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Crystal Structure of Levofloxacin Anhydrates: A High-Temperature Powder X-ray Diffraction Study Versus Crystal Structure Prediction Jennifer T.J. Freitas, Cristiane C. de Melo, Olímpia M. M. S. Viana, Fabio F. Ferreira, and Antonio C. Doriguetto Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.8b00363 • Publication Date (Web): 09 May 2018 Downloaded from http://pubs.acs.org on May 9, 2018

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Crystal Growth & Design

Crystal Structure of Levofloxacin Anhydrates: A High-Temperature Powder X-ray Diffraction Study Versus Crystal Structure Prediction Jennifer T. J. Freitas,† Cristiane C. de Melo, †,§ Olímpia M. M. S. Viana,† Fabio F. Ferreira, *,‡ Antonio C. Doriguetto*,†,§ †

Faculty of Pharmaceutical Sciences, Federal University of Alfenas; Rua Gabriel Monteiro da Silva, 700, Alfenas-MG, 37130-000, Brazil §

Institute of Chemistry, Federal University of Alfenas, Rua Gabriel Monteiro da Silva, 700, Alfenas-MG, 37130-001, Brazil

‡

Center for Natural and Human Sciences (CCNH), Federal University of ABC (UFABC), Av. dos Estados, 5001, Santo André-SP, 09210-580, Brazil

KEYWORDS. Levofloxacin. Dehydration. Variable-temperature PXRD. Thermal analysis.

ABSTRACT. Levofloxacin, the levo-isomer of the antibiotic ofloxacin, can crystallize into two different hydrate forms, the monohydrate and the hemihydrate. Although these forms are well

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characterized, little is known about their anhydrous structures besides the study of Singh and Thakur using crystal structure prediction. In order to solve this issue, this paper proposes the use of high-temperature powder X-ray diffraction to determine the anhydrous structures of the levofloxacin forms termed in the literature as α and γ, herein obtained by heating of the levofloxacin monohydrate and hemihydrate, respectively. These are the first levofloxacin anhydrates whose structures could be solved. To support our results, the dehydration process has been also evaluated by means of differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and hot-stage polarized microscopy (HSM). The resulting X-ray structures differ from the ones reported as the best-predicted solution, although the predicted work had predicted similar structures as lower ranked solutions.

1. INTRODUCTION Fluoroquinolones, including levofloxacin (Scheme 1), are recognized as effective therapeutic agents for the treatment of community-acquired pneumonia, complicated urinary tract infections, acute pyelonephritis, skin and tissue infections and nosocomial pneumonia.1-3 Compared with earlier quinolones, levofloxacin possesses a broad-spectrum of applications that includes not only gram-negative but also gram-positive and atypical pathogens. In addition, the antibacterial activity of levofloxacin is much higher than ofloxacin, since the levo-isomer is reportedly 8-128 times more active than the dextro-isomer of the racemic mixture.2 Levofloxacin and other fluoroquinolones act through inhibition of bacterial DNA topoisomerase II, also called DNA gyrase, an essential enzyme for the synthesis of bacterial mRNAs (transcription) and DNA replication.3


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Scheme 1. Molecular structure of levofloxacin with the adopted number scheme. Levofloxacin is found in two crystalline hydrate forms with determined crystal structures: the hemihydrate (C18H20FN3O4·1/2H2O)4-6 and the monohydrate (C18H20FN3O4·1H2O).4,6 The crystal structures of seven carboxylic acid salts,6,7 one magnesium salt dihydrate8 and one anhydrous perchlorate salt9 containing levofloxacin are also known. In addition, at least six solvated forms10 and one anhydrous form11 have been claimed in patents supported by powder X-ray diffraction, thermogravimetric, differential scanning calorimetry and infrared spectra data. Besides, four anhydrous polymorphic forms (α, β, γ, and δ) have been observed from the dehydration of the two known hydrated forms.4,5 The crystal structure of the chiral ofloxacin is also known.12,13 However, it is important to emphasize that levofloxacin is the pure (S)-(−)-enantiomer of the drug ofloxacin, which is marketed as another quinolone agent in an equimolar mixture of enantiomers. This large number of crystal forms of levofloxacin is relevant, since from a pharmaceutical perspective they can also be subject of commercialization and patent protection if they follow the patentability requirements: novelty, inventive step and industrial applicability.14 Levofloxacin is administered orally as its hemihydrate form (LH), which was approved by FDA


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in 1996 under the original brand name Levaquin, and nowadays has been marketed worldwide under several others trade names, e.g. Advaquin, Tavanic, Levomed, and Novotic.1,3 Although the literature mentions the existence of at least four levofloxacin anhydrous polymorphic forms, none of their crystal structures have been determined so far. This lack of information is due to the challenge of obtaining single crystals or pure polycrystalline phases of the anhydrous forms by recrystallization in solvent, as well as, the probable interconversion of the dehydrated samples to the original levofloxacin hydrate forms when they are returned to room temperature. To solve this issue, Singh and Thakur6 have employed a Crystal Structure Prediction (CSP) approach as an alternative method to obtain structural information of the levofloxacin anhydrous forms. One of the best predicted structures obtained from the CSP was pointed as the closest match to the experimental PXRD patterns previously attributed to the α form.6 However, no attempt was made to assign the others ranked solutions to the remaining anhydrous levofloxacin forms (β, γ, and δ). Therefore, the challenge of elucidating the levofloxacin anhydrous structures obtained from the dehydration of its hydrated forms has not yet been completely overcome. Besides that, there are still problems inherent to the use of CSP. The first is the search for stable structures in a multidimensional energy landscape, and the second and more difficult to solve, their ranking. Since crystallization is a process dominated by both thermodynamic and kinetic parameters, one of the major concerns is what criteria should be used for selecting the correct structure from the many others that are produced during CSP.15-16 Considering that experimental data are still necessary to prove the accuracy of CSP, we present here a variable-temperature PXRD study for elucidating the anhydrous levofloxacin structures obtained by heating the LH and LM. Additionally, the dehydration process has been also


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evaluated by means of differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and hot-stage polarized microscopy (HSM).

2. EXPERIMENTAL SECTION 2.1. Samples. LH was purchased from Sigma-Aldrich and Fagron and used as received. LM was obtained by recrystallization of LH from a solution of acetonitrile and water in accordance with the methodology described by Gorman et al.5 2.2. Thermal Analysis. Differential Scanning Calorimetry (DSC) curves were acquired using a TA Instruments Q100 model. The experiments were run in N2 flow (50 mL min-1). The samples (8 ± 1 mg) were placed in open aluminum pans and heated at a rate of 10 °C min-1 over a temperature range of 30-260 °C. Thermogravimetric analysis (TGA) was performed with a TA Instruments Q600, under N2 flow (50 mL min-1). The samples (≈ 10 mg) were placed in open alumina pans and heated from 25 to 600 °C at a rate of 10 °C min-1. 2.3. Hot-Stage Polarized Optical Microscopy (HSM). HSM experiments were performed on a Linkam LTS420 thermal stage coupled to a Leica DM2700M optical microscope. Single crystals of levofloxacin samples were heated at a constant rate of 10 °C min-1 over a temperature range from 30 °C until the melting of the crystals. Images were recorded using a CCD camera attached to the microscope at time intervals of 10 s. 2.4. Powder X-ray Diffraction. In situ powder X-ray diffraction (PXRD) data were collected for LH and LM samples at 25, 150, 160, 170, 180, 190 °C using a heating/cooling rate of 10 °C min-1. The heating/cooling protocol was as follow: The samples were heated from 25 (after the 1st PXRD measurement) to 150 °C (2nd PXRD measurement), cooled again to 25 °C (3rd PXRD measurement) and then heated to 150 (4th PXRD measurement), 160 (5th PXRD measurement),


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170 (6th PXRD measurement), 180 (7th PXRD measurement) and 190 °C (8th PXRD measurement). PXRD data were collected using STADI-P powder diffractometer (Stoe®, Darmstadt, Germany) equipped with a Stoe® furnace (model 0.653) in Debye-Scherrer geometry by using a CuKα1 (λ = 1.54056 Å) wavelength selected by a curved Ge(111) crystal, with a tube voltage of 40 kV and a current of 40 mA. The samples were hand-grinded in an agate mortar and then packed into 0.5-mm quartz capillaries (Hilgenberg®, Malsfeld, Germany), which were held spinning during data collection. The diffracted intensities were collected by a silicon microstrip detector, Mythen 1K (Dectris®, Baden, Switzerland), in the 2θ range from 5.000° to 61.685°, with 150 s of integration time at each 3.15°, thus producing data files with 2θ step sizes of 0.015°. 2.5. Determination of the Anhydrous LH (γγ form) and LM (α form) Crystal Structures by PXRD. Initially, PXRD data at room temperature (25 °C) have been collected (1st PXRD measurement) to check the purity of the LH and LM samples crystallized here. Then, we used the high temperature PXRD data collected at 150 °C for the LH and at 190 °C for the LM (the choice of a higher temperature for the LM is discussed further below) to determine their respective anhydrous structures. The first 20 reflections of the X-ray diffractograms were considered to index the patterns making use of the software TOPAS-Academic v.6.17 The peaks in this region were adjusted without considering corrections for the zero point of the diffractometer and the Lorentz-Polarization factor. After analyzing the systematic absences, we verified that the dehydrated LH (γ form) crystallizes in a monoclinic crystal system with space group C2, via a Pawley18 fit, with the follow unit cell parameters: a = 17.2789(5) Å, b = 7.0281(2) Å, c = 15.4632(7) Å, β = 104.172(3)°, and V = 1820.7(1) Å3. The same procedure was applied to the PXRD pattern of the dehydrated LM (α form), which yielded the following values


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for an orthorhombic unit cell with space group P212121: a = 15.6545(24) Å, b = 16.6850(15) Å, c = 6.8969(5) Å, and V = 1801.4(3) Å3. We used those values as input for the structure determination procedure, carried out by means of a simulated annealing procedure implemented into the DASH program.19 Detailed procedures may be found elsewhere.20-23 A 3D model of the levofloxacin molecule was created with the program MarvinSketch v. 17.13.0-7045, release year 2017 (http://www.chemaxon.com), which was then used in the simulated annealing procedure.2425

The full range of possible values of molecular positions and orientations as well as any flexible

torsion angles (three describing the positional coordinates, four, of which three are independent, describing the molecular orientation and two flexible torsion angles) were allowed to vary during the simulated annealing process. Fifteen runs (for a total of 3×108 movements) were globally optimized, and the best results were then considered in the final Rietveld refinement26-27of the structures using the program TOPAS-Academic v.6. Details about the refinement procedures may be found elsewhere.28 The molecular geometries (space group choice, unit cell parameters, bond lengths, angles and torsions) were verified with both the PLATON29 and MOGUL30 packages. The crystallographic information about the two dehydrated forms, such as the final unit cell parameters, goodness-of-fit indicator as well as R factors31 are shown in Error! Reference source not found..

3. RESULT AND DISCUSSION 3.1 Dehydration Solid-State Phase Transitions. LH and LM samples were characterized by thermal analysis (DSC, TGA/DTA), HSM and PXRD. In terms of thermal stability, previous studies have shown that the two levofloxacin hydrates present distinct behaviors. Kitaoka et al.4 have argued that the LH dehydration gives rise to a physical mixture of crystalline (α, β and γ)


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and amorphous forms, whereas the dehydration of the LM gives rise only to a single form (α). The authors have also discussed that the dehydration of the LH and LM occurs at comparable temperature range (50-90 °C) and that the resulting γ, β and α polymorphic forms melt at 227, 231, and 234 °C, respectively.4 More recently, Gorman et al.5 have observed a polymorphic enantiotropic conversion in the DSC around 54 °C involving the γ form and a new form denoted as δ, which was confirmed by Raman spectroscopy, PXRD, and solid-state NMR spectroscopy. Gorman et al.5 have also observed an exothermic event between the melting of the γ and β forms, which was attributed to the crystallization of the α or β forms from the melted γ. Therefore, Gorman et al.5 results contrast with the assumption that the dehydration of the LH results in a physical mixture of α, β and γ forms soon after the water released around 50-90 °C As expected, our DSC curves obtained for the LH and LM samples at a nitrogen atmosphere (Figure S1, Supporting Information) are similar to those ones reported in the literature.4,5 The TGA / DTA curves of the LH and LM samples (Figure S2, Supporting information) at a nitrogen showed the mass loss of 2.7% and 4.9%, respectively, after the endothermic dehydration events (DTA peaks at 78 and 72 °C, respectively) confirming the expected water:API stoichiometries. For a visual analysis of the thermal transformations occurring during the heating at synthetic air, hot-stage microscopy (HSM) was also carried out. The HSM microphotographs are shown in Figure 1. At room temperature and under polarized light, both LH and LM appear as colorless and birefringent single crystals. Further heating results in a loss of the birefringence, with the crystals becoming completely dark and opaque at about 100 °C, in agreement with the dehydration process. From these images, one can see that LH starts to melt (231 °C) at a temperature slightly lower than LM (237 °C), which are temperatures comparable to those ones observed in the DSC and TGA data.


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Figure 1. Hot-stage polarized microscopy of the LH and LM single crystals.

The experimental PXRD patterns of LH and LM samples (1st PXRD measurement at 25 °C prior the heating protocol) are depicted in Figures 2(a) and 2(b), respectively. The two hydrated forms were confirmed, since the experimental data show a good match (Rietveld refinement statistics criteria as well as a good visual fitting) with the theoretical ones calculated using as


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input the crystal structures (unit cell, space group and fractional coordinates) of the LH (CCDC code: YUJNUM02) and LM (CCDC code: YUJPAU01)6 forms deposited on CSD (Cambridge Structural Database).32 It is worth mentioning that the protocol of heating/cooling/heating (25 to 150 °C, cooled again to 25 °C and then reheated to 150, 160, 170, 180 and 190 °C) followed by in-situ PXRD measurements (See the experimental and Rietveld fitted patterns in Figures 2; S3 and S4 and the respective crystallographic data in Tables S1 and S2, Supporting Information) was established taking into account the data reported by Kitaoka et al.4 and Gorman et al.5 In fact, the LH and LM PXRD patterns measured after the dehydration do not match each other (Figures 2(c) and 2 (d)), which confirms that the anhydrous materials from LH and LM present different crystal structures. Moreover, the PXRD patterns of the dehydrate material obtained from LM and LH here are identical to those ones reported by Kitaoka et al.4 and Gorman et al.5 Therefore, adopting the literature labelling scheme,4,5 the anhydrous forms obtained here from LM and LH will be hereafter termed by α and γ, respectively.


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Figure 2. Rietveld analysis of the PXRD data from (a) LH and (b) LM at 25°C; and after their dehydration that result in the respective anhydrous (c) γ (at 150 °C) and α (at 190 °) forms.

Our PXRD data collected at 150 °C after the first heating of the LH in synthetic air (2nd PXRD measurement, Figure 2(c) and Table 1) shows that the γ form was obtained as a pure phase (without mixture of the α and β forms) in disagreement with Kitaoka et al.’s4 suggestion. Therefore, our results indicate that the α and β forms could be crystallized from the γ form and not directly from the LH during its dehydration processes. The PXRD pattern of the LH sample cooled again to 25 °C (3rd PXRD measurement, Figure S3(a), Supporting Information) besides


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showing the presence of characteristic peaks from the γ form as majority phase, also displays some extra peaks from another physical form as observed by Gorman et al.5 These authors have suggested that when the dehydrated levofloxacin material (pure γ form from LH) is cooled protected from moisture below 54 °C, the γ form converts to the δ form. The δ form, which structure remains unknown, was considered closely related to LH except to the water absence and changes in the conformation and orientation of the piperazine ring relative to the rest of the molecule.5 After the second heating cycle up to 150 °C (4th PXRD measurement, Figure S3(b), Supporting Information), once again the PXRD pattern indicates the γ form as a single phase. These results confirm the reversibility of the γ-δ phase transition. The Rietveld analyses of the PXRD patterns collected sequentially at 160, 170, 180 and 190 °C (Figure S3(c) – S3(f), Supporting Information) indicate that the γ form remained stable up to 190 °C. The Rietveld analysis of the PXRD pattern of the LM sample collected at 150 °C (2nd PXRD measurement, Figure S4(a), Supporting Information) shows that its dehydration can result in a binary physical mixture consisting of the α (~97%) and γ (~3%) forms (Table S2, Supporting Information). Alternatively, the LH can be a contaminant of the sample expected to have only the LM, which is respectively transformed into the γ and α forms during the dehydration. This coexistence of phases (without significant proportion change) is also observed in the PXRD patterns collected after the returning of the sample to 25 °C (3rd PXRD measurement, Figure S4(b), Supporting Information). This feature is kept when the sample is reheated at 150, 160, 170, and 180 °C (Figures S4(c) - S2(f), Supporting Information). Only in the PXRD pattern collected at 190 °C the α form appears as a single phase (Figures 2(d) which explain its choice for the crystal structure determination procedures (Table 1).


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Table 1. Crystal data and structure refinement parameters for the anhydrous levofloxacin structures.

Anhydrous LH (γ form)

Anhydrous LM (α form)

Empirical formula

C18H20FN3O4

C18H20FN3O4

Formula weight (g.mol−1)

361.37

361.37

Wavelength (λ)

1.54056

1.54056

Crystal system

Monoclinic

Orthorhombic

Space group

C2

P21 21 21

Temperature (°C)

150(2)

190(2)

a (Å)

17.2739(10)

16.6858(3)

b (Å)

7.0307(2)

15.6897(6)

c (Å)

15.4605(8)

6.88544(14)

α (o)

90

90

β (o)

104.204(5)

90

γ (o)

90

90

Volume (Å3)

1820.24(16)

1802.57(9)

Z, Z'

4,1

4,1

Density (calculated) (g cm-3)

1.31865(11)

1.33134(17)

Diffractometer

STADI-P, Stoe

STADI-P, Stoe

Monochromator

Ge(111)

Ge(111)

2θ range (o)

5-61.685

5-61.685


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Step size (o)

0.015

0.015

Time per 3.15o at 2θ (s)

150

150

Number of observations

3780

3780

Number of contributing reflections

333

364

Number of distance constraints

29

29

Structural parameters

105

103

Profile parameters

16

15

Rexp (%)

4.387

3.533

Rwp (%)

5.570

6.984

RBragg (%)

1.832

2.601

S

1.270

1.977

d-DW

0.700

0.427

CCDC number

1825455

1825456

Refinement

Number of refined parameters

Statistical parameters

3.2. Description of the Anhydrous LH (γγ form) crystal structure. This structure crystallizes in the monoclinic space group C2 with one levofloxacin molecule in the asymmetric unit (Z’ = 1) (Table 1). In spite of the differences in terms of number of independent molecules by symmetry in the asymmetric unit (LH also crystallizes in a C2 space group but with Z’ = 2) and unit cell dimensions (LH unit cell values: a = 29.127(3) Å, b = 6.8850(2) Å, c = 18.8490(8) Å, and β = 114.089(6)°)6, the packing of the host levofloxacin structure in the LH - if we consider the water as a guest molecule, is kept in the γ form (Figure 3). Indeed, the smaller unit cell of the γ form (Table 1) is related to the LH one by the matrix (0 0 1 / 0 1 0 / ½ 0 ½). In spite of the similar


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packing suggested by the perspective view depicted in Figure 3, the intermolecular chemical forces that stabilize the two structures are different due to the presence of the water molecules and two levofloxacin conformers in the LH against the water absence and just one levofloxacin conformer in the γ form.

Figure 3. Packing of the LH (left) and γ (right) form projected onto their respective ac planes. The matrix relating the two unit cells is also shown. The columnar staking with molecules related by the 21 screw axis along the unit cell b axis of the respective LH and γ form is highlighted. The two conformers present in LH are represented in blue and green and that one present in the γ form is represented in magenta. Hydrogen atoms were omitted for the sake of clarity. The two independent by symmetry water oxygen atoms of the LH are represented by yellow and red spheres.

The overlay of the single conformer (hereafter termed conformer C) present in the γ form with the two conformers (hereafter termed conformer ALH and BLH) present in the asymmetric unit of the LH is shown in Figure 4. As previously discussed by Gorman et al.,5 the conformers ALH and BLH differ by the torsion angle defined by the piperazine ring and the heterocyclic aromatic


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moiety. Now comparing them with that one determined for the γ form, it is observed that the main differences arise from the orientation of the methyl group (C13) in the oxazine ring and, as expected, the piperazine ring rotation around the C6-N2 bond (See Scheme 1 to help the atom labelling). In all structures, the oxazine ring is in a half-chair conformation; however, in the conformers ALH and BLH, the methyl group lies in the equatorial position, while in the conformer C it is axially positioned. Besides, it is observed a ring flipping since C12 atom is displaced upwards in the conformer C against downwards in both LH conformers. Moreover, they also differ with respect to the torsion angle defined by the atoms C5-C6-N2-C14 (probe the torsion between the piperazine ring and the aromatic planar moiety), which assumes the value of 133.2(5)°, 40.6(7)°, and 98(15)° in the conformers ALH, BLH and C, respectively. From another point of view (See Figure S5) it is observed that the piperazine and the aromatic planar moieties are rotated by ~45° but in opposite directions comparing the two LH conformers, whereas in the γ form the two moieties are rotated by ~90°. In addition, it is important to emphasize that the conformer C also differs from the others found in various crystalline forms of levofloxacin (maleate, malonate, oxalate and succinate)6 and from the eight conformers predicted by Singh and Thakur.6 To determine the most stable conformers, these authors have performed a screening, using relaxed potential energy surface (PES) scans of the torsion angle C5-C6-N2C14 for both axial and equatorial positions of the methyl group. In fact, the piperazine flexibility is associated to the dehydration process and with the loss of the water interactions. Studies of solid-state-NMR show that the piperazine ring undergoes dynamic motions once the water molecules are removed.5


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Figure 4. Views of an overlay matching the backbones of the conformers ALH (in green) and BLH (in blue) of the LH and the conformer C (in magenta) of the γ form. The structures were matched considering the planar bicyclic aromatic moiety.

As previously stated, in spite of the intramolecular differences, an analysis of the crystal packing reveals great similarities between the intermolecular geometries of the LH and the γ form. In the two structures, the levofloxacin molecules are stacked in helical columns along the b-axis (Figure 5). The stacked molecules are separated by half of the unit cell b-axis dimension of the respective structures, since they are related to each other by the 21 screw axis (introduced by the centering in C2 space group) parallel to b-axis. The three helical columns are stabilized by π-π interactions and non-classical H bonds. In the two independent by symmetry columns formed by the two conformers present in the LH, the π-π interaction (considering the rings centroids) are slightly stronger (~3.60 Å) than in the γ form (~3.96 Å). There is an in-common H-bond pattern having O3 as bifurcated H-bond acceptor to the C12 and C11 atoms stabilizing the two independent by symmetry columnar stacking of the LH. This pattern is not observed for the γ form, which has the O3 oxygen atom working as H bond acceptor to hydrogens of the methyl group (C13). The C13-H···O3 interaction explains in part the axial position of C13 highlighted in Figures 4 and S5, Supporting Information. On the other hand, there is no common


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H-bond pattern involving the carboxylic oxygen atoms comparing the three homologous columnar stacking (Figure 5). Each levofloxacin molecule in the columnar assembly formed by the conformer B in the LH is H bonded to neighboring levofloxacin molecules at the top and at the bottom using both carboxylic oxygen atoms – O1 and O2 are acceptor to C14 and C17, respectively. Differently, just O2 (having C17 as donor) or just O1 (having C14 as donor) help the stabilization of the columns formed by conformers A (in the LH) and C (in the γ form), respectively.


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Figure 5. Packing projection along the b-axis of the respective LH (columns built by the conformers ALH and BLH) and γ (column built by the conformers C) forms showing the intermolecular interactions operating in the stacking of the molecules. Hydrogen bonds and π···π interactions are highlighted by dotted cyan lines.


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The columns are themselves stacked along the respective [001] and [100] directions of the LH and γ form (Figure S6, Supporting Information). The columns formed by the conformers A and B in the LH are shifted by ~1.2 Å related to each other along the b-axis generating double layers separated by ~2.2 Å whether considering the upper and lower limit of neighbor double layers. On the other hand, no shift is observed for the γ form and the single layers are separated by 3.5154(1) Å (b/2). It is observed that C-H···F hydrogen bonds involving the methyl groups (C13) and the fluorine F1 atoms help the inter-columnar packing stabilization (Figure S6, Supporting Information). However, the way that one column is connected to two other ones is different when comparing the LH and the γ form. In the LH, a zig-zag chain H-bond pattern is formed along the [001] alternated by conformers ALH and BLH, whereas in the γ form the molecules are connected via cross-linked hydrogen bonds generating linear chains along [110] and [110]. The double (in the LH ) or single layer (in the γ form) features together with the CH···F bond patterns also explain the change of the equatorial conformation of C13 in the LH to the axial conformation in the γ form. If we consider the H bond patterns that stabilize the layers parallel to (010) plane in the LH and γ form, we can see that in the first the water molecules lie in a 2-fold axis and act as hydrogen bond donors, connecting two identical conformers through symmetrical interactions (OWAH···N3A and OWB-H···N3B) involving the nitrogen N3 atom of the piperazine rings (Figure 6). Additionally, the conformers ALH and BLH interact with each other along the c-axis via an R (16) homodimer33 formed by non-classical C-H···O hydrogen bonds (Table S3, Supporting Information). These interactions together with the C-H…F ones, which in turn define an R (8) homodimer, give rise to columnar layers parallel to the (010) plane. Actually, in the case of the LH double layers are created as previously discussed (see Figure S6, Supporting Information). In


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the γ form, the drug molecules from the adjacent columns interact by forming R (14) homodimers instead of R (16) ones. The reduction of the set-graph ring dimension is due to the occurrence of the C11-H···O2 interaction instead of the C13-H···O2 as observed in the LH packing (see Figure 6 inset). The homodimer R (8) is absent in the supramolecular assembly of γ form, in contrast to the observed for the LH. The resulting 3-D assembly is characterized by a close interlocking of the piperazine rings along the a-axis via van der Waals interactions.


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Figure 6. (a) View of the LH onto the ac plane showing the 2-D infinite network stabilized by intermolecular H bonds involving levofloxacin and water molecules. The homodimers R (16) and R (8) linking different conformers (···A···B···A···) and the water molecule linking the equivalent conformers (A···WA···A or B···WB···B) are highlighted. (b) View of the γ form onto the ac plane showing that the homodimer R (14) (instead R (16) as in LH) are not connected to each other by hydrogen bonds and thus breaking the 2-D hydrogen bond network observed for LH. The inset in the γ form packing highlights the set-graph ring dimension reduction due to the occurrence of the C11-H···O2 interaction instead of the C13-H···O2 as observed in the LH.


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Since the water loss implies a previous loss of the O-H···N interactions, the drug molecules, in particular the piperazine rings, have more freedom to move and occupy the water voids. For proving that, a search for possible voids has been carried out via Mercury software using a probe sphere of radius 1.2 Å. The calculated volume (Å3) of solvent accessible voids in the γ form corresponds to 2.9% of all unit cell volume, much less than the value found for the LH after the water-removing (ca. Å3, equal to 3.2% of the cell volume). In all cases (Figure S7, Supporting Information), these voids are not continuous, what means they are not connecting as a channel. In the LH, however, they are placed between the piperazine rings and the methyl groups in a cavity slightly polar than the anhydrous one. The fact that these voids in the γ form (Figure S7(b), Supporting Information) are situated only between the methyl groups (much less flexible than the piperazine rings), makes clear that it is impossible the re-entrance of the water molecules without the collapse of the lattice. 3.3. Description of the Anhydrous LM (α α Form) crystal structure. The anhydrous levofloxacin form (α form) obtained from LM was solved and refined in the orthorhombic space group P212121 with one molecule in the asymmetric unit (Z’ = 1) (Table 1). Therefore, the LM and α structures differ by the space group (LM crystallizes in P21), number of molecules independent by symmetry in the asymmetric unit (LM has Z’ = 2) and unit cell dimensions (the sub-cell determined for the α form (Table 1) is approximately related to the LH one (a = 6.745(5) Å, b = 13.836(9) Å, c = 18.393(12) Å, β = 95.380(11)°)6 by the matrix (0 0 1/ 0 1 0 / 1 0 0). However, as observed comparing the LH and γ structures, the main packing features of the LM is kept in the α form (Figures 7).


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Figure 7. Packing of the LM (a) and α form (b) projected onto their respective bc and ab planes. The matrix relating the two unit cells are also shown. The two conformers present in LM are represented in blue and green and that one present in α form is represented in orange. Hydrogen atoms were omitted for the sake of clarity. The two independent by symmetry water oxygen atoms of the LM are represented by spheres in yellow and red. Packing projection along (c) the a-axis of the LM (column built stacking alternately conformers A and B) and (d) along the c-axis of the α form (column built by the conformers D) showing the intermolecular interactions operating in the stacking of the molecules. Hydrogen bonds and π···π interactions are highlighted by dotted cyan lines.

The two conformers present in the LM structure (hereafter called conformer ALM and BLM) differ by the torsion angle defined by the piperazine ring and the heterocyclic aromatic moiety and are comparable to those ones present in the LH (i.e, ALH ~ ALM and BLH ~ BLM) (Figure


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8(a)). As expected, the single conformer of the α form (hereafter called conformer D) differ from those ones in the LM with respect to torsion angle C5-C6-N2-C14 (Figure 8(b)), which assumes the value of 39.3(8)° in the conformer ALM and 140.0(5)° in the conformer BLM, whereas in the conformer D this value is 91(2)°. It is noted that conformers C (in the γ form) and D (in the α form) present very similar oxazine ring conformations having both the methyl in axial position (Figure 8(c)), instead in equatorial one as observed for the conformers A and B in the LH and LM structures (Figure 8(a)). However, the torsion involving the piperazine ring and the heterocyclic aromatic moiety of the conformer C and D is different since both are rotated by ~90° but in different direction (clockwise vs. anticlockwise) (Figure 8(d)).

Figure 8. Overlays of the conformers ALH, ALM, BLH, BLM, C (γ form), and D (α form). In (a) is compared ALH, BLH, ALM, and BLM. In (b) is compared ALM and BLM, and D. In (c) and (d) is compared C and D in two perspective views. The structures were matched considering the planar bicyclic aromatic moiety. The packing similarities and differences about the LM and LH structures can be also easily observed comparing Figure 3, Figure 7 and Figure S8, Supporting Information. It is important to


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note that the packing of the A and B conformers generates two independent symmetry columns in the LH (···A-A-A-A··· and ···B-B-B-B···), whereas just one column (···A-B-A-B···) is observed in the LM (Figures 3, Figure 7 and Figure S8, Supporting Information). Thus, in the LM, the 21 symmetry along the unit cell a axis is broken due to the different conformation adopted by the conformers A and B (Figures 7(a) and 7(c)). In all remaining structures the molecules in the helical columnar network (···A-A-A··· or ···B-B-B··· in LH, ···C-C-C··· in γ, and ···D-D-D··· in α) are related to each other by the 21 screw axis present in their respective space groups. Two types of interactions stabilize the columnar assembly of LM and α form (Figures 7(c) and 7(d)): (1) non-classical C-H···O bonds and (2) π-π contacts involving the benzene rings. In the LM (Figures 7(c)), the O1, O2 and O3 oxygen atoms of each conformer act respectively as H bond acceptors to the C14, C17 and C11/C12 (bifurcately) carbon atoms of neighbor opposite conformers. In the α form the H bond involving C17 and O2 is absent and O3 is no more a bifurcated acceptor. The H bond pattern has O1 and O3 as acceptors of C14 and C13, respectively (Figures 7(d)). The C13-H···O3 interaction explains in part the change of C13 to the axial position in the α form (Figures 7(d) and Figure 8(b)). In spite of these differences, comparable distances separate the neighboring conformers A and B in the LM (interplanar distance of ∼ 3.5 Å) and neighbor conformer D (c/2 = 3.4427(1) Å) in the α form. These distances are also comparable to those ones discussed above to the LH and γ form. Instead of connecting two identical conformers (ALH···OWA···ALH or BLH···OWB···BLH), as seen in the LH (Figure 6), the water molecules in the LM structure connect to different conformers (···ALM···OWA···BLM···OWB···ALM···) in a head-to-tail fashion via O-H···O and OH···N hydrogen bonds involving not only the nitrogen N3-atom of the piperazine ring, but also


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the oxygen O2-atom of the carboxylic acid group (Figure S9, Supporting Information). Such interactions lead to the formation of ladder chains along the [101] direction. These chains are themselves connected along the b-axis through C18-H···F1 interactions (C18A···F1A = 3.228 Å and C18B···F1B = 3.134 Å) generating layers parallel to (011) plane (Figure 9(a)). Beyond the C18-H···F1 interactions, the layers in LM are also stabilized by H bonds having the OW oxygen atoms as either H bond donor (to O2) or trifurcated acceptor (from the C1, C12, and C13 carbon atoms), and O2 oxygen atom as H bond acceptor from the C13 carbon atom (Figure 9(a)). In contrast to the LH (Figure 6), in the LM, it is not observed the formation of the C-H···F and C-H···O homodimers (Figure 9(a)). Another difference is that in the LH each layer contains both A and B conformers (Figure 6), whereas in the LM the layer is formed by a unique conformer (A or B) stacked alternately (···A-B-A···) along the a-axis (Figure 9(a)).


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Figure 9. (a) View of the LM onto the bc plane showing the 2-D infinite network stabilized by intermolecular H bonds involving levofloxacin and water molecules. The inset highlights the layers stacking along the a-axis. (b) View of the α form onto the ab plane showing that the 2-D infinite network present in LM in broken given rise to zig-zag chains along [010] direction. The


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inset in the α form packing highlights the occurrence of the C12-H···O2 interaction instead of the C13-H···O2 as observed in the LM.

Comparing now the packing of the LM and α form, the C18-H···F1 interaction observed for LM is no more present in the α form in which the C18···F1 distance is 3.829 Å (longer than the sum of the fluorine and carbon van der Waals radius). Actually, just one intermolecular H-bond having C12 as H-bond donor to O2 is kept in the α form (Figure 9(b)) compared to homologous layers in the LM (Figure 9(a)). This H bond pattern generates a zig-zag chain along [010], which is itself connected along the [100] and [001] having C13 as H bond donor to F1 and O3 (Figure S10, Supporting Information). Such interactions lead to the formation of a new synthon, not reported in any other crystal form or predicted structures of levofloxacin.6 The partial packings depicted onto the respective ac planes of the LM and α form highlight their main structural differences (Figure S11, Supporting Information). LM´s columns are stacked in a fashion that results planar layers, whereas the neighbor columns in the α form are shifted relative to one another along the c-axis generating wavy layers (Figure S11, Supporting Information). These structural features are correlated to the water/voids sites, the C-H···F bond patterns and the axil/equatorial conformation of C13. Indeed, the α form adopts a packing more compact than the LM one (ca. 40.31 Å3, equal to 2.4% of the cell volume) and also than the γ form, since a search for accessible voids (using probe radius of 1.2 Å) has been indicated no space available. 3.4. Hirshfeld Surfaces. The intermolecular interactions are identified on the Hirshfeld Surfaces (HS) mapped with dnorm through a red-white-blue color scheme.34 The contacts involving atoms closer than the sum of their van der Waals radius are highlighted as red areas. The longer contacts are exhibited as blue and contacts equal or around their van der Waals radius


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are white (Figure S12, Supporting Information). The 2D fingerprint plots derived from the HS of the anhydrous structures are unique, once the molecules are conformationally and crystallographically distinct (Figure 10). The contribution of each interaction can be visualized in its individual 2-D fingerprint plot. In the γ form, the H···O/O···H contacts correspond to 18.5% of all HS and can be easily identified as two symmetrical spikes that spread to the minimum di + de = 2.30 Å. The upper spike (de > di) is associated to the donor (H···O) and the lower (de < di) to the acceptor (O···H) atoms. Note that the value of the shortest contact (minimum value of di + de) is almost identical to the distance reported for the C14-H···O1 and C13-H···O3 interactions. For the α form, the H···O/O···H contacts are represented by a longer pair of symmetrical spikes (minimum di + de = 1.80 Å), where the upper one represents the methyl carbon interacting with carbonyl oxygen (C13-H···O3) and the lower, those with the carbonyl oxygen acts as acceptor, O3···H-C13. The proportion of these contacts comprises 20.3% of all molecular surface (Figure 10). The proportion of the H···F/F···H contacts in the γ and α forms comprises 8.3 and 6.6%, respectively, of all HS area. They are the result of the C13-H···F1 hydrogen bonds which show up as a pair of symmetrical spikes in the γ form plot with the shorter distance (minimum value of di + de) around 2.5 Å (Figure 10). Another peculiarity of this plot is that is more compact, i.e. the scattered points are concentrated at short values of de, di, in contrast to the α form, where the points are diffuse and spread at higher values of de, di.


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Figure 10. 2-D fingerprint plots of the γ and α structures. Full fingerprint plots were shown in the first line and these are resolved into H···H, O···H/H···O and F···H/H···F contacts.

The proportion of the C···C contacts vary from 7.2% (in the γ form) to 6.4% (in the α form) and are mainly due to π-π stacking within the column. Such interaction seems to be more expressive in the γ form, where is observed a frontal stacking of the tree fused rings (Figure S13, Supporting Information). The H···H contacts are dominant in the crystal packing of the γ and α forms, contributing 55.5% and 54.4%, respectively, to the total area. In summary, these structures are sustained by


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van der Waals contacts existing mainly between the interlocking of the piperazine rings (Figure 10). Finally, the H···N/N···H and H···C/C···H contacts are the ones with the lowest contributions to the HS (Figure S13, Supporting Information). Furthermore, the scattered points distributed at higher values of de, di, shows that the H···N/N···H interactions are practically absent in both structures. A graphical summary with the relative contributions from the intermolecular contacts operating in both γ and α forms is shown in Figure S14, Supporting Information. 3.5. High-Temperature Powder X-ray Diffraction Study Versus Crystal Structure Prediction (CSP). The experimentally determined crystal structures of the anhydrous levofloxacin forms (α and γ) obtained from LH and LM are now compared to the crystal structures predicted by Singh and Thakur study.6 These authors have reported a set of most probable unit cells for the anhydrous form of LH, taking into account the occurrence of both Z’ = 1 and Z’ = 2. Considering the synthon information extracted from all structures of levofloxacin reported on CSD and the conformations of the eight predicted conformers, they have found a total of 20 solutions, ten for Z’ = 1 and ten for Z’ = 2. According to them, the rank 2 predicted structure with Z’ = 2 (P1 space group, cell parameters: 6.590, 14.181, 10.444 Å; 91.6, 116.1, 105.4°) is the one that has shown the best match with the experimental PXRD pattern of the α form reported by Kitaoka et al.4 The interesting fact is that this predicted structure is completely distinct from the other one reported by us (Table 1). Perhaps, because the authors have put so much weight on the contribution of the dimer, i.e. on the maintenance of the π-π interactions between two different conformers in the asymmetric unit. As mentioned before, in the crystal structure description part (section 3.2 and 3.3), such interactions are considerably weaker. Indeed, the α form crystal structure found here is almost identical to another lower ranked


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solution (rank 2 with Z’ = 1: P212121 space group, cell parameters: 16.405, 15.3636, 6.7255 Å) found by CSP approaches,6 although the conformation adopted by our molecule is different. While the conformers ALH/ALM and BLH/BLM (Figure 8) adopt a conformation similar to the ones predicted by Singh and Thakur, A1 = 118.4° and B2 = 28.8° respectively, conformer D (as conformer C in the γ form) differs from the eight conformers predicted by PES scans.6 None of the 20 solutions found by CPS was attributed as a possible structure for the γ form by Singh and Thakur.6 Interestingly, our γ form (Table 1) is very similar to the predicted rank 6 with Z’ = 1 (C2 space group, cell parameters: 17.5948, 6.8641, 14.8442 Å; 90, 75.17, 90°). The greatest divergence, however, lies on the synthons formed. Although they have reported the existence of three characteristic synthons (π-π, C-H···O and C-H···F) for this predicted solution, our structure just maintains two of them. In fact, this can be due to the conformation adopted by our structure (See Section 3.2), which does not match with the one of the rank 6 predicted solution (conformer from PES with a C5–C6–N2–C14 dihedral angle of 116.6°) and, as afore mentioned, with any one of the other conformers predicted by Singh and Thakur.6

4. CONCLUSIONS In spite of many studies on the levofloxacin dehydration, for the first time variable-temperature PXRD has been employed for elucidating the anhydrous structures of the LH and LM which correspond to the phases termed in the literature as the γ and α forms, respectively. As we suspected, our results differ from those reported by Singh and Thakur in their recent study using Crystal Structure Prediction (CSP). These authors report a structure with Z’ = 2, i.e., with two molecules of levofloxacin with conformations A1 and B2 in the asymmetric unit (space group P1, as the one more similar to the α form. They also state that the π-π dimer formed by


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these two conformers is the most stable mode of molecular association. From our results, the α form, formed by dehydration of LM, results in a PXRD pattern corresponding to a single molecule (Z’ = 1), instead of two, solved and refined in the orthorhombic space group P212121. In addition, the conformational adopted by the α form does not match with any one of the eight conformers predicted by them. The same occurs for the γ form; the polymorph formed by direct heating of LH in synthetic air atmosphere. The dehydration of LM gives rise to a PXRD pattern corresponding to a new structure, also solved with Z’ = 1, but belonging to the orthorhombic space group C2. These results agree well with the thermal analyses (DSC, TGA). The DSC and TGA curves show the existence of a unique phase for the direct dehydration process of LH and LM samples, which is also consistent with the HSM images. Besides the fact these are the first levofloxacin anhydrates whose structures could be solved, our results make clear that CSP is not always able to guarantee the best results and X-ray data will be for a long time, the most efficient method to determine a crystalline structure.

ASSOCIATED CONTENT Supporting Information.

Figures of the DSC, TG, DTA curves in the nitrogen atmosphere of LH and LM samples. Rietveld analysis of the PXRD data obtained after heating of LH and LM. Additional Figure with overlay matching the backbones of the conformers ALH and BLH of the LH and the conformer C of the γ form; Views of the LH an LM onto their respective ac and bc planes showing the packing of the conformers A and B. The calculated accessible-voids for the LH and its corresponding anhydrous form (γ form). Views of the LH an LM onto their respective ac and bc


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planes showing the packing of the conformers A and B. Figures of packing projections: onto bc and ac planes of the LM; the α form showing the 3-D H bond network having C13 as H bond donor to F1 and O3 atoms and C12 as donor to O2; of the LM and α forms projected onto their respective ac planes. Hirshfeld dnorm surfaces of the γ and α forms of the levofloxacin. Relative contribution of the different contacts to the Hirshfeld surface area of the levofloxacin anhydrates. Relative contributions from the intermolecular contacts to the Hirshfeld surface area of the levofloxacin anhydrates. Additional tables with refinement parameters of the LH and LM heating as well as geometric parameters of the hydrogen bonds in the levofloxacin structures (PDF).

ACS Publications website at DOI: Accession Codes CCDC 1825455 and 1825456 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/getstructures, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]; [email protected] Notes


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The authors declare no competing financial interests. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. † ‡ § These authors contributed equally.

ACKNOWLEDGMENT The authors acknowledge the Brazilian funding agencies FAPEMIG (RED-00010-14, APQ00273-14, APQ-02486-14, APQ-01931-16 and PPM-00533-16), FAPESP (2015/26233-7), CNPq (407289/2013-7, 448723/2014-0; 308162/2015-3, 307664/2015-5), FINEP (ref. 134/08) and CAPES (PNPD grant) for financial support. We also thank CNPq (A.C.D and F.F.F) and CAPES (J.T.J.F and C.C.M.) for the research fellowships.

ABBREVIATIONS

API, active pharmaceutical ingredient; PXRD, powder X-ray diffraction; DSC, differential scanning calorimetry; TGA, thermogravimetric analysis; HSM, hot-stage polarized microscopy; CSP, Crystal Structure Prediction; LH, levofloxacin hemihydrate; LM, levofloxacin monohydrate.

REFERENCES


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(25) van Laarhoven, P. J. M.; Aarts, E. H. L. Simulated Annealing: Theory and Applications. 4th ed. Mathematics and Its Applications, ed. P.J.M. van Laarhoven and E.H.L. Aarts. Dordrecht, Holland: Kluwer Academic Publishers. 1992, 198 p.

(26) Rietveld, H. M. Line profiles of neutron powder-diffraction peaks for structure refinement. Acta Crystallogr. 1967, 22, 151-152.

(27) Rietveld, H. M. A profile refinement method for nuclear and magnetic structures. J. Appl.Cryst. 1969, 2, 65-71.

(28) Ibiapino, A. L.; Seiceira, R. C.; Pitalunga Jr., A.; Trindade, A. C.; Ferreira, F. F. Structural characterization of form I of anhydrous rifampicin. CrystEngComm. 2014, 16, 8555-8562.


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"For Table of Contents Use Only" Crystal Structure of Levofloxacin Anhydrates: A HighTemperature Powder X-ray Diffraction Study Versus Crystal Structure Prediction Jennifer T. J. Freitas,† Cristiane C. de Melo, †,§ Olímpia M. M. S. Viana,† Fabio F. Ferreira, *,‡ Antonio C. Doriguetto*,†,§ †

Faculty of Pharmaceutical Sciences, Federal University of Alfenas; Rua Gabriel Monteiro da Silva, 700, Alfenas-MG, 37130-000, Brazil §

‡

Institute of Chemistry, Federal University of Alfenas, Rua Gabriel Monteiro da Silva, 700, Alfenas-MG, 37130-001, Brazil

Center for Natural and Human Sciences (CCNH), Federal University of ABC (UFABC), Av. dos Estados, 5001, Santo André-SP, 09210-580, Brazil

Synopsis The crystal structures of the levofloxacin anhydrous forms denoted as α and γ were determined from high-temperature powder X-ray diffraction data. These forms were obtained from the levofloxacin hydrates. The resulting X-ray structures were compared to those ones previously found by Crystal Structure Prediction approaches.


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Graphical Abstract 838x502mm (120 x 120 DPI)



Figure 1. Hot-stage polarized microscopy of the LH and LM single crystals. 100x242mm (120 x 120 DPI)


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Figure 2. Rietveld analysis of the PXRD data from (a) LH and (b) LM at 25°C; and after their dehydration that result in the respective anhydrous (c) γ (at 150 °C) and α (at 190 °) forms. 613x507mm (120 x 120 DPI)



Figure 3. Packing of the LH (left) and γ (right) form projected onto their respective ac planes. The matrix relating the two unit cells is also shown. The columnar staking with molecules related by the 21 screw axis along the unit cell b axis of the respective LH and γ form is highlighted. The two conformers present in LH are represented in blue and green and that one present in the γ form is represented in magenta. Hydrogen atoms were omitted for the sake of clarity. The two independent by symmetry water oxygen atoms of the LH are represented by yellow and red spheres. 867x306mm (120 x 120 DPI)


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Figure 4. Views of an overlay matching the backbones of the conformers ALH (in green) and BLH (in blue) of the LH and the conformer C (in magenta) of the γ form. The structures were matched considering the planar bicyclic aromatic moiety. 399x149mm (120 x 120 DPI)



Figure 5. Packing projection along the b-axis of the respective LH (columns built by the conformers ALH and BLH) and γ (column built by the conformers C) forms showing the intermolecular interactions operating in the stacking of the molecules. Hydrogen bonds and π···π interactions are highlighted by dotted cyan lines. 467x963mm (120 x 120 DPI)


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Figure 6. (a) View of the LH onto the ac plane showing the 2-D infinite network stabilized by intermolecular H bonds involving levofloxacin and water molecules. The homodimers R(_2^2)(16) and R(_2^2)(8) linking different conformers (···A···B···A···) and the water molecule linking the equivalent conformers (A···WA···A or B···WB···B) are highlighted. (b) View of the γ form onto the ac plane showing that the homodimer R(_2^2)(14) (instead R(_2^2)(16) as in LH) are not connected to each other by hydrogen bonds and thus breaking the 2-D hydrogen bond network observed for LH. The inset in the γ form packing highlights the setgraph ring dimension reduction due to the occurrence of the C11-H···O2 interaction instead of the C13H···O2 as observed in the LH. 704x704mm (96 x 96 DPI)



Figure 7. Packing of the LM (a) and α form (b) projected onto their respective bc and ab planes. The matrix relating the two unit cells are also shown. The two conformers present in LM are represented in blue and green and that one present in α form is represented in orange. Hydrogen atoms were omitted for the sake of clarity. The two independent by symmetry water oxygen atoms of the LM are represented by spheres in yellow and red. Packing projection along (c) the a-axis of the LM (column built stacking alternately conformers A and B) and (d) along the c-axis of the α form (column built by the conformers D) showing the intermolecular interactions operating in the stacking of the molecules. Hydrogen bonds and π···π interactions are highlighted by dotted cyan lines. 1087x624mm (120 x 120 DPI)


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Figure 8. Overlays of the conformers ALH, ALM, BLH, BLM, C (γ form), and D (α form). In (a) is compared ALH, BLH, ALM, and BLM. In (b) is compared ALM and BLM, and D. In (c) and (d) is compared C and D in two perspective views. The structures were matched considering the planar bicyclic aromatic moiety. 1576x604mm (120 x 120 DPI)



Figure 9. (a) View of the LM onto the bc plane showing the 2-D infinite network stabilized by intermolecular H bonds involving levofloxacin and water molecules. The inset highlights the layers stacking along the aaxis. (b) View of the α form onto the ab plane showing that the 2-D infinite network present in LM in broken given rise to zig-zag chains along [010] direction. The inset in the α form packing highlights the occurrence of the C12-H···O2 interaction instead of the C13-H···O2 as observed in the LM. 582x619mm (120 x 120 DPI)


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Figure 10. 2-D fingerprint plots of the γ and α structures. Full fingerprint plots were shown in the first line and these are resolved into H···H, O···H/H···O and F···H/H···F contacts. 186x370mm (96 x 96 DPI)


Crystal Structure of Levofloxacin Anhydrates: A ... - ACS Publications

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