Five solid forms of a potent imidazopyridazine antimalarial drug lead

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Five solid forms of a potent imidazopyridazine antimalarial drug lead: a preformulation study Terence James Noonan, Kelly Chibale, Peter Cheuka, Malkeet Kumar, Susan A. Bourne, and Mino R. Caira Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.9b00575 • Publication Date (Web): 17 Jul 2019 Downloaded from pubs.acs.org on July 21, 2019

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Crystal Growth & Design

Five solid forms of a potent imidazopyridazine antimalarial drug lead: a preformulation study Terence J. Noonan†, Kelly Chibale†,‡, Peter M. Cheuka†, Malkeet Kumar†, Susan A. Bourne† and Mino R. Caira†,* †Department

‡South

of Chemistry, University of Cape Town, Rondebosch 7701, South Africa

African Medical Research Council, Drug Discovery and Development Research Unit,

Department of Chemistry and Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa.

ABSTRACT

A novel antimalarial drug lead, 6-(3-(methylsulfonyl)phenyl)-3-(4-(methylsulfonyl)phenyl)imidazo[1,2-b]pyridazine (MMV652103), with good in vitro and in vivo antiplasmodial effects but poor aqueous solubility was investigated for preformulation beneficiation by supramolecular methods. Three polymorphs (Forms 1–3), an amorphous phase (Form 4) and a monohydrate (Form 5) were discovered and characterized by thermal analytical methods including hot stage microscopy (HSM), differential scanning calorimetry (DSC) and thermogravimetric analysis

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Crystal Growth & Design 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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(TGA), complemented by variable temperature powder X-ray diffraction (VTPXRD). Single crystals of polymorphic Form 1, Form 2 and a monohydrate of the drug lead were isolated, and their structures were elucidated by X-ray diffraction which enabled the respective molecular conformations, inter- and intramolecular interactions and packing arrangements to be determined. A schematic energy-temperature diagram incorporating the polymorphic forms and the amorphous form of the drug lead was constructed using data gleaned from thermal analysis, kinetic solubility experiments and observations of solvent-mediated transitions. The amorphous form of the drug lead displayed a significant increase in dissolution rate, yielding a maximum concentration of 3–4 times those of the crystalline forms after one hour.

INTRODUCTION At present, malaria caused by parasites of the Plasmodium spp. remains one of the leading global causes of death, primarily afflicting less developed regions of the world.1–2 The World Health Organization (WHO) estimates of malaria cases and deaths were 219 million and 435 000 respectively in 2017.2 The current WHO-recommended treatment includes artemisininbased combination therapy (ACT). The progress made in recent years in reducing global malaria cases has stalled and reported cases of resistance to the ACT regimens have become more frequent as of late and furthermore the rise of resistance to other antimalarial drugs with known mechanisms is a cause for concern. It is for these reasons that drugs with novel mechanisms of action are urgently needed to continue with the effective treatment of malaria.3–6 A novel antimalarial drug lead, 6-(3-(methylsulfonyl)phenyl)-3-(4-(methylsulfonyl)phenyl)-imidazo[1,2b]pyridazine (Scheme 1) (MMV652103, hereinafter referred to as MMV03), was developed by the University of Cape Town’s Drug Discovery and Development Centre (H3D) in collaboration with Medicines for Malaria Venture (MMV, Geneva, Switzerland). This drug lead displayed


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good in vitro potency (IC50: K1 = 6.3 nM, NF54 = 7.3 nM, where IC50 is the 50% inhibitory concentration) against multidrug resistant (K1) and sensitive (NF54) strains of the human malaria parasite Plasmodium falciparum and exhibited 98 % activity in the in vivo Plasmodium berghei mouse model in a 4-day test at 4 × 50 mg/kg po and high potency (ED90 = 1.5 mg/kg, where ED90 is the dosage required to produce the desired effect in 90% of the population) in a severe combined immunodeficient (SCID) mouse model7 against Plasmodium falciparum, comparable to marketed antimalarial drugs. An impeding feature of the development of this compound is, however, its poor aqueous solubility (< 5 μM at pH 6.5).8,

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Although a recent

study by Cheuka et al. identified derivatives of this compound with improved solubility, their in vitro antiplasmodium potency was compromised.10 Supramolecular methods were recently employed in our laboratory to explore potential solid-state forms of MMV03 with differing physicochemical properties such as improved aqueous solubility while maintaining its antiplasmodium effect.11 Several multi-component forms were produced including a series of cocrystals with pharmaceutically acceptable coformer molecules and one salt form. The other aspect of MMV03 that was explored was its propensity to form polymorphs and hydrates, which is the subject of the present study. As emphasised by Bernstein in his 2002 monograph,12 the occurrence of polymorphism is of pivotal importance in the pharmaceutical industry. A recent review addresses the challenge of solid-form selection for drug delivery with reference to polymorphs, amorphs and multi-component systems such as drug co-crystals.13 Differences in physicochemical properties can arise from variations in molecular conformations, crystal packing arrangements and non-covalent interactions that link molecules in different polymorphs.14 These differences, affecting properties such as stability during storage or handling, melting points, heat capacity, density, flowability and hardness for tableting,


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dissolution rate and solubility, are of considerable importance in the pharmaceutical field. It is thus crucial to have knowledge of both the existence of each form of an active pharmaceutical ingredient (API) as well as the specific experimental variables required to produce them, not merely for exploiting the obvious conceivable benefits, but also to prevent unexpected transitions to other forms during processing or storage, with potentially detrimental outcomes. Solvated forms of bioactive compounds, especially hydrates, feature prominently in the pharmaceutical context. A hydrated API generally has lower aqueous solubility than that of the API alone.15 Prudent analysis demands the determination of the relevant properties for each solvate and hydrate individually.16 In the present case, a very promising drug lead, rather than an API, is the focus of the investigation, the aim being to facilitate its development by early intervention. Three polymorphs, an amorphous phase and a hydrated form of MMV03 were discovered in the present study. The potential utility of any of these forms in the formulation of products intended for clinical trials motivated their comprehensive study. Accordingly, they were characterized with respect to their relative stabilities and potential interconversions by complementary methods such as solubility measurements, solvent-mediated transition experiments, thermo-analytical studies and X-ray structural analyses.

Scheme 1. The molecular structure of MMV652103 (‘MMV03’)


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EXPERIMENTAL SECTION Materials and Methods The synthesis of MMV03 was described previously by Le Manach et al.8 The solvents used in the present study were 1-butanol (99.4 %) and anhydrous acetonitrile (99.8 %), both purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany) and acetone (analytical grade), ethanol (99.9 %), ethyl acetate (analytical grade) and methanol (99.5 %), all obtained from KIMIX Chemical and Lab Supplies CC (Cape Town, South Africa). The various unsolvated solid forms of MMV03 are numbered by the chronology of their discovery from Form 1 to Form 4. Single crystals of Form 1 were obtained by recrystallization from solution. A 5 mg sample of analytically characterized MMV03 (raw material, RM) was dissolved in 1 cm3 of acetone while being stirred and heated to 45–50 C with a heating magnetic stirrer. The hot solution was filtered through a 0.45 μm nylon filter into a clean vial which was capped and placed on a benchtop. Crystals of Form 1 appeared after a few days. Their polymorphic purity was confirmed by the match between the experimental powder X-ray diffraction (PXRD) pattern of the RM and the simulated pattern based on the subsequent singlecrystal X-ray analysis of Form 1 (Figure S1, Supporting Information). Single crystals of a hydrated form were first observed in a recrystallization experiment in which 5 mg of MMV03 was dissolved in 6 cm3 of ethyl acetate, heated to 70 C while being stirred and filtered through a 0.45 μm nylon filter into a clean vial and placed on a benchtop. Initially acicular crystals of MMV03 Form 1 appeared and the hydrated crystals with a plate-like habit formed later in the same vial due to uptake of water from ambient air. Polymorphic Form 2 of MMV03 was serendipitously discovered in an attempt to grow co-crystals from solution. The conditions under which these crystals were first obtained were as follows. 5 mg of MMV03 (RM) and an


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equimolar amount of succinic acid (potential coformer) were dissolved in 5 cm3 of ethyl acetate at 60 C with stirring. The solution was filtered through a 0.45 μm nylon filter into a clean vial and placed in a desiccator. These conditions were found not to be necessary for crystallization of this form, since on subsequent occasions it precipitated from ethyl acetate and acetonitrile solutions in the absence of the co-crystal former. Polymorphic Form 3 and amorphous Form 4 were both discovered during the characterization of the other known forms; this is elaborated on below (Results and Discussion section). Subsequently, the method used to obtain the uncontaminated MMV03 monohydrate phase was as follows: 10 mg of MMV03 was dissolved in 5 cm3 of acetone that was heated to 50 C while being stirred. The hot solution was then filtered through a 0.45 μm nylon filter into a clean vial, to which was added 2 cm3 of water and the mixture was placed on a benchtop. After a few days the plate-like crystals of the monohydrate had formed. HSM was used to observe changes taking place during the heating of samples or to view solvent-mediated transformations. For these experiments, a Nikon SMZ-10 stereoscopic microscope was used, and micrographs were collected with a Sony Digital Hyper HAD colour video-camera fitted to the microscope. The software used was the Soft Imaging System program AnalySIS.17 A Linkam THMS600 hot stage was employed and a Linkam TP92 unit was used to control the temperature. A Q200 and a TA Discovery 25 instrument (TA Instruments) were used for DSC with sample masses in the range 1–2 mg, the samples being placed in vented aluminum pans. The heating rate for all DSC curves presented was 10 K min−1. All DSC runs were performed under dry nitrogen purge gas with a flow rate of 60 cm3 min−1 in the case of the Q200 and 40 cm3 min−1 for the TA Discovery 25. The software used for analysis of the DSC curves was TA instruments Universal


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Analysis 200018 for the Q200 and TRIOS19 for the TA Discovery 25. The latter instrument was also used for modulated differential scanning calorimetry (MDSC). Measurements were performed by conventional MDSC with an underlying heating rate of 3 K min−1, a modulation period of 60 s, an amplitude of  1 K, and sample masses ranging from 1.78 to 2.64 mg. A TA-Q500 instrument (TA Instruments) was used for TGA. These analyses were carried out to detect and quantify any solvents included in the crystal forms. The sample mass range was 1– 4 mg. For all experiments the heating rate was 10 K min−−1 and the flow rate of dry nitrogen was 60 cm3 min−1. The software used for the analysis of the results was TA instruments Universal Analysis 2000.18 Fourier transform infrared (FTIR) spectroscopic studies were carried out using a PerkinElmer Spectrum One FTIR spectrometer with attenuated total reflectance (ATR). Scans spanned the range 4000–400 cm−1. The data were analyzed using the program Spectrum.20 PXRD patterns were collected at constant or variable temperature on a BRUKER D8 Advance diffractometer equipped with a Lynxeye detector and using CuKα1 radiation (λ = 1.5406 Å). Samples were placed on a zero background holder with a rotating stage for samples maintained at constant temperature; for variable-temperature collections the samples were placed in a modular temperature chamber (Materials Research Instruments, Karlsruhe, Germany). The standard scanning range was 4–40 2 with 2260 steps, step sizes of 0.016 2θ and an exposure time of 20 minutes. X-ray settings were 40 mA and 30 kV. The program Mercury 3.921,

22

(Cambridge Crystallographic Data Centre, UK) was used to calculate PXRD patterns from refined crystal structures. Single-crystal X-ray diffraction data-collections were performed on a Bruker Kappa Apex II Duo diffractometer (Madison, Wisconsin) with graphite-monochromated MoKα radiation (λ =


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0.71073 Å). Crystals were mounted on cryoloops under Paratone oil (Exxon Chemical Company, Houston, Texas). The temperature was controlled using a Cryostream cooler (Oxford Cryosystems, Oxford, UK) to carry out data-collections at 173(2) K after initial unit cell dimensions had been recorded at 294(2) K to monitor possible phase changes occurring on cooling. Data reduction and unit cell refinement were performed with the program SAINT.23 All diffraction data were corrected for Lorentz-polarization effects and multi-scan absorption corrections were applied with SADABS.24 The crystal structures were solved by direct methods through the use of SHELXS.25 All non-hydrogen atoms were placed and refined with isotropic atomic displacement parameters (adps) initially and subsequently with anisotropic adps by fullmatrix least-squares with SHELXL.25 All hydrogen atoms were found in difference electron density maps and placed in idealised positions in a riding model and included with isotropic thermal parameters (Uiso) 1.2–1.5 times those of their parent atoms. In the case of MMV03 monohydrate, a DFIX command was used to restrain the O-H bond lengths of the water molecule to 0.84(2) Å. The absolute structure of the non-centrosymmetric crystal of MMV03 Form 1 was shown to be correctly assigned from the Flack parameter value which is very close to zero with a small e.s.d.26,27 To determine concentrations of MMV03 in solution, ultraviolet-visible light (UV-Vis) spectrophotometry on an Agilent Cary 60 UV-Vis spectrophotometer was performed. Spectra were recorded over the wavelength range 200–800 nm. The max values were measured at 310.5 nm in 50 % v/v ethanol in water and at 285 nm in methanol at a temperature of 25 ± 0.5 °C.


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A kinetic solubility experiment was carried out to compare the aqueous solubilities of the various polymorphic forms of MMV03 at 25 ± 0.5 °C. Due to the hydrophobic nature of the compound, a solvent medium of 25 % v/v of ethanol in water was used for this experiment. A standard curve was set up using UV-vis spectrophotometry employing a 50 % v/v ethanol in water solution, the compound being dissolved in pure ethanol initially and incrementally diluted with water to 50 % v/v to prevent precipitation of MMV03. The experiment was set up so that three separate vials containing an excess of the respective phase-pure samples were stirred at 500 rpm at 25  0.5 C to be assayed at intervals of 1.5, 24 and 72 hours. The resulting solutions were filtered through 0.45 μm nylon filters and diluted to a suitable concentration for triplicate absorbance measurements. The calibration curves are presented in the Supporting Information (Figures S2.1 and S2.2). The pertinent specifications are described in the relevant result sections.

RESULTS AND DISCUSSION Crystallographic data and X-ray structure refinement details for two polymorphs (Forms 1 and 2) and a hydrated form of MMV03 are listed in Table 1. The molecules comprising the respective asymmetric units are shown in Figure 1.

Table 1. Data-collection and refinement parameters for Form 1, Form 2 and the monohydrate of MMV03 MMV03 (Form 1)

MMV03 (Form 2)

MMV03 monohydrate

Molecular formula

C20H17N3O4S2

C20H17N3O4S2

C20H17N3O4S2 ·H2O

Formula weight (g mol−1)

427.48

427.48

445.50

Crystal system

Orthorhombic

Monoclinic

Triclinic


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Space group

P212121

P21/c

P1

a (Å)

5.4811(5)

9.7617(12)

8.2805(6)

b (Å)

16.5034(16)

8.5070(10)

9.8729(7)

c (Å)

20.9563(19)

23.799(3)

12.4456(9)

α (°)

90

90

101.036(2)

β (°)

90

97.621(2)

96.554(2)

γ (°)

90

90

90.363(2)

V (Å)3

1895.6(3)

1958.9(4)

991.70(12)

Z

4

4

2

Dc (g cm−3)

1.498

1.449

1.492

μ (Mo Kα) (mm−1)

0.315

0.305

0.308

F(000)

888

888

464

Data-collection temp. (K)

173(2)

173(2)

173(2)

Crystal size (mm3)

0.04  0.06  0.48

0.30  0.17  0.03

0.18  0.09  0.06

Range scanned θ (°)

1.57 – 28.37

1.73 – 28.34

1.68 – 28.31

Index ranges ±h, ±k, ±l

−7, 7; −22, 21; −27, 27

−13, 13; −11, 11; −31, −11, 11; −13, 13; −16, 16 31

Reflections (total)

24 852

38 374

29 794

Independent reflections

4746

4873

4920

Reflections with I > 2σ(I)

3715

3623

3798

Number of parameters

264

264

284

Rint

0.0719

0.0642

0.0507

S

1.020

1.011

1.015

R1 [I > 2σ(I)]

0.0420

0.0413

0.0385

Reflections omitted

1

2

5

wR2

0.0957

0.1078

0.0989

a, b in weighting scheme w = 1/[σ2(Fo2)+(aP)2+(bP)]

0.0447; 0.2128

0.0447; 1.1654

0.0450; 0.4001


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(Δ/σ)mean

< 0.001

< 0.001

< 0.001

Δρmin,max (e Å−3)

−0.34, 0.24

−0.38, 0.40

−0.34, 0.41

Flack parameter

0.018(96) by classical fit to all intensities;

1911941

1911993

0.031(51) from 1309 selected quotients (Parsons' method) 27 CCDC deposition number

1911934


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Figure 1. The asymmetric units and numbering schemes of MMV03 Form 1 (a), MMV03 Form 2 (b) and MMV03 monohydrate (c) with thermal ellipsoids drawn at the 50 % probability level.


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Clear differences are observed in the crystal packing arrangements and intermolecular interactions of the polymorphs (Form 1 and Form 2), as described in the following section. MMV03 Form 1 crystallizes in the space group P212121 and MMV03 Form 2 in P21/c. Relevant symmetry information and hydrogen bonding data are listed in Table S1 in the Supporting Information. The hydrogen bonding motifs are written as A–H⋯B, where the prime symbol is generic and represents the symmetry operator for atom B. The various conformers observed in Form 1, Form 2 and the hydrate are shown in Figure S3 in the Supporting Information. Four freely rotatable bonds are present in the MMV03 molecule, resulting in a somewhat flexible molecule as is evident from the variation of conformers overlaid in Figure S3. Torsion angles are listed in Table S2 in the Supporting Information. The occurrence in each of the presented conformers of intramolecular H-bonds C15-H15···N5 and C21-H21···N5 results in the phenyl rings C10C15 and C20C25 being roughly co-planar. When comparing the various conformers, some differences are visible, the most prominent being that in the case of the hydrated form, where the C20C25 ring is tilted at an angle of 28.7(2) relative to the imidazopyridazine ring. The interactions defined in the following section are written in the graph-set designation style proposed by Etter et al.28 A herringbone packing arrangement is found in MMV03 Form 1 and is visible when viewed down [001], as shown in Figure 2. The MMV03 molecules are layered in a head-to-tail manner, forming infinite ribbons propagating in the z-direction. Rings described as R22(21) are formed by the interactions C29-H29C···N1 and C7-H7···O18 which form these ribbons. The interactions C19-H19B···O17, C29-H29A···O27 with the graph-set designator R22 (32), and C14-H14···O17 form infinite columns propagating in the x-direction (Figure S4 in the Supporting Information). The H-bonds, C19-H19A···O27 and C24-H24···O28, link MMV03


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molecules to almost perpendicularly orientated surrounding MMV03 molecules in what could be misconstrued as different layers to form the herringbone pattern. Various interactions that play a role in the packing of this form are part of ‘ring’ motifs, some of which are elongated. The interactions connect molecules layered in the x-direction with other molecules layered in the ydirection and they are listed here with the motifs in parentheses: C7-H7···O18 and C14H14···O17 (R44(30)), C7-H7···O18 and C19-H19B···O17 (R44(32)), C7-H7···O18 and C29H29A···O27 (R44(48)), and C19-H19A···O27 with C14-H14···O17 (R44 (38)). These interactions are shown in Figure S5 in the Supporting Information.

Figure 2. Hydrogen bonding interactions found in the structure of MMV03 Form 1 viewed along [100] (a) and [001] (b).


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In the case of Form 2, infinite ribbons propagating in two different directions (Figure S6 in Supporting Information) are formed by the following interactions: C8-H8···N1 R22(8), C19H19A···O28 R22(32), C19-H19C···O18 R22(8), and C29-H29C···O17 R22(32) (Figure 3). These interactions are all assembled around inversion centers. The ribbons in Form 2 are formed by MMV03 molecules that are layered head-to-head and tail-to-tail. The interactions connecting the layered ribbons are C29-H29A···N1 R22(22) (Figure 3) and - interactions connecting the rings N4C9 and C10C15, generated at an inversion center by the symmetry operator 1-x,1-y,1-z (Figure S7, Supporting Information). The offset face-to-face centroid-to-centroid distance is 3.837 Å, the shortest interatomic distance between these rings being 3.328 Å (C8···C14). The next layer of molecules in the stack (generated by the symmetry operator 1−x,2−y,1−z) is not connected by - interactions and successive layers are thus not continuously -stacked; instead the sequence is interrupted in this direction to form alternating stacks (Figures S6 and S7, Supporting Information). The interactions connecting these stacks to form ribbons are C23H23···O17 and C19-H19B···O27 R22(9) (Figure 3) as well as a ring, R24(36), formed by the bifurcated H-bond comprising C19-H19B···O27 and C29-H29B···O27 (Figure S8, Supporting Information).


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Figure 3. Hydrogen bonding interactions in the structure of MMV03 Form 2 illustrated from different perspectives. The MMV03 molecules in the monohydrate phase are arranged in a head-to-tail manner and form slightly sloped layers. The topology of such a layer is presented in Figure S9 (Supporting Information). These layers are stacked in an alternating fashion according to the direction in which the molecules are orientated. Various interactions play a role in interweaving the layers, though the structure is held together by water molecules (located in isolated sites) that are strongly H-bonded and which act as linkers to attach MMV03 molecules within the same layers. The crystal structure is relatively stable as a result of the multiple H-bonds connecting the water molecules,

namely

O30-H30A···N1,

O30-H30B···O17,

C23-H23···O30

and

C29-

H29B···O30; C29-H29C···O30 (Figure 4). Consequently, these crystals do not indicate water


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loss upon storage at ambient conditions, as confirmed by the thermal results in subsequent sections. Several ring motifs are observed in the interactions connecting the layers. One of these rings is centred around an inversion center and is formed by C11-H11···O27 and its designation is R22 (24). - interactions also play a role in connecting the stacked molecules forming the different layers. The rings (C20C25) (Figure S10 in Supporting Information) are connected at a center of inversion through the symmetry operator 1−x, 1−y, −z and have an offset face-to-face centroid-centroid distance of 3.666(1) Å, the shortest atom to atom distance being that between C23 and C25 (3.383(3) Å). The rings N4C9 and C10C15, connected through the symmetry operator 2−x,1−y,1−z, have an offset face-to-face centroid-to-centroid distance of 3.781(1) Å, the closest contacts being between C8 and C15 (3.341(2) Å). There are also ring motifs present in which water plays the role of a linker (see e.g. the interactions C29-H29B···O30 and C23H23···O30 R12(7) (Figure 4).


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Figure 4. Hydrogen bonding interactions found in the structure of MMV03 monohydrate viewed from different perspectives. Two-dimensional fingerprint plots29 derived from the Hirshfeld surfaces of Form 1 and Form 2 are presented in Figure 5. The plots are very similar as a result of the shared main features of these forms. In both cases the most prominent features are the symmetrically shaped spikes, commonly observed with cyclic interactions, indicated in the plots formed by the shortest contacts, specifically those of the O⋯H and N⋯H bonds. These contact distances are slightly shorter in the case of Form 1 than in Form 2, in accord with the values reported in the H-bond interaction Table S1 in the Supporting Information. The comparable donor-hydrogen···acceptor angles reported in the tables relate to the similarly shaped, sharp spikes. An obvious dissimilarity is indicated in Figure 5 where there is a short H⋯H contact in Form 2, between the atoms labelled H19C and H8, but not in Form 1. These atoms are related by inversion and are in


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relatively close proximity (2.64 Å apart). The relative contributions of various interactions for the two polymorphic forms of MMV03 are shown in Figure 6. The comparative effects of O⋯H and N⋯H interactions are very similar, though Form 1 has a significantly higher contribution value for C⋯H and Form 2 has a higher value for H⋯H. The ratios of C⋯H/ C⋯C contributions are of interest since they reflect the nature of -interactions. These interactions are of even greater importance in cases where no strong hydrogen bond donors are present in the molecule of interest as they could be the determining factor influencing the final crystal packing arrangement.30 The C⋯H/C⋯C ratio is 7.9 (26.1 % / 3.3 %) for Form 1 and 2.6 (16.9 % / 6.5 %) for Form 2. The so-called ‘wings’ visible in the plots of both forms are due to C-H⋯ interactions that occur in the two polymorphs as shown in the Supporting Information (Figures S11 and S12). The ⋯ interactions present in Form 2 result in a smaller C⋯H/C⋯C ratio (Figure S12 in Supporting Information). It is important to bear in mind that there are nitrogen atoms in the rings N1-C2-C3-N4-C9 and N4-N5-C6-C7-C8-C9 and this will affect the C⋯H and C⋯C contributions. Another noteworthy difference is that the plot for Form 1 is more compact and the one for Form 2 is spread out over the top right corner, which correlates well with what is expected, as Form 1 has the higher density and should thus be more densely packed.


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Figure 5. Two-dimensional fingerprint plots for MMV03 Form 1 (top) and MMV03 Form 2 (bottom). Parameters de and di are the distances from a point on the surface to the nearest atom nucleus exterior and interior to the surface.

Figure 6. Relative contributions to the Hirshfeld surfaces for the close intermolecular contacts of MMV03 Form 1 and Form 2.


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Thermal analysis results obtained by DSC and MDSC are summarized in Table 3.

Table 3. Thermal data for different forms of MMV03 obtained by DSC and MDSC. Datum

Form 1

Form 2

Form 3

Form 4

Melting point onset [°C]

245.0  0.5

233.5  0.1

193.8  0.8

-

Enthalpy of fusion [kJ mol−1]

48  3

49.0  0.8

b

-

Entropy of fusiona [J K−1 mol−1]

92  10

97  2

b

-

Glass transition (midpoint) [°C]

-

-

-

104  3

a

Calculated from ΔSf = ΔHf /Tm. b Unobservable.

A single sharp endothermic peak in the DSC curve of MMV03 Form 1 (Figure 7) with an onset temperature of 245.0  0.5 C (n = 3), a peak at 247.0  0.3 C (n = 3), and an enthalpy related to the event of 112  7 J g−1 (n = 3) was visible. As was the case with Form 1, a single sharp endothermic peak was observed for Form 2 (Figure 7) with a lower onset of 233.5  0.1 C (n = 3), a peak at 235.6  0.1 C (n = 3) and an enthalpy of 115  2 J g−1 (n = 3). If traces of Form 1 were present in the sample of Form 2 the DSC curve showed an additional endothermic peak that is interrupted by an exothermic peak (Figure S13 in Supporting Information). It is possible that varying amounts of Form 1 could be present in each sample as the different forms demonstrate a tendency to crystallize concomitantly under non-ideal conditions. It was however shown that at a heating rate of 10 K min−1, pure Form 2 melts without any recrystallization and in the presence of small quantities of Form 1, recrystallization and a second endothermic peak correlating with the melt of Form 1 are observed. This would suggest that the presence of ‘seeds’ of Form 1 results in nucleation that facilitates the recrystallization of the melt to the higher melting Form 1.


21


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Figure 7. Stacked DSC curves for different forms of MMV03 obtained by DSC. The HSM results for MMV03 monohydrate are presented in Figure 8.1. The crystal becomes opaque from roughly 100 C with the onset of fragmentation and bubbles of water vapour forming simultaneously above 160 C. The formation of needle-like crystals occurs concurrently with melting of the original crystals around 255 C as is visible in the HSM Figures 8.1 and 8.2. The melting continues and is nearly complete by 265 C. The DSC result for MMV03 monohydrate is presented in Figure 9, which shows at least four distinct events. The first (labelled A) is a broad endothermic peak spanning a range from roughly 70 to 120 C and is consistent with the temperature range in which mass loss is visible in the TGA curve (Figure 10). The observed thermal results indicate the loss of water from the crystals as the most plausible explanation. The mass loss determined by TGA was 3.8  0.2 % (n = 3) and this equates to one molecule of water for every molecule of MMV03 (calculated percentage 4.0). A likely explanation for the high temperature associated with the mass loss is that the water molecule is extensively hydrogen-bonded (as was evident from the X-ray crystal structure) thus playing a


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substantial role in the structural integrity of the crystals. The event labelled B in Figure 9 commences with an endothermic peak which is curtailed by an exothermic peak. No events were observed at this temperature for any of the other forms and it was presumed that the phase present after the dehydration of the hydrated form was a new phase. In order to characterize and compare the various forms present during the heating of MMV03 monohydrate, a variabletemperature PXRD experiment was carried out and the result is presented in Figure 12. To facilitate the comparison of the known forms with the result of the variable-temperature PXRD experiment, the PXRD pattern of each individual form is presented in Figure 11. The difference between the pattern recorded at 150 C and 25 C in Figure 12 is apparent, though at first glance the pattern resembles that of Form 2. On closer inspection the pattern was found to have clear differences, including new peaks at 8.3 and 27.3 2 that were absent in the pattern of Form 2, though some high intensity peaks are present in both, such as those at 14.0, 15.2, and 16.5 2. After seeing this result it was thought that the form present from 130 to 190 C, after dehydration of the starting hydrated phase, was either a new phase or a mixture consisting of a new phase and a sizeable portion of Form 2. This new form, hereafter referred to as Form 3, is elaborated on in a later section.

The PXRD pattern recorded at 210 C matches that of Form 2 and suggests that a transition occurs to this form at the event labelled B in Figure 9. The PXRD trace recorded at 240 C matches that of Form 1 and indicates that a transition to this form occurred at the endothermic event, curtailed by an exothermic event labelled C in Figure 9. This result was also observed in DSC curves of Form 2 where traces of Form 1 were present (Figure S13 in Supporting Information). The final event (labelled D in Figure 9) is observed at the same temperature as


23


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where the pure Form 1 melts. The HSM result at 240 C shows that tiny needle-like crystals had formed and the PXRD pattern at this temperature matches that of the pure Form 1. These results serve as evidence that a transition to Form 1 had occurred before the onset of melt for this form.

Figure 8.1. HSM photographs of MMV03 monohydrate crystals immersed in silicone oil at various temperatures.

Figure 8.2. Enlarged image of the HSM photograph recorded at 255 C.


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Figure 9. Representative DSC curve for MMV03 monohydrate.

Figure 10. Representative TGA of MMV03 monohydrate.

Figure 11. Calculated PXRD patterns for MMV03 Form 1, Form 2 and the monohydrate, based on their single-crystal X-ray analyses.


25


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Figure 12. Variable-temperature PXRD showing the sequence of events starting with MMV03 monohydrate. As was described in the previous section, the metastable polymorphic form referred to as Form 3 was serendipitously discovered during a DSC experiment of the monohydrated form. A method of heating the latter form in an open DSC pan at 20 K min−1 to a temperature of 135 C was employed in an attempt to scale up the production of this form for use in subsequent characterization. The product of this method turned out to contain a mixture consisting of mostly Form 3, but with small traces of Form 2 being present. It was hypothesized that the transition from the hydrate to Form 2 was caused by heating and a new method was devised in order to avoid this thermally induced transition. The hydrated sample was freeze-dried with a VirTis® BenchTop™ “K” Series Freeze Dryer (SP Industries, Inc., Warminster, Pennsylvania). The method entailed cooling the sample to −60 C under vacuum in order to dehydrate the sample without applying heat. The product was determined to be pure Form 3 by PXRD analysis. This method did not, however, produce any crystals of adequate size and quality for use in structural elucidation by single-crystal X-ray diffraction.


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HSM results for Form 3 are presented in Figure 13. The onset of melting of the powder occurs at the periphery of the images at approximately 200 C. At 208 C the powder near the periphery has solidified and the powder near the center has begun to melt. The reason for this transition occurring from the outside inward is that the center of the image is situated above a window that allows stage illumination and is at a slightly lower temperature than the surrounding heating block. The transition appears to be complete near 215 C. The solid product of the first transition starts to melt around 250 C and is completely melted by 260 C.

Figure 13. HSM micrographs of MMV03 Form 3 recorded at various temperatures. The first event visible in the DSC results for MMV03 Form 3 (Figure 7) has an onset temperature of 193.8  0.8 C (n = 3), comparable to the onset of melting observed by HSM. It was not possible to measure accurately the enthalpy of this event as the endothermic peak appears to be abruptly curtailed by the onset of an exothermic event. The next visible feature is a sharp

endothermic

peak

with

an

onset

at

244.8  0.2 C (n = 3)

and

a

peak

at

246.9  0.1 C (n = 3). These data are consistent with the melting behavior observed for Form 1. It is possible to deduce from the combination of the DSC and HSM results that the Form 3 powder starts to melt and subsequently recrystallizes into Form 1, this event being followed by the melting of Form 1. No sign of Form 2 was observed in this DSC curve, reaffirming the claim that Form 3 is a distinct polymorphic form. The absence of the Form 2 peaks rules out the


27


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possibility of the common features present in the PXRD patterns being due to the samples simply being a mixture of the forms. The similarities could thus merely be coincidental and are assumed to be due to similar crystal packing features. Forms 1 and 2 displayed negligible mass loss with TGA up to the onset of decomposition, indicating that these are not solvated/hydrated. A small mass loss was, however, observed during heating in the case of Form 3 (Figure S14, Supporting Information). The onset appears to coincide with the commencement of heating and the total measured mass loss during heating to 100 C was 0.9  0.1 % (n = 2). Possible explanations for this occurrence are that after the freeze-drying process, some residual water could be present in the crystals or that moisture from the air could have been adsorbed onto the highly hygroscopic surface of the powder. It was found that melting and subsequent cooling of any of the forms described in the previous sections results in the formation of a brittle glass by vitrification. This amorphous form is referred to as Form 4 hereafter. No signs of recrystallization were observed in DSC curves when the molten sample was cooled and the onset of decomposition of MMV03 was determined by TGA to be much higher than the melting temperatures of the known forms. The method used to produce this amorphous form in sufficient quantity for further characterization was to heat the starting material in an open DSC pan to a temperature of 255 C at a heating rate of 50 K min−1 and the product was then left to cool at an uncontrolled rate. It was found that this material has a good glass-forming ability and it was possible to exceed the critical cooling rate for forming a glass without any further steps being required. The experimental PXRD pattern for MMV03 Form 4 is presented in Figure 14. The result is typical for a phase lacking long-range order and the broad, low intensity hump, devoid of distinguishing features is commonly referred to as a diffuse “halo”.


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Figure 14. Experimental PXRD pattern of MMV03 Form 4. HSM results for Form 4 are presented in Figure 15. The micrographs at 140 C show what appears to be a phase change from a brittle solid to a fluid state. Resolidification commences near 150 C and continues until completion around 200 C. The onset of melting is visible at 250 C and is completed at 258 C.

Figure 15. HSM photographs of MMV03 Form 4 at various temperatures. The DSC result for MMV03 Form 4 is presented in Figure 16. The event labelled A has the signature appearance of a glass transition having a midpoint calculated at half height of 104  3 C (n = 3). This event is also visible in the HSM micrographs. The thermal history of a glass affects the enthalpy of a glass transition due to release of energy over time as the system approaches equilibrium and becomes more ordered. The rate of enthalpic


29


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relaxation will increase at higher temperatures and decrease at lower temperatures down to a minimum at the Kauzmann temperature (TK).31,32 The latter parameter is the temperature at which most translational molecular motion will be slowed to a point at which the chemical and physical changes of pharmaceutical products are deemed negligible over experimental timescales. Many methods exist for calculating TK including a simple approximation stated as Tg −50 K.33 MMV03 Form 4 has a relatively high Tg and should thus be thermally stable when maintained at or below room temperature. It is important to note that other factors such as humidity could also affect transition rates to more stable forms. The experimental heating rate used will also significantly affect Tg as the macromolecular motion is time-dependent and more enthalpic relaxation could occur at slower heating rates, where the samples are exposed to higher temperatures for longer periods. Glasses can be categorized as ‘strong’ or ‘fragile’, where evidence for the latter is a significant heat capacity change at Tg, while the former is more difficult to detect as the change is considerably smaller.33 MMV03 Form 4 is expected to have high fragility, relying on a “rule of thumb” described by Kaushal and Bansal,33 who proposed that a Tm/Tg ratio of less than 1.5 indicates fragility and a ratio higher than 1.5 suggests strong glass characteristics. The second observed event (labelled B in Figure 16) is exothermic and has an onset temperature of 161  7 C (n = 3), a peak at 174  9 C (n = 3), and an enthalpy related to the event of −73  2 J g−1. Recrystallization was observed at this temperature in the HSM micrographs. The next event (labelled C) is endothermic with an onset temperature of 243  2 C (n = 3), a peak at 246  1 C (n = 3), and an enthalpy of 95  4 J g−1. This event corresponds to the melting observed in the HSM micrographs for this form, which is also consistent with the melting


30

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temperature of MMV03 Form 1. The suggested account for the events is that the glass undergoes a glass transition at A, recrystallizes into the higher melting Form 1 at B and finally melts at C.

Figure 16. Representative DSC curve for MMV03 Form 4. Calculating thermodynamic transition points (Tt) Numerous examples in the literature34–37 demonstrate how one can approximate the order of transition points of different solid forms and determine whether these forms are monotropically or enantiotropically related through the use of expression (1), provided that sufficiently accurate thermochemical data are available. The required data include melting points, heat capacities, and enthalpies of fusion. 𝑻𝐭 =

∆𝑯𝐟,𝟐 ― ∆𝑯𝐟,𝟏 + (𝑪𝐩,𝐥𝐢𝐪 ― 𝑪𝐩,𝟏)(𝑻𝐟,𝟏 ― 𝑻𝐟,𝟐) ∆𝑯𝐟,𝟐 𝑻𝐟,𝟐

―

∆𝑯𝐟,𝟏 𝑻𝐟,𝟏

𝑻𝐟,𝟏

+ (𝑪𝐩,𝐥𝐢𝐪 ― 𝑪𝐩,𝟏)𝒍𝒏(𝑻 )

(1)

𝐟,𝟐

where Tt is the transition temperature (K), Tf is the melting point (K), ΔHf is the enthalpy of fusion (kJ mol−1), and Cp is the heat capacity (kJ K−1 mol−1). The difference in heat capacity of the liquid and that of the higher melting form (Cp,liq-Cp,1) can be replaced with an empirical correction term kΔfusH1 if highly accurate heat capacities are unavailable. This results in a simplified expression (2). A typical range of values for k is 0.001 to 0.007 K−1 according to Yu,36 while Burger and Ramberger34,35 reported a maximum value of


31


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0.005 K−1. The k value used for calculations in subsequent sections of the present account was 0.003 K−1. 𝑻𝐭 =

∆𝑯𝐟,𝟐 ― ∆𝑯𝐟,𝟏 + 𝒌∆𝑯𝐟,𝟏(𝑻𝐟,𝟏 ― 𝑻𝐟,𝟐) ∆𝑯𝐟,𝟐 𝑻𝐟,𝟐

―

∆𝑯𝐟,𝟏 𝑻𝐟,𝟏

𝑻𝐟,𝟏

(2)

+ 𝒌∆𝑯𝐟,𝟏𝒍𝒏(𝑻 ) 𝐟,𝟐

Solubility experiments A kinetic solubility experiment was carried out initially in a 25 % v/v of ethanol in water with Forms 1 to 3 (Figure S15, Supporting Information). The results obtained were surprising in that Form 1 demonstrated the highest solubility and Form 3 the lowest, contrasting the expected results presumed from the stability ranking determined from the thermal experiments. It was hypothesized that solvent-mediated transformations may have been playing a role in this outcome and the products of the various vials were therefore analyzed by PXRD to determine which phases were present at each stage of the experiment. At 72 hours the Form 1 and Form 2 vials had remained phase-pure, while Form 3 had transitioned into the hydrated form during the course of the experiment. The transition from Form 3 to the hydrate was unsurprising as this is a metastable dehydrated form of the monohydrate and the medium used for this solubility experiment is mostly aqueous. The surprising result was that almost complete conversion from Form 3 to the hydrate had taken place after a mere 1.5 hours, with traces of Form 2 also being present. In order to prevent transformations to the hydrated form, the experiment was repeated with a solvent medium in which the water content was minimized. The same experiment was thus repeated with methanol (99.5 %) as solvent medium and in this case the first time-interval was taken at one hour instead of 1.5 hours and the third was taken after five days instead of three. The result of this experiment is presented in Figure 17. The first experiment was carried out before the discovery of Form 4, but the latter was added to the list of tested forms in this experiment. As expected the amorphous form had the highest concentration at the one hour


32

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mark. The measured concentration was much higher for Form 4 with values 4.0, 4.9, and 3.1 times those of Forms 1, 2, and 3 respectively. After stirring for 24 hours the concentration of Form 4 was lower than at the one hour mark and had dropped to a concentration nearer to those of the other forms. After 5 days of stirring the concentration of Form 4 was even lower, specifically just higher than the values measured for Forms 1 and 2 and slightly lower than that of Form 3. At each of the assessed intervals Form 3 had the highest concentrations of the known crystalline forms (roughly 1.3 and 1.5 times that of Form 1 and Form 2 respectively). Unsurprisingly, the measured concentrations were higher after 24 hours of stirring than after 1 hour. The crystalline forms showed roughly the same ratio of solubility during the course of this experiment. In order to assess whether solvent-mediated phase transitions had taken place, the products of each of the vials that had been stirred for five days were tested using PXRD (Figure S16, Supporting Information). Near complete conversion to Form 2 of the contents of the Form 1, Form 3 and Form 4 vials was observed while the contents of the Form 2 vials remained the same. The lower solubility of Form 2 and the observed transitions indicate that at a temperature of 25 ± 0.5 °C, Form 2 has the highest stability of the forms tested.


33


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Figure 17. Chart displaying MMV03 concentrations in methanol at 25 ± 0.5 °C after 1 h, 24 h, and 5 days of stirring.

Solvent-mediated transformation experiments Solvent-mediated transformation experiments were employed to establish the stability ranking of Forms 1 and 2 at different temperatures. The method involved placing phase-pure samples in vials with selected solvents (methanol/1-butanol) which were left to stir for a period of five days at 500 rpm. The solid products of these experiments as well as the products from the solubility experiments described in the preceding section were then inspected by PXRD analysis to determine which form was present (Figure S17, Supporting information). The solubility experiments were carried out with methanol as solvent at 25 ± 0.5 °C. The same setup was used for the solvent-mediated transformation experiment carried out at 50 ± 0.5 °C. In order to carry out the same experiment at higher temperatures, a different solvent with a higher boiling point was required and for this reason 1-butanol was used to carry out the experiments at 75 ± 0.5 °C


34

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and 100 ± 0.5 °C. As discussed previously, Form 1 had completely converted to Form 2 after stirring at 25 °C and Form 2 remained unchanged. At 50 °C and at 75 °C the result was the same. However, complete conversion of Form 2 to Form 1 was observed when carried out at 100 °C, with Form 1 in this case remaining the same. This result indicates that the temperature at which the free energies for these forms are equal lies between 75 and 100 °C and that Form 2 is the more stable form up to this temperature, while Form 1 is stable at higher temperatures.

Energy/temperature diagram The information acquired from the different characterization methods was used to create a schematic energy/temperature diagram (Figure 18). The thermodynamic stability ranking deduced from the kinetic solubility and solvent-mediated transition experiments indicated that at 25 °C the order from highest to lowest is Form 2 > Form 1 > Form 3 > Form 4. It is assumed that the ranking will be the same when the temperature is extrapolated to 0 K. Form 4 should have the highest free energy at any temperature as glasses naturally have higher free energy than crystalline forms. At temperatures below Tg the free energy isobar for Form 4 is higher than that of the liquid because of an excess of enthalpy and configurational entropy caused by the drastically reduced molecular motion resulting in the sample not being able to reach an equilibrium at experimental rates. During heating the molecular mobility of the sample will increase rapidly at Tg with increases of up to 10-fold with increases in temperature of 3 to 5 K just above Tg.38 The increase in mobility at Tg allows the glass to reach equilibrium with its surroundings and the sample will have properties of a supercooled liquid above this temperature. This is represented in the diagram as a merging of the glass and liquid isobars. A glass transition is however a kinetic process and will depend on the crossing of timescales of molecular


35


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rearrangement and experiments performed. Forms 1 and 2 were found to be more stable than Form 3 at 25 °C, with Form 3 having the lowest Tf of the crystalline polymorphic forms. The ΔHf of Form 3 is presented as an estimation in Figure 18 since the exact value was unobservable due to the incongruent nature of this event. Form 3 has a lower melting temperature than the other forms and no transitions to Form 3 were observed. The G isobars would thus not cross and this would indicate that Form 3 is monotropically related to Forms 1 and 2. Form 2 has a lower Tf than Form 1 and has the higher stability of the two forms at room temperature. The G isobars for these two forms would thus have to cross between these temperatures. Calculations were employed [making use of expressions (1) and (2) the calculated Tt values were 73.2 and 77.7 °C respectively] to determine the Tt of Form 2 to Form 1 as the ΔHf values were too close to make use of these values alone to establish the relationship of these forms in terms of enantiotropy/monotropy with confidence. The solvent-mediated transition experiments also indicated that Tt of Form 2 to 1 takes place between 75 and 100 °C.

Figure 18. A schematic energy-temperature diagram for the various polymorphs of MMV03.


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Stability ranking of the various forms The heat of fusion rule states that in the case of enantiotropically related polymorphs the higher melting form has the lower heat of fusion; if not, they are monotropically related.34 The melting points of Form 1 and Form 2 showed a significant difference (greater than 10 C), Form 1 having the higher melting onset. However, the heats of fusion were experimentally determined to be very similar and when taking standard deviations into consideration these differences are not statistically significant. The heat-of-fusion and entropy-of-fusion rules34 can thus not be applied with confidence in this instance. Form 3 has a melting point with an onset approximately 40 C lower than that of Form 2. The incongruent nature of this event prevented the measurement of the heat of fusion. Overlap of events, namely the melt of Form 3 and recrystallization of the more stable Forms 1 and 2, render the separation and quantification of the heats of fusion of the individual events impossible. Attempts to prevent recrystallization by increasing the heating rate to 150 K min−1, with the notion that the melt would not have sufficient time to recrystallize, were unsuccessful. Recrystallization to Form 1 by heating any of the other forms (Forms 2, 3, 4, and the monohydrate) was, however, thought to be incomplete at these heating rates as the observed heat of fusion was significantly lower in each case than for the pure Form 1. It is possible to calculate the recrystallized fraction from the ratio of these enthalpies as illustrated by Lefort et al.39

FT-IR analysis The FT-IR spectra of the various forms are presented in Figure S18 in the Supporting Information. Clear differences in intensity, peak positions, and multiplicity are visible in the fingerprint region as well as in the C-H and O-H stretching regions. Absorption due to the


37


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presence of water is evident from the broad and intense peaks in the 3000–3500 cm−1 region in the case of the hydrated form and to a much lesser extent in the spectrum of Form 3. When compared with the spectra of the crystalline forms, that for Form 4 displays broader peaks with lower intensity, as expected for an amorphous form with its wider distributions of interatomic distances and energies of molecular interactions. Distinguishing the various solid forms of MMV03 would thus be possible by inspecting the FT-IR spectra alone.

CONCLUSION This study was based on the premise that early screening for possible solid-state forms of a promising new drug lead and comprehensive characterization of such products are desirable in order to (a) generate a multiplicity of phases from which the best candidate may be selected for eventual formulation and, (b) pre-empt problems (e.g. unforeseen polymorphic transitions) that might arise during more advanced stages of its development. Five novel solid-state forms of the antimalarial drug lead MMV03 were isolated. The three polymorphic forms of MMV03 were characterized with reference to their PXRD profiles and physicochemical properties, including solubility and stability rankings. The two polymorphs whose single-crystal X-ray structures were determined crystallize in the space groups P212121 (Form 1) and P21/c (Form 2). Methods were developed to scale up all solid forms reproducibly and rapidly identify them by FT-IR or PXRD. The amorphous phase, MMV03 Form 4, displayed a significant improvement in the dissolution rate (a factor of 3 to 4 times the concentration at 1 h of stirring) compared to the crystalline polymorphs. A monohydrate form of MMV03 was also produced and characterized. While this form might not be the preferred one to use in formulation of a drug product, the knowledge of its existence is nevertheless essential, as are the available reliable references for PXRD and FTIR comparison and understanding of the measures required to prevent its formation during


38

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processing of the other forms. The aim of the study was to produce new solid-state forms of MMV03 with improved physicochemical properties, the most important of which being improved solubility, to be used in the formulation of new drug products. This goal was accomplished and any of these solid forms could in principle be used to formulate new products of the drug lead. In order to select the best form to be used in formulation, further experiments such as accelerated stability testing under elevated temperature and humidity and the products generated will also require analysis to confirm that no transitions take place during the manufacturing processes involved.

ASSOCIATED CONTENT Supporting Information. The following file is available free of charge on the ACS Publications website at DOI: ***************. UV calibration curves, PXRD patterns, H-bond and torsional parameter data, crystal packing motifs, FTIR spectra, solubility data (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; Telephone: +27 21 650 3071; Fax: +27 21 650 5195. 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.


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Funding Sources These include the University of Cape Town, South African Medical Research Council, and South African Research Chairs Initiative of the Department of Science and Technology, administered through the South African National Research Foundation (NRF, Pretoria).

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The funders listed above are gratefully acknowledged for support (K.C., SARChI grant 64767). M.R.C. thanks the University of Cape Town and the South African National Research Foundation (NRF) for research support (grant number 80825). Any opinion, findings and conclusions or recommendations expressed above are those of the authors and therefore the NRF does not accept any liability in that regard.

REFERENCES 1. S. Bhatt, D. Weiss, E. Cameron, D. Bisanzio, B. Mappin, U. Dalrymple, K. Battle, C. Moyes, A. Henry and P. Eckhoff. The effect of malaria control on Plasmodium falciparum in Africa between 2000 and 2015. Nature, 2015, 526, 207. 2. World

Health

Organization

(WHO),

World

Malaria

Report

http://www.who.int/malaria/publications/world-malaria-report-2018/report/en/,

2018, Geneva,

Switzerland (accessed 04 December 2018).


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For Table of Contents Use Only Five solid forms of a potent imidazopyridazine antimalarial drug lead: a preformulation study Terence J. Noonan, Kelly Chibale, Peter M. Cheuka, Malkeet Kumar, Susan A. Bourne and Mino R. Caira*

Synopsis Five novel solid-state forms of the antimalarial drug lead MMV03 were isolated during preformulation experiments. Polymorphic forms were characterized with reference to their physicochemical properties, including solubility and stability rankings. An amorphous phase displayed a significant improvement in the dissolution rate compared to the crystalline polymorphs. The produced forms are viable options for use in formulation of drug products.


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Figure 1. The asymmetric units and numbering schemes of MMV03 Form 1 (a), MMV03 Form 2 (b) and MMV03 monohydrate (c) with thermal ellipsoids drawn at the 50 % probability level. 98x259mm (300 x 300 DPI)


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Figure 2. Hydrogen bonding interactions found in the structure of MMV03 Form 1 viewed along [100] (a) and [001] (b). 85x116mm (300 x 300 DPI)



Figure 3. Hydrogen bonding interactions in the structure of MMV03 Form 2 illustrated from different perspectives. 85x118mm (300 x 300 DPI)


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Figure 4. Hydrogen bonding interactions found in the structure of MMV03 monohydrate viewed from different perspectives. 85x104mm (300 x 300 DPI)



Figure 5. Two-dimensional fingerprint plots for MMV03 Form 1 (top) and MMV03 Form 2 (bottom). Parameters de and di are the distances from a point on the surface to the nearest atom nucleus exterior and interior to the surface. 65x125mm (300 x 300 DPI)


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Figure 6. Relative contributions to the Hirshfeld surfaces for the close intermolecular contacts of MMV03 Form 1 and Form 2. 91x46mm (300 x 300 DPI)



Figure 7. Stacked DSC curves for different forms of MMV03 obtained by DSC. 62x51mm (300 x 300 DPI)


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Figure 8.1. HSM photographs of MMV03 monohydrate crystals immersed in silicone oil at various temperatures. 70x26mm (300 x 300 DPI)



Figure 8.2. Enlarged image of the HSM photograph recorded at 255 ○C. 13x17mm (300 x 300 DPI)


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Figure 9. Representative DSC curve for MMV03 monohydrate. 76x53mm (300 x 300 DPI)



Figure 10. Representative TGA of MMV03 monohydrate. 79x63mm (300 x 300 DPI)


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Figure 11. PXRD traces for MMV03 Form 1, Form 2 and the monohydrate, based on their single-crystal X-ray analyses. 106x78mm (300 x 300 DPI)



Figure 12. Variable-temperature PXRD showing the sequence of events starting with MMV03 monohydrate. 100x74mm (300 x 300 DPI)


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Figure 13. HSM micrographs of MMV03 Form 3 recorded at various temperatures. 53x26mm (300 x 300 DPI)



Figure 14. Experimental PXRD trace of MMV03 Form 4. 107x76mm (300 x 300 DPI)


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Figure 15. HSM photographs of MMV03 Form 4 at various temperatures. 36x18mm (300 x 300 DPI)



Figure 16. Representative DSC curve for MMV03 Form 4. 79x58mm (300 x 300 DPI)


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Figure 17. Chart displaying MMV03 concentrations in methanol at 25 ± 0.5 °C after 1 h, 24 h, and 5 days of stirring. 127x69mm (300 x 300 DPI)



127x101mm (300 x 300 DPI)


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Scheme 1. The molecular structure of MMV652103 (‘MMV03’) 79x59mm (300 x 300 DPI)



not applicable 88x34mm (600 x 600 DPI)


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Five solid forms of a potent imidazopyridazine antimalarial drug lead

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