Discovery of a New Polymorph of Dehydroepiandrosterone

CRYSTAL GROWTH & DESIGN

Discovery of a New Polymorph of Dehydroepiandrosterone (Prasterone) and Solution of Its Crystal Structure from X-ray Powder Diffraction Data

2006 VOL. 6, NO. 4 925-932

G. Patrick Stahly,* Simon Bates, Mark C. Andres, and Brett A. Cowans SSCI, Inc., 3065 Kent AVenue, West Lafayette, Indiana 47906 ReceiVed September 16, 2005; ReVised Manuscript ReceiVed February 13, 2006

ABSTRACT: A new polymorph of dehydroepiandrosterone, also known as DHEA or prasterone, was found and designated form FVI. Form FVI is anhydrous and nonsolvated, and is less thermodynamically stable than the known form FI under ambient conditions. The X-ray powder diffraction patterns of FI and FVI are so similar that this technique is inadequate to determine if samples contain pure FI, pure FVI, or mixtures of the two. However, the forms can be differentiated by solid-state NMR spectroscopy. The crystal structure of FVI was determined from X-ray powder diffraction data using a combination of proprietary SSCI software and the Cambridge Crystallographic Data Centre’s program DASH. Introduction Dehydroepiandrosterone, also known as DHEA or prasterone (Figure 1) is a naturally occurring adrenal steroid hormone. Prasterone is under investigation by Genelabs Technologies, Inc. (Redwood City, CA) as a treatment for systemic lupus erythematosus (SLE) in women. The solid-state chemistry of prasterone has been studied in detail. The crystal forms that were reported are listed in Table 1. Kuhnert-Brandsta¨tter found four crystal forms by thermomicroscopy, reporting their melting points as 149-153 °C (I), 139-141 °C (II), 137-140 °C (III), and 130-136 °C (IV).1 Chang et al reported the isolation and characterization of seven crystal forms and thermomicroscopic observation of a form melting at 146.5-148 °C, which was designated as form FV.2 The crystal structures of forms FI,3 FII,4 S1,3 S2,5 and S43 were reported. The seven crystalline forms studied by Chang et al were well characterized using differential scanning calorimetry (DSC), thermogravimetry (TG), X-ray powder diffractometry (XRPD), infrared (IR) spectroscopy, and solution calorimetry.2 XRPD and IR spectroscopy were found to be particularly useful in differentiating the forms, and XRPD was suggested to be the best technique to “determine modification purities.”2 Multiple prasterone samples were provided by Genelabs to SSCI for analysis of polymorphic content and were accompanied by analytical data generated by various techniques, including IR spectroscopy, DSC, optical microscopy, and solid-state, carbon-13 nuclear magnetic resonance (ssNMR) spectroscopy. Review of these data revealed that XRPD and ssNMR spectroscopy provided inconsistent results. XRPD indicated that certain samples contained one (or two) forms, while ssNMR indicated that the same samples contained two (or three) forms. All the samples provided for this study had been through a drying process, so that no hydrates or solvates were expected to be present. A study was undertaken to understand the inconsistencies and elucidate the solid form content of samples provided to Genelabs by the manufacturers.

Figure 1. The structure of prasterone. Table 1. Known Prasterone Crystal Forms designation FI FII FIII FIV FV S1 S2 S3 S4 c

nature

melting range (°C)

same as KuhnertBrandsta¨tter formb

anhydrate anhydrate anhydrate unknowna unknowna 1/4 hydrate monohydrate monohydrate hemi-methanolate

149-150c 140-141c 137-138c 130-136 146.5-148 127-128c unknown unknown unknown

I II III IV

a Not isolated; observed by thermomicroscopy only. b Reference 1. Reference 2.

Table 2. Production of Known Prasterone Forms target form FI FII FIII S1 S2 S3 S4 a

form obtaineda

procedure used evaporated a 2-propanol solution in a nitrogen stream evaporated a THF solution in a nitrogen stream kept S4 over P2O5 at high vacuum for 3 days ground a methanol slurry of FI and FII agitated a slurry of FI and FII in water for 1 day kept S4 under 58% RH for 2 days induced crystallization of a methanol solution by cooling

FI FII FIII + S4 S1 S2 FIII + S3 or S4 S4

Based on comparison of the XRPD patterns to those reported by Chang.2

Experimental Section Preparation of Form FI. A mixture of 1.6 g of prasterone and 21 mL of anhydrous 2-propanol was heated until the solid dissolved. The solution was allowed to cool to ambient temperature under a stream of * To whom correspondence [email protected].

should

be

addressed.

E-mail:

dry nitrogen, and then was kept under a stream of dry nitrogen for 3 days to evaporate the solvent. The remaining solid was shown to be form FI by XRPD analysis. Diagnostic ssNMR peaks are shown in Table 3. Preparation of Form FII. Addition of 1.5 g of prasterone to 7.2 mL of anhydrous tetrahydrofuran resulted in a solution. The solution was kept under a stream of dry nitrogen for 12 h to evaporate the

10.1021/cg050479c CCC: $33.50 © 2006 American Chemical Society Published on Web 03/17/2006

926 Crystal Growth & Design, Vol. 6, No. 4, 2006 Table 3. ssNMR Data for Known Prasterone Forms form

carbon no. 18 (ppm)

carbon no. 6 (ppm)

carbon no. 17 (ppm)

FI FII FIII S1 S2 S4

14.0, 14.8a 13.1 13.1, 15.4 12.8, 14.3a 12.6a,b 13.9

118.9, 120.4a 119.9 120.1 119.5, 121.2a 120.4, 123.4a 119.1

222.1, 224.2a 224.1 216.6, 225.1 225.3a,b 224.8, 226.7a 218.5

a Known to have two crystallographically independent molecules in the structure.3,5 b It is assumed that the carbon resonances of the two crystallographically independent molecules are coincident.

solvent. The remaining solid was shown to be form FII by XRPD analysis. Diagnostic ssNMR peaks are shown in Table 3. Attempted Preparation of Form FIII. Form S4, made as described below, was kept under 58% relative humidity at ambient temperature for 2 days and under vacuum (mechanical pump) over phosphorus pentoxide for 3 days. XRPD analysis showed the solid to be a mixture of forms S3 and/or S4, along with some FIII. Diagnostic ssNMR peaks are shown in Table 3. Preparation of Form S1. Some prasterone (a mixture of forms FI and FII) and methanol were added to a mortar. The mixture was ground by hand using a pestle for about 30 min. Methanol was added occasionally to keep the mixture the consistency of paste. The mixture was allowed to dry in the air to give a solid that was shown by XRPD analysis to be form S1. Diagnostic ssNMR peaks are shown in Table 3. Preparation of Form S2. A slurry of prasterone forms FI and FII in distilled water was stirred at 37 °C overnight. The solids were collected by vacuum distillation and allowed to dry in the air. XRPD analysis showed the solid to be form S2. Diagnostic ssNMR peaks are shown in Table 3. Attempted Preparation of Form S3. Form S4, made as described below, was kept under 58% relative humidity at ambient temperature for 2 days. XRPD analysis showed the solid to be a mixture of forms S3 and/or S4, along with some FIII. Diagnostic ssNMR peaks are shown in Table 3. Preparation of Form S4. A saturated solution was made by adding prasterone to methanol at 60 °C with stirring until solids persisted and filtering the resulting slurry at 60 °C. The filtrate was allowed to cool to ambient temperature with stirring, and the material that crystallized was collected by vacuum filtration. The recovered crystals were shown to be the hemi-methanolate (form S4) by XRPD analysis. Diagnostic ssNMR peaks are shown in Table 3. X-ray Powder Diffraction. XRPD analyses were carried out on either a Siemens D-500 X-ray powder Diffractometer-Kristalloflex or a Shimadzu XRD-6000 X-ray powder diffractometer, both using Cu KR radiation. The Siemens instrument utilizes an IBM-compatible interface and DIFFRAC AT software. Slits I and II were set at 1°, and the radiation was electronically filtered by a Kevex Psi Peltier cooled silicon detector with slits III at 1° and IV at 0.15°. A θ-2θ continuous scan at 6°/min (0.4 s/0.04° step) from 4 to 40° 2θ was used. Silicon was used as the calibration standard. The Shimadzu instrument is equipped with a long fine focus X-ray tube. The tube voltage and amperage were set to 40 kV and 40 mA, respectively. The divergence and scattering slits were set at 1°, and the receiving slit was set at 0.15 mm. Diffracted radiation was detected by a NaI scintillation detector. A θ-2θ continuous scan at 3 °/min (0.4 s/0.02° step) from 2.5 to 40° 2θ was used. Silicon was used as the calibration standard. Solid-State, 13C NMR. ssNMR spectra were obtained on a General Electric Omega PSG, 400 MHz spectrometer using high-power proton decoupling and cross-polarization with magic-angle spinning at approximately 5 kHz. The magic angle was adjusted using the Br signal of KBr by detecting the sidebands as described by Frye and Maciel.6 Approximately 50 mg of sample packed into a zirconia rotor were used for each experiment. The chemical shifts were referenced externally to the CH resonance of adamantane at 29.50 ppm as described in the literature.7 Differential Scanning Calorimetry. The DSC analysis was carried out using a TA Instruments differential scanning calorimeter 2920. The sample was placed into an aluminum DSC pan, and the weight was accurately recorded. The pan was covered with a lid and then crimped.

Stahly et al. The sample cell was heated from about 20 to 192 °C under a nitrogen purge at a rate of 20 °C/min. Indium was used as the calibration standard. Thermogravimetry. The TG analysis was performed using a TA Instruments 2950 thermogravimetric analyzer. The sample was placed in an aluminum sample pan and inserted into the TG furnace. The furnace was heated from about 20 to 346 °C under a nitrogen purge at a rate of 10 °C/min. Nickel and Alumel were used as calibration standards.

Results and Discussion Samples 1 and 2 were analyzed by XRPD and ssNMR spectroscopy. The diffractograms are shown in Figure 2, along with the patterns of forms FI and FII. The XRPD pattern obtained from sample 1 matches the FI pattern quite well. Evidence that the sample contained a small amount of form FII is the peak at about 8.8 °2θ. While there are some differences between the sample 1 and FI patterns, they occur in regions of high peak density, such as between 16 and 20 °2θ. Note that all free-standing peaks in each diffractogram correspond very closely. The relatively minor differences observed could be the result of the normal Poisson distribution of intensity variations inherent in data generated by Bragg-Brentano diffractor geometry, or the result of particle size or preferred orientation effects. There are no peaks in either the FI pattern or the sample 1 pattern which do not overlap peaks in the other. In addition, the sample 1 pattern contains no evidence for the presence of form FIII based on the characteristic peaks denoted by Chang. Thus, the XRPD data suggest that sample 1 contained FI and a very small amount of FII. Sample 2 exhibited an XRPD pattern that matched the pattern of FII reasonably well. However, peaks diagnostic of FI are present at about 15.1, 17.9, and 18.8 °2θ. No peaks are evident in the sample 2 diffractogram that do not overlap peaks in the patterns of FII or FI. The XRPD data suggest that sample 2 consists of a mixture containing FII as the major component and FI as the minor component. As with sample 1, the XRPD pattern of sample 2 contained no evidence of FIII. The ssNMR spectra of samples 1 and 2 are shown in Figure 3. Interpretation of the spectra was based on peak assignments for the solution carbon-13 NMR spectrum,8 as well as the fact that FI is known to have two crystallographically independent molecules in the crystal structure.3 The latter might result in doubling of ssNMR peaks that arise from a single atom. Resonances that occur in relatively clean areas of the spectrum are those for the methyl carbon atom adjacent to the carbonyl group (C18 at about 13-15 ppm), the olefinic carbon atom bearing a proton (C6 at about 118-120 ppm), and the carbonyl carbon atom (C17 at about 222-224 ppm). Sample 1 exhibited three ssNMR peaks of similar intensity in the methyl region (14.2, 14.4, and 14.8 ppm), three peaks of similar intensity in the olefinic region (118.5, 118.9, and 120.4 ppm), and two peaks in the carbonyl region (222.1 and 224.4 ppm), the downfield peak approximately half the intensity of the upfield peak. Since sample 1 contains mostly FI (by XRPD), and FI contains two crystallographically independent molecules, at most two peaks of approximately equal intensity should be observed for each atom. Instead, three peaks are observed for the methyl and olefinic atoms. Although a small amount of FII appears to be present by XRPD, comparison of relative peak intensities in the XRPD and ssNMR data sets suggests that a form other than FI and FII is present in sample 1. For example, in the XRPD pattern of FI the intensity ratio of the peaks at 8.0 and 20.4 °2θ is about 4, in the XRPD pattern of FII the intensity ratio of the peaks at 8.8 and 21.0 °2θ is about 0.1, and in the

New Polymorph of Dehydroepiandrosterone

Crystal Growth & Design, Vol. 6, No. 4, 2006 927

Figure 2. The XRPD patterns of samples 1 and 2 from Genelabs (top) and of forms FI and FII (bottom). The location of form FI peaks in sample 2 are shown by arrows.

Figure 3. The ssNMR spectra of prasterone samples 1 and 2 (* denotes spinning sideband).

XRPD pattern of sample 1 the ratio of the peaks at 8.1 and 20.4 °2θ is about 7 (see Table 4). These ratios indicate that only a small amount of FII is present. In the ssNMR spectrum of sample 1 the intensity ratio of the olefinic peaks at 120.4, 118.9, and 118.5 ppm is approximately 1:1.3:1.6. Thus, the material responsible for the third ssNMR peak, whichever one is not assignable to FI, appears to be present in much greater quantity than would be expected from the amount of FII which appears to be present based on the XRPD data. If a significant amount of FII were present the XRPD peak in the region of 20-21 °2θ would be expected to be much more intense. Sample 2 exhibited three peaks in the methyl region (12.9, 14.2, and 14.8 ppm), possibly four peaks in the olefinic region (118.5, 118.9, 119.9, and 120.4 ppm), and two peaks in the carbonyl region (222.1 and 224.0 ppm). Peak intensity evalu-

ations similar to those described above for sample 1 led to the supposition that a form other than FI and FII was also present in sample II. For each sample, the ssNMR data suggested the presence of more solid forms than did the XRPD data. To understand this apparent inconsistency, and to be able to elucidate the solid form composition of samples of prasterone provided in the future, we made samples of the known solid forms that had previously been isolated and measured their ssNMR spectra. The forms were produced based on the procedures reported by Chang,2 with the exception of S1. Chang obtained S1 by allowing dichloromethane solvent to evaporate from a solution under 50-60% relative humidity (RH);2 we obtained that form by grinding a methanol slurry of anhydrous forms using a mortar and pestle under ambient conditions. The forms produced were

928 Crystal Growth & Design, Vol. 6, No. 4, 2006

Stahly et al.

Table 4. Positions of Characteristic XRPD Peaksa FI 8.0 (53) 15.1 (88)b 15.5 (100)b 16.0 (30) 16.9 (16) 17.8 (73)b 18.2 (33)b 18.7 (46)b 19.2 (16) 19.5 (14) 20.4 (12) 24.8 (10) 25.1 (11) 26.9 (12) 29.5 (37)

FII

FVI

sample 1

sample 2

8.1 (2) 8.8 (5)

8.0 (17)

8.1 (33)

14.8 (98) 15.4 (38) 16.2 (15) 16.5 (22) 17.7 (100)

15.1 (100) 15.6 (61) 16.1 (18) 16.8 (12) 17.9 (59)

8.1 (9) 8.8 (6) 15.1 (33) 15.6 (93) 16.1 (100)

18.8 (23) 19.4 (29)

18.8 (13)

19.0 (59)

20.4 (5)

21.0 (32)

15.6 (88)b 16.1 (100)b

21.0 (35)b

17.9 (29)

25.1 (16) 27.1 (16)b 27.4 (10)

27.1 (18) 27.4 (10) 29.1 (22) 29.4 (18)

29.5 (33)

Figure 4. The XRPD pattern of form FVI.

29.6 (14)

a Positions are in °2θ; relative intensities are in parentheses. b Peaks denoted by Chang as characteristic.2

identified based on their XRPD patterns (Table 2). Attempts to make FIII and S3 involved desolvation and water-for-methanol exchange procedures, respectively, starting with S4. In each case, a mixture was obtained that consisted of FIII and either S3 or S4. The ssNMR spectrum of each form prepared was measured. On the basis of the data, the positions of the diagnostic peaks were determined for each form except S3. The results are shown in Table 3. Assignment of the ssNMR peaks for FI, FII, S1, S2, and S4 was straightforward. When S4 was kept under vacuum over phosphorus pentoxide for 3 days in an attempt to make FIII, conversion was incomplete and a mixture of S4 and FIII was obtained. The fact that S4 was one of the components (rather than S3, see below) was inferred from the water-free experimental conditions used. Comparison of the ssNMR spectrum of this mixture to the spectrum of pure S4 allowed assignment of the FIII peaks. FIII exhibited two peaks in the methyl and olefinic diagnostic regions, suggesting that the crystal structure of this form consists of two crystallographically independent molecules of prasterone. The fact that only one resonance was found in the carbonyl carbon atom region was attributed to the fortuitous coincidence of the carbonyl carbon signals from the two independent molecules. When S4 was kept under 58% relative humidity for 2 days in an attempt to make S3, a mixture of FIII and S3 or S4 was obtained. The similarity of the XRPD patterns of S3 and S4 made it difficult to determine if S3 or S4 were present and thus prevented unequivocal assignment of the ssNMR peaks of S3. However, S3 peak assignments are not critical to this study as the samples being investigated were all dried to remove water and solvent. Comparison of the ssNMR data of the known forms of prasterone (Table 3) with the data obtained from samples provided by Genelabs (Figure 3) showed that the samples exhibited peaks that were not accounted for by the known forms. In sample 1 peaks corresponding to FI (14.2, 14.8, 118.9, 120.4, 222.1, and 224.4 ppm) and a very small peak corresponding to FII (about 13 ppm) were identified. In sample 2, peaks corresponding to FI (14.2, 14.8, 118.9, 120.4, and 222.1 ppm) and FII (12.9, 119.9, and 224.0 ppm) were identified. However, each sample exhibited peaks in the methyl and olefinic regions that were assigned to a new polymorph of prasterone designated as form FVI. It was concluded that sample 1 consisted of a mixture of FI, FII, and FVI, and that sample 2 consisted of a mixture of FI, FII, and FVI. Note that peak intensities reinforce this conclusion. In the ssNMR spectrum of sample 1, the 13 ppm FII

Figure 5. The ssNMR spectrum of form FVI (* denotes spinning sideband).

peak is much weaker than any of the other methyl peaks, but the 118.5 FVI peak is stronger than any of the other olefinic peaks. Clearly, the peaks assigned to FVI cannot be accounted for by FII. Since ssNMR could be used to differentiate forms FI, FII, and FVI, Genelabs provided a series of samples to SSCI for determination of their polymorphic content using this technique. To our surprise, one such sample was found to be essentially pure form FVI. The XRPD pattern of FVI is shown in Figure 4. Table 4 contains listings of characteristic peaks from the XRPD patterns of FI, FII, FVI, and samples 1 and 2. Every FVI peak of significant intensity (relative intensity greater than 15) occurs within 0.4 °2θ of an FI peak. Consequently, visual inspection of standard, laboratory XRPD patterns is not a good method for differentiation of FI and FVI, and would be expected to be particularly ineffective for analyses of mixtures containing a low concentration of either. The lack of adequate specificity is clearly shown by comparison of the sample 1 and 2 peak positions to the FI and FVI peak positions (Table 4). This is why our initial XRPD analyses of samples 1 and 2 suggested that they contained only FI and an FI/FII mixture, respectively. The ssNMR spectrum of FVI (Figure 5) exhibits the signals assigned to this form based on analysis of the spectra of samples 1 and 2; a single methyl carbon resonance at 14.3 ppm and a single olefinic carbon resonance at 118.3 ppm. The carbonyl carbon atom resonates at 222.1 ppm, a position coincident with


Figure 6. The DSC trace of FVI.

the carbonyl signal of FI. This coincidence is the reason that the FVI carbonyl carbon resonance could not be observed in the spectra of the sample 1 and 2 mixtures. Form FVI is an anhydrous, nonsolvated polymorph of prasterone. Thermogravimetric analysis indicated no weight loss below about 200 °C. Differential scanning calorimetry measurements of FVI obtained at a heating rate of 20 °C/min provided the trace shown in Figure 6. The small endotherm at about 139 °C likely results from the conversion of FVI to FI, either by melt-recrystallization or solid-state transformation. The endothermic event at about 151 °C is likely the melting of FI. Form FVI is different from other known prasterone forms besides FI. Since it is anhydrous and nonsolvated it is not S1, S2, S3, or S4. It is not FIII based on comparison of XRPD data; diagnostic FIII peaks described by Chang (particularly those at 11.99 and 12.99 °2θ)2 are clearly not present in the FVI pattern. Finally, it does not appear to be FIV or FV based on melting points. To test the relative thermodynamic stability of form FVI, a mixture of solid forms FI, FII, and FVI in prasterone-saturated


ethyl acetate was agitated at ambient temperature for one week. The solids were removed by filtration, analyzed by ssNMR, and found to be FI. Thus, FI is more thermodynamically stable at ambient temperature than either FII or FVI. A similar experiment was conducted at ambient temperature in which the solvent was water containing 0.5% sodium lauryl sulfate, the latter to increase the solubility of prasterone. The solids remaining after only five minutes of agitation at ambient temperature were found to be hydrate S2. These results are consistent with Chang’s finding that the most stable anhydrate is FI and the most stable hydrate is S2. The sample of form FVI obtained from Genelabs has been kept in a closed jar under ambient conditions for five-and-a-half years without converting to FI. Therefore, FVI appears to be stable to transformation to the more stable form FI in the absence of a solvent. The sample of form FVI that we received consisted of small crystallites which were unsuitable for study by the single-crystal X-ray technique. Thus, the crystal structure of form FVI was elucidated from XRPD data. The first step was to download the known crystal structures of FI and FII from the Cambridge Crystallographic Database9 and refine the data with respect to the measured XRPD patterns of FI and FII using the Rietveld program MAUD (Figures 7 and 8).10 Each structure is defined by a chiral space group. The refined unit cell parameters, volumes, and densities are shown in Table 5. Table 5. Unit Cell Parameters for Forms FI and FII form I II

class

space group

a (Å)

b (Å)

c (Å)

β (°)

volume density (Å3) (g/cm3)

monoclinic P21 6.1819 44.261 6.241 107.148 1520 orthorhombic P212121 6.6154 11.376 21.937 90 1651

1.17 1.16

Packing diagrams of form FI are shown in Figure 9. FI contains two crystallographically independent molecules, with packing described by space group P21, giving four molecules in the complete unit cell. Hydrogen-bonding requirements cause the molecules to line up head-to-tail along the unique monoclinic 2-fold axis [010]. The hydrogen bond involving the carbonyl oxygen atom favors the 21 screw operation to fill space while satisfying hydrogen bonding angle requirements. The screw operation results in twisting of the molecules through 180° and

Figure 7. The form FI XRPD patterns measured (solid) and calculated from crystal structure data (dotted). The difference trace is shown at the bottom. Lattice parameters were refined to account for thermal expansion.


Stahly et al.

Figure 8. The form FII XRPD patterns measured (solid) and calculated from crystal structure data (dotted). The difference trace is shown at the bottom. Lattice parameters were refined to account for thermal expansion.

Figure 9. The packing diagrams of form FI, showing the [010],[001] plane (left) and the [100],[010] plane (right). In the left image the b axis is vertical and the c axis is horizontal. In the right image the b axis is vertical and the a axis is horizontal.

Figure 10. The packing diagram of form FII, showing the [010],[001] plane. The c axis is vertical and the b axis is horizontal.

continues the head-to-tail hydrogen bonding sequence with a translation of b/2. This gives one pair of head-to-tail molecules the same orientation of the methyl groups and the neighboring pair the opposite (180°) orientation of the methyl groups. This screw behavior continues along the b-axis. As the P21 space group has no further symmetry operations, aside from lattice translation, the molecules in the ac-plane all have the same orientation of the methyl groups. A packing diagram of form FII is shown in Figure 10. FII contains one crystallographically independent molecule, with packing described by space group P212121, giving four molecules in the complete unit cell. As in FI, the hydrogen bond involving the carbonyl oxygen atom favors the 21 screw operation. However, in FII the molecules are aligned along the

c-axis and, with only one molecule per unit cell, neighboring molecules along the [001] head-to-tail chain have their methyl groups oriented 180° with respect to each other. The next step in the solution of the FVI structure was to index its XRPD pattern. Indexing was carried out using proprietary software developed at SSCI, which provides a high rate of indexing success using data from common laboratory diffractometers.11 The method is based on a reverse Monte Carlo approach where unit cells are randomly generated within constraints derived from allowed molecular packing motifs. The constraints can be determined automatically based on space group packing rules or manually derived using methods such as those described below. At each iteration, the indexing program increases the number of molecules per asymmetric unit


until a statistically acceptable sampling of unit cells has been completed or until an optimal solution has been found. For each unit cell that satisfies the physical constraints, a powder pattern is calculated using the LeBail method, and its suitability is scored using a least sum of squares error estimation with respect to the measured XRPD pattern. Constraints on the indexing search space were derived as follows. The ssNMR spectrum of FVI does not exhibit the crystallographic splitting which is evident in the spectrum of FI, suggesting that FVI contains only one crystallographically independent molecule. On the basis of the structures of FI and FII, it seemed likely that FVI has at least one 21 screw symmetry operation along the long axis of the molecule. Since prasterone is chiral its packing will be described by a chiral space group. These structural features, coupled with consideration of the most common space groups describing organic crystals, limit the possible space groups to describe FVI to monoclinic P21 or orthorhombic P21P21P2 or P21P21P21. A P21 solution can be assumed to have a upper volume limit of 875 Å3, defined by the fact that there would be two molecules in the unit cell. Furthermore, because of the head-to-tail molecular packing, it is possible to give some limits to the expected unit cell parameters. For the P21 solution, the single molecule must be aligned along the monoclinic axis with the 21 screw giving two molecules having a head-to-tail orientation in the unit cell. Using the predictive rule that the lattice parameter x in a specific real space direction can be approximated by nL-3 < x < nL + 5, where L is the length of the molecule in the specific lattice direction and n is the number of molecules in the symmetric unit aligned along the same direction,12 then 19 < b < 27 Å. In the same way, the lattice parameters for a and c can be given realistic limits of 4 < a, c < 9 Å. Each orthorhombic solution (P21P21P2 or P21P21P21) would have four molecules in the unit cell. Using the same reasoning described above, the target volume is 1650 to 1750 Å3 and the unit cell lengths are 4 < a < 9 Å, 19 < b < 27 Å, and 5 < c < 14 Å. XRPD data obtained under standard conditions on a Shimadzu XRD-6000 diffractometer were indexed.13 An initial indexing pass using all 10 visible peaks below 20 °2θ combined with the eight free-standing peaks between 20 and 30 °2θ yielded no viable solutions, even with a relaxed 2θ error of 0.25°. A secondary indexing pass looking for only orthorhombic solutions used all 15 free-standing peaks below 30 °2θ with an allowed 2θ error of 0.21°. The best LeBail fit to the measured XRPD pattern was achieved by a P21212 unit cell with a ) 6.128 Å, b ) 11.953 Å, c ) 22.001 Å and a volume of 1612 Å3. The R factor for this fit was 0.15 with a normalized, weighted, chi-squared error of 5.2. A final indexing pass looking for only monoclinic solutions using the same default peak list described above identified a P21 solution with a unit cell having a ) 6.268 Å, b ) 21.931 Å, c ) 6.435 Å, β ) 107.745°, and a volume of 843 Å3. The R factor for this fit was 0.17 with a normalized, weighted, chisquared error of 5.3. Close inspection of the calculated LeBail patterns for the P21 and P21212 solutions with respect to the measured XRPD pattern shows that two overlapped peaks at 16.8 and 18.6 °2θ are not described by either solution. These unmatched peaks, which were included in the initial indexing search that failed, correspond to form FI peaks and thus can be associated with lowlevel contamination by FI. The final test of any indexing solution is the ability to pack the molecule into the chosen unit cell and approximate the


Figure 11. The packing diagrams of form FVI, showing the [010],[001] plane (left) and the [010],[101] plane (right). In the left image the b axis is vertical and the c axis is horizontal. In the right image the b axis is vertical, the a axis is horizontal to the left, and the c axis is horizontal to the right.

measured XRPD peak intensities. DASH14 was used to pack a rigid prasterone molecule in the two successful indexing solutions. No specific allowance was made for hydrogen-bond requirements during packing, and the carbon atom closest to the center of mass of the molecule was used as a center of rotation. The termination criteria for each packing iteration was either 5 × 105 steps or the profile error for the complete pattern was twice the profile error (∼25) of the Pawley refinement for the strongest free-standing peaks. The orthorhombic P21212 unit cell could not be packed with a rigid prasterone molecule to give an XRPD pattern that matched the measured pattern for Form VI. The best fit to the data gave a profile error of over 20 times the Pawley profile error with the resulting molecular packing having interlocking molecules centered on high symmetry points. The monoclinic P21 unit cell was successfully packed with the best fit giving a profile chi-squared error of 59.6 and an intensity chi-squared error of 46.2. The profile error is higher than the target of 50 because the sample was contaminated with low levels of form FI. The resulting molecular packing satisfies the asymmetric hydrogen bond requirement with sheets of prasterone molecules in the ac-plane aligned head-to-tail along the monoclinic axis and the methyl groups rotated 180° from one molecule to the next due to the 21 screw (Figure 11). The resulting crystal structure was loaded into the Rietveld program MAUD for final refinement of the molecule. Even in the presence of the FI contamination MAUD was able to refine the complete molecular structure of prasterone without breaking the molecule. The best model gave an R value of 0.1906 for the monoclinic P21 solution having a ) 6.261 Å, b ) 21.920 Å, c ) 6.432 Å, β ) 107.81°, and a volume of 860.4 Å3 (Figure 12). The fractional atomic coordinates are shown in Table 6.


Stahly et al.

Figure 12. The form FVI XRPD patterns measured (solid) and calculated (dotted) from prasterone packed into the indexed unit cell. The difference trace is shown at the bottom. Table 6. Fractional Atomic Coordinates of Form FVI atom

x

y

z

O1 O2 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13

0.3956 0.3948 0.5016 0.5386 0.3493 0.3300 0.3008 0.1319 0.0928 0.2994 0.3956 0.4764 0.5753 0.5086 0.4375

0.4079 0.8894 0.5761 0.5088 0.4705 0.4804 0.5470 0.5651 0.6293 0.6694 0.6576 0.5905 0.7040 0.7710 0.7791

0.9819 0.5167 0.0020 0.0625 0.9218 0.6828 0.6199 0.4531 0.3717 0.4651 0.7117 0.7606 0.8302 0.7725 0.5260

Conclusion The discovery of prasterone form FVI illustrates the importance of using multiple analytical techniques in polymorphism studies. In this case, ssNMR spectroscopy allowed differentiation of FVI from FI where XRPD did not. The similarity of the FI and FVI XRPD patterns indicates that their crystal structures are similar and that was found to be the case by solution of the structure from XRPD data. The solution was made possible by the combination of SSCI’s proprietary software for indexing of standard-quality laboratory XRPD patterns and CCDC’s DASH software. Note that details of sample preparation methods were and remain unavailable to the authors of this manuscript. We have not carried out the work necessary to define a process by which form FVI can be prepared.

Acknowledgment. The authors would like to thank Mr. James Fowler of SSCI for obtaining the DSC and TG data, Ms. Ann McKenzie for performing XRPD analyses on the Siemens diffractometer, Purdue University for the use of the ssNMR spectrometer and the Siemens diffractometer, and the Cambridge Crystallographic Data Centre for generously allowing us to use DASH. We are particularly indebted to Genelabs Technologies, Inc., who provided the prasterone samples. References (1) Kuhnert-Brandsta¨tter, M. Thermomicroscopy in the Analysis of Pharmaceuticals; Pergamon Press: Oxford, 1971. (2) Chang, L.-C.; Caira, C. M. R.; Guillory, J. K. J. Pharm. Sci. 1995, 84, 1169-1179. (3) Caira, M. R.; Guillory, J. K.; Chang, L.-C. J. Chem. Crystallogr. 1995, 25, 393-400. (4) Bhacca, N. S.; Fronczek, F. R.; Sygula, A. J. Chem. Crystallogr. 1996, 26, 483-487. (5) Cox, P. J.; MacManus, S. M.; Gibb, B. C.; Nowell, I. W.; Howie, R. A. Acta Cryst. 1990, C46, 334-336. (6) Frye, J. S.; Maciel, G. E. J. Magn. Reson. 1982, 48, 125-131. (7) Earl, W. L.; VanderHart, D. L. J. Magn. Reson. 1982, 48, 35-54. (8) Pouchert, C. J. The Aldrich Library of NMR Spectra; Aldrich Chemical Co.: Milwaukee, 1983; compound number 12, 578-4. (9) ZOYMOP (FI) and ZOYMOP01 FII. (10) http://www.ing.unitn.it/∼luttero/maud/. (11) Patents pending. (12) Gavezzotti, A. Acc. Chem. Res. 1994, 27, 309-314. (13) The pattern indexed is that shown in Figure 3, which was obtained from the sample of FVI provided by Genelabs. (14) DASH, version 2.2; Cambridge Crystallographic Database Centre: Cambridge, UK.

CG050479C

Discovery of a New Polymorph of Dehydroepiandrosterone

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