CRYSTAL GROWTH & DESIGN
Increased Chemical Purity Using a Hydrate
2004 VOL. 4, NO. 3 539-544
Simon N. Black,*,# Murray W. Cuthbert,# Ron J. Roberts,# and Birgitta Stensland‡ AstraZeneca, Macclesfield, SK10 2NA, England, and AstraZeneca, So¨ derta¨ lje, S151-85, Sweden Received November 17, 2003;
Revised Manuscript Received January 28, 2004
ABSTRACT: Crystallization is usually a highly selective process, and this selectivity is exploited in the pharmaceutical and other industries as a method of chemical purification. Relatively little is known about how polymorphism and solvate formation can influence the selectivity of crystallizations. We describe a piperidene pharmaceutical intermediate that can crystallize as either a hydrate or an anhydrous form, with very different purities. The exploitation of this effect is described, and an explanation in terms of the crystal structures is proposed. Introduction Crystallization is widely used as a method of purification in the pharmaceutical and other industries.1 Typically, the selectivity during crystal growth is very high, and crystallization is a very successful method of purification. However, in some cases, crystal structures are less discriminating, and partial or complete solid solutions are formed.2,3 Purification by crystallization is then doomed to fail. Changing the crystallization and washing procedures will be ineffective in such cases, and the only remedy is to change the crystal structure. Polymorphism and the related phenomena of hydrate and solvate formation are receiving increasing attention in the pharmaceutical industry because of their influence on product performance.4-6 Less attention has been paid to polymorphism of intermediates in a synthetic route, which may often go unnoticed. Some impurities inhibit crystallization, and more specifically some additives will selectively inhibit undesired polymorphs.7,8 For intermediates in a chemical synthesis, the focus is on the degree of purity obtained during a crystallization, rather than the polymorph obtained. Yet, there is little known about how polymorphism affects crystal purity. “Piperidene” (Figure 1) is an intermediate in the manufacture of a novel antibacterial pharmaceutical. After initial toxicity studies on the final product had been performed, an impurity, that had previously been obscured in the HPLC method by another known impurity, was detected in piperidene. Attempts to remove this impurity by recrystallization gave inconsistent results. This triggered a structured investigation in four parts. First, the chemical nature of the impurity was established, and chemical means of impurity removal were studied. The physical purity of the samples from inconsistent recrystallizations was studied using solid-state techniques including single-crystal structure determination. A correlation between physical and chemical purities was sought. Finally, attempts were made to * To whom correspondence should be addressed: Dr. Simon Black, AstraZeneca, Maccclesfield, SK10 2NA, England. Tel: 44 1625 514948. Fax: 44 1625 500780. E-mail:
[email protected]. # AstraZeneca, England. ‡ AstraZeneca, Sweden.
Figure 1. Piperidene (I), a pharmaceutical intermediate.
rationalize the observed difference in chemical purity of the two structures. Experimental Section Crystallizations. The synthesisis of 4-(2,6-difluoro-4-{(5R)5-[(3-isoxazolyl]-2-oxo-1,3-oxazolan-3-yl}phenyl)-1,2,3,6-tetrahydropyridinium chloride (hereafter piperidene, Figure 1) is described elsewhere.9 Crystallizations were performed on scales from 5 to 500 mL by adding 1 part of piperidene to 2.5 parts by weight of solvent, warming to 70 °C, holding for 10 min and cooling to 10 °C overnight. Experiments at the 500mL scale were carried out in a 500-mL jacketed vessel fitted with a 60-mm diameter retreat curve impeller and connected to a Haake C1 circulating/heating bath. The vessel was inerted with nitrogen prior to charging the reagents. The suspension was filtered in a Buchner funnel, displacement washed with water (0.5 parts) and air-dried to constant weight. Chemical Composition. The NMR spectra were recorded in DMSO using a Brucker DRX 500 NMR spectrometer. Water contents were determined using a Mitsubishi CA-106 Moisture meter. The levels of the impurity in piperidene samples were determined by HPLC using a Hewlett-Packard HP 1100 HPLC with a UV detector. Optical Microscopy. Solid samples were suspended in paraffin oil between glass slides and viewed with partially crossed polars using a ×40 objective in an Olympus BH2 optical microscope. Images were recorded digitally via a DP11 camera. Thermal Analysis. Thermogravimetric analysis (TGA) was carried out selected samples using a Mettler TGA850 with TSO801RO robot system (Mettler-Toledo). Approximately 5 mg of each sample was added to a 70 µL alox (aluminum oxide) crucible, and the accurate weight was recorded. The samples were heated over the temperature range 25-325 °C at a constant heating rate of 10 °C/min with a purge gas of helium (flow rate of 50 mL per minute). A blank alox pan was also run as a standard and deducted from the measurement run to compensate for balance drift due to increasing temperature. The data analysis was performed using the Mettler STARe software. X-ray Powder Diffraction. Samples were prepared on zero background silicon wafers by pressing approximately 2 mg of each sample over the wafer with a glass slide. The samples were run on a D5000 diffractometer (Brucker AXS).
10.1021/cg034222v CCC: $27.50 © 2004 American Chemical Society Published on Web 04/20/2004
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Figure 2. Reaction scheme for piperidene (1) and N-linked impurity (3). The samples were spun at 30 rpm to improve counting statistics. X-rays were generated using a copper long-fine focus tube operated at 40 kV and 40 mA, having a wavelength of 1.5418 Å. The data for each sample were collected using a standard scintillation detector. The collimated X-ray source was passed through an Automatic Variable Divergence Slit set at V20 (20-mm path length) and the reflected radiation was directed through a 2-mm antiscatter slit; the secondary soller slit was in place. Each sample was exposed for 4 s per 0.02° 2-theta increment (continuous scan mode) over the range of 2-40° 2-theta in theta-theta mode. A Dell Optiplex 686 NT 4.0 workstation operating with Diffrac+ was used for control and subsequent data analysis. Single-Crystal Studies. Diffracted intensities were collected at room temperature (anhydrate) and at 200 K (hydrate) with monochromatized MoK(R)-radiation on a KappaCCD single-crystal X-ray diffractometer, equipped with an κ-axis goniometer and a CCD area detector. The diffraction raw data was processed within the Denzo-SMN software package, and the structure was solved with direct methods, taking advantage of SIR92. The structure was refined with full-matrix leastsquares calculations within the MaXus program system. All non-H atoms were refined anisotropically, whereas the hydrogen atoms were placed in geometrical relevant positions at a distance 0.96 Å from the parent atom. The hydrogen-atom positions were verified from subsequent Fourier electron density maps and supplied with isotropic thermal displacement factors, U(iso) ) 0.05 Å2. No absorption correction was done. Molecular Modeling. Visualization of the two structures was performed on a Silicon Graphics Indigo2 Workstation using Cerius software version 2.3 (MSI, now Accelrys). The hydrate structure was built using four cells along the b-axis (water hydrogen-bond direction) and the hydrogen bonded chain was abstracted. One of the molecules in the chain was easily changed to the impurity. The isoxazolyl ring with the N-linkage was rotated until the distance between the carbonyl and the oxygen of the oxazolanyl ring and the ether respectively were at a minimum, e.g., maximum repulsion. This was done by monitoring the hydrogen-bond length by enabling automatic recalculation within the edit hydrogen bonding function in Cerius.
Results and Discussion Chemical Purity. Using 2D correlation NMR the impurity was identified as the N-linked regio-isomer of piperidene (1). The “N linked” impurity precursor (2) is formed during the Mitsunobu coupling of the ambulent 3-hydroxyisoxazole nucleophile and hydroxyoxa,9 and carries through to piperidene, as the oxaisoxazole (4) is not isolated in the process. Piperidene as prepared by the established process contained up to 4.5% of N-linked impurity (3). Subsequent trials showed that this N-linked regioisomer carried through to the final product. As the impurity was not toxicologically qualified, bridging studies would have been required unless this impurity could be removed.
Piperidene is a relatively insoluble material and the free base could not be isolated as a crystalline form. Out of the initial screen of solvent mixtures only aqueous acetone achieved a reduction in the level of the N-linked impurity to less than 1.5%. It was observed during the investigation that piperidene would dissolve ready in water at room temperature, crystallize at around 40 °C, and then redissolve as heating is continued. Crystallizing piperidene containing 4.5% of N-linked impurity from water in the laboratory surprisingly reduced the level of 3 to 0.40.5% with a recovery of ∼80%. Thus, the N-linked impurity could be removed by a separate aqueous crystallization step. However, this introduced extra process costs, time, and yield losses. The preferred solution was to obtain this degree of purification in the crystallization from the reaction. Accordingly, the reason for variable purification from different solvents was investigated. Physical Purity. Optical microscopy, using partially polarized light, of several samples of piperidene crystallized from different solvents and solvent mixtures showed that two different morphologies could be obtained, as shown in Figure 3. Figure 3 shows two very different morphologies of piperidene, which were described as “needles” and “lumps”, respectively. The absence of any intermediate morphologies, and the occurrence of both morphologies in some samples, suggested that two different structures were present. This was investigated further by powder XRD. The two powder patterns are quite different, suggesting that the lumps and needles have quite different crystal structures. Confirmatory evidence was sought from TGA of the two forms. Single-crystal structure studies of both structures were also carried out. The structural information is summarized in Table 2. The powder XRD patterns calculated for these structures are in good agreement with the powder XRD patterns shown in Figure 4. The suggestion from the optical micrographs that two different structures are present is confirmed by the powder XRD patterns, which are completely different. The TGA trace for the needles suggests that this is a hydrate containing 4.06% water. From the molecular weight of piperidene, the predicted weight loss for dehydration of a monohydrate is 4.17%, which is in good agreement. The suggestion that the needles are a monohydrate is confirmed by the single crystal study. The needles and lumps are respectively monohydrate and anhydrate forms of piperidene.
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Figure 3. Two different morphologies of piperidene: needles (top) and lumps (bottom). The width of each image corresponds to 0.9 mm. Table 1. Recrystallizations of Piperidene solvent composition
volume (mL) impurity % recovery %
water/acetone 1/1 water/acetone 9/1 water/acetone 0.5/9.5 water/butanol 0.5/9.5 water/iso-butanol 0.5/9.5 water/ethanol 1/9 water/iso-propanol 1/9 water water water
6 6 6 20 20 6 6 5 2.5 3
0.7 1.1 2.1 2.4 1.9 1.7 0.8 0.4 0.5
0 54.3 67 71.7 79 69 78 66.5 80.5 81.4
Table 2. Summary of Single-Crystal Structure Determinations for Needles and Lumps designation stoichiometry space group unit cell a b c β Z density packing fraction Rw
“needles”
“lumps”
hydrate (200K) C18H18F2O4N3+Cl-‚H2O C2 21.675(1) 5.897(1) 16.129(1) 115.10(1) 4 1.540(1) 72.4% 0.0425
anhydrate 2x[C18H18F2O4N3+Cl-] P21 12.770(1) 9.931(1) 15.560(1) 107.95(1) 4 1.4642(1) 68.0% 0.0374
This investigation into the physical purity of piperidene showed two different forms with fixed stoichiometry. This is not sufficient to account for the variable impurity contents reported in Table 1. A further investigation was carried out to combine the data on physical and chemical data to seek correlations.
Correlating Physical and Chemical Purity. Fifteen further crystallizations were carried out using a range of aqueous and nonaqueous solvents. To allow for the effect of varying impurity levels in the starting material, a “segregation coefficient”1 was defined as the ratio of (weight % impurity in product)/(weight % impurity in starting material). A segregation coefficient of 0 indicates complete purification in the crystallization, whereas a segregation coefficient of 1 indicates no purification. The segregation coefficient was plotted against the water content of samples prepared from different solvent compositions. In addition, samples were examined by polarized light microscopy and the physical form was characterized. The results are given in Figure 6: The data fall into three groups. Nine samples showed the hydrate morphology, good purification (segregation coefficient e 0.3), and water content 3.0-4.2%. Three samples showed the anhydrate morphology and had lower water content and higher segregation coefficients. Three samples were physical mixtures of hydrate needles and anhydrate lumps. The results are consistent with the data from the previous section, showing that the piperidene exists in two crystal forms: a hydrate and an anhydrate. The behavior of the anhydrate is variable, but it is capable of accommodating up to 4% of the N-linked impurity. Some recystallizations result in physical mixtures of the two forms, with intermediate impurity levels. The segregation coefficient for the hydrate is 0.1-0.3. Hence, the hydrate is the preferred form for achieving purification of the piperidene, provided that the following stage in the synthesis can tolerate water. Solvent composition can be crucial in determining whether a hydrate or anhydrate is thermodynamically favored in crystallizations from aqueous solvents.10 In this study, the anhydrate was obtained from acetone/ water mixtures when the solvent water content was below 10%. The hydrate was obtained from acetone/ water mixtures containing more than 10% water. Variable results were obtained from acetone/water mixtures containing 10 vol % of water, suggesting that at this solvent composition the thermodynamics are finely balanced, and the outcome of crystallization is under kinetic control. The crystal structure of the hydrate discriminates between piperidene molecules and the N-linked impurity, whereas the anhydrous structure can accommodate the N-linked impurity. The two crystal structures were studied in more detail to account for this difference. Comparison of Anhydrate and Hydrate Structures. Crystal Packing Fraction. The crystal packing fraction (% of space filled by the van der Waals volume) of the hydrate is 72.4%, compared with 68.0% for the anhydrous form. For comparison, the packing fraction for monodisperse spheres is 74.0%. Hence, there is more “free space” in the anhydrate structure for it to accommodate molecules of slightly different shapes. Conformations. There are two different conformations of piperidene in the anhydrous structure, and the conformation in the hydrated structure is slightly different again, as shown in Figure 7. In all three conformations, the middle two rings are coplanar. The conformations differ chiefly in the orientations of the first
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Figure 4. Powder XRD of lumps (upper trace) and needles (lower trace).
Figure 5. TGA of lumps (upper trace) and needles (lower trace).
(isoxazolyl) and fourth (tetrahydropyridinium) rings relative to this plane. Intermolecular Interactions. Piperidene is a hydrochloride salt containing two aromatic rings, two hydrogen bond donors, and nine hydrogen bond acceptors per molecule. Thus, there are several options for intermolecular interactions. The two crystal structures were examined in detail to select the key bonding motifs. The anhydrate structure contains dimers indicating strong π-π interactions between the two central rings. These dimers are oriented “head to tail” as shown in Figure 8. This feature does not appear in the hydrated structure. In the hydrate, the oxygen of the water molecule is between two chlorine ions, suggesting a bridging role.
Initially, it was not possible to locate the hydrogen atoms of the water molecule. Hence, the structure was redetermined at 200 K to establish the hydrogen atom positions and hydrogen bonding unequivocally. This confirmed the role of hydrogen bonding in bridging neighboring chlorine atoms. This facilitates a completely different packing of the piperidene molecule in the hydrate structure. There is also an addition hydrogen bond between the water molecule and the oxygen of the isoxazolyl ring, as shown in Figure 9. Selectivity of the Hydrate and Anhydrate Crystal Structures. The chemical structures of piperidene and the N-linked impurity are shown in Figure 10. There is no possibility of intramolecular hydrogen bonds in either molecule. Both molecules consist of four rings,
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Figure 9. Hydrogen bonding in the hydrate.
Figure 6. Comparison of segregation coefficient, water content, and morphology.
Figure 10. Chemical structures of piperidene (I) and N-linked impurity (II).
Figure 7. The three conformations of piperidene in the crystal structures, viewed perpendicular to the plane of the middle two rings.
Figure 8. Dimers in the anhydrate structure, viewed along the plane of the two central rings.
and they only differ in the configuration of the fourth ring to the right. The conformations of the piperidene molecule are dominated by the common plane of the two central rings, and the flexibility of the two rings at
either end. These features are shared with the N-linked impurity, which also shares the absolute configuration at the chiral center. The strongest intermolecular interactions in the crystal will be the attractive and repulsive Coulombic interactions between the piperidene nitrogen and the chlorine ions. These features are at the opposite end of the molecule from the end at which the N-linked impurity differs. On the basis of these simple considerations, it is not clear on what basis either structure would reject molecules of N-linked impurity. Therefore, the specific structural consequences of replacing the isoxazolyl ring of the piperidene with an N-linked ring were considered. An important structural feature are the dimers shown in Figure 8. The π-π interactions involve the two central rings only. Therefore, the N-linked impurity could also take part in dimer formation. In the hydrate structure, there is a specific hydrogen bond shown in Figure 9 between the water molecule and the isoxazolyl ring. The N-linked impurity could not form this hydrogen bond. The molecule is fully extended to enable the oxygen atom to take part in this hydrogen bond, and if the N-linked ring is substituted it is not possible to place a nitrogen or oxygen atom this close to the water molecule. A further attempt was made to “fit” the N-linked impurity into the hydrate structure, as shown in Figure 11. The van der Waals fit is good, with no clashes. The figure shows a C-H‚‚‚O interaction between neighboring piperidene molecules. However, when the N-linked impurity is introduced this favorable interaction is replaced by two unfavorable O‚‚‚O contacts. To summarize the structural considerations: these two molecules are very similar. There is no conformational basis to account for preferential incorporation of
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There are examples in the literature in which the impurity levels control which structure crystallizes. In this case, it was possible to select the crystal structure to control the fate of the impurity. Acknowledgment. We thank Anne Kavanagh for XRD and TGA analysis, Ian Jones for NMR studies, Linda McCulloch for the HPLC determinations, and Danny Levin for helpful comments. Supporting Information Available: The two CIF files for the crystal structures of the hydrate and anhydrate, as well as further details of the NMR and HPLC determinations are available free of charge via the Internet at http://pubs.acs.org.
References Figure 11. A molecule of N-linked impurity inserted into the hydrate structure.
the impurity in the anhydrate structure. Consideration of the strong and weak hydrogen bonding in the hydrate structure suggests that this may be the origin of the enhanced selectivity of this structure. Conclusion This study was only undertaken because it was noticed that routine observations of impurity levels and crystal morphology were giving variable results. These observations have been correlated, and an attempt has been made to provide an explanation based on crystal chemistry. As a result of this work, the solvent for this crystallization was specified to ensure that the hydrate structure was obtained, and the procedure was operated several times at larger scales. Over 500 kg of piperidene was prepared, with the level of N-linked impurity at acceptable levels of e 0.3%.
(1) Davey, R. J.; Garside, J. From Molecules to Crystallisers: An Introduction to Crystallisation, Oxford Chemistry Primers No 86; Oxford University Press: UK, 2000. (2) Dorset, D. L. Macromolecules 1990, 23, 623-633. (3) Davey, R. J.; Black, S. N.; Logan, D.; Maginn, S. J.; Fairbrother, J. E.; Grant, D. J. W. J. Chem Soc. Faraday Trans. 1992, 88, 3461-3466. (4) Byrn, S. R.; Pfeiffer, R. R.; Stowell, J. G. Solid State Chemistry of Drugs, 2nd ed.; SSCI Inc.: West Lafayette, IN, 1999. (5) Chemburkar, S. J.; Bauer, J.; Deming, K.; Spiwek, H.; Patel, K.; Morris, J.; Henry, R.; Spanton, S.; Dziki, W. P.; Quick, J.; Bauer, P.; Donaubauer, J.; Narayanan, B.A.; Soldani, M.; Riley, D.; McFarland, K. Org. Proc. Res. Dev. 2000, 4, 413417. (6) Bernstein, J. Polymorphism in Molecular Crystals; Oxford Clarendon Press: New York, 2002. (7) Davey, R. J.; Blagden, N.; Potts, G. D.; Docherty, R. J. Am Chem. Soc. 1997, 119, 1767-1772. (8) Weissbuch, I.; Leiserowitz, L.; Lahav, M. Adv. Mater. 1994, 6, 952-956. (9) Levin, D.; Howells, G. E.; Cuthbert, M. W. Patent Application WO 02/096890 A2, 2002. (10) Zhu, H.; Yuen, C.; Grant, D. J. W. Int. J. Pharm. 1996, 135, 151-160.
CG034222V
