted to 43 and 36 °C, respectively. How their experiments were conducted was not described, but they did construct a phase diagram of the system (Figure 1). We have adopted the phase nomenclature that they used in the phase diagram (Figure 1). The latest study on the structural and thermal aspects of SS-EB2HCl by Gamberini et al.8 reports an enantiotropic phase transition at 72.7 °C between Forms II and I with ∆H ) 5.74 kJ mol-1 and a second phase transition at 47.8 °C on cooling with ∆H ) 3.61 kJ mol-1 as determined by DSC with a heating rate of 5 °C/min. We have analyzed SS-EB2HCl using DSC with a heating rate of 10 °C/min. Unfortunately, the cooling rate could not be controlled, and thus the ambient cooling rate was used. Initial results agree well with those reported by Brancone and Ferrari.5 On heating the sample, we observed an endotherm at 72 °C, which produced a hysteresis curve on cooling and reheating. The ∆H for this phase transition was ca. 5.70 kJ/mol. This endotherm was always observed unless melting occurred; thereafter, it was not observed. On cooling from the melt two exothermic peaks were observed; the largest was thought to be that of recrystallization, based

Figure 2. Phase transformation from Form II (a) at room temperature to Form I (c) above 74 °C, as observed on a hot stage Kofler microscope with crossed polarized light; (b) shows partial conversion from Form II (the colored region) to Form I (the colorless region). W A movie in .mpg format is available.

Figure 3. DSC thermogram of several heating and cooling cycles of SS-EB2HCl; the initial sample was Form II, which was heated with a rate of 10 °C/min but with an uncontrolled cooling rate.

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Rubin-Preminger et al.

Figure 4. DSC thermogram of the heating (conversion from Form IV to Form III) and cooling (conversion from Form III to Form IV) cycle of the phase transformation in SS-EB2HCl.

on observations from parallel hot stage microscopy experiments. A new peak was seen on cooling at 36.4 °C. It has been shown by Brancone and Ferrari5 that the peak at 72 °C corresponds to the reversible phase transition from Form II to Form I. This has been confirmed by experiments carried out on a Kofler hot stage microscope with crossed polarizers. The phase transition is observed as a front moving from one end of the needle shaped crystal to the other end and could only be observed under polarized light (Figure 2). There is no large hysteresis between the temperatures of the phase transition on heating and cooling of the material; in fact, it is possible to hold the temperature constant and thus to maintain conversion at an intermediate stage. After melting, the recrystallized product is Form III, and this explains the absence of the endotherm at 72 °C on reheating. In other words, the cooled melt does not recrystallize back to the form obtained from Sigma (Form II). The peak at 36.4 °C was observed, but it was very small (∆H ca. -0.39 kJ/mol); it is indicative of a second phase transformation either to a new polymorph, Form IV, or back to Form II. We have not observed the double peak reported by Kuhnert-Brandsta¨tter and Moser7 at 40 and 46 °C. In the DSC thermogram, the phase transition at 74 °C can be clearly seen in the first heating cycle (Figure 3). However, it is absent in subsequent heating cycles. This is true regardless of the number of heating and cooling cycles run on the same material. It should be noted that the size and position of the melting and recrystallization peaks decrease as the number of heating and cooling cycles increases. This may be due to degradation of the sample15 or the presence of impurities.16 However, the NMR results do not indicate the presence of any additional material. The reversible phase transformation from Form IV to Form III was most clearly seen on samples freshly prepared in open containers, by heating a sample on a hot stage microscope and allowing it to cool to room temperature. Samples that were allowed to stand overnight before measurement were found to have converted back to Form II. Therefore, the freshly prepared material was immediately placed in an Al-pan, sealed, and the heating and cooling cycle over the range 30 to 80

°C was measured. Thus, the peak for this phase transition was observed (Figure 4). It should be noted that the large difference in peak height is caused by the large difference in the heating (20 °C/min) and cooling (under ambient conditions); the cooling peak is circled in red. Because of the large difference in scale, accurate measurement of ∆H values for this phase transition has not yet been obtained. The value found from the thermogram (Figure 4) was 0.4 kJ/mol, but this varied considerably from experiment to experiment. Observations of this phase transformation on a hot stage microscope are in good agreement with the DSC results. Recrystallization from the melt (generally occurring at 124 ( 5 °C) produces a thin film of crystals (Form III as determined by DSC) that undergo a gradual darkening over 15 °C and half an hour on cooling from about 50 °C and then a rapid phase transition at 32 °C, darkening completely to Form IV. These dark crystallites can then be reheated to return to Form III. The phase transformation then tends to occur over about 10 min. A physical mixture of Forms III and IV appears to be stable over quite a large temperature range, although by 60 °C it has generally all converted to Form III (Figure 5). Thermodynamic Analysis. The thermodynamic relationships between the four polymorphic forms of SSEB2HCl are summarized in an energy-temperature diagram26,27 (Figure 6). Energy-temperature diagrams are the graphical semiquantitative solution of the Gibbs-Helmholtz equation (∆G ) ∆H - T∆S) for a system. Spectral Methods of Analysis. The variable temperature solid-state 13C NMR spectra reveal the presence of four polymorphic forms of SS-EB2HCl. Again we consider these from the perspective of two pairs of polymorphs with an enantiotropic phase transformation between the two components of each pair. The atom numbering used throughout is as indicated in Scheme 1. The differences between the spectra of Forms I and II can clearly be seen in Figure 7. The reversibility of this phase transformation was confirmed by running spectra on cooling to ambient temperature again. The initial and final spectra obtained at room temperature were essentially identical. The heating and cooling cycle



Figure 5. Phase transformation from Form IV (a) at room temperature to Form III (c) 60 °C, as observed on a hot stage Kofler microscope with crossed polarized light; (b) shows partial conversion from Form IV (the dark region) to Form III (the light region), moving from the lower left to the upper right of the figure. W A movie in .mpg format is available.

Figure 8. The 13C CP/MAS NMR spectra taken in the vicinity of 74 °C with a deliberate temperature gradient to show the transformation between (a) Form II (indicated by X) and (b) Form I (atoms indicated by X′, where X is the number of the atom numbering according to Scheme 1) where the average temperature for (b) is slightly higher than for (a). Table 1. Peak Positions in the 13C NMR Spectra of Four Polymorphic Forms of [S,S]-Ethambutol Dihydrochloride, Together with Solution-State Data

Figure 6. The semiempirical energy-temperature diagram of the thermodynamic relationships among the four polymorphic forms of SS-EB2HCl. L indicates the melt; TIITI indicates the phase transformation from Form II to Form I at 74 °C; TIVTIII indicates the phase transformation from Form IV to Form III at 36 °C; Tf,I indicates the melting point of Form I observed at ca. 200 °C; Tf,II is the virtual melting point of Form II; Tf,III is the virtual melting point of Form III; and Tf,IV is the virtual melting point of Form IV.

Figure 7. The 13C CP/MAS NMR spectra of (a) Form II at 50 °C (indicated by X) and (b) Form I at 80 °C (atoms indicated by X′, where X is the atom numbering according to Scheme 1).

was repeated several times with good agreement between the results of these repetitions. The 13C CP/MAS NMR spectra were also measured in the vicinity of 74 °C with a deliberate temperature gradient to show the transformation between Forms I and II (Figure 8). The relative proportion of Form II is larger at lower temperatures (Figure 8a) and smaller at higher temperatures Figure 8b). The phase transformation from Form II to Form I is very rapid, taking no more than 10 s to complete. Thus, this is the only way in which the phase transformation itself can be followed directly.

δc/ppm assignment

C1

C3

C2

C4

solution (D2O) Form II 23 °C, 200 MHz Form I 80 °C, 200 MHz Form IV 23 °C, 200 MHz Form III 50 °C, 200 MHz

60.87

57.26

40.22

19.85

8.49

C5

58.9 br18

54.2

22.7

10.8

61.0

56.2

22.7

10.3

59.4

55.3

39.6/ 40.2 v.br19 41.2/ 41.8 v.br 40.4 br.

60.8

56.0

40.9 br.

22.8 and 25.0 22.7

11.6 and 12.1 10.7

The chemical shifts are reported in Table 1. The differences between the values for the two temperatures (Figure 7) give a clear indication of the existence of the two polymorphic forms, reflecting changes in the crystalline environment of the atoms resulting from the phase change. These differences in chemical shift can most clearly be seen in the peaks representing C5, C3, and C1 (Scheme 1). The peaks from C2 and C1 are broadened by residual dipolar coupling to the quadrupolar 14N nucleus20 (Table 1). The number of peaks strongly suggests that for both polymorphic forms the asymmetric unit is half a molecule. This conclusion is consistent with the subsequent result from singlecrystal X-ray diffraction experiments. For a complete molecule in the crystallographic asymmetric unit 10 peaks would be expected. Differences between the chemical shifts in the solidstate spectra and those observed in D2O solution are quite significant, and are likely to be the result of conformational mobility in solution, particularly internal rotation about the CH2-CH3 and CH2-OH bonds.

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Figure 9. 13C CP/MAS NMR of the phase transformation of SS-EB2HCl phases (III/IV) from the forms at ambient probe temperature (assignments indicated by the atomic numbering as in Scheme 1 and discussed in the text) to Form III (indicated by X′) on heating, and back to Form IV (the room temperature phase indicated by X and where necessary a subscript indicating a doubling of the peak) on cooling.

Form IV was obtained by melting commercially available SS-EB2HCl and allowing it to cool from the melt under ambient conditions. Only one phase transformation in the temperature range 30-55 °C was observed, the phase transition from Form IV to Form III. An initial spectrum was acquired at room temperature (23 °C). The sample was heated in situ to 90 °C and annealed for approximately 1 h before acquisition of the spectrum at that temperature. Spectra (Figure 9) were acquired during the cooling cycle, after allowing the sample to equilibrate to the desired temperature for 30 min following each cooling stage. A final spectrum was recorded at room temperature (23 °C). The reversibility of the transition is shown by a comparison between the initial and final room temperature spectra (Figure 9). It is clear from the spectra at 30 and 23 °C that a mixture of forms exists at these temperatures. However, it is not possible to rule out the alternative explanation that there is structural disorder in Form IV. The peaks at 25.0 and 59.4 ppm for C4 and C1, respectively, may be assigned to Form IV, as can the methyl carbon signal at 12.1 ppm. Other signals are not at significantly different positions from those of Form III, and it would seem that the C1 and C4 signals of Form III remain in the ambient temperature spectrum. A spectrum obtained on another occasion at ca.


-20 °C is similar to that at ambient temperature which seems to suggest that the III T IV transition may not be as simple as indicated by the other techniques discussed here. Instances of Forms II and III existing side by side below the III f IV transition temperature are known. However, it should be realized that the nature of the sample used is very different for NMR, which uses bulk microcrystalline material confined in a rotor, with substantial pressures in parts of the sample during magic-angle spinning. This could result in differences in kinetic behavior from those occurring for thermal techniques or powder XRD. The C5 region in the room temperature spectrum (Figure 9, top and bottom) are puzzling, since there seem to be four signals present. We have no explanation for this fact. It is possible that Form IV has a whole molecule in the crystallographic asymmetric unit, in principle splitting each signal into two. The large remaining intensity of the peak at 22.8 ppm in the room temperature spectrum may support such a conclusion but is by no means certain. The number of peaks seen for Form III strongly suggests that it has only half a molecule in its crystallographic asymmetric unit. Some of the signals appear to have somewhat broader resonances underlying them, which may be accounted for by the presence of some proportion of amorphous material in the sample, due to incomplete crystallization. In fact, on one occasion, heating to ca. 100 °C resulted in a gross broadening of all signals (except C5), which may indicate formation of a glassy material. The chemical shift for C3 (and to a lesser extent C1) for Form III is temperature-dependent, increasing by ca. 1 ppm on heating from 30 to 90 °C (Figure 9). As for Forms I and II, the C2 peak is broadened for Forms III and IV due to residual dipolar coupling to the quadripolar 14N nucleus. The C1 peak is also affected by this factor but to a lesser extent. Table 1 lists the peak assignments and chemical shifts of the 13C NMR spectra for the four polymorphic forms of SS-EB2HCl. The spectra of Forms I and III are very similar. This indicates that the overall chemical environments of the molecules in the two forms are very similar. As noted above, the biggest differences between the spectra for forms III and IV are for C1 and C4. Diffraction Methods of Analysis. Powder X-ray diffraction studies (Figure 10) confirm the existence of four polymorphic forms of SS-EB2HCl. The diffraction pattern of Form II was measured at room temperature and then at 60 °C. The material was then heated in situ to above the phase transition temperature (>74 °C), and the diffraction pattern of Form I was obtained. Melting and recrystallization by cooling to room temperature ex situ produced Form IV, which was then heated in situ to 70 °C and the diffraction pattern of Form III was obtained. Both phase transformations have been confirmed as being enantiotropic by cooling back to room temperature and recollecting the data. Although the diffraction patterns of the four forms exhibit some similarities (Figure 10), there are a number of distinct differences with clearly characteristic peaks for each polymorphic form. The background in the diffraction patterns of Form IV and Form III is higher than that of Form II and Form I; the shape of the



Figure 10. The powder X-ray diffraction patterns of the four polymorphic forms of SS-EB2HCl. Table 2. Positions of Five Characteristic Peaks of the Four Polymorphic Forms of SS-EB2HCl (•20)

1 2 3 4 5

Form II (60 °C)

Form I (80 °C)

Form III (70 °C)

Form IV (30 °C)

14.1 20. 6 22.4 32.7 41.6

17.1 23.4 26.5 39.6 43.1

13.1 24.2 29.3 31.8 37.1

14.6 22.7 24.0 28.1 33.2

background is consistent with the contribution of some amorphous material contained in the recrystallized samples.

Five characteristic peaks of each phase may be chosen for the rapid identification of each of the crystal forms (Table 2). These peaks are unique to each form, although they may not necessarily be the strongest peaks in the diffractograms. The powder patterns of Forms II and I have been analyzed to check the phase purity of the samples under investigation. As the preferred orientation effects in the sample were quite severe, the analysis was performed by Pawley fitting.21 The parameters refined included unit cell parameters, sample displacement, background terms, pseudo-Voigt profile function, and a peak shape asymmetry term. The final plots obtained for the powder patterns of Forms II and I (Figures 11 and 12, respectively) show that the materials observed are phase pure, i.e., that Form II fully converts to Form I under these experimental conditions. The crystal structures of Forms I and II have been determined from single-crystal X-ray diffraction studies (crystal structures assigned numbers 221758 and 221759 by the CCDC). The results for the room temperature crystal form (Form II) are in good agreement with that described in the literature (Table 3) by Hamalain et al.13 This form grows as colorless needles, and single-crystal measurements conducted between -100 and 60 °C attest to its stability. Form I was obtained by a singlecrystal-to-single-crystal phase transition from Form II at 74 ( 2 °C on heating. The diffraction data of this form were collected at 82 °C on the same crystal used for the room temperature measurement.

Figure 11. Pawley fit profile for SS-EB2HCl Form II: observed (blue), calculated (red), and difference (gray).

Figure 12. Pawley fit profile for SS-EB2HCl Form I: observed (green), calculated (red), and difference (gray).

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Table 3. Comparison of the Literature and Experimental Crystallographic Data for SS-EB2HCl Form II

space group a (Å) b (Å) c (Å) V (Å3)

Form I

literature data9

experimental data 295 K

experimental data 355 K

P21212 6.555 (5) 23.18 (2) 5.176 (5) 787.0 (1)

P21212 6.626 (7) 23.380 (3) 5.196 (6) 805.0(2)

P21212 6.845(4) 22.80(1) 5.210(3) 813.0(8)

Although a superficial comparison of the unit cell parameters (Table 3) at the two temperatures might suggest a simple thermal expansion of the unit cell, the crystal structures differ sufficiently to permit visual distinction between them (Figures 13 and 14). In a manner similar to the changes in the R,S-diastereomer above and below the phase transition,10 the a and c axes decrease in length and the b axis increases in length. The most visible difference, however, is between the calculated unit cell volumes. The crystal structures of both polymorphs are orthorhombic, in space group P21212 with Z ) 2. The packing efficiencies of the two forms are quite high,24 with the packing coefficient decreasing slightly in the high-temperature form. This is as expected for a transformation from a more stable form (more densely packed) to a less stable (less densely packed) form, according to Burger’s Density Rule.8 Attempts to grow crystals of Forms III and IV are continuing, and we are investigating the possibility of structure determination from powder diffraction as well. For both Forms I and II, the two ends of each molecule are related by a 2-fold symmetry axis, so the asymmetric unit is half a molecule, in agreement with the conclusion from the MAS NMR spectra. The molecular chains are approximately in an all s-trans stretched conformation from the methyl carbon at one end to the methyl carbon at the other, with the CH2OH groups offset to mutually opposite sides. In passing from Form II to Form I, this long molecular axis appears to rotate about the midpoint of the C2-C2′ bond by 14.7° away from the b-axis (indicated by the blue lines in Figure 14). Using the program PLATON23 the positions of voids in the unit cells were determined.25 The unit cell of Form II possesses nine voids, eight on the corners of the unit cell and one in the center (Figure 14). Form I possesses the analogous nine voids and an additional four voids within its unit cell. All of these voids have a radius of approximately 1.1 Å. The addition of more free space within the unit cell for Form I (in comparison to Form II) is consistent with a larger volume, lower density, and lower stability by the Density Rule. The most significant structural differences between the molecules in the two polymorphs of SS-EB2HCl are found in the chain length, which is reduced from 11.331 (4) Å in Form II to 11.192 (6) Å in Form I and in most of the torsion angles (Table 4), e.g., around the C1-C3 bond. Indeed, the dihedral angles generally differ significantly from the ideal staggered 60° and 180°. The small differences in torsion angles between the two forms are cumulative, resulting in the net shortening of the chain length for Form I. As one of the reviewers of this paper pointed out, it is curious to find two polymorphs with very similar unit cell dimensions and very similar packing in the same

Figure 13. SS-EB2HCl as viewed down the c-axis of (a) Form II and (b) Form I.

Figure 14. PLATON plots of the unit cells of Forms I (b) and II (a) wherein the yellow circles represent voids, and the green circles indicate the chloride ions. Again the view is down the c-axis in both forms. Table 4. Torsion Angles of Forms I and II C1-N-C2-C2′ C2-N-C1-C4 C2-N-C1-C3 N-C1-C4-C5 C3-C1-C4-C5 C4-C1-C3-O N-C1-C3-O

Form II/(°)

Form I/(°)

165.5(4) 171.5(6) 46.1(7) 168.5(6) -68.4(8) -62.6(6) 58.6(6)

164.9(4) 176.2(8) 46.3(9) 168.8(10) -64.8(13) -70.4(10) 54.2(10)

space group, along with very similar torsion angles along the molecular backbone, yet possessing a sharp phase transition. However, variable temperature single crystal diffraction studies above and below the phase transition reveal no sharp or sudden change in any of the unit cell parameters, nor in the atomic coordinates. As such, the barrier to conversion is still not fully understood. Conclusions Using a range of techniques involving variable temperature, we have conclusively shown that [S,S]-etham-


butol dihydrochloride exists as two pairs of enantiotropically related polymorphs. The transformations between the components of each pair occur in single-crystal-tosingle-crystal mode, and this has enabled us to solve the structure of Form I (the stable high-temperature form) for the first time, as well as that of Form II. Carbon-13 NMR chemical shifts and powder XRD patterns are reported for all four forms. We have established the energetic relationships between the four forms (summarized in Figure 6). However, the precise mechanisms of the phase transformations in terms of the reaction coordinate have not yet been elucidated. Also, we have no evidence to support or to contradict the mechanism proposed by Sorensen and Simonsen10 (1989) for the transition in the similar structures of the [R,S]-derivative. Acknowledgment. We would like to thank Dr. David Apperley for his assistance in the NMR work, and Prof. Judith Howard for the kind use of her facilities for the powder diffraction work. This research was partially supported by the U.S.-Israel Binational Science Foundation and by the European Polymorphism Network (MORPH) of the European Science Foundation. We thank the U.K. Engineering & Physical Sciences Research Council for Grant RG/N05635, which underpinned the NMR work. One of us (R.K.H.) is grateful to the Leverhulme Trust for the provision of an Emeritus fellowship. References (1) Gamberini, G.; Ferioli, V.; Rustichelli, C.; Vezzalini, F. Il Farmco 1994, 49, 415-419. (2) Wilkinson, R. G.; Shepard, R. G.; Thomas, J. P.; and Baughn, C. J. Am. Chem. Soc. 1961, 83, 2212-2213. (3) Blessington, B.; O’Sullivan, J. Chem. Br. 1994, 566569. (4) Wilkinson, R. G.; Cantrall, M. B.; Shepard, R. G. J. Med. Pharm. Chem. 1962, 5, 835-845. (5) Brancone, L. M.; Ferrari, H. J. Microchem. J. 1966, 10, 370392.

Crystal Growth & Design, Vol. 4, No. 3, 2004 439 (6) Ferrari, H.; Grabar, D. G. Microchem. J. 1971, 16, 5-13. (7) Kuhnert-Brandsta¨tter, M.; Moser, I. Mikrochim. Acta 1979, I, 125-136. (8) Burger, A.; Ramberger, R., Mikrochim. Acta 1979, II, 259272. (9) Hamalainen, R.; Lehtinen, M.; Ahlgren, M. Arch. Pharm. 1985, 318, 26-30. (10) Sørensen, A. M.; Simonsen, O. Acta Crystallogr. 1989, C45, 506-509. (11) Bruker AXS: SAINT+, Release 6.22. Bruker Analytical Systems, Madison, Wisconsin, USA, 1997-2001. (12) Sheldrick, G. M. SHELXS-97. Program for the Solution of Crystal Structures, University of Gottingen, Germany, 1997. (13) Sheldrick, G. M.; Schneider, T. R. SHELXL-97. Program for the Refinement of Crystal Structures, University of Gottingen, Germany, 1997. (14) Sheldrick, G. M. SHELXTL-Plus, Release 6.10. Bruker Analytical Systems, Madison, Wisconsin, USA, 2000. (15) Kofler, L.; Kofler, A. Thermal Micromethods: For the Study of Organic Compounds and Their Mixtures; Translated by McCrone, W. C.; McCrone Associates, Inc.: Chicago, IL, 1952; pp 68-86. (16) Giron, D. Thermochem. Acta 1995, 248, 1-59 (17) Amber, M. M.; Tawakkol, M. S.; Ismaiel, S. A. J. Pharm. Belg. 1976, 31, 80-88. (18) br. indicates that the peak is broad. (19) v.br. indicates that the peak is very broad. (20) Harris, R. K.; Olivieri, A. C. Prog. Nucl. Magn. Reson. Spectrosc. 1992, 24, 435-456 and references therein. (21) Pawley, G. S. J. Appl. Crystallog. 1981, 14, 357. (22) The reported a and b axes have been interchanged to facilitate comparison of the literature and experimental data. (23) Spek, A. L. Acta Crystallogr. 1990, A46, C34. (24) Kitaigorodskii, A. I. Organic Chemical Crystallography; Originally printed in Russian in Moscow by the Press of the Academy of Sciences of the USSR, 1955; Authorized translation, Consultants Bureau: New York; pp 106-109. (25) van der Sluis, P.; Spek, A. L. Acta Crystallogr., Sect. A: Found. Crystallogr. 1990, 46, 194-201. (26) Grunenberg, A.; Henck, J. O.; Siesler, H. W. Int. J. Pharm. 1996, 129, 147-158. (27) Bernstein, J. Polymorphism in Molecular Crystals; Clarendon Press: Oxford, 2002; pp 32-34.

CG0341959

Variable Temperature Studies of a Polymorphic System Comprising

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