Investigation into the Solid and Solution Properties of Known and

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An Investigation into the Solid and Solution Properties of Known and Novel Polymorphs of the Antimicrobial Molecule, Clofazimine Pauric Bannigan, Jacek Zeglinski, Matteo Lusi, John O'Brien, and Sarah P. Hudson Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.6b01411 • Publication Date (Web): 07 Nov 2016 Downloaded from http://pubs.acs.org on November 8, 2016

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Crystal Growth & Design

An Investigation into the Solid and Solution Properties of Known and Novel Polymorphs of the Antimicrobial Molecule, Clofazimine Pauric Bannigan1,2, Jacek Zeglinski1,2, Matteo Lusi1, John O’Brien3 and Sarah P. Hudson1,2* 1

Department of Chemical Sciences, Bernal Institute, University of Limerick, Castletroy, Co.

Limerick, Ireland. 2

Synthesis and Solid State Pharmaceutical Centre, Bernal Institute, University of Limerick,

Castletroy, Co. Limerick, Ireland. 3

School of Chemistry, Trinity College Dublin, Dublin, Ireland.

KEYWORDS Clofazimine, polymorphs, thermodynamic stability, solubility, molecular modelling, antimicrobial resistance, crystal structure, solid state NMR ABSTRACT Clofazimine is an anti-mycobacterial agent used as part of a multidrug treatment for leprosy. Recently clofazimine has shown promising activity against multidrug resistant tuberculosis. Clofazimine has been previously known to exist in two different crystal forms, or polymorphs, which are triclinic (F I) and monoclinic (F II) in crystal structure. The thermodynamic relationship between, and the solubility of, these different crystal structures of clofazimine has not previously been characterised. In this work, their solid and solution 1 ACS Paragon Plus Environment


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properties are studied and as a result, two novel polymorphs of clofazimine (an orthorhombic crystal polymorph and a high temperature polymorph with a monoclinic structure) are reported. The properties of these new solid forms are compared and contrasted with those of the two previously reported polymorphs using thermal, spectroscopic and microscopic techniques. Molecular modelling studies were also carried out to predict the relative thermodynamic relationship and the crystal morphology of the polymorphs. There was an excellent correlation observed between the aforementioned experimental and molecular modelling results, allowing for the unequivocal determination of the thermodynamic relationship between all four polymorphs of clofazimine.

Introduction: Clofazimine (CFZ) is a Biopharmaceuticals Classification System (BCS) class II drug substance, which is recommended by the World Health Organisation (WHO) for the treatment of Leprosy [1]–[4]. CFZ has been used to treat Leprosy since it was first marketed as the multi-drug treatment Lamprene® in 1969 by Novartis [5]. CFZ has also shown good in vitro activity against other members of the mycobacterium family, both slow and fast growing species, as well as activity against most other Gram-positive bacteria species; including strains that are multidrug resistant with minimum inhibitory concentration (MIC) in the range 0.5 – 2 mg/L in most cases [6]–[15]. In contrast, Gram-negative bacteria are uniformly resistant to CFZ [6], [9]. Despite the impressive antimycobacterial activity in vitro, the clinical use of CFZ has been limited [6]. This is most likely due to the extremely low aqueous solubility of CFZ which prevents the MIC being achieved in vivo. The poor aqueous solubility of CFZ is often attributed to the bioaccumulation of the hydrophobic CFZ molecules inside fatty tissue in the body, resulting in pigmentation of the skin and an extremely long half-life in humans of almost 70 days [16]–[18]. Recently it has shown that in 2 ACS Paragon Plus Environment

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mice CFZ massively accumulates as crystal-like drug inclusions (CLDIs) in subcellular spaces inside macrophages when administer over long periods of time [19]. CFZ crystals extracted from in resident tissue macrophages in mice have been identified as a hydrochloride salt form of the drug substance and thus deduced that CFZ administered orally as the free base converts into a hydrochloride salt form [20]. Several other clofazimine salts have been isolated and characterized recently in the literature [21]. Recently there has been a renewal of interest in CFZ. The molecule has been shown to have potential in the treatment of multidrug resistant (MDR) infections, most notable is its’ reported in vitro activity against MDR-tuberculosis (TB) [6]. The potential of CFZ in treating MDR-TB has led to it being recommended by the World Health Organization (WHO) for use in patients with extensively drug resistant (XDR) TB, despite a lack of clinical evidence for its use in the treatment of MDR-TB [6], [9]. In recent times CFZ has also been shown to have anti-inflammatory properties, an ability to inhibit the growth of Babesia and Theileria parasites in vitro and in vivo as well as good chemotherapeutic potential against various cancer cell lines in vitro and in vivo [7], [22]–[26]. CFZ, like many pharmaceutical compounds, exhibits polymorphism [27]–[29], i.e. the chemical entity can exist in different crystalline forms. Different polymorphs of the same drug substance can have different physical properties, such as melting point, density solubility and dissolution rate [28], [29]. Thus polymorphism is of particular interest to the pharmaceutical industry as those properties have a great impact on the bioavailability of drugs [28]–[31]. At the same time, the thermodynamically most stable polymorph, under ambient conditions, is usually the most desired crystalline form for the development of an oral drug delivery system. A polymorph which is less stable than the thermodynamically most stable polymorph (metastable) can undergo solid-phase conversions during isolation, manufacture, storage and dissolution in the body [32]. The driving force for these solid-phase 3 ACS Paragon Plus Environment


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conversions is the reduction of Gibbs free energy. Ultimately metastable polymorphs are more difficult to manufacture reproducibly, and to store effectively [27]. For this reason extensive polymorph screening has become an FDA requirement for new marketable drugs [33]. CFZ was originally marketed before the effects of polymorphism on the physical properties of drug substances were fully understood and thus has been on the market for many years without the solid and solutions properties of its various polymorphs being studied or the thermodynamically more stable polymorph being known. Cases like this also arise when the more stable polymorph is not initially known and found later in the drug development process, e.g. Ritonavir [34]. In this study the solid and solution properties of two previously reported CFZ polymorphs have been studied [35]. The thermodynamic relationship between these and two novel polymorphs is reported using a combination of molecular modelling and experimental approaches. Polymorphs have been characterised experimentally by thermal, spectroscopic and microscopic techniques. Experimental results and conclusions regarding thermodynamic stability and observed particle morphologies are supported by molecular modelling studies. These were carried out using both quantum-chemical and force-field computational methods, along with structural analysis using the Cambridge Crystallographic Structural Database (CCSD) and Hirshfeld surface and fingerprint plots [36], [37].

Experimental Section: Materials CFZ (CAS registry number 2030-63-9) was purchased from Beijing Mesochem Technology Co., Ltd, purity > 99.9%. Organic solvents: methanol (MeOH), acetonitrile (ACN), diethyl ether (Et2O), chloroform (amylene stabilised), toluene and tetrahydrofuran 4 ACS Paragon Plus Environment

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(THF) were of HPLC grade and were all purchased from Sigma-Aldrich (Ireland) and used as received without further purification. Preparation of CFZ polymorphs: CFZ received from the supplier was verified to be chemically pure using solution NMR and then confirmed to be the triclinic polymorph (F I) by powder X-ray diffraction (PXRD). Both the monoclinic polymorph (F II) and one new crystal form (F III) were prepared by slurry methods. Solvents used in the slurry experiments to prepare F II and F III were selected based on solution mediated solid form transformations observed during solubility measurements of F I or F II. 5 g of CFZ F I (as received) was slurried in 50 mL of Et2O, at room temperature and for 24 h at 800 rpm to produce F II. The resulting slurry was then removed and filtered under vacuum; any remaining solvent was removed by drying in an oven at 313.15 K overnight. F III (new polymorph) was prepared by slurry of 5 g of CFZ F I in 50 mL of toluene for 24 h at 800 rpm and at 313.15 K. The resulting slurry was then removed from the waterbath and filtered under vacuum; any remaining solvent was removed by drying in an oven at 313.15 K overnight. Purity and structural identity of the product was confirmed via 1

H solution NMR and PXRD respectively. Crystal growth Single crystals of F III were grown in toluene by cooling crystallization. Cooling

crystallization experiments were performed in 30 mL glass vials with around 20 mL of toluene and an excess of solute (F III), agitated at 600 rpm over a 24 h period to reach equilibrium at 303.15 K. The saturated solution was then filtered into clean, preheated vials and heated to 10 K above the saturation temperature. The solution was then cooled to 300.15 K and seeded with F III prepared from a slurry in toluene (mentioned earlier) and further cooled to 293.15 K at a rate of 0.5 K per hour. Single crystals were harvested from at 293.15 5 ACS Paragon Plus Environment


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K after 24 h. Temperatures were selected for these crystal growth experiments based on the solubility data generated during this study. Slurry stability experiments Mixtures of pure polymorphs F I, F II and F III were slurried in a 1:1:1 ratio with each other, in 30 mL glass vials, filled with 20 mL of solvent, containing PTFE-coated magnetic stir bars and stirred at 800 rpm. Slurries were performed in MeOH, ACN and Et2O, at 278.15 K and 310.15 K, over a period of three weeks. The solid phase present in the slurry was analysed at different intervals over this period; solid samples were removed from the solution, filtered under vacuum and then quickly characterised using reflection PXRD. Similarly noncompetitive slurry experiments were carried out for each pure polymorph of CFZ in MeOH, ACN, Et2O and toluene (15 mL) at the same temperatures. Solubility experimental setup Solubility experiments and measurements were carried out using a gravimetric method and experimental set up was consistent with that reported by Cheuk et al [38]. To verify that 24 h was sufficient to achieve equilibrium, saturated solutions of CFZ were prepared in triplicate at 298.15 K for each solvent and stirred for 48 h and the results compared with samples taken after 24 h. After equilibrium was reached, stirring was stopped and the vials were left for 3 h in the waterbath to allow the excess solid to settle. Syringes (10 ml) and syringe filters (PTFE, 25 mm, pore size 0.2 µm, VWR) were heated/cooled to a temperature that was 5 K above the temperature of the water bath. These were then used to filter off the supernatant into preweighed glass vials, which were weighed immediately following addition of the supernatant. The samples were dried in a well-ventilated fume hood at room temperature. Finally the samples were placed in an oven for 12 h at 313.15 K to ensure any remaining solvent had fully evaporated. At each temperature the solids in equilibrium with the solution 6 ACS Paragon Plus Environment

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were sampled, filtered and quickly analysed via reflection PXRD to ensure that no transformation occurred during the drying process. The solids were also checked for the presence of solvent or decomposition using solution 1H NMR. HPLC Analysis HPLC analysis was carried out using a Shimadzu SPD-6A UV spectrophotometric detector (285 nm), along with a Knauer K-501 HPLC pump (1.5 mL.min-1) and a Hewlett Packard data integrator (HP 3396 series II). A mobile phase of 80:20 MeOH: 0.1 M monobasic sodium phosphate was used. Separation was carried out with a Walters C18 column (3.9 x 300mm). Good linearity (R2 = 0.998) was seen for calibration samples, with a limit of detection of 0.01 mg/L. Solid state characterisation: Powder X-ray Diffraction (PXRD) Reflection PXRD was performed using an Empyrean diffractometor (PANalytical, Phillips) with Cu Kα1,2 radiation (γ = 1.5406 Å) operating at 40 kV and 40 mA and at room temperature. Samples were scanned from 4º to 35º (2θ) with 0.0131º (2θ) step size and 48.195 seconds per step, on a flat stage that was spinning at 4 rpm. Variable temperature powder X-ray diffraction VT-PXRD was carried using a PANalytical X’pert MPD Pro with Cu Kα1,2 radiation (γ = 1.5406 Å) operating at 40 kV and 40 mA and equipped with an Anton-Paar TK 450 hot stage. Samples were scanned from 4º to 40º (2θ) with a scan step size of 0.004º (2θ) and 15.20104 seconds per step. Scans where carried out for each polymorph between 298.15 K and 483.15 K (which was the lowest possible scan prior to melting of the samples) under a nitrogen

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atmosphere; diffractograms were collected at predetermined temperatures on both the heating and cooling cycles. The structure for F IV was solved by simulated annealing of a powder pattern between 4 and 40º collected at 453 K with the DASH suite of programs. The Kα2 radiation was stripped off from the pattern and the unit cell metrics were determined with the DICVOL 6 software. The final solution was refined using the rigid body constrains in the Rietveld refinements software GSAS. Single crystal X-ray diffraction

SCXRD measurements for F III were collected at room temperature (299.86 K), on a Bruker Quest D8 Mo Sealed Tube (λ = 0.71073 Å), equipped with CMOS Photon Detector. Data were corrected for absorption using empirical methods (SADABS) based upon symmetry-equivalent reflections combined with measurements at different azimuthal angles. Crystal structures were solved and refined against all F2 values using the SHELX interfaced with the X-SEED program. Non-hydrogen atoms were refined anisotropically and hydrogen atoms were placed in calculated positions refined using idealized geometries (riding model) and assigned fixed isotropic displacement parameters. The Crystallographic Information Files are available from the CCDC (deposition numbers CCDC 1504595-1504596). Scanning electron microscopy SEM imaging was performed using a JEOL CarryScope scanning electron microscope JCM-5700. Samples were mounted on aluminum stubs with carbon tape tabs and coated by an ultrathin gold layer prior to analysis, using a gold sputtered (EMITECH K55) and the particles were imaged at a voltage of 5 kV. Solid State Nuclear Magnetic Resonance (SSNMR) 8 ACS Paragon Plus Environment

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A Bruker 400 MHz wide bore magnet was used to collect 13C solid state NMR spectra measured at a proton frequency of 400.14 MHz and a carbon frequency of 100.6 MHz in a 4 mm triple resonance probe in double resonance mode. The magic angle was optimised using a rotor packed with KBr and spun at 5 kHz. Using a rotor packed with adamantane and directly exciting the carbons, with decoupling from protons, the magnetic field was adjusted to set the chemical shift of adamantane’s low field peak to 38.48 ppm. The magnet was shimmed such that this low field peak was symmetric and had a peak width of less than 2 Hz. Glycine was then used to check that the signal to noise ratio was acceptable. CFZ polymorphs were packed into 4 mm zirconia rotors for solid state NMR analysis. Samples were spun at a rate of 10 kHz. Proton spin lattice relaxation times (T1) were determined using a direct saturation recovery pulse sequence. 13C CPMAS experiments with arrays to optimise contact time for cross polarisation conditions were run. Cross polarisation conditions were conducted at a ramp of 50 % - 100 % and spinal64 decoupling at 100 %. 13C CPMAS spectra were collected using the optimised contact times and relaxation delays (at least 1.4 x T1 values) for each sample (Table 1). Solution Nuclear Magnetic Resonance (NMR) 1

H and 13C NMR spectra were measured at 600.13 MHz and 150.9 MHz using a 600 MHz

Bruker Avance II with a 5mm TCI cryoprobe in deuterated chloroform (99.8% deuteration, 0.03% TMS). 2D edited HSQC (Heteronuclear Single-Quantum Correlation) and HMBC (Heteronuclear Multiple Bond Correlation) experiments were run to correlate the observed chemical shifts with the CFZ carbon nuclei found in the solid state NMR (data not shown). Thermal analysis Thermogravimetric analysis



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TGA was carried out under nitrogen atmosphere (50 mL.min-1) using a Perkin Elmer TGA 4000 instrument. Experiments were carried out using alumina crucibles and a temperature ramp of 20 K·min-1 from 303.15 K up to 773.15 K. The TGA data was analyzed using Pyris 6 software version 11.1.10492. Differential scanning calorimetry DSC was carried out under a nitrogen environment (30 mL.min-1) using a Perkin Elmer instruments Pyris 1 DSC. Experiments were carried out using platinum pans with pinholes (as manufactured), sealed by a crimping press. The temperature range 303.15 K – 513.15 K was studied using various ramp rates (20 K.min-1for melting point determinations and 10 K .min-1 to monitor transformations). The instrument was calibrated using samples of indium and lead. Molecular modelling Density functional theory (DFT) calculations were applied using a Gaussian 09 package [39] to investigate the strength of interactions of the two stacked CFZ molecules (dimers). The dimers were extracted from the respective crystal structures of CFZ F I (DAKXUI01), F II (DAKXUI), and F III (a new polymorph), being subsequently optimized in isolation (gasphase). The equilibrium geometry of the dimer is calculated with a B97-D3 Grimme’s functional [40], and a Gaussian-type 6-31G(d,p) basis set [41]. The (1:1) binding energy in a dimer is calculated as per equation 1 (eq.1). ∆Ebind = ECFZ-CFZ – 2ECFZ

(eq.1)

Where ECFZ-CFZ is the energy of the CFZ dimer and ECFZ is the energy of an isolated CFZ molecule, both being in fully relaxed gas phase geometries. The DFT energies are calculated using a double hybrid B2PLYP-D3 functional [42], which combines exact Hartree-Fock


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exchange with an MP2-like correlation and long-range dispersion corrections; here we use a basis set of quadruple-ζ valence quality (def2-QZVPP)[43]. The vacuum morphology was predicted using a growth morphology algorithm, in which the growth rate of each crystal face is assumed to be proportional to the attachment energy, viz. the energy released upon attachment of a growth slice to a given crystal face. Calculations were done with Materials Studio 7.0 from Accelrys Inc., and the COMPASS II force field, applicable for organic molecules, including heterocyclic systems [44]. Using the crystallographic information files for each of the polymorphs; F I, F II and F III, Hirshfeld surfaces of these three polymorphs were calculated and analysed with CrystalExplorer 3.1 [36] [37].

Results and discussion F I and F II crystallise into triclinic and monoclinic modifications respectively and these structures were previously reported (CCSD reference code: DAKXUI01 and DAKXUI) [35]. F III was first discovered during independent slurry experiments of both F I and F II in toluene. Analysis of the solid phase present in these slurries, via PXRD, indicated a new solid form was present. Obtaining single crystals of F III, to determine its crystal structure, proved to be difficult initially as saturated solutions of F III at, and above, room temperature crystallised into F II using the solvent evaporation technique. Thus a combination of solid state and solution NMR was used confirm that solid phase present in solutions of toluene was a new polymorph. Solution NMR studies allowed for the assignment of the

13

C chemical shifts of CFZ

molecules in solution, (Figure 1a, b). A comparison of the solid state 13C CPMAS spectra of F I, F II and F III confirmed F III to be a new polymorph of CFZ, (Figure 1c). The different chemical shifts of the carbon atoms are an indication of the different intermolecular bonding 11 ACS Paragon Plus Environment


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environments of carbons atoms within these polymorphs. The chemical environments of the carbons involved in the pi stacking within F I, F II and F III varied and thus the

13

C peak

positions varied significantly from polymorph to polymorph in the region 110-145 ppm, (Figure 1c). In addition, the isopropyl methyl groups are in two distinct environments in F II and F III while in F I, a single, slightly broader peak is observed indicating that there may be a chemical equivalence in the methyl groups in this polymorph. Only in F III does it appear that the C2 carbon and the tertiary carbon between C4 and C5 on the phenazine nucleus have distinctive chemical shifts – in F II these two peaks are slightly resolved but are even less resolved in FI. The absence of multiple peaks for each carbon atom indicates one CFZ molecule per asymmetric unit in each polymorph. Single crystals of F III were eventually isolated via seeded, slow cooling crystallisation of a saturated solution of F III. However it was found that if the supersaturation exceeded 1.5, the solution would favor the nucleation and growth of F II instead of F III. The crystal structure of FIII was determined to be orthorhombic with a Pbca space group with 8 symmetrically equivalent molecules in the unit cell Table 2. A comparison of the unit cells of F I, F II and F III shows they contain 2, 4 and 8 CFZ molecules respectively. In F III, CFZ packs in dimers that are similar to those observed in F I and II, with only minor conformational differences that are most visible in the torsion angle of the chloro-substituted benzene rings (about 34º vs -23º for F III and F II respectively). Melt-decompositions of FI, FII and FIII were confirmed using a combination of TGA, DSC and 1H NMR (data not shown). F II was found to reversibly convert into a second novel polymorph, F IV (a high temperature polymorph), at 364.3 K, Figure 2. The enantiotropic phase transformation between FII and FIV was confirmed by VT-PXRD during heating and cooling cycles and this is shown in Figure 2. Neither F I or F III showed a change in crystal structure during similar VT-PXRD experiments (see ESI). F IV was only seen during high 12 ACS Paragon Plus Environment

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temperature PXRD experiments and attempts to isolate this polymorph using different cooling rate on the DSC and by quenching DSC pans in liquid nitrogen have been unsuccessful, resulting in F II in every case. The crystal structure of F IV was determined to be monoclinic by simulated annealing of the PXRD pattern collected at 453 K (details are shown in the experimental section and the associated content). Figure 3 shows a comparison of this high temperature diffractogram with the PXRD patterns of F I, F II and F III. The most relevant difference between F II and F IV are the values of the dihedral angles between the central “phenazine” moiety and the benzene rings (from about 82º to 111º and from -34º to 30º) and the isopropyl group (from 81º to 139º). Indeed the reduced unit cells for the two forms are comparable (See Table 2). Thus the melting point of F II was thermodynamically unobtainable and the shallow endothermal peak for the FII/FIV transition suggests that there could be a kind of order-disorder relationship between the two forms, Figure 4. The onset and enthalpy change for this transformation, as well as the melting of F I, F IV and F III is summarised in Table 3 and DSC profiles are shown in Figure 4. This thermal analysis showed F III to have the highest melting onset, while F I exhibited the lowest. As FIII also has the higher enthalpy of fusion, it can be assumed it is monotropically related to both FI and FIV, according to the Burger-Ramberger rules [45].

CFZ has hydrogen bonding capabilities but it is almost insoluble in water, due to the large hydrophobic regions of the molecule. Solvents for solubility studies were selected to vary properties such as polarity and H-bonding ability. The solvents chosen were; tetrahydrofuran (THF), toluene, chloroform, diethyl ether, acetonitrile, methanol and water (good aqueous solubility is critical for any orally administered drug). Solution mediated transformations of the added solid form (be it FI, FII or FIII) of CFZ in different solvents made it difficult to obtain solubility data for all polymorphs in all solvents. F IV was found to only be stable at 13 ACS Paragon Plus Environment


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high temperatures and could not be isolated, thus it was excluded from solubility and slurry experiments. In solution, F I was found to spontaneously transform into F II in non-competitive slurry experiments in methanol, acetonitrile and diethyl ether at all temperature screened, within 24 h. Thus it was found that the kinetic stability of F I was so low that it was not possible to determine solubility values for this polymorph using gravimetric means. Under the same conditions and in the same solvents F II was found to be kinetic stable and remained in solution following 24 h. Slurry experiments in toluene resulted in the conversion of F I into F II at low temperatures (275.15 K), while F II remained stable in solution at these temperatures. When F I was slurried in toluene at higher temperatures (288.15 – 293.15 K) a mixture of both F II and F III was observed in the solid phase (after 24 h). For slurry experiments above 293.15 K, both F I and F II were seen to undergo complete conversion into F III after 24 hours. Figure 5 summarises the results of non-competitive slurry experiments of F I. Non-competitive slurry experiments of F I and F II in chloroform (CHCl3) and tetrahydrofuran (THF), resulted in the formation of CFZ solvates with the respective solvents (see ESI). Competitive slurry experiments of F I, F II and F III in methanol, acetonitrile and diethyl ether resulted in pure F III being present in solution after one week at 308.15 K, while at 278.15 K a mixture of F II and F III was observed in situ after one week (see ESI). When this experiment was run for three weeks, only F III was present at 278.15 K (data not shown). Thus F III catalysed the conversion of F I and F II into F III (similar to the afore mentioned case of Ritonavir [34]). The conversion proceeded slower at lower temperatures, taking almost 3 week at 278.15 K.


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Solubility measurements were obtained for the various solid forms of CFZ both directly and indirectly due to the previously mentioned solution mediated transformation in various solvents. The average solubility (expressed in g of CFZ per 100 g of solvent) of the polymorphs F II and F III is summarised in Table 4. Aqueous solubility values for the three room temperature stable polymorphs of CFZ were unobtainable via the gravimetric method. Even the attempt of determining the water solubility by HPLC failed since the concentration of the dissolved solute for each polymorph was below the limit of detection (> 0.01 mg/L). Overall the solubility of CFZ in these various solvents follows the trend; THF > toluene > chloroform > diethyl ether > acetonitrile ≈ methanol >> water. Thus the solubility of CFZ was highest in non-polar solvents, as would be expected for such a highly hydrophobic molecule. Polymorph solubility could not be measured in THF due to the formation of a solvate, but the high solubility of the solvate was unexpected as THF is usually considered a polar aprotic solvent. However THF also has a significant hydrophobic character due to the four carbon long chain in the ring, which could disrupt the pi stacking observed between CFZ molecules in all polymorph structures. The ether group of THF is electron donating, sending electron density from the oxygen atom to the adjacent carbons. The dipole formed here could potential interact with benzene substituted chlorine molecules of CFZ, disrupting the lattice bonding in the CFZ polymorphs. The crystal structure of the CFZ-THF solvate shows such dipole-dipole interacts (see ESI). Thus the high solubility of CFZ in THF can be explained by the ability of THF molecules to disrupt bonding interactions between CFZ molecules within crystals and as a result higher solubility was seen in THF than in any other solvent. Toluene, a non-polar solvent, has no potential to form hydrogen bonds but does have non-polar properties and this can disrupt the pi-pi stacking in the CFZ crystals mentioned previously, and form non-polar interactions with the hydrophobic moieties present. Fairly high solubility was also noted in chloroform and this is accounted for by an ability to form dipole-dipole interactions with CFZ, similar to THF mentioned previously. Like THF, CFZ formed a 15 ACS Paragon Plus Environment


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solvate with chloroform through these dipole interactions. With the decrease in non-polar character from THF, toluene and chloroform to diethyl ether, an almost 10-fold decrease of CFZ solubility is seen. Methanol and acetonitrile are solvents which readily form hydrogen bonds with themselves and with other molecules, and thus could potentially hydrogen bond to CFZ in the crystal lattice. These solvents have very little non-polar bonding character and are unable to sufficiently disrupt the intermolecular bonding between CFZ molecules. Thus solvation and solubility is very low in methanol and acetonitrile. Water, the final solvent studied, which only has hydrogen bonding potential, showed no detectable level of CFZ in solution using HPLC. The solubility of CFZ F II and F III was comparison in methanol and acetonitrile to determine the relative free energy of these polymorphs. In these solvents, the monoclinic polymorph F II was found to exhibit higher solubility that F III, Figure 6 6. As mentioned earlier the poor kinetic stability of F I made solubility data unobtainable. Similarly the poor thermal stability of F IV made further experimental measurements impossible. Molecular modelling of F I, F II and F III To verify the results of thermal and solubility experiments, the crystal structures of polymorphs F I, F II and F III were compared and contrasted at the molecular level using the unit cell representation as well as its multiplication (supercell) (Figure 7). F IV was found to only be stable at high temperatures, thus it was excluded from modelling studies. A feature common for the three polymorphs is lack of strong intermolecular hydrogen bonding. The most distinct difference in the molecular arrangement of these crystals is in the presence of centrosymmetric dimers of CFZ in F I and the columnar parallel arrays of stacked CFZ molecules in F II and F III (Figure 7). The relatively large surface of interaction of the stacked CFZ molecules appears to be the major contributor to the total interaction/lattice 16 ACS Paragon Plus Environment

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energy in the three crystal forms. Thus, quantification of the interaction strength between a pair of stacking CFZ molecules across the different crystal lattices may allow for estimating a thermodynamic stability order of the different polymorphs. The strength of the interactions between the CFZ molecules in the different crystal forms has been quantified with DFT for the isolated dimer structures extracted from their respective crystal lattices (Figure 8). The binding strength for the F I and F II dimers is quite similar, being slightly higher (by 1.1 kJ/mol) for the latter. The binding energy of -27.8 kJ/mol calculated for the form III-like dimer was seen to be larger than for either F I or F II-like dimers (by 4.3 and 3.2 kJ/mol respectively). This suggests that the strength of interaction of the two overlapping CFZ molecules within these crystal structures follows the order F III > F II > F I. These calculated binding energies are relatively low, considering the high surface of interaction between the stacked molecules. In comparison, binding energy in the smaller dimer of salicylic acid, calculated with a similar method, is -64.5 kJ/mol [46]. The main reason for such a difference can be the lack of strong intermolecular H-bonding between the CFZ molecules, as opposed to the presence of strong H-bond interaction between carboxylic groups in the salicylic acid dimer. Taking into account that stacking of CFZ molecules is an important aspect of the molecular arrangement in the crystal forms, the observed stronger interaction between the two stacked CFZ molecules in the F III-like dimer may indicate higher thermodynamic stability of the F III as compared to F I and F II. If thermodynamic stability can be judged from the binding energy of these CFZ dimers, then it should follow that stability is of the order F III > F II > F I. Hirshfeld surface analysis for the polymorph series, (excluding F IV whose structure was determined at elevated temperatures), showed that the surface of F III has a larger proportion 17 ACS Paragon Plus Environment


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of potentially stronger interactions compared to F I and F II. For example Cl --- H and N---H contacts represent respectively 19.2% and 3.9% of the Hirshfeld surface in Form III, higher than that in F I (12.1% and 4.5%) and F II (18.5% and 3.5%). Moreover the fingerprint plots of the surfaces show that in F III these contacts are shorter (see ESI) suggesting a closer interaction between the molecules of CFZ in the unit cell of F III than in the other polymorphs. These observations support the proposed thermodynamic stability of F III > F II > F I deduced from molecular modelling studies. FIII crystals have a crystal habit of thick needles with a deep red colour, Figure 9. In contrast, the experimentally observed particles of F I have a brownish red, flat, parallelogramlike shape and F II crystals have distinctly different thin needle shape and are a bright orange colour. The crystal morphology of the F I crystal, predicted with the attachment energy method, is parallelogram-like and generally matches its macroscopic crystal habit, Figure 9. Also, the needle-like crystals of both F II and F III are reasonably matched by the predicted rod-like morphologies. In addition to the morphological information, an insight into actual molecular packing taking place in the predicted crystalline particles has been obtained. The packing of CFZ molecules in F I crystals facilitates quite uniform interactions in the 3D space; this yields the observed blocky crystalline particles. On the other hand, in the F II and F III crystals, the molecular packing is more directional, with columns of stacked CFZ molecules placed parallel to the longitudinal axis of the predicted particle; this arrangement yields the experimentally observed elongated needle-like crystals for both polymorphs.

Conclusions Two new polymorphs of CFZ have been identified, F III and F IV. Using a combination of solution and solid state NMR, F III was confirmed as a new polymorph of CFZ prior to its crystal structure being identified. This highlights the use of NMR in polymorph screening. 18 ACS Paragon Plus Environment

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The crystal structure of F III was then obtained and solved using SCXRD and the structure of F IV was solved using simulated annealing of a high temperature PXRD pattern, collected at 453 K. The thermodynamic relationship between all four polymorphs of CFZ (F I, F II, F III and F IV) has been established here using a combination of slurry experiments, thermal analysis, solubility measurements and X-ray diffraction methods. F III, F II and F I exist in a series of monotropic relationships with thermodynamic stability in the order F III > F II > F I by solubility measurements. Thermal analysis (DSC and VT-PXRD) showed an enantiotropic relationship existed between F II and F IV, with a conversion at 364.3 K. DSC analysis also indicated a thermal stability order of F III > F IV > F I. Combining these results gave an overall stability order of FIII > FIV > FI above 364.3 K and F III > F II > F I below 364.3 K. CFZ solubility in the organic solvents screened here was of the order: THF > toluene > chloroform > diethyl ether > methanol ≈ acetonitrile >> water. The order of CFZ solubility in these solvents can be accounted for according to the properties of the solvents and the ability of these solvents to interfere with non-polar CFZ-CFZ interactions causing solvation. Using molecular modelling, it was found that the DFT-calculated binding energy between the CFZ-CFZ dimers taken from the F I, F II and F III crystals correlate to the experimentally determined thermodynamic stability of these solid forms. Computational predictions of crystal morphology of the different polymorphs were found to be consistent with experimentally observed crystal shapes of the relevant forms. The presented modelling approaches may be applicable in predicting the morphology and thermodynamic stability of chemical entities being structurally similar to CFZ.



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FIGURES

(b)

(a)

CFZ FIII

(c)

CFZ FII

CFZ FI

Figure 1 (a)

13

C chemical shift assignments from solution NMR (blue) and atom labels

(black) (b) 13C solution NMR spectrum of CFZ measured at 150.9 MHz and (c) 13C CPMAS spectra of FI (bottom), FII (middle) and FIII (top) measured at 100.6 MHz.


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Figure 2: VT-PXRD cycle for CFZ F II with temperatures of scans indicated.

Figure 3: Comparison of the experimentally derived diffractograms of F I (black) and F II (red), F III (blue) and F IV (green).



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Figure 4: Thermal events of the CFZ polymorphs seen on the DSC.

Figure 5: Results of non-competitive slurry experiments of F I in various solvents at 305.15 K. Solution mediated solid form transformations into F II and F III respectively.


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Figure 6: Overlay of the solubility curve of CFZ F II and F III obtained in methanol and acetonitrile. Curves fitted with second order polynomial best fit lines.



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Figure 7: Comparison of the crystal structures of CFZ F I, F II, and F III. A pair of overlapping CFZ molecules (a dimer) in each of the polymorphs is highlighted in yellow.


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Figure 8: DFT binding energies calculated for dimers of clofazimine being extracted from the crystal structures of F I, F II and F III, subsequently optimised in isolation. Calculations performed at B97-D3/6-31G(d,p) level (geometry) and B2PLYP-D3/def2-QZVPP level (energy).



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Figure 9: Comparison of the vacuum morphology (computational prediction) of CFZ crystal F I, F II and F III (left panel) with macroscopic crystals (SEM images) of the three polymorphs (right panel). An arrangement of the CFZ molecules in each of the simulated crystalline particles is also highlighted.


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TABLES Table 1: Optimised parameters for SSNMR analysis of CFZ polymorphs

Polymorph

Contact time (ms)

Relaxation delay (s)

No. of scans

FI

3

8

128

FII

2.5

3

128

FIII

2

6

128

Table 2: Summary of the crystallographic data obtained from SCXRD, for FIII and FIV, compared with the previously reported data for the monoclinic and triclinic polymorphs of CFZ.

Crystal system Space group [ref.] Description

FI Triclinic P-1 [35] red, prism

a, Å b, Å c, Å α, deg β, deg γ, deg Volume, Å3 Z Dcald, g cm-1 Dexp, g cm-1 R-Factor (%) Temp. (K)

10.705 (4) 12.852 (12) 9.601 (2) 95.96 (4) 97.22 (1) 69.73 (6) 1204.01 2 1.306 1.29 6.2 283-303

F II Monoclinic P 21/a [35] orange, needle 7.788 (14) 22.960 (13) 13.362 (7) 90 98.58 (12) 90 2362.55 4 1.331 1.3 3.6 283-303

F III Orthorhombic Pbca this work Deep red, needles 23.2336 (15) 8.1100 (5) 25.5806 (16) 90 90 90 4820.01 8 1.305 ---5.09 299.86

FIV* Monoclinic P 21/c this work n/a 12.9083 23.3031 8.3092 90 95.1697 90 2489.27 4 n/a n/a n/a 453

*Structure obtained from high temperature PXRD pattern

Table 3: Summary of thermal analysis of CFZ polymorphs Melting points: CFZ F I CFZ F III CFZ F IV Average onset (K) 498.86 ± 0.23 504.11 ± 0.29 500.2 ± 0.31



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ΔH (kJ mol-1)

71.290 ± 0.5290

79.093 ± 1.396

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71.627 ± 0.423

F II F IV 364.3 ± 0.25 19.109 ± 2.65

Transformations: Average onset (K) ΔH (kJ mol-1)

Table 4: Summary of solubility data for CFZ solid forms in various solvents, between 278.15 K and 310.15 K Temp 278.15 283.15 288.15 291.65 293.15 298.15 303.15 308.15 310.15 (K)

CFZ F II (g/100 g of solvent) MeOH ACN Et2O

0.0630 ±0.0051

0.0665 ±0.0067

0.0820 ±0.0026

-

0.0932 ±0.0009

0.1041 ±0.0011

0.1160 ±0.0022

0.1360 ±0.0029

-

0.0539 ±0.0023

0.0569 ±0.0026

0.0666 ±0.0027

-

0.0844 ±0.0014

0.1014 ±0.0005

0.1234 ±0.0045

0.1452 ±0.0038

-

0.8766 ±0.0199

0.8985 ±0.0172

1.0750 ±0.0015

-

1.1100 ±0.0025

1.3569 ±0.0240

1.3876 ±0.0361

1.4750 ±0.0414

-

CFZ F III (g/ 100 g of solvent) MeOH

0.0355 ±0.0039

-

-

0.0581 ±0.0031

-

-

-

-

0.1132 ±0.0024

ACN

0.0294 ±0.0039

-

-

0.0538 ±0.0031

-

-

-

-

0.1199 ±0.0024

-

-

-

-

-

9.5640 ±0.0129

11.4373 ±0.0712

12.7281 ±0.1469

-

toluene


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AUTHOR INFORMATION Sarah P. Hudson *Department of Chemical and Environmental Sciences, Synthesis and Solid State Pharmaceutical Centre, Bernal Institute, University of Limerick. Email: [email protected] Notes: The authors declare no competing financial interest. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Supporting Information. Solution 1H NMR spectrum, comparison of calculated and experimental PXRD diffractograms of F I, F II and F III, variable temperature PXRD diffractograms for F I and F III, PXRD diffractograms of F III and F IV at 483.15 K and additional PXRD comparisons of polymorphs from competitive slurry experiments as well as crystallographic data for FIII, FIV and THF and chloroform clofazimine solvates and Hirshfeld fingerprint plots are presented in supporting information.

Funding Sources and Acknowledgements This project was funded directly by Science Foundation Ireland through 13/CDA/2122 and we also acknowledge the Program for Research in Third-Level Institutions (PRTLI) Cycle 5 for its role in funding certain elements of this project. The Science Foundation Ireland (SFI) and Higher



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Education Authority funded Irish Centre for High End Computing (ICHEC) is acknowledged for access to computational facilities.

ABBREVIATIONS ACN, acetonitrile; BSC, Biopharmaceutics Classification System; CAS, Chemical Abstracts Service; CCSD, Cambridge Crystallographic Structure Database; CFZ, Clofazimine; CPMAS, Cross Polarisation Magic Angle Spinning; DFT, Density Functional Theory; Et2O, diethyl ether; F, form (polymorphic form); HPLC, High Pressure Liquid Chromatography; MDR, Multidrug Resistant; MeOH, methanol; MIC, Minimum Inhibitory Concentration; NMR, Nuclear Magnetic Resonance, PTFE, Polytetrafluoroethylene; PXRD, powder x-ray diffraction; SCXRD, single crystal x-ray diffraction; SSNMR, Solid-State Nuclear Magnetic Resonance; TB, tuberculosis; THF, tetrahydrofuran; UV, Ultraviolet; WHO, World Health Organisation; XDR, Extensively drug-resistant.

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[46] D. Khamar, J. Zeglinski, D. Mealey, and Å. C. Rasmuson, “Investigating the Role of Solvent − Solute Interaction in Crystal Nucleation of Salicylic Acid from Organic Solvents,” J. Am. Chem. Soc., vol. 136, pp. 11664–11673, 2014.



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For Table of Contents Use Only: An Investigation into the Solid and Solution Properties of Known and Novel Polymorphs of the Antimicrobial Molecule, Clofazimine Pauric Bannigan1,2, Jacek Zeglinski1,2, Matteo Lusi1, John O’Brien3 and Sarah P. Hudson1,2* 1

Department of Chemical and Environmental Sciences, Bernal Institute, University of Limerick,


Synthesis and Solid State Pharmaceutical Centre, Bernal Institute, University of Limerick,


School of Chemistry, Trinity College Dublin, Dublin, Ireland.

Solid and solution state studies of clofazimine, a potent antimicrobial compound, uncovered two novel polymorphs (F III and F IV) in addition to the two previously known polymorphs (F I and F II). A stability order of FIII > FIV > FI above 364.3 K and F III > F II > F I below 364.3 K was proven.


Investigation into the Solid and Solution Properties of Known and

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