Reviewing the Manifold Aspects of Ganciclovir ... - ACS Publications

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Reviewing the Manifold Aspects of Ganciclovir Crystal Forms José A. Fernandes,† Simona Galli,*,† Giovanni Palmisano,† Paolo Volante,‡ Ricardo F. Mendes,§ Filipe A. Almeida Paz,§ and Norberto Masciocchi† †

Dipartimento di Scienza e Alta Tecnologia, Università dell’Insubria, via Valleggio 11, 22100 Como, Italy Trifarma SpA, Ceriano Laghetto Unit, via Industrie 6, 20816 Ceriano Laghetto, Italy § Department of Chemistry, CICECO-Aveiro Institute of Materials, University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal ‡

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S Supporting Information *

ABSTRACT: Ganciclovir, an active pharmaceutical ingredient against cytomegalovirus, is known to crystallize in the form of anhydrous or hydrated phases. The crystal and molecular structures of two hydrated forms, the hydrochloride salt, and two pro-drug derivatives (i.e., the triN2,O,O-acetyl and di-O-acetyl) have been successfully unveiled from powder X-ray diffraction (PXRD) or single-crystal X-ray diffraction studies. The thermal behavior of the hydrates has been unraveled by juxtaposing the results of our thermal analysis and variable-temperature PXRD experiments to those previously reported on the topic. On the whole, this investigation revisits and complements the somehow incomplete and scattered information present in the literature. It is envisaged that this study can open the way to systematic PXRD qualitative and quantitative analysis of ganciclovir mixtures formed during synthesis, isolation, or processing.

1. INTRODUCTION

can satisfy their curiosity by reading the excellent reviews collectively quoted in the works of Cruz-Cabeza11 or Griesser.12 The first “form” of ganciclovir, proposed as a hydrate, was prepared in 1982 by Verheyden & Martin and tested for its potential antiviral activity.13 Later, an anhydrous form (labeled form II by Sarbajna et al.14) of this API was prepared.15 A very recent work16 was devoted to the analytical and physicochemical characterization of a GCV sample obtained from a pharmaceutical industry and claimed to contain “hydrated GCV” slightly contaminated by “polymorph III”. Commercial and scientific interests eventually led to the discovery and characterization of a number of polymorphs: as for early 2016, four forms of ganciclovir have been described in the literature, namely, the anhydrous forms I14,17 and II,18 and the hydrated forms III and IV, which have been identified and formulated as hemi- and monohydrate, respectively, in 2011.14 Witnessing the importance of polymorphs identification and quantification, qualitative and quantitative analysis of binary and ternary mixtures of “three pure polymorphs” of GCV was successfully carried out already in the 1990s by attenuated total reflectance Fourier transform IR spectroscopy.19 To the best of our knowledge, form I is accessible only through chemical pathways from the tri-N 2,O,O-acetyl precursor (GCVA3, Chart 1)17 and cannot be obtained by

The monosodium salt of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (Chart 1), an acyclic guanine nucleoside analogue known as ganciclovir (GCV) (CAS RN 82410-32-0), is an active pharmaceutical ingredient (API) against cytomegalovirus (CMV). GCV monosodium salt, currently marketed only by a handful of pharma industries for intravenous treatment, is used for (i) sight-threatening CMV retinitis in severely immunocompromised and AIDS affected patients, and the (ii) prevention and cure of CMV pneumonitis in bone marrow transplant recipients.1,2 A topical ophthalmic gel preparation of this API has been recently approved to treat acute herpes simplex keratitis.3 As most of the APIs the structural features of which have been reported in the literature,4−7 ganciclovir is known to present polymorphism. Investigating, characterizing, and controlling this phenomenon is of crucial importance for the pharmaceutical industry, as different polymorphs of a given substance may possess different physical and chemical properties, which can severely impact stability, solubility, suspensibility, formulation, shelf life, and bioavailability. 8 The consequences of polymorphism, largely neglected or underestimated for many years, have raised the interest of many companies: intellectual protection of the discovery, nature, and properties of the polymorphic forms of a given API is now common practice. Patent litigations on the subject have entered the forensic field as early as in the 1970s.9,10 Interested readers © 2016 American Chemical Society

Received: April 22, 2016 Revised: May 13, 2016 Published: May 18, 2016 4108

DOI: 10.1021/acs.cgd.6b00617 Cryst. Growth Des. 2016, 16, 4108−4118

Crystal Growth & Design

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Chart 1. Molecular Structure of (a) 9-(1,3-Dihydroxy-2-propoxymethyl)guanine, Ganciclovir (GCV), (b) 9-(1,3-Diacetoxy-2propoxymethyl)guanine, GCVA2, and (c) N2-Acetyl-9-(1,3-diacetoxy-2-propoxymethyl)guanine, GCVA3

following, chemical shifts are reported in part per million (δ, ppm) downfield from TMS. Splitting patterns are described as singlet (s), doublet (d), triplet (t), multiplet (m), and broad (br). The values given for coupling constants (nJ) are first-order and quoted in Hz. All 1 H- and 13C NMR assignments were supported by 2-D 1H−1H COSY and 2-D 1H−13C HETCOR experiments, respectively. 2.2. Isolation of Form III as Bulk Powders. In the present work, ganciclovir form III was isolated by following a path different from that previously reported.14 In a typical preparation, as-received GCV (200.0 mg, 0.784 mmol) was let under magnetic stirring, at ambient temperature, in water (60 mL) for 1 h. The white solid was then isolated by filtration, washed with water, and dried under ambient conditions overnight, yielding pure ganciclovir form III as a white polycrystalline powder (96.0 mg, 0.376 mmol, yield 48%). Elem. Anal. Calc. for C9H13N5O4·2H2O (FW = 291.29 g mol−1): C, 37.11; H, 5.88; N, 24.54%. Found: C, 37.70; H, 5.60; N, 23.95%. As it appears here and will be clarified below, the polycrystalline batches of form III we isolated showed a water content different from that originally proposed.14 For a detailed comment on this topic, the reader is referred to section 3.1. 2.3. Isolation of Form IV as Bulk Powders. In the present work, ganciclovir form IV was isolated by following a path different from that previously reported.14 KCl (Merck, 99.999%) (95.4 mg, 1.28 mmol) was added under magnetic stirring to a suspension of as-received GCV (111.1 mg, 0.4353 mmol) in water (5 mL) at 60 °C. Partial dissolution was observed. For a complete dissolution of the pristine solid, water was slowly added up to a total volume of ca. 9 mL. Stirring was then stopped, and the solution was left in open air and cooled down to ambient temperature. After 16 h, form IV, contaminated by form III (13 wt %), was recovered by decantation (49.8 mg) and dried under ambient conditions. Elem. Anal. Calc. for C9H13N5O4·H2O (FW = 273.23 g mol−1): C, 39.56; H, 5.53; N, 25.63%. Found: C, 39.07; H, 5.98; N, 25.12%. 2.4. Isolation of Forms III and IV as Single Crystals. Single crystals of forms III and IV were typically obtained after a slow decrease of the temperature of an aqueous solution (15 mL) of asreceived GCV (33.0 mg, 0.129 mmol) stirred at 40 °C for 1 h. Form III consisted of long needles, as already described by Sarbajna and colleagues,14 partially suitable (see section 2.6) for the acquisition of single-crystal X-ray diffraction data. On the contrary, form IV was isolated in the form of platelets (Sarbajna and colleagues spoke of thin flakes14). Grinding portions of the isolated batches enabled us to ascertain, by PXRD, the presence of both forms. Grinding a sample of platelets after manual separation from the needles enabled us to establish that they were representative of form IV. In spite of numerous single crystal X-ray diffraction experiments on the platy crystals, none suitable for this technique were ever obtained. 2.5. Synthesis of the Hydrochloride Salt (GCV·HCl) as Bulk Powders. As-received GCV (106.1 mg, 0.416 mmol) was suspended in ethanol (25 mL) at 50 °C, and aqueous hydrochloric acid (2.0 M, 0.5 mL) was then added dropwise. During the addition, dissolution of the pristine solid was observed concomitantly with the precipitation of a new white solid. After drying under ambient conditions, pure

recrystallizing, heating, or moisturizing other forms of ganciclovir.14,17 Conversely, besides direct crystallization of commercially available ganciclovir from an oxygen-containing organic solvent (a list of potential solvents is provided in refs 15 and 17), pure form II was obtained also by dehydration of forms III and IV at temperatures above 230 °C.14 On the other hand, forms III and IV were isolated from aqueous solutions of ganciclovir at ambient temperature and from water/methanol (1:1 v/v) solutions of ganciclovir at 50−60 °C, respectively.14 For the sake of completeness, we report here that two forms of the monosodium salt, an anhydrous and a hydrated one, are also known.20−22 Remarkably, despite the deep interconnection between API crystal and molecular structure and physicochemical behavior, to date only the crystal structure of form II of ganciclovir has been determined, by single crystal X-ray diffraction.18 In the past few years, some of us have been involved in unveiling the crystal and molecular structures of several APIs, such as theophylline23 and ibuprofen24 cocrystals, difluprednate,25 bupropion,26 nortryptiline,27 benfluorex,28 diflorasone acetate,29 nicardipine,30 and doripenem,31 mostly by using powder X-ray diffraction (PXRD) coupled to solid state nuclear magnetic resonance, scanning electron microscopy (SEM) imaging, differential scanning calorimetry, and variable-temperature PXRD. Pursuing our interest in shedding light on APIs polymorphism, with this contribution we disclose the main structural features of ganciclovir forms III and IV, ganciclovir hydrochloride (GCV·HCl), and two acetylated pro-drugs (i.e., the tri-N2,O,O-acetyl, GCVA3, and the di-O-acetyl, GCVA2, Chart 1). The thermal behavior of the hydrated forms has been investigated by complementing the evidence previously reported with our thermal analysis and variable-temperature PXRD experiments. For the sake of clarity, throughout the manuscript we will adopt the labels introduced by Sarbajna et al.14

2. EXPERIMENTAL SECTION 2.1. Materials and Methods. Samples of ganciclovir (found to contain a mixture of phases II, III, and IV; see section 3.1), as well as of the two acetylated pro-drug derivatives GCVA2 and GCVA3 were provided by P. Volante. Elemental analyses were carried out with a CHN PerkinElmer 2400 analyzer at the University of Milan. Thermogravimetric analyses and differential scanning calorimetry were performed simultaneously with a Netzsch STA 409 instrument under N2, from 20 °C up to 300 °C, increasing the temperature with a rate of 10 °C/min. Unless otherwise stated, 1H and 13C NMR spectra were acquired at 298 K in DMSO-d6 with a Bruker AVANCE 400 spectrometer operated at 400 and 100 MHz, respectively. In the 4109



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in TOPAS-R,39 provided approximate unit cell parameters for all the compounds. With respect to form III, there was a wide uncertainty about the space group: based on the observed Bragg’s reflections, we could not discern between Pcnn, Pccn, Pnan, and Pbnn, among others. For this reason, the acquisition of single-crystal data was fundamental. For all of the compounds except form III (section 2.6), structure solution was performed by a combined Monte Carlo/Simulated Annealing approach using a rigid body, letting the position of its center of mass, its orientation within the unit cell, and the torsion angles τ1−τ9 (Table S1 and Chart S1 in the Supporting Information) to be refined. The rigid body was built up through a Z-matrix formalism, starting from the good-quality single-crystal data of form II,18 to which all hydrogen atoms were added in idealized positions. For GCVA2 and GCVA3, the acetyl moieties were modeled based on molecular structures derived from the Hic-Up database.40 Besides τ1−τ9, two additional torsion angles groups (τ10 and τ11 in Chart S1 in the Supporting Information) were let to refine for GCVA3 due to the presence of the COCH3 moiety. Protonation at N7 (pKBH+25° 2.2) in GCV·HCl was suggested by theoretical calculations,41 inferred from the well-known 1H NMR deshielding of proton(s) (i.e., H8; 0.85 ppm) attached to carbon atoms adjacent to protonated nitrogen atoms, and found to be consistent with the presence of a hydrogen bond with the chloride anion (vide inf ra). In the case of form III, to build the rigid group we adopted the Cartesian coordinates of the API as obtained by single-crystal X-ray diffraction (disorder of the 1,3dihydroxypropan-2-yloxy branch included, see section 2.6), letting the position of its center of mass, its orientation within the unit cell, and the torsion angles τ1−τ9 (Table S1 and Chart S1 in the Supporting Information) to be refined, together with the position and site occupation factors of the three water molecules found. These three site occupation factors reached the value of 1, to which they were finally fixed. Structure refinements were carried out by the Rietveld method, maintaining the rigid bodies used at the structure solution stage. The background was modeled by a polynomial function of the Chebyshev type, while peak profiles were described by the Fundamental Parameters Approach.42 A common, refined isotropic thermal factor was attributed to all atoms, except to the chloride anion in GCV·HCl, to which the isotropic thermal factor Biso(Cl) = Biso(C,H,N,O)−2.0 (Å2) was assigned. A spherical harmonics description of the Lorentzian peak broadening caused by anisotropic crystal size effects was found to be beneficial for all the compounds. A correction for preferred orientation was applied, adopting the March-Dollase model,43,44 for GCVA2 and forms III and IV. As form IV was contaminated with form III (13 wt %), the structural model of the latter was added during the Rietveld refinement in a multiphase description of the observed PXRD trace. The final Rietveld refinement plots are shown in Figures S1 and S2 of the Supporting Information. Crystal Data for III. C9H13N5O4·3H2O, FW = 309.26 g mol−1, orthorhombic, Pccn, a = 11.3992(5) Å, b = 35.427(1) Å, c = 6.9312(4) Å, V = 2799.0(2) Å3, Z = 8, ρ = 1.47 g cm−3, F(000) = 1312, RBragg = 0.033, Rp = 0.099, and Rwp = 0.134, for 5051 data and 46 parameters in the 4−105° (2θ) range. CCDC No. 1475570. Crystal Data for IV. C9H13N5O4·H2O, FW = 273.23 g mol−1, orthorhombic, Pbca, a = 29.049(1) Å, b = 11.1914(4) Å, c = 7.2115(2) Å, V = 2344.4(1) Å3, Z = 8, ρ = 1.55 g cm−3, F(000) = 1152, RBragg = 0.055, Rp = 0.072 and Rwp = 0.098, for 5051 data and 44 parameters in the 4−105° (2θ) range. CCDC No. 1475571. Crystal Data for GCV·HCl. C9H13N5O4·HCl, FW = 291.68 g mol−1, triclinic, P1̅, a = 6.7975(4) Å, b = 9.8332(6) Å, c = 11.7084(7) Å, α = 124.569(4)°, β = 91.380(5)°, γ = 76.311(4)°, V = 620.17(7) Å3, Z = 2, ρ = 1.56 g cm−3, F(000) = 304, RBragg = 0.042, Rp = 0.054 and Rwp = 0.073, for 4826 data and 40 parameters in the 8.5−105.0° (2θ) range. CCDC No. 1475569. Crystal Data for GCVA2. C13H17N5O6, FW = 339.30 g mol−1, monoclinic, P21/c, a = 17.0205(3) Å, b = 11.5544(4) Å, c = 8.4042(3) Å, β = 105.568(2)°, V = 1592.14(8) Å3, Z = 4, ρ = 1.42 g cm−3, F(000) = 712, RBragg = 0.026, Rp = 0.073 and Rwp = 0.101, for 5026 data and 41 parameters in the 4.5−105.0° (2θ) range. CCDC No. 1475572. Crystal Data for GCVA3. C15H19N5O7, FW = 381.34 g mol−1, monoclinic, P21/c, a = 13.4303(3) Å, b = 14.4722(4) Å, c = 9.2821(4)

ganciclovir hydrochloride was recovered (65.8 mg, 0.226 mmol, yield 54%). Mp 163−240 °C (melting overlapping with decomposition). 1H NMR [DMSO-d6-D2O (2:1)] 3.29 (2H, AA′ part of AA′BB′X system; 2 J 11.5, 3J 6.5, 3J 4.4), 3.42 (2H, BB′ part of AA′BB′X system; 2J 11.5, 3 J 6.5, 3J 4.5), 3.63 (1H, m, HC-O), 5.56 (2H, s, NCH2O), 8.92 (1H, s, H-8*). 13C NMR 61.3 (CH2OH), 74.0 (NCH2O), 82.3 (CH−O), 109.4 (C-5*), 138.0 (C-8*), 150.4 (C-4*), 154.3 (C-6*), 155.9 (C2*). *Purine numbering system (see Chart 1). Elem. Anal. Calc. for C9H13N5O4·HCl (FW = 291.69 g mol−1): C, 37.56; H, 4.84; N, 24.01%. Found: C, 37.15; H, 4.68; N, 23.66%. 2.6. Single-Crystal X-ray Diffraction Structure Determination of Form III. Single-crystal X-ray diffraction data collection of form III was performed at the University of Aveiro, Portugal. Several crystals of form III picked out directly from the mother liquor, containing both forms III and IV (see section 2.4), diffracted poorly, as a consequence of what we believe is the intrinsic instability of this hydrated form under ambient conditions (see section 3.1). Several needles were immersed in FOMBLIN Y and mounted on a Hampton Research CryoLoop with the help of a Stemi 2000 stereomicroscope equipped with Carl Zeiss lenses.32 Preliminary X-ray diffraction data were collected at 180(2) K on a Bruker D8 QUEST equipped with a Mo Kα sealed tube (λ = 0.71073 Å), a multilayer TRIUMPH X-ray mirror, a PHOTON 100 CMOS detector, and an Oxford Instruments Cryostrem 700+ Series low temperature device controlled by the APEX2 software package.33 On the basis of the quality of these tests, one of the crystals was chosen for a complete data collection. The acquired data were processed using the software package SAINT+34 and were corrected for absorption by the multiscan semiempirical method implemented in SADABS.35 The assignment of the space group based on systematic absences was not straightforward. However, attempts of solving the crystal structure by direct methods produced a plausible solution only when the space group Pccn was adopted. The structure was solved by direct methods, as implemented in SHELXS97,36 which allowed the location of the guanine moiety. All the remaining non-hydrogen atoms were located from difference Fourier maps calculated from successive full-matrix least-squares refinement cycles on F2 using SHELXL-2014.37 The electron density around the 1,3-dihydroxypropan-2-yloxy branch was ill defined, which hampered the refinement of anisotropic thermal displacement parameters. Additionally, one of the OH groups was found to be disordered into two positions, while the water molecule was distributed among four distinct crystallographic sites. Hydrogen atoms bound to carbon and nitrogen atoms were placed at their idealized positions using HFIX instructions and described with isotropic thermal displacement parameters fixed at 1.2Ueq of the atom to which they were bound. Single-Crystal Data for Form III. C9H13N5O4·H2O, FW = 273.23 g mol−1, colorless needle of dimensions 0.17 × 0.04 × 0.03 mm3, 18390 collected data, orthorhombic, Pccn, a = 11.319(2) Å, b = 35.200(5) Å, c = 6.872(1) Å, V = 2738(7) Å3, Z = 8, ρ = 1.316 g cm−3, F(000) = 1136, Rint = 0.824, R1 = 0.2237, and wR2 = 0.5704, for 2557 observed [I > 2σ(I)] data in the 2.3−25.6° θ range. The low quality of the single-crystal structural model, as reflected by the values of R1 and wR2, prompted us to complement it with what emerged from a PXRD structure refinement (see section 2.7). 2.7. X-ray Powder Diffraction Structural Characterization. Gently ground powders of III, IV, GCV·HCl, GCVA2, and GCVA3 were deposited in the hollow of a silicon zero-background plate 0.2 mm deep (supplied by Assing Srl, Monterotondo, Italy). Data acquisitions were performed on a vertical-scan Bruker AXS D8 Advance θ:θ diffractometer, equipped with a Lynxeye linear positionsensitive detector, primary beam Soller slits (2.5°), divergence slit (1 mm), antiscatter slit (8 mm), and Ni-filtered Cu−Kα radiation (λ = 1.5418 Å). The generator was set at 40 kV and 40 mA. After preliminary acquisitions for fingerprinting analysis, typically performed in the 3−35° 2θ range, diffraction data sets for a full structure determination were collected up to 105° 2θ, with steps of 0.02°, with an overall scan time of approximately 16 h. A standard peak search, followed by profile fitting, enabled us to estimate the low-to-medium-angle peak maximum positions which, through the Singular Value Decomposition algorithm38 implemented 4110



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Å, β = 86.791(2)°, V = 1801.3(1) Å3, Z = 4, ρ = 1.42 g cm−3, F(000) = 800, RBragg = 0.043, Rp = 0.083 and Rwp = 0.108, for 4826 data and 41 parameters in the 8.5−105.0° (2θ) range. CCDC No. 1475573. 2.8. Variable-Temperature Powder X-ray Diffraction. The thermal evolution of form IV was investigated by variable-temperature powder X-ray diffraction.45 A 20-mg sample of form IV, contaminated by form III (13 wt %), was heated in air from 20 °C up to 240 °C, with steps of 10 °C. A PXRD pattern was acquired at each step, covering the 2θ range 4−30° (0.02° per step, 0.4 s per step, for a total of ca. 10 min per acquisition, carried out under isothermal conditions) using a custom-made sample heater (Officina Elettrotecnica di Tenno, Ponte Arche, Italy).

Also in the case of form IV, the original crystallization procedure14 invariably resulted into the isolation of batches contaminated by non-negligible amounts of form III (PXRD evidence). We found that adding KCl in the reaction medium was beneficial not only to increase the solubility of pristine GCV, but also to favor the precipitation of purer batches of form IV. This follows the same principle which regulates the socalled salting out procedure employed to precipitate proteins from their solutions. Incidentally, isolation of bulk powders of forms III and IV enabled us to reinterpret the PXRD pattern of the first “form” of ganciclovir ever reported: the batch isolated in 1982 and proposed as a hydrate13 is indeed a mixture of forms III and IV. In the present work, forms III and IV could be isolated also in the form of single crystals. Similar attempts were already carried out by Sarbajna and colleagues,14 who admitted they were unable to isolate specimens suitable for structure determination. This is true for form IV also in the present case (see section 2.4). Regarding form III, we were able to isolate specimens which allowed only a partial structural characterization with data acquired at 180 K (see sections 2.4 and 2.6). These partial single-crystal X-ray diffraction results had to be complemented with PXRD studies (see sections 2.7 and 3.2.2). On the whole, the lack of structural characterization of the multifaceted system of ganciclovir could be traced back also to the difficulties faced in obtaining suitable single crystals. The hydrochloride salt, GCV·HCl, which, to the best of our knowledge, has never been reported before, was isolated in the form of polycrystalline powders in a straightforward manner, by adding aqueous HCl to an ethanolic solution of as-received GCV at 50 °C.47 3.2. Structural Characterization. The reader is referred to Tables S2−S8 of the SI for the values of selected distances and angles of the supramolecular interactions of all the compounds described in the following. 3.2.1. Crystal and Molecular Structure of Form II. For the sake of completeness, we propose the reader a brief description of the main structural features of form II, characterized in the recent past, using single-crystal X-ray diffraction, by Kawamura & Hirayama. 18 Ganciclovir form II crystallizes in the monoclinic space group P21, with an asymmetric unit containing one molecule of API. The most striking feature of this crystal structure is a net of hydrogen bonds lying approximately parallel to the (102̅) plane. The net is formed by neighboring ganciclovir molecules through their N1 and N2 atoms as donors,48 and N7 and O1 atoms as acceptors, respectively, which define a graph set motif of the type R22(9)49 (Figure 1 and Table S2 in the SI). As a result, 1-D zigzag chains of API molecules run parallel to the [010] crystallographic direction. An O−H···N intramolecular interaction is also present (Figure 1 and Table S2 in the SI), involving the O3(H) group of the pending arm and the heterocyclic nitrogen atom N3 of the guanine moiety. Along the (102̅) plane, N2 is involved in another intermolecular N−H···O interaction with the atom O4 of a neighboring molecule (Figure 1 and Table S2 in the SI). Finally, out of the (102)̅ plane, an O−H···O interaction between neighboring pending arms is present (not shown in Figure 1; Table S2 in the SI). 3.2.2. Crystal and Molecular Structure of Form III. The following description is based on the structural model retrieved by PXRD (see section 2.7). Ganciclovir form III crystallizes in the orthorhombic space group Pccn. The asymmetric unit contains one molecule of API and three molecules of water, in

3. RESULTS AND DISCUSSION 3.1. Isolation of the Different Crystal Phases. A preliminary PXRD check of as-received GCV enabled us to ascertain the presence of forms II, III, and IV. Starting from this mixture, ganciclovir forms III and IV were isolated as bulk powders by following slightly different paths (sections 2.2 and 2.3) with respect to those reported in the literature. In the case of form III, the originally proposed crystallization procedure14 always resulted into the isolation of a mixture of forms III and IV (PXRD evidence). Only by washing the precipitate with water after filtration enabled us to recover batches of pure form III (see Figure S3 in the SI for the low-angle portion of the PXRD pattern). A comment on the water content of this form is necessary at this stage. On the basis of a thermogravimetric analysis, form III was originally proposed as a hemihydrate:14 a two-step mass loss was observed by Sarbajna et al. in the range 47−145 °C, but only the second loss (3.4 wt %, 65−145 °C) was attributed to crystallization water, while the first one (6.6 wt %, 47−65 °C) was interpreted as the loss of surface moisture. The PXRD pattern reported by Flores et al.16 and attributed to “hydrated GCV” (to which they assigned the PXRD peaks at ca. 5, 10, 11, 14, 15, and 20° 2θ) slightly contaminated by “polymorph III” (the presence of which was inferred from the PXRD peaks at ca. 7 and 8° 2θ) can now be reinterpreted as belonging to form III, to which all of the observed peaks can be ascribed but the small one at ca. 7°, pertaining to form II. The TGA trace of Flores’ sample was characterized by a two-step mass loss (overall amounting to 2.5 wt %) in the temperature range 25− 190 °C, which is consistent with a water content of only 0.3 molecules of water per formula unit (f.u.). According to elemental analysis, our bulk phase contains two molecules of water per molecule of API. Our TGA, performed on the same batch 15 days later, is consistent with 1.4 molecules per f.u. (see section 3.3). On the other hand, structure determination from single crystal at 180 K suggests the presence of one water molecule per f.u., while the match between the observed and calculated PXRD patterns is satisfactory only if three water molecules per f.u. are introduced to describe the electronic density within the channels (section 3.2.2). Despite this difference in water content, in all of the cases form III was unambiguously identified using PXRD, as forms III, IV, and II show distinguishable diffraction patterns (Figure S3 in the SI). Hence, form III is a nonstoichiometric hydrate,46 the water content of which can vary without significant changes of the crystal structure. Incidentally, this evidence suggests that the two-step mass loss observed in the TGA trace reported by Sarbajna et al. could be due exclusively to the loss of crystallization water: if this was the case, they would have dealt with a batch of form III possessing 1.5 molecules of water per f.u. 4111



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moieties are engaged in π···π stacking interactions along the [001] direction between their pentagonal and hexagonal rings (distances between centromers 3.508(3) and 3.883(3) Å, Figure S5b in the SI). As in form III, also in IV the close packing of molecules generates 1-D channels parallel to the caxis hosting water molecules of crystallization (Figure S5c in the SI). Nevertheless, in the present case, the channels are considerably smaller than in form III: in IV, the empty volume, calculated with Mercury CSD 3.751 after removal of the water molecules, corresponds to only 4.2% of the unit cell volume with channels dimension of only 3.8 × 3.8 Å2. Comparing Figures 2c and S5c (in the SI) should help the reader appreciating this remarkable difference. The smaller dimension of the channels in IV is consistent with the small temperature range covered for the complete release of the water molecules of crystallization, as observed by TGA by Sarbajna et al.14 In the channels of IV, the water molecules take part in an intricate net of hydrogen bonds with some oxygen atoms of the ganciclovir molecule (Table S4 in the SI). 3.2.4. Crystal and Molecular Structure of Ganciclovir Hydrochloride (GCV·HCl). Ganciclovir hydrochloride crystallizes in the triclinic space group P1.̅ The asymmetric unit contains one protonated API molecule and one chloride anion, in general position. The crystal structure is dominated by the presence of an intricate net of hydrogen bonds, as depicted in Figure 3a (see also Table S5 of the SI). Two organic molecules close pack forming a tubular centrosymmetric dimer: while the guanine moieties define the longest edges of this motif, the 1,3dihydroxypropan-2-yloxy branches define the shortest ones. The intermolecular interactions at work within this dimer are one hydrogen bond between N1 and O4 [N1···O4xvii 2.830(8) Å, N1−H1X···O4xvii 158°; (xvii) −x, 2 − y, −z] plus π···π stacking interactions between the hexagonal rings [Figure 3b; distance between centromers 3.573(3) Å]. Adjacent dimers interact, along the [001] direction, by means of hydrogen bonds mediated by the chloride anions, forming a 2-D slab. The most remarkable graph set motif within this hydrogen bond net is a ring of the type R46(12)49 (highlighted in violet in Figure 3a), involving two chloride ions, two NH2 groups, and two OH groups (Table S5 of the SI). No strong intermolecular interactions are present between adjacent slabs along the [010] direction (Figure 3b). As anticipated in section 2.7, protonation occurs at N7, as witnessed by the short N7···Cl distance of 3.10(1) Å. 3.2.5. Crystal and Molecular Structure of Compound GCVA2. Compound GCVA2 crystallizes in the monoclinic space group P21/c. The asymmetric unit contains one molecule of GCVA2. Remarkably, the crystal structure of this compound shares, with forms II, III, and IV, the 1-D chain of hydrogen bonds between adjacent guanine moieties, describing a graph set motif of the type R22(9)49 (Figure 3a can be adopted as a good model; see also Table S6 in the SI). Notably, in spite of the similarity of the 1-D motif described by the guanine moieties in II, III, IV, and GCVA2 (Figures 2, 3a, and S5a in the SI), there is a certain variability in the disposition of the 1,3dihydroxypropan-2-yloxy arm, as can be qualitatively appreciated from Figure 6 and, in a more quantitative manner, from the values of the torsion angles τ1−τ9 (Chart S1 and Table S1 in the SI). This occurrence, inter alia, concurs to define the different shape and dimension of the 1-D channels in III vs IV. Back to GCVA2, other hydrogen bonds are at work between the N2(H) group of one molecule and the oxygen atom (O5) of the carbonyl group belonging to a neighboring molecule

Figure 1. Representation of a portion of the crystal structure of form II, showing the 1-D hydrogen-bonded chain running parallel to the [010] direction. The hydrogen bond interactions have been depicted with yellow dashed lines. Symmetry codes: (i) −x, −1/2 + y, −z; (ii) 2 − x, −1/2 + y, 1 − z. See Table S2 in the SI for details upon hydrogen bond distances and angles.

general positions. In spite of the different space group, forms II and III share the same arrangement of guanine moieties along 1-D zigzag chains (Figure 2a)though, in the case of III, they run parallel to the [100] direction. As a matter of fact, also in form III neighboring API molecules interact by means of hydrogen bonds (Table S3 in the SI) forming a graph set motif of type R22(9).49 The hydrogen bond between N2 and O2W [N2···O2Wv 2.93(4) Å, N2−H2X···O2Wv 124°; (v) 1 + x, y, z] mimics the N2···O4 interaction present in form II [N2···O4ii 3.085(11) Å, N2−H2X···O4ii 141°; (ii) 2 − x, −1/2 + y, 1 − z]. Inter alia, this hydrogen bond is bifurcated, as N2 shares its hydrogen atom H2X also with an oxygen atom (O4) belonging to the pendant arm of a neighboring molecule [N2···O4vi 3.05(3) Å, N2−H2X···O4vi 140°; (vi) 1.5 − x, y, −1/2 + z]. Along the [001] direction, adjacent guanine moieties mutually interact via π···π stacking between their pentagonal and hexagonal rings [distances between centromers alternatively 3.66(3) and 3.42(3) Å, Figure 2b].50 The close packing of individual molecules leads to the formation of 1-D ellipsoidal channels running parallel to the [001] direction and occupied by water molecules (Figure 2c). The channels correspond to 16.7% (465 Å3) of the unit cell volume and have dimensions of approximately 4.3 × 13 Å2 (calculated with Mercury CSD 3.751 after removal of the water molecules). On the basis of this structural feature, the variable water content, and the rather ample temperature range covered for the complete release of the water molecules of crystallization (as observed by means of TGA by Sarbajna et al.14 and by us; see section 3.3), III can be classified as a nonstoichiometric channel hydrate. Within the channels, the water molecules are arranged in a quasi-planar tape perpendicular to the [100] direction (Figure S4 and Table S3 in the SI) and are involved in hydrogen bonds of different strength with each other and with the atoms N2 and O3P of the pendent arm of the API (not shown; see Table S3). 3.2.3. Crystal and Molecular Structure of Form IV. Ganciclovir form IV crystallizes in the orthorhombic space group Pbca. The asymmetric unit is composed of one molecule of API and one water molecule of crystallization in general positions. Form IV shares with forms II and III the same arrangement of guanine moieties along the 1-D zigzag chains (Figure S5a and Table S4 in the SI) though, in form IV, the chains run parallel to the [010] direction. Adjacent guanine 4112



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Figure 2. Representation of the crystal structure of form III, as retrieved by PXRD. For the sake of clarity, only one of the two conformations adopted by the 1,3-dihydroxypropan-2-yloxy branch has been depicted. (a) Portion of the 1-D hydrogen-bonded chain running along the [100] direction. The hydrogen bond interactions have been depicted with yellow dashed lines. (b) View, along the [100] direction, of the π···π stacking interactions present between adjacent guanine moieties along the [001] direction. The violet dashed lines have been added to guide the eye. (c) Crystal packing, viewed in perspective along the [001] direction, emphasizing the 1-D channels hosting water of crystallization. Symmetry codes: (iv) 1/2 + x, 1 − y, 1.5 − z; (v) 1 + x, y, z; (vi) 1.5 − x, y, −1/2 + z. See Tables S3 and S8 in the SI for details upon supramolecular interaction distances and angles.

R22(7)49 (Figure 4a). Although the guanine moieties stack parallel to the [001] direction (Figure 4b), π···π interactions are not present due to an unfavorable arrangement of the molecules. 3.2.6. Crystal and Molecular Structure of Compound GCVA3. Compound GCVA3 crystallizes in the monoclinic space group P21/c, with one molecule in the asymmetric unit.

(not shown, see Table S6 in the SI). The other carbonyl moiety is engaged (through O6) in a weak C−H···O interaction [C12···O6xxv 3.311(8) Å, C12−H12A···O6xxv 141°; (xxv) 2 − x, −1/2 + y, 1/2 + z] with an OCH group from a neighboring branch. Closely, another weak C−H···O interaction [C17xxv ··· O3 3.456(9) Å, C17xxv−H17Cxxv···O3 157°; (xxv) 2 − x, −1/2 + y, 1/2 + z] is present forming a graph set motif of the type 4113



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Figure 3. Representation of the crystal structure of GCV·HCl. (a) Detail of the intricate net of hydrogen bonds (yellow dashed lines) and π···π stacking interactions (violet dashed lines) viewed along the [21̅0] direction. (b) Crystal packing, viewed in perspective along the [010] direction, showing the dimers formed by adjacent API molecules. Symmetry codes: (xvii) −x, 2 − y, −z; (xviii) −x, 3 − y, −z; (xix) x, 1 + y, z; (xx) x, y, −1 + z. See Tables S5 and S8 in the SI for details on supramolecular interaction distances and angles.

the melting point of GCVA3 was reported as 175 °C by Boryski and Golankiewicz53). The TGA of form III was carried out on the same batch employed for the elemental analysis, but 15 days later. The TGA trace of III (Figure 6) shows a mass loss of 9% in the temperature range of 30−110 °C, which can be interpreted as the loss of 1.4 molecules per f.u. The difference in water content for the same batch at different stages further supports the nonstoichiometric nature of this hydrate. The endothermic peak centered at 184 °C can be ascribed to a phase transition toward form II (which is consistent with the observations of Sarbajna et al.14 and Flores et al.16), the melting of which begins at 253 °C. To complement these observations, a VT-PXRD experiment was carried out, in air, by heating a batch of form IV contaminated by form III (13 wt %) (Figure 8, cyan patterns). As already observed,14 at 70 °C form IV starts undergoing a phase transition (Figure 8, orange patterns). The growing phase, originally interpreted as form III,14 is indeed a new phase (form V in the following), the PXRD pattern of which is clearly distinguishable from that of III. Further rising of the temperature promotes the progressive transformation of IV

Similarly to forms II, III, IV, and GCVA2, adjacent guanine moieties form hydrogen-bonded 1-D chains (Table S7 of the SI). However, due to the lower number of acidic hydrogen atoms, the spatial arrangement of the molecules is different (Figure 5a). The oxygen atom (O7) of the carbonyl group is involved in one intra- and one intermolecular interactions with two nearby N−H moieties. As in compound GCVA2, also in GCVA3 one weak C−H···O interaction, involving one ester group, is present (not shown; C11···O5xxvii 3.281(2) Å, C11− H11···O5xxvii 131°; (xxvii) x, 2.5 − y, 1/2 + z). The packing forces induce an alternation of the pending arms along the [010] direction (Figure 4b). Once again, π···π interactions are not at work. 3.3. Thermal Behavior. The results of the thermogravimetric analysis performed on form IV agree with those previously reported.14 According to the TGA traces (Figure S6 in the SI), GCV·HCl, GCVA2, and GCVA3 do not undergo phase transitions up to the melting point (163−240 °C, melting overlapping with decomposition, 235−243 °C and 173−182 °C, respectively. Previously, the melting point of GCVA2 was assessed in the temperature range of 237−239 °C,52 whereas 4114



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Figure 4. Representation of the crystal structure of GCVA2. (a) Weak C−H···O supramolecular interactions (cyan dashed lines), viewed along the [101] direction. (b) The crystal packing, viewed along the [010] direction. Symmetry code: (xxv) 2 − x, −1/2 + y, 1/2 − z. See Table S6 in the SI for details upon supramolecular interaction distances and angles.

4. CONCLUSIONS In this work, we have described the rich crystal chemistry of ganciclovir. In the absence of single crystals of suitable quality, we extensively resorted to PXRD to unveil the structural features of a nonstoichiometric (form III) and a stoichiometric hydrate (form IV) of GCV, the hitherto unknown hydrochloride (GCV·HCl), the di-O-acetyl (GCVA2) and the triN2,O,O- acetyl (GCVA3) pro-drug precursors. Coupling the results of our thermal analysis and variable-temperature PXRD to information previously reported enabled us to individuate the transformation paths among the hydrated and anhydrous forms, as well as to trap a metastable, previously unknown form (form V). Overall, this study reviews and complements the somehow incomplete data appeared on this system up to now, paving the way to straightforward PXRD qualitative and quantitative analyses of ganciclovir mixtures formed during synthesis, isolation, or processing. Indeed, in the academic and industrial realms, many are the analytical techniques currently

into V (Figure 8, orange patterns); the transformation is complete at 140 °C. Form III is expected to disappear at about 84 °C, as observed through DSC by Sarbajna et al.14 Here, the disappearance of the characteristic peak of form III at 5° 2θ is concomitant to the appearance and growth of a peak of form V just a few tenths of degrees before. Form V survives up to 180 °C (Figure 8, dark red patterns) as, at 190 °C, form II appears. The transformation V-to-II is complete at 210 °C (Figure 8, green patterns). Incidentally, a deep analysis of the previously reported DSC traces14 of forms III and IV enabled us to individuate a very small endothermic peak (amounting to less than 10 kJ/mol) which could be interpreted as the transformation into form V. Worthy of note, if left under ambient conditions for half a day, form V undergoes a phase transformation into III (PXRD evidence). As a consequence, and also based on the low crystallinity of the batches of form V, we isolated, no structural characterization could be attempted, either at ambient conditions, or at high temperature. 4115



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Figure 5. Representation of the crystal structure of compound GCVA3. (a) Portion of the hydrogen bonding network (yellow dashed lines) viewed along the [100] direction. (b) Crystal packing viewed in perspective along the [001] direction. Symmetry code: (xxvi) x, 1.5 − y, 1/2 + z. See Table S7 in the SI for details upon supramolecular interaction distances and angles.

Figure 6. Comparison of the disposition adopted by the 1,3dihydroxypropan-2-yloxy branch with respect to the mean plane of the guanine moiety, in III (yellow), IV (fuchsia), GCV·HCl (red), GCVA2 (blue), and GCVA3 (green).

Figure 7. TGA (green) and DSC (blue) traces for form III: the mass loss of 9.0% in the rather wide temperature range of 30−110 °C is due to the loss of 1.4 molecules of water per molecule of API. The endothermic peak centered at 184 °C is due to the phase transformation into form II. The exothermic peak centered at 256 °C is due to melting of form II, immediately followed by decomposition.

employed to characterize, at the molecular level, the polymorphs of a given API, namely, solubility tests, thermal analysis, microscopy, NMR and (FT)IR spectroscopy. Yet, PXRD provides a more complete landscape, at both the crystal and molecular level, allowing for a more accurate identification and quantification of polymorphic forms. 4116



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thank Fundaçaõ para a Ciência e a Tecnologia (FCT, Portugal), the European Union, QREN, FEDER through Programa Operacional Factores de Competitividade (COMPETE), and CICECO-Aveiro Institute of Materials, POCI-01-0145FEDER-007679 (FCT ref. UID/CTM/50011/2013), financed by national funds through the FCT/MEC and when appropriate cofinanced by FEDER under the PT2020 Partnership Agreement. FCT is also gratefully acknowledged for the Ph.D. Grant No. SFRH/BD/84231/2012 (for R.F.M.).

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(1) Faulds, D.; Heel, R. C. Drugs 1990, 39 (4), 597−638. (2) Crumpacker, C. S. N. Engl. J. Med. 1996, 335 (10), 721−729. (3) Wilhelmus, K. R. Cochrane Database Syst. Rev. 2010, No. 12, CD002898. (4) Bernstein, J. Cryst. Growth Des. 2011, 11 (3), 632−650. (5) Brittain, H. G. Polymorphism in Pharmaceutical Solids; Informa Healthcare: New York, 2009. (6) Bernstein, J. Polymorphism in Molecular Crystals; International Union of Crystallography: Oxford, 2007. (7) Polymorphism: in the Pharmaceutical Industry; Hilfiker, R., Ed.; Wiley-VCH: Weinheim, 2006. (8) Desiraju, G. R. J. Chem. Sci. 2010, 122 (5), 667−675. (9) Cabri, W.; Ghetti, P.; Pozzi, G.; Alpegiani, M. Org. Process Res. Dev. 2007, 11 (1), 64−72. (10) Bernstein, J. Polymorphism and Patents from a Chemist’s Point of View [1], Polymorphism: in the Pharmaceutical Industry; Hilfiker, R., Ed.; Wiley-VCH: Weinhem, 2006, pp 365−384. (11) Cruz-Cabeza, A. J.; Reutzel-Edens, S. M.; Bernstein, J. Chem. Soc. Rev. 2015, 44 (23), 8619−8635. (12) Griesser, U. J. The Importance of Solvates, Polymorphism: in the Pharmaceutical Industry; Hilfiker, R., Ed.; Wiley-VCH: Weinheim, 2006; pp 211−233. (13) Verheyden, J. P.; Martin, J. C. 9-(1,3-Dihydroxy-2propoxymethyl)guanine as antiviral agent. US4355032, 1982. (14) Sarbajna, R. M.; Preetam, A.; Devi, A. S.; Suryanarayana, M. V.; Sethi, M.; Dutta, D. Mol. Cryst. Liq. Cryst. 2011, 537, 141−154. (15) Chan, T. W.; Nguyen, H. T. Anhydrous crystalline 9-(1,3dihydroxy-2-propoxymethyl)guanine. US4642346, 1987. (16) Flores, R. L. R.; Á vila, S. G.; Oliveira, J. F.; Carvalho, F. M. S.; Araújo, G. L. B.; Matos, J. R. In Caracterização Fisico-Quı ́mica e Analı ́tica do Ganciclovir; Abratec: Serra Negra, 2014. (17) Parthasaradhi, R. B.; Rathnakar, R. K.; Muralidhara, R. D.; Raji, R. R.; Vamsi, K. B.; Subash, C. R. K.; Venkata, R. R. I. Process for the isolation of ganciclovir intermediate. WO20111114336A1, 2011. (18) Kawamura, T.; Hirayama, N. X-Ray Struct. Anal. Online 2009, 25, 51−52. (19) Salari, A.; Young, R. E. Int. J. Pharm. 1998, 163, 157−166. (20) Mao, W.; Wu, P. Unhydrous crystal form of ganciclovir sodium and preparation method thereof. CN 103570716, 2013. (21) Mao, W.; Wu, P.; Zhang, Q.; Cui, J. Crystal type ganciclovir sodium hydrate and preparing method thereof. CN103524505, 2014. (22) Volante, P.; Michelini, P. Ganciclovir sodium salt in crystalline form. WO/111049 A1, 2003. (23) Fernandes, J. A.; Sardo, M.; Mafra, L.; Choquesillo-Lazarte, D.; Masciocchi, N. Cryst. Growth Des. 2015, 15 (8), 3674−3683. (24) Vladiskovic, C.; Masciocchi, N. J. Pharm. Biomed. Anal. 2015, 107, 394−402. (25) Giacobbe, C.; Palmisano, G.; Giovenzana, G. B.; Giovannelli, L.; Negri, R.; Masciocchi, N. J. Pharm. Biomed. Anal. 2015, 102, 305−313. (26) Vladiskovic, C.; Masciocchi, N. Cryst. Growth Des. 2014, 14 (7), 3603−3611. (27) Vladiskovic, C.; Masciocchi, N.; Cervellino, A. J. Pharm. Sci. 2012, 101 (12), 4481−4489. (28) Barrio, M.; Maccaroni, E.; Rietveld, I. B.; Malpezzi, L.; Masciocchi, N.; Céolin, R.; Tamarit, J. J. Pharm. Sci. 2012, 101 (3), 1073−1078.

Figure 8. PXRD patterns acquired during the VT-PXRD experiment carried out in the temperature range of 20−240 °C, on a batch of form IV (contaminated by form III; see the peak indicated by an asterisk). Cyan patterns, 20−60 °C: form IV contaminated by form III. Orange patterns, 70−140 °C: copresence of forms III, IV, and V. Dark red patterns, 150−180 °C: pure phase V. Green patterns, 190−210 °C: copresence of forms V and II. Fuchsia patterns, 220−240 °C: pure phase II. The peak marked by an asterisk (belonging to form III) disappears between 70 and 150 °C, concomitant to the formation and growth of a peak of form V centered at a slightly lower angle. This is in agreement with the DSC data reported by Sarbajna et al.,14 who observed loss of water with an endothermic peak centered at 84 °C (onset near 60 °C).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.6b00617. Final Rietveld refinement plots for GCV form III, GCV form IV, GCV·HCl, GCVA2, and GCVA3; graphical representation of the crystal structure of GCV form IV; actual values of selected torsion angles of the molecule in GCV form III, GCV form IV, GCV·HCl, GCVA2, and GCVA3; selected hydrogen bond distances and angles for GCV form III, GCV form IV, GCV·HCl, GCVA2, and GCVA3; TGA and DSC traces for GCV·HCl, GCVA2, and GCVA3 (PDF) Accession Codes

CCDC 1475569−1475573 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The University of Insubria is acknowledged for providing a grant (Junior Assignee) to J.A.F. Angelo Maspero and Alessandro Cimino (University of Insubria) are acknowledged for performing part of the analytical measurements. We wish to 4117



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(29) Maccaroni, E.; Giovenzana, G. B.; Palmisano, G.; Botta, D.; Volante, P.; Masciocchi, N. Steroids 2009, 74 (1), 102−111. (30) Moreno-Calvo, E.; Muntó, M.; Wurst, K.; Ventosa, N.; Masciocchi, N.; Veciana, J. Mol. Pharmaceutics 2011, 8 (2), 395−404. (31) Colombo, V.; Masciocchi, N.; Palmisano, G. J. Pharm. Sci. 2014, 103 (11), 3641−3647. (32) Kottke, T.; Stalke, D. J. Appl. Crystallogr. 1993, 26, 615−619. (33) APEX2. Data Collection Software, Version 21-RC13; Bruker AXS: Delft, 2006. (34) SAINT+. Data Integration Engine v 723a; Bruker AXS: Madison, Wisconsin, USA. (35) Sheldrick, G. M. SADABS v201, Bruker/Siemens Area Detector Absorption Correction Program; Bruker AXS: Madison, Wisconsin, USA, 1998. (36) Sheldrick, G. M. Acta Crystallogr., Sect. A: Found. Crystallogr. 2008, 64, 112−122. (37) Sheldrick, G. M. Acta Crystallogr., Sect. C: Struct. Chem. 2015, 71 (1), 3−8. (38) Coelho, A. A. J. Appl. Crystallogr. 2003, 36 (1), 86−95. (39) TOPAS-R, version 3.0, Bruker, Karlsruhe, Germany 2005. (40) Kleywegt, G. J.; Jones, T. A. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1998, 54 (6), 1119−1131. (41) Jang, Y. H.; Noyes, K. T.; Sowers, L. C.; Goddard, W. A., III; Hwang, S.; Chung, D. S. J. Phys. Chem. B 2003, 107, 344−357. (42) Cheary, R. W.; Coelho, A. J. Appl. Crystallogr. 1992, 25 (2), 109−121. (43) March, A. Z. Kristallogr. - Cryst. Mater. 1932, 81, 285−297. (44) Dollase, W. A. J. Appl. Crystallogr. 1986, 19 (4), 267−272. (45) When comparing the TGA and VT-PXRD results, the reader must be aware that the thermocouple of the VT-PXRD setup is not in direct contact with the sample, this determining a slight difference in the temperature at which the same event is detected by the two techniques. The TGA temperatures have to be considered more reliable. (46) Morris, K. R. In Polymorphism in Pharmaceutical Solids; Brittain, H. G., Ed.; Marcel Dekker: New York, 1999; pp 125−181. (47) For the sake of completeness, we add here that, based on the peak position maxima reported in ref 20, we can propose a tentative space group and unit cell parameters for the anhydrous monosodium salt: triclinic, P1̅, a = 7.32 Å, b = 7.87 Å, c = 10.80 Å, α = 100.3°, β = 108.6°, γ = 105.1°, V = 545.3 Å3, Z = 2, ρ = 1.69 g/cm3. Because of the paucity of peak position maxima in ref 21, we cannot suggest a tentative unit cell for the hydrated monosodium salt. (48) The labels of the atoms reported in ref 18 have been changed in order allow direct comparison with the results presented in this work. (49) Grell, J.; Bernstein, J.; Tinhofer, G. Acta Crystallogr., Sect. B: Struct. Sci. 1999, 55 (6), 1030−1043. (50) The existence of hydrogen bonded chains along the b axis and π−π stacking along the c axis, vs water molecules-mediated hydrogen bond interactions between the chains along the a axis, might concur to explain the existence of preferred orientation along the [100] direction. (51) Macrae, C. F.; Edgington, P. R.; McCabe, P.; Pidcock, E.; Shields, G. P.; Taylor, M.; van de Streek, J.; Towler, M. J. Appl. Crystallogr. 2006, 39, 453−457. (52) Martin, J. C.; Tippie, M. A.; McGee, D. P.; Verheyden, J. P. J. Pharm. Sci. 1987, 76, 180−184. (53) Boryski, J.; Golankiewicz, B. Synthesis 1999, 625−628.

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