A Combined Solid-State NMR and X-ray Powder Diffraction Study of a

Jul 27, 2005 - Beverly, Massachusetts 01915. Yuegang Zhang, Peter L. Lee, and Robert B. Von Dreele. Advanced Photon Source (APS), Argonne National ...
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A Combined Solid-State NMR and X-ray Powder Diffraction Study of a Stable Polymorph of Paclitaxel James K. Harper, Dewey H. Barich, Elizabeth M. Heider, and David M. Grant* Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112

CRYSTAL GROWTH & DESIGN 2005 VOL. 5, NO. 5 1737-1742

Roland R. Franke and James H. Johnson Natural Pharmaceuticals, Inc. (NPI), 100 Cummings Center, Suite 414G, Beverly, Massachusetts 01915

Yuegang Zhang, Peter L. Lee, and Robert B. Von Dreele Advanced Photon Source (APS), Argonne National Laboratory, Argonne, Illinois 60439

Brian Scott, Darrick Williams, and Gerald B. Ansell Los Alamos National Laboratory (LANL), Los Alamos, New Mexico 87545 Received September 10, 2004

ABSTRACT: Solid-state NMR (SSNMR) and X-ray powder diffraction (XRPD) allow a study of a novel and stable polymorph of paclitaxel (Taxol) with two molecules per asymmetric unit (Z′) in the P212121 space group. The asymmetric unit volume is 2167 Å3, about four times larger than that previously characterized in combined XRPD/ SSNMR studies. The method, employing SSNMR constraints, allows the XRPD Rietveld analysis to establish many of the lattice details that otherwise would be unavailable. NMR structural constraints are provided by isotropic shifts and three-dimensional (3D) chemical shift tensors (CST), which are determined by ab initio quantum mechanical calculations. CST data give highly sensitive information on short-range structural features such as intra-atomic distances (particularly for proton positions that are undetermined with XRPD methods) and short-range valence angles that exhibit relatively poor sensitivity in reasonably large microcrystalline powders. Conversely, space group symmetry, unit cell volumes, long-range cell dimensions, and dihedral angles of extended chains are estimated with XRPD measurements. Corroboration of many structural parameters by combined quantum mechanical, SSNMR, and XRPD results indicate the efficacy of these combined approaches in relatively sizable microcrystalline powders. The population of the asymmetric unit, Z′ ) 2 is clearly observed even in the one-dimensional isotropic 13C spectra, which also confirmed the stability of the polymorph over a three-year period. This structural determination depends specifically on the agreement between previous SSNMR CSTs and single crystal results for baccatin, the rigid part of paclitaxel. Hence, CST data provide a reasonable initial model for the early iterative steps of a Rietveld analysis of XRPD data for a new polymorph of Taxol. I. Introduction Structural elucidation of polymorphs, while significantly important to the drug design, is inhibited in the absence of single-crystal data. In some instances, the combined use of solid-state NMR (SSNMR), ab initio quantum mechanically computed structures, and synchrotron X-ray powder diffraction (XRPD) data provide high-quality crystallographic lattice and structural parameters. Unfortunately, these methods break down in overly abundant local energetic minima. This is especially true for large or flexible molecules in which computational explorations are impaired because of the large number of local minima. 13C and/or 15N SSNMR chemical shift tensors (CST) recently have supplied structural constraints that reduce the conformational degrees of freedom and furnish reasonable initial XRPD structures of polymorphs and other unobtainable single crystals. In turn, the XRPD results provide synergistic long-range lattice parameters needed to optimize the molecular structure, to corroborate the CST data, and to assist the NMR calculation of long-range electrostatic lattice fields.

The merging of SSNMR and XRPD methods has been reported by others for moderately sizable asymmetric unit volumes (Vasym ) Vunit/Z), where Z is the number of asymmetric cells within the unit cell volume, Vunit. Thus, Vasym are calculated in anhydrous theophylline1 as 798.56/4 ) 199.64 Å3; in cimetidine2 as 1281/4 ) 320.38 Å3; in N-(p-tolyl)-dodecyl-sulfon-amide3 as 2026.1/4 ) 506.53 Å3; and in ambuic acid4 as 907.79/2 ) 453.90 Å3. In each of these cases, only one molecule per asymmetric unit (Z′ ) 1) is observed. A recent quantum mechanical and XRPD study of the 1:1 inclusion complex of β-cyclo-dextrine and mefenamic acid5 with an asymmetric unit volume of 3637.7/2 ) 1818 Å3 is larger than the present SSNMR and XRPD study. However, β-cyclo-dextrine is a moderately rigid cage into which the mefenamic acid is insertedsthe complexity of this inclusion complex is reduced from the other similar molecules. The paclitaxel polymorph, described in this report, is a relatively large molecule important in chemistry, biology, and medicine. This new polymorph contains only trace impurities (likely H2O) and exhibits remark-

10.1021/cg030074x CCC: $30.25 © 2005 American Chemical Society Published on Web 07/27/2005

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Figure 1. Paclitaxel and baccatin molecular numbering schemes. In baccatin, the H atoms occupy positions of sidechain segments highlighted for paclitaxel. Positions 21 through 24, respectively, are the C21, C21-ipso, and two carbons in the acetyl group (AcO) attached to C4.

able stability. The special significance of this work arises in part from the relatively large asymmetric volume found in paclitaxel (i.e., 8659.81/4 ) 2167.73 Å3). This study is made difficult because of the large number of conformational degrees of freedom, but the Z′ ) 2 value complicates the powder analysis. Previously, a singlecrystal polymorph of paxlitaxel, which includes solvent, was reported6 (see below). Recent work in pharmaceutical formulation has emphasized polymorphism. Unique polymorphs have been recognized as promising structures that deserve the legal protection of a patent.7 Hence, the ability to discover and to verify new forms has assumed greater significance in light of recent efforts in developing polymorphs, e.g., Zoloft.8 TransForm Pharmaceuticals conducted over 6200 crystallization trials, resulting in the discovery of one structural form and several salts of Zoloft. Similarly, pharmaceuticals such as Zantac,9 Norvasc,10 and others11 have faced competition from companies supplying new polymorphs. Because approximately 90% of all pharmaceuticals are marketed as solids,10 the conformation of the polymorphic structure often is of central importance to this industry, and SSNMR methods greatly augment diffraction data especially when a single crystal is unavailable. The stable paclitaxel polymorph12 is unusually well suited for this study because of its medicinal importance, purity, size, and otherwise unavailable structure. Since the initial discovery of paclitaxel, an anticancer drug (see Figure 1) from the Pacific Yew in 1971,13 drug trials14 have demonstrated it to be “one of the most significant advances in cancer therapy.”15 The limited botanical supply of paclitaxel has been alleviated by semi-synthetic methods.16 Paclitaxel is used to treat ovarian, breast, and lung cancers and AIDS-related Kaposi’s sarcoma. Additional applications are under investigation. Presently, the only reported single-crystal structure of paclitaxel6 contains several solvent molecules (i.e., dioxane and water) per unit cell, making it susceptible to solvent loss and subsequently its decomposition. This likely affects the stability of this well-characterized polymorph. Indeed, it was noted that the single-crystal suffered, “signs of decomposition with time...most likely due to...solvent loss”.6 Thus, the stability of a molecule and its various solid polymorphs is vital for storage and

Figure 2. MAS 13C-spectra of the C-O region in paclitaxel. The sample illustrates stability over a three-year period, i.e., A (2004) and B (2001). The doubling of observed lines is due to two molecules in the asymmetric unit. Thus, the nine positions yield 13 peaks with five additional peaks obscured by overlap. The small shoulders with asterisks (*) at 81 and 84 ppm suggest the presence of minor polymorphic impurities.19

maintenance of purity. Polymorphs, which are substantially solvent-free, appear to be more stable. When properties such as color, solubility, and shelf life are dependent upon lattice arrangements, polymorphic characterizations with SSNMR and XRPD methods are beneficial. These techniques are feasible for microcrystalline powders despite the larger size of the asymmetric unit. This precludes the need for crystals of sufficient size necessary for single-crystal methods when such are unavailable. A highly stable polymorph of paclitaxel, prepared by Natural Pharmaceuticals, Inc., was available for this study.12 This sample has less than 0.42% water, which is less than 0.4 water molecules for the asymmetric unit of two paclitaxel molecules. This noninteger ratio suggests that the water may be absorbed on the microcrystalline surfaces and/or in some of the interstitial sites in the powder. Minor polymorphic impurities also may be present (see caption of Figure 2 below). SSNMR confirms the absence of organic solvents. When the emphasis shifts to sample preparation and long-term stability, the methods, discussed herein, for structural identification of various polymorphs take on even greater importance to the pharmaceutical industry.

SSNMR and XRPD of a Stable Polymorph of Paclitaxel

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II. SSNMR Analysis of the Stable Paclitaxel Polymorph SSNMR studies fall into two types: one dimensional (1D) magic angle spinning (MAS) spectra and CST data, which may be obtained using the FIREMAT method.17 MAS spectra provide only the isotropic chemical shift terms, but this is sufficient to determine Z′ and the sample’s purity. In Figure 2, the 1D isotropic MAS 13C-spectra18 of the C-O region taken over a three-year period demonstrate that the lattice is very stable with no detectible chemical shift or spectral changes over an extended time. This relatively reactive region would be among the first to show chemical decomposition. The narrow lines are indicative of a microcrystalline powder sample. Doubling of lines clearly establishes Z′ ) 2, notwithstanding the substantial signal overlap observed for some pairs of lines in Figure 2. Further, the absence of solvent lines in Figure 2 establishes that the polymorph differs from the single-crystal structure that includes dioxane and water. In contrast, SSNMR CST methods give detailed threedimensional (3D) information for each molecular position in solid samples even though only microcrystalline powder samples (not suitable for single-crystal study) are available. CST19 from SSNMR methods establish constraints for direct bond distances, valence, and some of the dihedral angles; identify mixtures of polymorphs; verify sample crystallinity; and provide the number (i.e., Z′) of molecules per asymmetric unit. All of this information is useful in processing XRPD data. Computational modeling alone is unsuitable for structural analysis of paclitaxel due to the large number of iso-energetic conformations that are present in a molecule of this size.20 CST data provide experimental information that greatly reduces the number of conformations to be considered in the structural permutations of a good theoretical model. When Z′ ) 2, pairs of chemically equivalent carbon atoms (termed congruent molecules, atoms, or nuclei) place the molecules in different lattice environments. This creates a number of problems for both the XRPD and the SSNMR methods. Such complexity increases the difficulty of structural determination by standard X-ray powder methods, as one must have a docking model to locate the two structures within the asymmetric unit. Two molecules per asymmetric unit also complicate the assignment of congruent CSTs. While it is a relatively simple matter to identify the pairs of congruent nuclei for the two molecules in an asymmetric unit, the ad hoc method for designating typical shift tensors to a specific A or B molecule makes the process much more difficult without additional independent data. In some cases, the variations between the congruent sets of shift components is either accidentally or nearly degenerate as seen in Figure 3 for major portions of the two baccatin moieties in Taxol. Variations in the Baccatin Moieties of Taxol. Highly similar types of carbon atoms typically are found only in the baccatin or phenyl moieties in paclitaxel, indicating that pairs of congruent carbon atoms in the asymmetric unit have similar short-range structures. Sufficiently small differences, fortunately, leave the assignment of congruent nuclear resonances irrelevant

Figure 3. The correlation of Taxol and baccatin CST Components are given above as ppm. The attachment carbons (C10 and C13) and their immediate neighbors (C9, C11, C12, and C14) show side chain substituent shifts not attributable directly to the basic structure of the baccatin molecule.

(see filled circles, b, in Figure 3) because the two structures indicate the same structures. The two sets of congruent CST from C1-C8 and C15-C24 yield a root-mean-square difference (rms ∆TT′) between the two Taxol moieties that is 1.49 ppm. The root-mean-square difference (rms ∆BT) between the 13C CST in the parent baccatin and corresponding carbons in both moieties of Taxol is 2.78 ppm. This confirms our previous study21 that the baccatin structure is rigid and essentially invariable for the two congruent baccatin moieties in the asymmetric unit of paclitaxel. The positions C9C14 designated by open circles (see O in Figure 3) are near the side chain attachment sites in the rigid baccatin structure. These perturbed carbons, which differ appreciably, reflect steric and inductive effects from the side chains. The statistical data for the open circles in Figure 3 indicate that the side chains give rise to differences between congruent baccatin moieties in Taxol, primarily in the cyclohexene ring of baccatin where the side chains attach. These differences between baccatin moieties are clearly measurable with the rms ∆TT′ ) 3.61 ppm for the C9 to C14 set of shifts, a value that is larger than the experimental uncertainty by a factor of about 2-fold. On the other hand, variation in the C9 to C14 baccatin tensor components relative to the parent molecule (shown on the x-axis in Figure 3) is given by rms ∆BT ) 7.77 ppm. The best explanation for this relatively large scatter attributes these variations in the baccatin moiety to inductive and/or steric perturbations of the shift tensor components by the side chains. This supposition is preferable to suggesting differences in the rigid baccatin structure of paclitaxel.21 Variations in the Side Chains of Paclitaxel. While this portion of paclitaxel is relatively independent of any shifts in the baccatin parent molecule, the Z′ ) 2 allows us to compare the shifts between the two congruous sets of paclitaxel side chains. Such information indicates where the conformational structure varies the most. These deviations include torsional, steric, and electrostatic perturbations of the CST. We first compare

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the carbons of the two congruous acetyl side chains of paclitaxel attached at the C10 oxygen (see Figure 1). The value of the rms ∆TT′ ) 1.33 ppm for the acetyl carbons (a methyl and a carboxyl carbon) is less than the rms ∆TT′ ) 1.49 ppm for the filled circles in Figure 3 but only barely larger than the experimental uncertainties. Thus, the structures of both C10 acetyl groups, relative to the attached baccatin system, must be essentially identical. Considerable variation is noted in the rms ∆TT′ ) 7.54 ppm for the 13C and 15N atoms in the principal side chain containing the amide group. This variation between the two conformational isomers in the larger side chain is likely the most interesting feature in paclitaxel as these differences indicate major conformational changes in the structure. Such variations for the congruent 13C and 15N CST involve N, C1′, C2′, C3′ and the ipso carbons attached to C3′ and C4′. The CST values for C3′ and C4′ are somewhat tentative because these resonances broaden by the adjacent 14N quadrupolar nucleus. Work is underway to resolve the diversity that we find in both XRPD and SSNMR results. While most of the structural features in these side chains are comparable, a few differences remain that require better refinement in several of the carbons of the side chains. The serious CST problem in assigning two or more pairs of congruent atoms to either the A or B molecules continues to exist. These uncertainties cannot be resolved until we have better diffraction data, on which the NMR method depends. The XRPD treatment of the side chains has so far failed to provide a satisfactory structural analysis. Six aromatic benzenoid rings exist within the Z′ ) 2 asymmetric unit. The ipso carbons are resolvable and assignable, but the remaining ortho, meta, and para carbon atoms overlap greatly. Thus, it is presently impossible to characterize these corresponding CST. Further, CST data indicate that the phenyl groups are moving and coalescing into broader peaks. Line broadening in SSNMR with temperature variations and thermal motion parameters in XRPD data indicate motion in these aromatic carbons. It is likely due to librations of ortho and meta carbons in some phenyl groups. The effect is minimal for ipso and para carbon positions that lie along the liberation axis. Fortunately, the phenyl rings are rigid, and typical aromatic structural parameters constrain the structure of these groups in Taxol. III. Preliminary X-ray Studies of Paclitaxel XRPD Analysis. Powder methods determine accurately the unit cell of the space group and many longrange lattice parameters. Some of the XRPD molecular parameters become apparent after applying the SSNMR constraints. Before imposing these NMR constraints upon the paclitaxel asymmetric unit, the XRPD analysis was unable to differentiate among several similar symmetry point groups. This improves the XRPD fitting and Rietveld analysis when the crystal structure of the baccatin moieties are constrained to the baccatin singlecrystal structure.21 The structure determination process was undertaken using the DASH software22 and an initial structural model taken from the prior single-crystal data.6 The

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Figure 4. Comparison of paclitaxel XRPD histograms of different crystal polymorphs. The upper trace is a superposition of the experimental data (+) and the Le Bail fit (s) of the P212121 orthorhombic phase. The middle curve is a high-quality residual between these two superimposed curves. The bottom curve is the simulated XRPD pattern of the P21 monoclinic polymorph generated from single-crystal parameters.1

best-fit model for Rietveld refinement used GSAS software with bond distances and bond angles restrained using the theoretical models.23 Each fragment of the refined molecules was individually set to zero occupancy, and the difference Fourier maps were calculated and viewed with Swiss PDB viewer24 to check whether the fragment fits the electron density sufficiently well to avoid ambiguity. Normally, such types of iterations between the Rietveld refinement and the Fourier difference maps are repeated until a reasonable fit of the model and electron density is achieved. The structural fitting then is allowed to converge. Work25 on a microcrystalline powder of human insulin prepared by grinding a single crystal of the compound confirmed the stereochemical parameters obtained from the single crystal data. The initial model for docking two molecules of paclitaxel was obtained by theoretically optimizing with a Gaussian program.26 The docking computation remains tentative pending the existence of more advanced structural models. Successful results27 on a new polymorph of hen egg-white lysozyme with three molecules per unit cell support these Monte Carlo methods, but a more successful crystal structure for paclitaxel awaits additional constraints and possibly improved software capabilities for docking the two congruent molecules. Diffractometer at the Advanced Photon Source (APS). Synchrotron XRPD data from the 1-BM beam line28 at the APS, Argonne National Laboratory (ANL), were used in concert with SSNMR data to study the structure of the new stable polymorph of paclitaxel obtained from NPI. Observed CST typically are quantum mechanically modeled26 with density functional theory (DFT) before these data can be used to obtain structural information.4 The monoclinic polymorph of paclitaxel, with a P21 space group,6 had previously been studied by singlecrystal methods. Its simulated XRPD pattern is given in the bottom histogram of Figure 4. This structure contains two molecules of paclitaxel per asymmetric unit with mobile dioxane and water in the unit cell. CST

SSNMR and XRPD of a Stable Polymorph of Paclitaxel

calculations for paclitaxel based on the X-ray data taken on the original single crystal6 imply that the CSTs in the single crystal and in the powder sample differ by more than the differences between the two molecules per asymmetric unit.19 Without CST data on the original single crystal, no longer extant, these differences cannot be duplicated nor studied. The 1-BM beam-line at APS29 yields high-quality XRPD experimental data from the synchrotron for the stable polymorph of paclitaxel. The pattern was indexed as an orthorhombic cell by the Dicvol program30 and confirmed as a P212121 space group using Le Bail fitting.31 The top trace in Figure 4 is a superposition of both the experimental and the fitted histograms for the P212121 form. A residual curve between the two superimposed (i.e., calculated and experimental) traces is provided in the middle trace of Figure 4. The relative error of the fit is about 5.0%, sufficient to confirm the P212121 space group and give unit cell dimensions: a ) 28.7386(5) Å, b ) 9.5916(2) Å, c ) 31.4162(6) Å for the new polymorph. Reasonable crystallographic densities [i.e., the experimental value is 1.344(1) g/cm3 with a calculated value of 1.310 g/cm3] are obtained when this orthorhombic crystal contains two molecules (Z′ ) 2) per asymmetric unit cell in agreement with the SSNMR results. The relatively sharp diffraction lines in the experimental XRPD histogram once again confirm the microscopic crystallinity of the sample. When the simulated histogram of the single-crystal polymorph is compared, significant differences exist between the structures of the two polymorphs, verifying a new polymorphic structure. Continuing refinements32 will use direct spacing methods constrained by improved CST presently being refined experimentally and theoretically. The Rietveld method33 uses the GSAS suite of programs.34,35,36 The model has been refined by iteratively switching between CST and XRPD data until the internuclear distances and structural angles for both NMR and X-ray data are self-consistent. Often one set of data will improve significantly for minor lattice changes, while the other set of data remains essentially insensitive and vice versa. CSTs are at times insensitive to periodic rotational energy minimums in conformational angles, much like the failure of powder diffraction methods to locate hydrogen atoms. More serious is the instance in which one approach achieves a moderately better fit, while the other method deteriorates seriously. Consistently, when this happens, untenable structural parameters are usually encountered that can be readily recognized. One either has inadvertently found an unreasonable local minimum or else has encountered a situation in which the sensitivity of the method has failed to differentiate between structural alternatives (e.g., when two hydrogen atoms are located very close to one another with distances considerably below the sum of acceptable van der Waals radii). The XRPD data fail to detect such inconsistencies involving hydrogen atoms, whereas CSTs are very sensitive to such information. To obtain a complete crystal structure for a molecule of paclitaxel’s size, XRPD data from the synchrotron and CST information must combine in concert to yield a better structure. Otherwise, it is impossible to take full advantage of the unique structural degrees of freedom of the two methods. NMR CSTs

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are very sensitive to proton positions and similar local structural parameters involving C, N, and O atoms. In relatively large molecules, XRPD is relatively insensitive to moderate errors in short-range parameters (especially proton positions) but provides high sensitivity for indexing the space group of the crystal system and for obtaining long-range lattice order parameters such as unit cell dimensions. As the size of the asymmetric unit increases, all methods suffer from the accumulation of errors, but dissimilar sensitivity of the two physical methods is based on various degrees of freedom for both XRPD and SSNMR data. Correspondingly, the accumulation of errors in a XRPD (and for that matter in SSNMR studies) begins to limit the precision with which one may study details of short-range order in microcrystalline powders, respectively. To succeed under these conditions, independent constraints increase the chance of completing the analysis. Thus, a combination of XRPD and computationally directed SSNMR methods augments the determination of accurate crystal structures. Very diverse physical methods provide precise structural information that often exceeds the accuracy of each individual method used independently. This benefit has the consequence of increasing the amount of structural information in the study of paclitaxel. IV. Summary While the two methods discussed in this study enhance the fitting process, XRPD may differentiate at times between local conformational minima not clearly distinguished by corresponding CST data when certain rotational symmetry obtains. Conversely, the CST data provide quite accurate short-range data not readily available from XRPD results. The synergism between these two methods is clearly exhibited in this study. While a complete structure of the polymorph has yet to be found, the relative fitting error is reduced to about 5%. This structure yields sufficient agreement with the powder pattern to give us reliable lattice parameters and establishes the point group symmetry of the crystal. Such data are required to explore structural models derived from quantum mechanics augmented by SSNMR results. Acknowledgment. This work was supported by the Institute of General Medical Sciences at NIH under GM 08521-42 and the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences and by the Advanced Photon Source (W-31-109-Eng-38). References (1) Smith, E. D. L.; Hammond, R. B.; Jones, M. J.; Roberts, K. J.; Mitchell, J. B. O.; Price, S. L.; Harris, R. K.; Apperley, D. C.; Cherryman, J. C.; Docherty, R. J. Phys. Chem. B 2001, 105, 5818-5826. Pna21, Vunit ) 798.54 Å3, Z ) 4, Z′ ) 1. (2) Middleton, D. A.; Peng, X.; Saunders: D.; Shankland, K.; David, W. I. F.; Markvardsen, A. J. Chem. Comm. 2002, 1976-1977. P21/n, Vunit ) 1281.51 Å3, Z ) 4, Z′ ) 1. (3) Rajeswaran, M.; Blanton, T. N.; Zumbulyadis, N.; Giesen, D. J.; Conesa-Moratilla, C.; Misture, S. T.; Stephens, P. W.; Huq, A. J. Am. Chem. Soc. 2002, 124, 14450-14459. P21/c, Vunit ) 2026.1 Å3, Z ) 4, Z′ ) 1.

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(4) Harper, J. K.; Zhang, Y.; Lee, P. L.; Von Dreele, R. G.; Grant, D. M. Work completed and manuscript in preparation. Zhang, Y. Presented at the Pharmaceutical Powder X-ray Difrraction Symposium, Hilton Head Island, South Carolina, February 23-25, 2004. P21, Vunit ) 907.79 Å3, Z ) 2, Z′ ) 1. Manuscript in final preparation. (5) Pop, M. M.: Goubitz, K.; Borodi, G.; Bogdan, M.; DeRidder, D. J. A.; Peschar, R.; Schenk, H. Acta Crystallogr. 2002, B58, 1036-1043. P21, Vunit ) 3637.7 Å3, Z ) 2, Z′ ) 1. We appreciate the referee’s suggestion that this rather large asymmetric unit cell represents the present state of the art for a combined ab initio quantum and XRPD study of cyclodextrin (CD) and mefenamic acid. One should take note of the rather large number of bond and angle constraints that are found for CD and the relative percent errors of the Rietveld fit of about 10%. (6) Mastropaolo, D.; Camerman, A.; Luo, Y.; Brayer,. G. D.; Camerman, N. Proc. Natl. Acad. Sci., U.S.A. 1995, 92, 6920-6924. We acknowledge Donald Mastropaolo and the late Arthur Camerman for providing the coordinates. The space group is P21 and the unit cell parameters are β ) 99.730°, a ) 9.661 Å, b ) 28.275 Å, c ) 19.839 Å. (7) Slowik, H. In Vivo: Bus. Med. Rep. 2003, 21, 75-82. (8) (a) Almarsson, O ¨ .; Hickey, M. B.; Peterson, M. L.; Morissette, S. L.; Soukasene, S.; McNulty, C.; Tawa, M.; MacPhee, J. M.; Remenar, J. F. Cryst. Growth Des. 2003, 3, 927-933. (b) Remenar, J. F.; MacPhee, J. M.; Larson, B. K.; Tyagi, V. A.; Ho, J. H.; McIlroy, D. A.; Hickey, M. B.; Shaw, P. B.; Almarsson, O ¨ . Org. Proc. Res. Dev. 2003, 7, 990-996. (9) Ranitidine hydrochloride, marketed by Glaxo plc under the name Zantac is known to be polymorphic. In a recent patent litigation [Glaxo Inc. (1993), Plaintiff v. Novopharm Ltd. Defendant, No. 91-759-CTV-% BO United States District for the Easter District of California, Raleigh Division 1993 U. S. Dist. LEWIS 13928], Glaxo’s patent protection on this polymorph has been challenged. (10) Bugay, D. E. Pharm. Res. 1993, 10, 317-327. (11) A discussion of the issues involved in the case of Paxil also can be found at the Glaxo Smith Kline website. As websites change regularly, the reader is left with the task of finding this and other relevant future websites. (12) Johnson, J. H. Natural Pharmaceuticals, Inc. Drug master file # 15,583. (13) Wani, M. C.; Taylor, H. L.; Wall, M. E.; Coggon, P.; McPhail, A. T. J. Am. Chem. Soc. 1971, 93, 2325-2327. (14) Kris, M. G.; O’Connell, J. P.; Gralla, R. J.; Wetheim, M. S.; Parente, R. M.; Schiff, P. B.; Young, C. W. Cancer Treat. Rep. 1986, 70, 605-607. (15) Nicolaou, K. C.; Yang, Z.; Liu, J. J.; Ueno, H.; Nantermet, P. G.; Guy, R. K.; Claiborne, C. F.; Renaud, J.; Couladouros, E. A.; Paulvannan, K.; Sorensen, E. J. Nature 1994, 367, 630-634. (16) (a) Denis, J. N.; Greene, A. E.; Guenard, D.; GueritteVoegelein, F.; Mangatal, L.; Potier, P. J. Am. Chem. Soc. 1988, 110, 5917-5919. (b) Holton, R. A. Midwest Regional American Chemical Society Meeting, Cleveland, OH, June 1989; (c) Holton, R. A. Method for preparation of taxol using an oxazinone. U.S. Patent 5,015,744, May 14, 1991. (d) Ojima, I.; Sun, C. M.; Zucco, M.; Park, Y. H.; Duclos, O.; Kuduk, S. Tetrahedron Lett. 1993, 34, 4149-4152. (e) Commercon, A.; Bezard, D.; Dernard, F.; Bourzat, J. D. Tetrahedron Lett. 1992, 33, 5185-5188. See additional references contained in these citations. (f) Hauser, Inc., U.S. Patents 5,679,807 (1997) and 5,808,113 (1998). (17) Alderman, D. W.; McGeorge, G.; Hu, J. Z.; Pugmire, R. J.; Grant, D. M. Mol. Phys. 1998, 95, 1113. (18) Isotropic chemical shifts are used in Figure 2 for the sake of simplicity and because they illustrate the symmetry features and asymmetric unit populations necessary to obtain unit cell dimensions and distances. Refinement of specific angular and distance constraints in the baccatin moiety requires an iterative sequence of fitting together the CST and XRPD data. (19) Harper, J. K.; Barich, D. H.; Heider E. M.; Grant, D. M. Manuscript in preparation. Preliminary results were presented by D. M. Grant at the International Society of Magnetic Resonance meeting in Rhodes, Greece, August 19-23, 2001.

Harper et al. (20) Lakdawala, A.; Wang, M.; Nevins, N.; Liotta, N.; RusinskaRoszak, D.; Lozynski, M.; Snyder, J. P. BMC Chem. Biol. 2001, 1, Article 2, 13-20. (21) Harper, J. K.; Facelli, J, C.; Barich, D. H.; McGeorge, G.; Mulgrew A. E.; Grant, D. M. J. Am. Chem. Soc. 2002, 124, 10589-10595. In this reference, both the conformational structure and the chemical shift tensors are shown to be relatively invariant in a variety of baccatin molecular systems. This earlier shift tensor information from a study of various baccatin moieties provides a critical constraint on the paclitaxel fit of the XRPD data in this study. (22) David, W. I. F.; Shankland, K.; Shankland, N. DASH software, Routine determination of molecular crystal structures from powder diffraction data. Chem. Commun. 1998, 931-932. (23) Larson, A. C.; Von Dreele, R. B. General Structure Analysis System (GSAS), Los Alamos National Laboratory Report LAUR 86-748, Los Alamos National Laboratory: Los Alamos, NM, 1994. (24) Guex, N.; Peitsch, M. C. SWISS-MODEL and the SwissPdbViewer: An environment for comparative protein modeling. Electrophoresis 1997, 18, 2714-2723. (25) Von Dreele, R. B.; Stephens, P. W.; Smith, G. D.; Blessing, R. H. Acta Crystallogr. 2000, D56, 1549-1553. A larger XRPD crystal structure involving 1630 atoms has been exhibited for the protein human insulin-zinc complex by utilizing independent stereochemical constraints. This study on paclitaxel uses a similar approach except that the constraints are provided by the CST/SSNMR data from Baccatin and Taxol. (26) Gaussian 98 and Gaussian 03: Frisch, M. J.; Trucks, G. W.; Schlegel, H. B. et al. Gaussian, Inc.; Pittsburgh, PA, 1998 and 2003. (27) Von Dreele, R. B.; Zhang, Y.; Lee, P. L. Structure of the Sea Urchin Phase of HEW Lysozyme by Powder Diffraction, Acta Crystallogr D., manuscript submitted. (28) Lang, J. C.; Srajer, G.; Wang, J.; Lee, P. L. Rev. Sci. Instrum. 1999, 70, 4457-4462. (29) XRPD data were collected at 1-BM beamline at APS with wavelength of 0.619 Å, calibrated with a NIST Silicon standard. Data were collected with the Debye-Scherrer geometry using a Mar345 image plate detector. Sample-todetector distance, calibrated with a NIST LaB6 standard, was 835 mm. The paclitaxel sample was contained at room temperature in a 0.8-mm capillary and spun at 2 Hz. Data were averaged with five 20-s exposures, and the pattern was integrated for an azimuthal angle of 30° by the program Fit2d. The calculated density is obtained from the molecular weight, Z, Z′, and the unit cell volume, while the experimental density was obtained with the Archimides’ method. The Le Bail fitting was carried out by GSAS program and gave wRp 3.36% and Rp 3.51%. The good quality fitting confirmed the P212121 space group. The requirements of Z′ ) 2, a chiral space group, density, and good Le Bail fitting mitigated against other possible indexing or space groups. (30) Boultif A.; Loue¨r, D. J. Appl. Crystallogr. 1991, 24, 987993. (31) Le Bail, A.; Duroy H.; Fourquet, J. L. Mater. Res. Bull. 1988, 23, 447-452. (32) Harper, J. K.; Zhang, Y.; Heider, E. M.; Lee, P. L.; Von Dreele, R. B.; Grant, D. M., work to be published. (33) Rietveld, H. M. Acta Crystallogr. 1967, 22, 151-152. Rietveld, H. M. J. Appl. Crystallogr. 1969, 2, 65-71. (34) Larson, A. C.; Von Dreele, R. G. Los Alamos Laboratory Report LA-UR-86-748; Los Alamos National Laboratory: Los Alamos, NM, 1987. (35) David, W. I. F.; Shankland, K.; Shankland, N. Chem. Commun. 1998, 931-932. (36) Pagola, S.; Stephens, P. W. Mater. Sci. Forum 2000, 321324, 40-45.

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