Contraction of

Publication Date (Web): April 14, 2010 ... Methanol forms a hydrogen bond with oxygen of the phosphonyl group of TDF (b) top view of TDF form B-MeOH: ...
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DOI: 10.1021/cg1000667

Characterization and Anisotropic Lattice Expansion/Contraction of Polymorphs of Tenofovir Disoproxil Fumarate

2010, Vol. 10 2314–2322

Eun Hee Lee,† Daniel T. Smith,† Phillip E. Fanwick,§ and Stephen R. Byrn*,† †

Department of Industrial and Physical Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, Indiana 47907, and §Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana 47907 Received January 18, 2010; Revised Manuscript Received March 30, 2010

ABSTRACT: Tenofovir disoproxil fumarate (TDF) is a nucleotide reverse transcriptase inhibitor that is used to treat HIV/ AIDS. According to the literature, TDF is known to have three polymorphic forms: I, A, and B. Crystals of all three polymorphs were grown following literature procedures and characterized in the solid state using optical microscopy, single crystal X-ray crystallography, powder X-ray diffraction, Fourier transform infrared spectroscopy, Raman, solid-state NMR, differential scanning calorimetry, thermogravimetric analysis, and organic vapor sorption isotherms. In the course of the analysis, we found that (1) the stoichiometric composition of TDF form A (TD/FA = 2:1) is different from that of TDF forms B and I (TD/FA = 1:1), and therefore TDF form A should not be regarded as a polymorph of TDF form I or TDF form B, (2) TDF forms B and I are actually desolvated forms, (3) anisotropic lattice contraction/expansion occurs between (i) hemimethanolate of TDF form B (TDF form B-MeOH) at low temperature and TDF form B-MeOH at room temperature, (ii) TDF form B and TDF form I, and (iii) TDF form I and TDF form I solvate. Crystal structure analysis was conducted to explore lattice expansion/contraction upon heating/cooling. We believe that the layered structure of TDF form B-MeOH is responsible for lattice expansion/ contraction as a function of temperature.

Introduction The crystal structure obtained by single crystal X-ray analysis is regarded as perhaps the most definitive characterization of a compound in the solid state. Information from single crystal X-ray analysis is critical for an analysis of structure-property relationships as well as for developing strategies for crystal engineering. The single crystal structure provides parameters that allow the calculation of the X-ray powder pattern. This calculated pattern is typically used as a reference to identify the polymorphic form and to determine the phase purity among polymorphic forms. However, a study of the effect of temperature on the lattice parameters by Stephenson et al. showed that the calculated powder X-ray diffraction (PXRD) pattern obtained at low temperature did not match well with the PXRD pattern obtained at room temperature.1 Single crystal X-ray analysis is commonly conducted at low temperature in order to increase the quality of the data and prevent desolvation/dehydration during analysis. In practice, experimental PXRD patterns are obtained at room temperature. The difference between calculated versus experimental PXRD patterns can be attributed to the temperature at which data are collected. The study by Stephenson reported that generally one or two lattice parameters expand and the other lattice parameter contracts. Thus, the overall unit cell volume decreases by an average of 0.978% at low temperature. It is believed that lattice parameters normally change anisotropically due to the lack of structural symmetry. However, there has been little study on structural aspect of the anisotropic lattice contraction/expansion of pharmaceutical compounds.

*Corresponding author. E-mail: [email protected]. Fax: (þ1) 765-494-6545. Tel: (þ1) 765-494-1460. pubs.acs.org/crystal

Published on Web 04/14/2010

In this work, we studied anisotropic lattice contraction/ expansion of hemimethanolate of tenofovir disoproxil fumarate (TDF) form B (TDF form B-MeOH) and investigated the crystal structure which enables the lattice contraction/expansion of TDF form B-MeOH and the transformation from TDF form B to TDF form I. Tenofovir disoproxil fumarate (TDF), the HIV/AIDS drug, was used as a model compound (Figure 1). In the course of an ongoing study within our laboratories, it became necessary to characterize the polymorphs of TDF. TDF is known to have three polymorphic forms (I, A, and B),2 and TDF form I is known to be unsolvated.3 However, solid-state characterization of TDF polymorphs demonstrated that TDF form A is not a polymorph of TDF form I or form B, and TDF forms I and B are actually desolvated solvates. Another interesting aspect of the system is that Fourier transform infrared (FT-IR) spectra of TDF form B is same as that of TDF form I, while solid-state NMR (SSNMR) spectra of the two forms are very different. Solid-state characterization of TDF forms A, B, and I was conducted by optical microscopy, single crystal X-ray crystallography, powder X-ray diffraction (PXRD), FT-IR, Raman, SSNMR, differential scanning calorimetry (DSC), and organic vapor sorption. Anisotropic lattice contraction/expansion of TDF form B-MeOH was investigated using thermogravimetric analysis (TGA), PXRD, and single crystal X-ray analysis. Materials and Methods Materials. TDF was kindly provided by the Clinton Healthcare Access Initiative (Boston, MA). Methanol and isopropyl alcohol were purchased from Sigma-Aldrich (St. Louis, MO). Water was double-distilled and filtered with a Milli-Q ultrapure water purification system (Billerica, MA). r 2010 American Chemical Society

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Figure 1. Molecular structure of tenofovir disoproxil fumarate (TDF) with atomic numbering. Powder X-ray Diffraction (PXRD). PXRD analysis was performed on TDF crystals using a Siemens X-ray diffractometer D 5000 (Bruker AXS Inc., Madison, WI) equipped with Cu KR radiation. Samples were analyzed over a 2θ range of 4-40° at the rate of 4°/min. Differential Scanning Calorimetry (DSC). A TA Instruments differential scanning calorimeter (model 991100.901) (TA Instruments, New Castle, DE) was used to investigate the thermal behavior of the TDF polymorphs. The temperature scale was calibrated by measuring the onset temperature of melting of an indium standard. The aluminum pans were hermetically sealed, and the heating rate was 10 °C/min from 40 to 165 °C. Data were acquired and analyzed using Universal Analysis - NT software. Thermogravimetric Analysis (TGA). The weight change of TDF solvates was measured using a TA Instruments thermogravimetric analysis system (TGA 2050 Thermogravimetric analyzer) (TA Instruments, New Castle, DE). A sample of less than 10 mg in weight was placed on the sample pan. The heating rate was 10 °C/ min from 40 to 150 °C. Data were acquired and analyzed using Universal Analysis - NT software. Organic Vapor Sorption Analysis (VTI). A symmetrical gravimetric analyzer (SGA-100) (VTI Corporation, Hialeah, FL) at 25 °C was used for gravimetric sorption analysis. Fifteen to twenty milligrams of TDF sample was used for analysis. Equilibrium criterion for the step was 0.01% w/w in 5 min with a maximum step time of 30 min. During the measurements, the sample was exposed to increasing relative pressure of methanol from 5 to 90% with a 5% increment in relative pressure. Fourier Transform Infrared Spectroscopy (FT-IR). FT-IR measurements of TDF forms A, B, and I were done using a Bio-Rad FTS 6000 spectrometer with a DTGS detector and KBr beam splitter (Varian, Inc., Palo Alto, CA). The scan range was set from 500 to 4000 cm-1 with 4 cm-1 resolution, and each spectrum was obtained by coadding 128 scans. A Specac ATR sample accessory with a diamond window was used for these measurements. Raman Spectroscopy. FT-Raman spectroscopy was conducted using a Perkin-Elmer Spectrum System 2000 (PerkinElmer, Inc., Shelton, CT) equipped with a diode pumped near IR Nd:YAG 1064 nm laser with a power of 1000 mW. The Raman spectra were measured at 1.0 cm-1 intervals with a spectral resolution of 4 cm-1. 128 accumulations were averaged to obtain spectra for each sample. Solid-State NMR (SSNMR). Spectra were obtained using a Chemagnetics CMX-400 spectrometer (Varian, Inc., Palo Alto, CA) equipped with three high-power RF channels and a sample temperature control unit. 13C cross-polarization with a double resonance magic angle spinning (MAS) probe was obtained at 100.6 MHz (400.3 MHz = 1H). Powder sample was placed inside a 4 mm rotor. The spin rate was 6.00 kHz. Spectra were collected using a 90° pulse (4.00 μs) with proton decoupling and a pulse delay of 3.0 s. X-ray Structure Determination. A TDF crystal was mounted on a fiber in a random orientation. Data collection was performed Cu KR radiation (λ = 1.54184 A˚) on a Rigaku Rapid II equipped with confocal optics. The data were collected at a temperature of 150(1) K. Cell constants for data collection were obtained from least-squares refinement. Refinement was performed on a LINUX PC using SHELX-97. Lorentz and polarization corrections were

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applied to the data. The remaining atoms were located in succeeding difference Fourier syntheses. Hydrogen atoms were included in the refinement but restrained to ride on the atom to which they are bonded. The structure was refined in full-matrix least-squares. Lattice Parameter Simulation Using TOPAS. The simulated data in Figure 15 was calculated using Topas (Bruker AXS v3.0, 1999, 2000). The crystal structure of TDF form B-MeOH at low temperature was used as the input structure. Using a Rietveld-like procedure, the crystal structure of TDF form B-MeOH was refined in order to achieve a good fit to the measured TDF form B-MeOH ambient powder pattern. The information content in the TDF form B-MeOH powder pattern is insufficient to attempt a full Rietveld structure refinement, so the initial refinement was limited to the unit cell lattice parameters. Refining only the unit cell lattice parameters is a reasonable approach in this specific situation as the measured TDF form B-MeOH powder pattern has a similar distribution of diffraction peak intensities as the powder pattern calculated from the TDF form B-MeOH crystal structure. The main difference between the powder patterns is a shift in the peak positions. With the lattice parameters optimally refined, there remained some peak intensity differences between the measured and simulated powder patterns. These intensity differences are most likely due to slightly different relative molecular positions and orientation between form B-MeOH at low temperature and form B-MeOH at room temperature. The intensity difference could be modeled using a fourth-order spherical harmonic. While the use of the spherical harmonic intensity modification blurs any structural difference between form B-MeOH at low temperature and form B-MeOH at room temperature, it does allow for a more precise determination of the unit cell lattice parameters shown in Table 3.

Results and Discussion Crystallization of Polymorphs. For solid-state characterization of TDF polymorphs, (1) TDF form I was used as received, (2) TDF form B was crystallized from methanol (MeOH), and (3) TDF form A was crystallized from isopropyl alcohol (IPA). TDF form A was prepared by dissolving 0.5 g of TDF form I in 5 mL of IPA at 50 °C. The solution was cooled from 50 to 4 °C (10 °C/h) and produced TDF form A. The resulting solids were filtered and dried in an oven at 40 °C. TDF form B-MeOH/TDF form B was prepared by dissolving 2.5 g of TDF form I in 15 mL of MeOH. The solution was slowly cooled from 50 to 4 °C (10 °C/h) and produced form B-MeOH. The form B-MeOH solids were filtered and used for TDF form B-MeOH analysis. TDF form B was obtained by drying TDF form B-MeOH in an oven at 40 °C. For single crystal analysis, TDF form A crystals were grown by dissolving 0.1 g of TDF form I in 10 mL of IPA at 50 °C and letting the resulting solution cool to room temperature. In the case of TDF form B-MeOH, 0.1 g of TDF form I was dissolved in 2 mL of methanol/water mixture (60/40, v/v %) at 40 °C and the solution was cooled in a refrigerator for 1 day, and left at room temperature for several months until TDF form B-MeOH crystals grew suitable for single crystal X-ray analysis. Solid-State Characterization of TDF polymorphs A, B, and I. Solid-state characterization of TDF polymorphs A, B, and I was conducted using optical microscopy, PXRD, FT-IR, Raman, and SSNMR spectroscopy, and DSC. Optical microscopy data showed three distinct morphologies for the TDF polymorphs (Figure 2). The morphology of form A was a rod, and that of form B was a square plate. However, the morphology of form I was irregular. Form B had a welldefined shape, but the crystals tended to be small in size and grew on top of each other making single crystal X-ray analysis impossible when methanol was used as a solvent. Therefore, TDF form B-MeOH was grown in the mixture of

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Figure 2. Micrographs of TDF forms I (left), A (middle), and B (right).

Figure 4. FT-IR spectra of three polymorphic forms of TDF showing that FT-IR spectra of forms I and B are exactly the same while form A exhibits the distinct FT-IR spectra. Figure 3. PXRD patterns of three polymorphic forms of TDF showing that TDF form A has distinct PXRD pattern while PXRD patterns of TDF forms B and I are similar.

methanol/water for single crystal X-ray analysis. TDF form B-MeOH crystals grown from the mixture was rectangularshaped crystals. The PXRD patterns of the three polymorphs are distinct as shown in Figure 3. However, the PXRD patterns of form B and form I are somewhat similar. Both forms exhibit a sharp peak at around 5° 2θ. In addition, both forms have three large peaks at high 2θ (around 20°, 25°, and 30° 2θ). The distinct difference between the forms is that those peaks at high 2θ of TDF form B are shifted toward high 2θ compared to those of TDF form I. This would indicate a smaller d-spacing for TDF form B than that of TDF form I. Figure 4 shows the FT-IR spectra of three TDF polymorphs. It is interesting to note that the FT-IR spectrum of TDF form I is same as that of TDF form B. The spectrum of TDF form A is different from those of forms I and B in that TDF form A has generally sharper peaks. Raman spectra of the three polymorphic forms of TDF are shown in Figure 5. Unlike the FT-IR spectra, the Raman spectrum of TDF form I is slightly different from that of TDF form B: (1) TDF form I has a peak at 1256 cm-1 but TDF form B has a peak at 1263 cm-1, (2) TDF form B has three distinct peaks around 1450 cm-1, however, TDF form I has two distinct peaks, one of which has a shoulder, and (3) in a region around 1520 cm-1, the peak shape of TDF form I is slightly different from that of TDF form B. As was seen in the FT-IR results, the Raman spectrum of TDF form A is distinct among the three polymorphs. As opposed to FT-IR spectra, SSNMR spectra of TDF forms, I and B are different. SSNMR is often used as a

Figure 5. Raman spectra of three polymorphic forms of TDF showing the difference in highlighted areas between TDF form I and TDF form B, while the FT-IR spectra of forms I and B are exactly the same.

complementary technique for the solid-state characterization of pharmaceutical compounds. The anisotropic nature of solids as compared to solution makes it useful to differentiate the polymorphs of the same compound by means of SSNMR. TDF form I has generally sharper peaks with more separation especially in the regions around 140-147 ppm, 117 ppm, 62 ppm, 23 ppm, and 15 ppm (boxed regions in Figure 6) than TDF form B. Similar to the FT-IR and the Raman spectra, TDF form A has generally sharper and wellseparated peaks as compared to TDF forms I and B. TDF forms B and I are the examples of the case where the FT-IR spectroscopic technique does not differentiate between

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Figure 6. SSNMR spectra of TDF forms A (top), B (middle), and I (bottom) showing distinct SSNMR spectra.

Figure 7. DSC thermogram of TDF forms A, B, and I showing that TDF form A has the highest melting point at 118.11 °C, followed by TDF form I at 113.77 °C, and TDF form B melts below 100 °C, recrystallizes, and remelts at 111.02 °C.

Figure 8. Organic vapor sorption isotherm of TDF form B as a function of relative pressure: approximately a half of methanol molecule absorbed on one molecule of TDF at 0.9 relative pressure.

polymorphs, whereas the SSNMR technique is able to differentiate between polymorphs. The DSC thermograms of the TDF polymorphs showed that TDF form A had the highest melting point at 118.11 °C, followed by TDF form I at 113.77 °C. TDF form B melted below 100 °C, recrystallized, and remelted at 111.02 °C (Figure 7). TDF form B when heated on a hot plate at 116 °C gave a PXRD pattern that was the same as that of form I indicating that TDF form B transformed to TDF form I at high temperature. During our characterization work, it was noted that crystals of TDF form B became transparent as soon as they were introduced to a solvent. During solvent removal and

drying, the crystals would undergo a transformation to an opaque white color. On the basis of these observations, it was suspected that TDF form B may be a desolvated solvate. Organic vapor sorption of TDF form B suggested that TDF form B can absorb approximately 0.5 mol of methanol per TDF molecule (the maximum absorption of MeOH was 2.317 w/w% at 0.9 relative pressure). One thing noticed here is that the absorption behavior of methanol on TDF form B solids is similar to that of nonstoichiometric hydrate (Figure 8). There was no stepwise increase in weight but rather a gradual weight gain as a function of relative pressure of methanol. This gradual weight gain indicates the

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Table 1. Crystallographic Data of TDF Form A and TDF Form B-MeOH from Single-Crystal X-ray Analysis parameter

TDF Form A

chemical formula

2(C19H30N5O10P) 3 C4H4O4

formula weight space group crystal system a (A˚) b (A˚) c (A˚) R (°) β (°) γ (°) V (A˚3) Z temperature R-factor (%)

1153.963 P21 monoclinic 9.7774 (2) 22.2104 (5) 12.5000 (3) 90.00 95.1689 (1) 90.00 2703.46 2 -123 °C 4.3

TDF Form B-MeOH C19H31N5O10P 3 C19H30N5O10P 3 C4H3O4 3 C4H4O4 3 CH4O 651.543 P21212 orthorhombic 18.438 (10) 34.057 (2) 9.907 (6) 90.00 90.00 90.00 6221.03 8 -123 °C 7.2

nonstoichiometric behavior of TDF and a further indication of the existence of a channel in the crystal structure. Structural Analysis of TDF Polymorphs A, B, and B-MeOH. 1. Single Crystal X-ray Analysis of TDF Form A and TDF Form B-MeOH. Crystallographic data for TDF form A and TDF form B-MeOH are summarized in Table 1. TDF form A crystallized in the monoclinic space group P21, having the unit cell parameters of a = 9.774(2) A˚, b = 22.2104(5) A˚, c = 12.5000(3), and β = 95.1689(1)°. Unit cell volume is 2703.46 A˚3. TDF form B-MeOH crystallized in the orthorhombic space group P21212, having the unit cell parameters of a = 18.438(10) A˚, b = 34.057(2) A˚, and c = 9.907(6) A˚. Unit cell volume of TDF form B-MeOH is 6221.03 A˚3. It is interesting to note that the stoichiometric composition of TDF form A is 2:1 (TD/FA) while, TDF form B-MeOH is 1:1:0.5 (TD/FA/methanol). Actually, TDF form A and form B-MeOH should not be regarded as polymorphs since polymorphs should have the “same” chemical composition but different crystal packing. Also, TDF form B-MeOH is a hemimethanolate with a 1:1 ratio of TD/fumaric acid (FA). Single crystal X-ray data for TDF form I is not available at this point due to the difficulty of growing crystals suitable for a single crystal X-ray analysis. TDF forms I and B can be regarded as at least pseudopolymorphs when they are solvated and polymorphs when they are desolvated. Crystal packing diagrams of TDF form A are shown in Figure 9. Each of two carboxylic acids of FA forms a hydrogen bond with one molecule of TD. One pair of two purine rings of TD and FA is aligned perpendicular to another pair forming a herringbone pattern. One end of carboxylic acid of FA forms a hydrogen bond with the 6-amino group attached to the purine base of TDF. The 6-amino group attached to the purine base of TDF also forms hydrogen bonds with the phosphonyl oxygen of TDF. The other end of carboxylic acid of FA does the same so that one molecule of FA has hydrogen bonds with two molecules of TD. Crystal packing diagrams of TDF form B-MeOH are shown in Figure 10. Fumaric acid and the purine base of TDF are connected by hydrogen bonds to form planes, which are parallel to the ac plane. Chains attached to the 6-amino purine moiety of TDF sit between the planes forming a lamellar-type layered structure. Top view, perpendicular to the b axis, of TDF form B-MeOH is shown in Figure 10b. Fumaric acid forms hydrogen bonds with both N7 of the 6-amino purine moiety of TDF and 6-amine

Figure 9. Crystal packing diagrams of TDF form A showing hydrogen bonds between fumaric acid (FA) and the purine base: (a) a carboxylic acid of FA forms hydrogen bonds with the 6-amino group attached to the purine base. The 6-amino group attached to the purine base of TDF also form hydrogen bonds with the phosphonyl oxygen of TDF and (b) one pair of two purine rings of TD and FA is aligned perpendicular to another pair forming a herringbone pattern.

attached to the 6-amino purine moiety of TDF and the N7 and N1 of 6-amino purine moiety of TDF forming zigzag patterns. Each carboxylic acid of FA is involved in two different types of hydrogen bonds with TD so that one molecule of FA requires one molecule of TD. As shown in Figure 10b, the (010) faces form planes consisting of a hydrogen bond network. Two crystallographically independent molecules constitute the building block of the crystal structure of TDF form B-MeOH. Figure 11a shows that the torsion angle between O15-C16 and P1-O23 bonds of (a) TDF is 135.48°, and the two chains are tilted with a similar angle with respect to the purine base of the TDF. A torsion angle between O150 -C160 and P10 -O230 bonds of (b) TDF is 77.15°. One of the two side chains is nearly parallel to the purine base, while the other is almost perpendicular to the purine base. This TDF bonds to methanol by a hydrogen bond. TDF molecules with these two conformations form the building block for the crystal. Two side chains are sandwiched between the purine bases which are hydrogen-bonded to FA. The sandwiched structure forms one lamella and the distance between lamellae is approximately 3.2 A˚. 2. Thermogravimetric Analysis (TGA) of TDF Form B-MeOH. In addition to organic vapor sorption isotherm analysis of TDF form B, weight loss of TDF form B-MeOH was conducted by TGA ( Figure 12). Overall weight loss was

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Figure 10. Crystal packing diagrams of TDF form B-MeOH: (a) FA and the purine base of TDF connected by hydrogen bonds form planes which are parallel to the ac plane. Chains attached to the purine base of TDF are sandwiched between the planes forming lamellar-type layered structure. Methanol forms a hydrogen bond with oxygen of phosphonyl group of TDF (b) top view of TDF form B-MeOH: FA forms hydrogen bonds with both N7 of the purine base of TDF and the 6-amine attached to the purine base of TDF and N7 and N1 of the purine base of TDF forming zigzag patterns.

1.85%, which corresponds to a stoichiometry of approximately 1:0.5 (TDF/MeOH), and the gradual removal of methanol as the temperature increases indicates the presence of channels in the crystal structure of TDF form B-MeOH. Single crystal X-ray also suggests the 0.5 mol of methanol per 1 mol of TDF in the crystal structure. However, on the basis of the single crystal X-ray analysis, methanol forms hydrogen bonds with phosphonyl oxygen of TDF molecules in the crystal structure. The result obtained by single crystal X-ray analysis contradicts the formation of channel suggested by the result based on VTI and TGA analysis. However, it needs to be pointed out that unlike single crystal structure analysis, VTI and TGA experiments were conducted at room temperature and the elevated temperature, respectively. 3. Anisotropic Lattice Contraction/Expansion of TDF Form B-MeOH. PXRD patterns of calculated TDF form A and TDF form A powder obtained experimentally at room temperature are shown in Figure 13. Calculated PXRD patterns were based on the single crystal X-ray data obtained at -123 °C. There was no significant difference in peak

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positions as well as intensities of peaks showing no significant changes in lattice parameters with respect to temperature. PXRD patterns of calculated TDF form B-MeOH at low temperature and TDF form B-MeOH powder, TDF form B powder, TDF form I powder, and TDF form I soaked with IPA (TDF form I-IPA) obtained experimentally at room temperature are shown in Figure 14. Calculated TDF form B-MeOH was obtained using Mercury 2.2. Preferred orientation on the (020) face was applied with a March-Dollase parameter of 0.4. It is apparent that the PXRD pattern of TDF form BMeOH calculated from single crystal X-ray analysis is different from the PXRD pattern of the powder obtained at room temperature. The intensities of the PXRD patterns of TDF form B-MeOH and TDF form I are generally not strong except for peaks at 2θ = 5.20, 20.88, 26.16, and 31.52. These strong peaks show shifts toward low 2θ from the PXRD pattern at the bottom to the PXRD pattern at the top (Figure 14). Shifts of the first peaks are small but apparent, and the differences between the strong peaks become larger as 2θ increases. It is also interesting to note that the second peak which is much smaller than the first peak does not show significant shifts among PXRD patterns shown in Figure 14. On the basis of a single crystal X-ray analysis, the peaks shifted toward low 2θ were indexed as the (020), (060), (080), and (0100) faces of TDF form B-MeOH. The peak shifts at a specific lattice parameter indicates the anisotropic lattice contraction (along the b axis in our case). As shown in Figure 9, TDF form B-MeOH forms a lamellar-type layered structure along the b axis. Interestingly, single crystal X-ray analysis was done at -123 °C, which is a very low temperature when compared to room temperature at which the PXRD experiment was conducted. Therefore, the difference between the calculated PXRD pattern and experimentally obtained PXRD pattern can be attributed to the anisotropic lattice contraction. The lattice contraction along the b axis is expected at low temperature. However, it should be pointed out that the PXRD pattern of TDF form B-MeOH and TDF form B at room temperature are similar in the sense that the peaks, which show significant shifts among various forms of TDF, do not move significantly. Therefore, we can conclude that the desolvation process of TDF form B-MeOH to TDF form B did not induce a significant lattice contraction/expansion along the b axis in spite of the flexibility of the lattice. Only thermal changes induced anisotropic lattice contraction/expansion of TDF form B-MeOH. As stated previously, the DSC thermogram of TDF polymorphs (Figure 7) showed that TDF form B underwent a solid-state polymorphic transformation to TDF form I when TDF form B was heated on a hot plate at 116 °C. In Figure 14, the similarity of PXRD patterns between TDF form I and TDF form B is clear: four large peaks appeared at similar 2θ except slight changes in peak position of these four peaks: 5.12, 20.44, 25.60, and 30.84° 2θ for TDF form B at room temperature and 5.04, 20.08, 25.12, and 30.24° 2 θ for TDF form I (Table 2). Also, TDF form I-IPA shows further shifts toward low 2θ (4.84, 19.40, 24.28, 29.24° 2θ for TDF form I-IPA) indicating the flexibility of cell parameters. However, it is noticed that unlike TDF form B-MeOH/TDF form B, the lattice expansion/contraction of TDF form I-IPA/TDF form I occurs during solvation/desolvation process. This phenomenon related to TDF form I will be published in detail elsewhere.

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Figure 11. ORTEP drawings of two crystallographically independent molecules of TDF form B-MeOH: (a) a torsion angle between O15-C16 and P1-O23 bonds of the other TDF is 135.48°, and two chains has tilted with a similar angle with respect to the purine base of the TDF, and (b) a torsion angle between O150 -C160 and P10 -O230 bonds of the TDF is 77.15°. One of the two side chains is nearly parallel to the purine base, while the other is almost perpendicular to the purine base. TDF bonds to methanol by a hydrogen bond.

Figure 12. TGA trace of TDF form B-MeOH: overall weight loss was 1.85%, which corresponds to a stoichiometry of approximately 1:0.5 (TDF/MeOH), and the gradual removal of methanol, as the temperature increases, indicates the presence of channels in the crystal structure of TDF form B-MeOH.

Figure 13. PXRD patterns of calculated TDF form A (blue, bottom), TDF form A powder obtained at room temperature (red, top) showing no significant difference between PXRD pattern obtained at low temperature and PXRD pattern obtained at room temperature.

Figure 14. PXRD patterns of calculated TDF form B-MeOH at -123 °C (blue), TDF form B-MeOH powder obtained at room temperature (red), TDF form B at room temperature (green), TDF form I (pink), and TDF form I-IPA (purple) showing nearly identical peaks for TDF form B-MeOH and TDF form B while shifts of peaks among other samples in boxed areas.

Lattice parameters of TDF form B-MeOH at room temperature were obtained by both simulation using TOPAS and single crystal X-ray analysis and were essentially the same obtained by the both methods (Table 3). Lattice expansion along the b axis was significant (2.45%), while lattice expansions along the a and c axes were negligible (0.62% and 0.15%, respectively). Lattice expansion along the b axis was expected because peaks indexed as (020), (060), (080), etc. in PXRD pattern were shifted toward low 2θ (Figure 14). Single crystal X-ray analysis of TDF form B-MeOH at room temperature was attempted but we were unable to assign all atoms crystallographically. Two distinctly different void spaces were identified and are shown in yellow color in Figure 16: one interacting with phosphonyl moiety and the other on the (020) faces. The void space next to phosphonyl moiety was also observed at low temperature (Figure 9). However, the large void space on the (020) face

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Table 2. Summary of 2θ of Four Large Peaks of TDF Form B-MeOH at Low Temperature and TDF form B-MeOH, TDF Form B, TDF Form I and TDF Form I-IPA at Room Temperature Showing Shifts of Peaks toward Low 2θ from Left Column to the Right Column TDF form B-MeOH (at -123 °C)

TDF form B-MeOH (at room temperature)

TDF form B (at room temperature)

TDF form I (at room temperature)

TDF form I-IPA (at room temperature)

5.20 20.88 26.16 31.52

5.12 20.48 25.68 30.92

5.08 20.44 25.60 30.84

5.04 20.08 25.12 30.24

4.84 19.40 24.28 29.24

Table 3. Lattice Parameters of TDF Form B-MeOH Obtained from Single Crystal X-ray Analysis and TDF Forms B-MeOH at Room Temperature Using TOPAS and Single Crystal X-ray Analysis: Changes in Lattice Parameters Are Shown in Parentheses parameter a (A˚) b (A˚) c (A˚) cell volume crystal system

TDF form B-MeOH (at -123 °C)

TDF form B-MeOH (at room temperature)

18.4381 34.057 9.9074 6221.32 orthorhombic

18.5518 (0.62%) 34.8910 (2.45%) 9.9224 (0.15%) 6422.68 (3.23%) orthorhombic

Figure 16. Crystal packing diagram of TDF form B-MeOH at room temperature obtained by single crystal X-ray analysis showing the void space in which anything can be fitted.

(Figure 11b) and the hydrogen bond between methanol and TDF. The intramolecular hydrogen bond forms between O15 and O18 (or O23 and O28) of TDF (Figure 1). The other of the two side chains of TDF (Figure 11a) does not have any intra- or intermolecular interactions. Also, the layered lamella structures are stacked along the b axis without any interactions. Therefore, we believe that the layered lamella structure of TDF form B-MeOH gives the crystal structure the flexibility, prone to the lattice expansion/contraction along the b axis as a function of temperature. Figure 15. Simulated PXRD of TDF form B-MeOH at room temperature (bottom) and experimentally obtained PXRD patterns of TDF form B-MeOH (middle) and TDF form B (top) showing good matches between experimental and simulated PXRD patterns.

was only identified at room temperature. At this point, we did not conduct the molecular simulation to obtain the crystal structure of TDF form B-MeOH at room temperature because the crystal structure at room temperature appeared to be disordered and thus we do not believe that the simulation will give any additional information. Some inorganic or hybrid materials are known to have a layered structure.4 The layered structure allows guest molecules to intercalate between layered spaces without altering the crystal structure of a host compound. The guest molecules can be water, organic materials, ionic materials, or solvent. Recent studies showed that a pharmaceutical compound, compound 1, can expand as much as 20% when the compound is exposed to 90% relative humidity.5 TDF form B-MeOH also showed a layered structure. As mentioned in discussion of TDF form B-MeOH crystal structure, FA and the purine base of TDF connected by hydrogen bonds form planes which are parallel to the ac plane. The hydrogen bonds may prevent the lattice expansion/contraction upon heating/cooling along the ac plane. However, there is no strong bond along the b axis, except only one intramolecular hydrogen bond of one of the two side chains of TDF

Conclusions The solid-state characterization of known TDF polymorphic forms A, B, and I, was conducted. We found that (1) the stoichiometric composition of TDF form A (TD/ FA = 2:1) is different from that of TDF forms B and I (TD/FA = 1:1) and therefore, TDF form A should not be regarded as a polymorph of TDF form I or TDF form B, (2) TDF forms B and I are actually desolvated forms, (3) anisotropic lattice contraction/expansion occurs between (i) TDF form B-MeOH at low temperature and TDF form B-MeOH at room temperature, (ii) TDF form B and TDF form I, and (iii) TDF form I and TDF form I-IPA. Crystal structure analysis revealed that TDF form B-MeOH forms the layered lamella structure at low temperature and the large void spaces within the crystal structure at room temperature. We believe that the layered lamella structure of TDF form B-MeOH is responsible for the anisotropic lattice expansion/contraction as a function of temperature. The result from this study highlights the importance of a full solid state characterization for each step of a drug’s development. Acknowledgment. We would like to thank Dr. Simon Bates (SSCI, an Aptuit Company, 3065 Kent Avenue, West Lafayette) for simulation studies and Dr. Carl Murphy (Purdue University, Department of Chemistry) for assistance with SSNMR studies.

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Crystal Growth & Design, Vol. 10, No. 5, 2010

Financial support for this project was provided by the Clinton Healthcare Access Initiative (Boston, MA).

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Lee et al. CODEN: PIXXD2 WO 2007013086 A1 20070201 CAN 146: 190527:115705 CAPLUS. (3) Munger, J. D., Jr., Alviso; Rohloff, J. C., Mountain View; Schultze, L. M., San Carlos, all of Calif. Assignee, Gilead Sciences, Inc.: Foster City, CA, Patent No: 5,935,946, 1999. (4) Megaw, H. D. Proc. R. Soc. A 1993, 142, 198–214. (5) Kiang, Y. H; Xu, W.; Stephens, P. W; Ball, R. G.; Yasuda, N. Cryst. Growth Des. 2009, 9, 1833–1843.