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Evaluating a crystal energy landscape in the context of industrial polymorph screening Salima Z Ismail, Clare Anderton, Royston C Copley, Louise Susan Price, and Sarah L. Price Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/cg400090r • Publication Date (Web): 28 Mar 2013 Downloaded from http://pubs.acs.org on April 8, 2013

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Crystal Growth & Design

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Evaluating a crystal energy landscape in the context of industrial polymorph screening Salima Z. Ismail1, Clare L. Anderton1,*, Royston C. B. Copley1, Louise S. Price2, Sarah L. Price2 1

2

GlaxoSmithKline, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, United Kingdom

University College London, Department of Chemistry, 20 Gordon Street, London, WC1H 0AJ, United Kingdom

To evaluate how the calculation of a crystal energy landscape can be used in the solid-form screening of pharmaceuticals, a Knowledge Transfer Secondment between GlaxoSmithKline and University College London was established to carry out computational crystal structure prediction (CSP) and further guided experimentation on a molecule from GSK’s compound collection. The molecule chosen was 6-[(5-chloro-2-([(4-chloro-2fluorophenyl)methyl]oxy)phenyl)methyl]-2-pyridinecarboxylic acid (GSK269984B) since the preliminary thermodynamic form screening had only identified one anhydrate, Form I. The calculations confirmed that Form I is the most thermodynamically stable form. The thermodynamically competitive computed structures all had very different conformations of GSK269984B and further experiments were designed to attempt to generate these conformations in solution and hence the crystalline solid. The experimental screening generated four novel solvates which all eventually transformed to Form I, two of which could also be structurally characterized by single crystal Xray diffraction. The molecular conformation (apart from the position of the polar proton) in all three crystal structures was, however, very similar. GSK269984B appears to have an unusually small number of solid forms because there is no kinetic barrier to crystallizing in the most stable conformation which corresponds to the most thermodynamically stable and densely packed structure.

*

Author to whom correspondence should be addressed. Dr Clare L. Anderton, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, United Kingdom. Tel +44 (0)1438 768510. Fax +44 (0)1438 764869. Email [email protected] ACS Paragon Plus Environment


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Evaluating a crystal energy landscape in the context of industrial polymorph screening Salima Z. Ismail1, Clare L. Anderton1,*, Royston C. B. Copley1, Louise S. Price2, Sarah L. Price2 1

2

GlaxoSmithKline, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, United Kingdom

University College London, Department of Chemistry, 20 Gordon Street, London, WC1H 0AJ, United Kingdom

[email protected]

Abstract: To evaluate how the calculation of a crystal energy landscape can be used in the solid-form screening of pharmaceuticals, a Knowledge Transfer Secondment between GlaxoSmithKline and University College London was established to carry out computational crystal structure prediction (CSP) and further guided experimentation on a molecule from GSK’s compound collection. The molecule chosen was 6-[(5-chloro-2-([(4-chloro-2fluorophenyl)methyl]oxy)phenyl)methyl]-2-pyridinecarboxylic acid (GSK269984B) since the preliminary thermodynamic form screening had only identified one anhydrate, Form I. The calculations confirmed that Form I is the most thermodynamically stable form. The thermodynamically competitive computed structures all had very different conformations of GSK269984B and further experiments were designed to attempt to generate these conformations in solution and hence the crystalline solid. The experimental screening generated four novel solvates which all eventually transformed to Form I, two of which could also be structurally characterized by single crystal Xray diffraction. The molecular conformation (apart from the position of the polar proton) in all three crystal structures was, however, very similar. GSK269984B appears to have an unusually small number of solid forms because there is no kinetic barrier to crystallizing in the most stable conformation which corresponds to the most thermodynamically stable and densely packed structure.

*

Author to whom correspondence should be addressed. Dr Clare L. Anderton, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, United Kingdom. Tel +44 (0)1438 768510. Fax +44 (0)1438 764869. Email [email protected] ACS Paragon Plus Environment

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Introduction The development of Crystal Structure Prediction (CSP) techniques was partly inspired by the promise of useful applications in the field of industrial pharmaceutical development.1-4 The crystal energy landscape,5 the set of the most stable computer generated structures, can be examined for potential polymorphs, with the focus not restricted to seeking the thermodynamically most stable structure. Work on smaller molecules has previously been carried out, where the screening in an academic institution complemented developing the methodology and gave vital insights.5,6 Recent success in the CCDC blind test7 for a highly flexible and drug-like molecule [benzyl-(4-(4-methyl-5-(p-tolylsulfonyl)-1,3-thiazol-2-yl)phenyl)carbamate (XX)] suggested it was timely to evaluate how CSP might fit into industrial solid form screening.8 This was done by a secondment of an industrial particle scientist (S. Ismail) into the academic laboratory. A molecule that had previously been under development within GSK was chosen (for which a preliminary form screen had been performed on the neutral molecule) in order to simulate the process of further screening guided by the crystal energy landscape as a joint academic-industrial study. The molecule chosen was 6-[(5-chloro-2-([(4chloro-2-fluorophenyl)methyl]oxy)phenyl)methyl]-2-pyridinecarboxylic acid, which will be referred to throughout this paper by the GSK identifier for this compound, GSK269984B (Figure 1Figure 1). The sodium salt of this compound has been reported as an EP(1) receptor antagonist for the treatment of inflammatory pain.9 The preliminary thermodynamic form screen had already been done, as is usual, prior to first time tests in man (phase 1). This molecule had crystallized so readily that the structure of Form I had already been determined using single crystal X-ray diffraction, and the thermodynamic screen suggested this was the thermodynamically most stable form.

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O3

C 20 O 2

θcarboxylic C4

θΟΗ

C5

C3

H1

N1 C2

C1

θpyridine

C6 C12 Cl 1

C7

θOPhCl

C11 C10

θCPhCl

C8 C9

O 1θ

C19

CO

C 13 C 14

θPhF

C18 C17

Cl 2

C 15 C16

F1

Figure 1. 6-[(5-Chloro-2-([(4-chloro-2-fluorophenyl)methyl]oxy)phenyl)methyl]-2-pyridinecarboxylic acid (GSK269984B) with atom labels used in computational work. The torsion angles considered as flexible in the computational work are defined as θpyridine (C2_C1_C6_C7), θCPhCl (C1_C6_C7_C8), θOPhCl (C7_C8_O1_C13), θCO (C8_O1_C13_C14), θPhF (O1_C13_C14_C15), θcarboxylic (C4_C5_C20_O2) and θOH (C5_C20_O2_H1). Although GSK269984B has fewer atoms than the drug like molecule used in the CCDC blind test (XX), it provides additional challenges. The carboxylic acid group is expected to form the strongest hydrogen bonding interactions, though the formation of these has to be balanced with the ability to pack the bulk of the molecule. Since the carboxylic acid is ortho to a pyridine nitrogen, there is the possibility of either intramolecular or intermolecular hydrogen bonds, a situation that is particularly demanding of the method of evaluating the lattice energy.10 The ortho substitution of the C7-C12 ring suggests a more complex conformational surface than XX with the flexibility to adopt quite a range of three dimensional conformations. The primary aim was to determine whether the crystal energy landscape helped to validate experimental observations. A secondary aim was to evaluate how the process of calculation and interpretation of the crystal energy landscape could be made most useful to the scientists involved in the solid-form screening. In this paper the following are reported: the crystal structure resulting from the previous thermodynamic polymorph screening; the calculation of the low energy structures of the molecule combined with a more tailored metastable form screen; and the solvate single crystal structures generated through this. This helps understand the extent to which the ACS Paragon Plus Environment

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calculations might affect the comprehensive screen that would be undertaken for final development and how computational methods can be used to complement experimental screening data.

Method Calculation and analysis of the crystal energy landscape (Crystal Structure Prediction) The crystal energy landscape was generated using crystal structure prediction (CSP) methods1 based on quantum mechanical calculations on the single molecule, to provide conformational energy differences ∆Eintra, and the molecular charge distribution. This molecular charge distribution is analyzed to give the model for the intermolecular electrostatic forces, which in combination with an empirical exp-6 repulsion-dispersion model is used11 to evaluate the intermolecular lattice energy Uinter. The relative stability of the different crystal structures is estimated from the lattice energy, Elatt=Uinter+∆Eintra, which is the energy of the static crystal relative to a gas of the molecules in their lowest energy conformation. The search for possible crystal structures has to consider the entire range of conformations which the molecule could adopt to give a favorable crystal structure, balancing specific intermolecular interactions such as hydrogen bonding with the dispersion stabilization from a densely packed crystal structure. Ab initio calculations on a single GSK269984B molecule showed that the accessible conformational space naturally separated into 9 regions separated by barriers, as defined in Figure 2. Crystal structures in each conformational region were generated using CrystalPredictor,12 with one molecule in the asymmetric unit cell (Z′ =1) in the 12 most common space groups observed for molecules of this size8 in the Cambridge Structural Database (CSD).13 Approximately 500 unique low energy structures of the million generated were optimized to minimize their lattice energy, Elatt, with CrystalOptimizer14,15 allowing all the torsion angles shown in Figure 1Figure 1 to change along with the crystal cell parameters and relative positions and orientations of the molecules. As a final refinement of the lattice energy, the effect of an average organic crystalline environment16 was approximated by using a Polarizable Continuum Model (PCM)17 with ε=3, to calculate the molecular charge density and conformational energy ∆Eintra. Further details are in SI Section 6.1. Mercury18 was used to analyze the resulting crystal structures, including graph set19 analysis of the hydrogen bonding and other close contacts, and void space calculations. The similarity of pairs of crystal structures and the molecular conformations in them were measured by calculating the root mean square deviation (RMSDn) of the n ACS Paragon Plus Environment


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molecule overlay20 using the Crystal Packing Similarity tool. The Logit Hydrogen Bond Propensity method 21,22 was also applied to GSK269984B to determine which hydrogen bonds between the functional groups were most likely to occur based on statistics taken from the crystal structures in the CSD.

θCO = 90

θpyridine θCPhCl

θCO = 180

θOPhC

Intra

θPhF

InterA

θCO = 270

InterB Figure 2. Definition of the nine different conformational regions used as separate searches with the arrows indicating the other torsion angles that were allowed to vary during the search. The overall range of conformations covered in the search is shown in SI Section 6.1.2. The three carboxylic acid conformations were each combined with the three θCO regions, and the search name (e.g. 180InterA) used as a classification of the structures.

Experimental The known anhydrate structure has been named according to the Kofler notation using Roman numerals (i.e., the highest melting anhydrate is named Form I). All solvates, hydrates, metastable forms or solids with unique Raman spectral or XPRD data (regardless of the extent of characterization) have also been described as Forms but with Arabic numeral labels (e.g. Form 2). Further details of all experimental techniques, including instrumentation and ACS Paragon Plus Environment

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characteristic spectra are provided in the SI Sections 1-4. The crystallization methods used to obtain Forms 2 and 6 in the screen were adapted in order to obtain phase pure and/or better quality crystals which could be used for single crystal X-Ray analysis to determine the structures. Details of the X-ray crystal structure determinations of Forms I, 3 and 6 can be found in SI Section 3.4, together with the unit cell dimensions measured for Form 2.

Thermodynamic Polymorph Screen An automated thermodynamic polymorph screen of GSK269984B Form I with high chemical purity had been carried out before this joint study. The screen involved four different crystallization modes (slurry ripening, slow cool and evaporation, and vapor diffusion) and its purpose was to find the most stable form (SI Section 1.1).23 In order to explore a wider experimental space during the joint project, the potential of microemulsion crystallization24 in experimental screening was also investigated (SI Section 1.2). It has previously been useful in obtaining stable polymorphs which had not crystallized from bulk solution due to a high nucleation barrier and/or slow growth rate and so provides a test of the conclusions on form stability.

Metastable Polymorph Screen The metastable screen included four modes of crystallization (fast evaporation, rapid cooling, anti-solvent addition and thermal crystallization) which were selected to encourage rapid nucleation and crystallization of kinetic forms rather than the thermodynamically most stable form (SI Section 2). GSK269984B Form I of high chemical purity was used as input to this screen. Following the results of the computational work, solvents were selected that were thought might encourage intra- or intermolecular hydrogen bonding of GSK269984B, in addition to those which gave potential new forms in the thermodynamic form screen.

Results Computational Modeling Results The CSP study considered the whole range of low energy conformations of GSK269984B, as illustrated in SI Section 6.1.2 and in summary in Figure 2, which can be classified into three distinct carboxylic acid conformations (where the



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main difference is in the position of the only classically hydrogen bonding proton) each combined with a wide range of relative orientations of the aromatic rings.

Figure 3. Summary plot of the crystal energy landscape of GSK269984B. Each point represents a crystal structure classified by the search in which it was found, i.e. by θCO (90, 180 or 270°) and acid conformation (Intra, InterA or InterB) as defined in Figure 2. The calculated crystal energy landscape (Figure 3) had as the global minimum in lattice energy a structure that matched the X-ray single crystal result for Form I (Figure 4), both in reproducing the fifteen molecule coordination cluster (RMSD15 = 0.25 Å) and the molecular conformation (RMSD1 = 0.21 Å).


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Figure 4. Overlay of experimental Form I (colored by element) and calculated global minimum (blue) crystal structures. The internal hydrogen bond is highlighted (purple dotted line). The calculated crystal energy landscape (Figure 3) shows that almost three hundred structures were within 18 kJ mol-1 of the most stable structure. Detailed analysis, including the torsion angles, hydrogen bonding graph sets and void space analysis, was performed on the 38 lowest energy structures (within 7.5 kJ mol-1 of the most stable) and is reported in SI Tables 10 and 11. Form I is notable for being the densest of the lower energy structures found in the search. It also stands out as having the lowest conformational energy (SI Table 10). Thus, the conformation observed in Form I is not only a particularly low energy conformation in isolation, but this also gives one of the most dense, as well as energetically favorable crystal structures.

(a) (b) Figure 5. Contrasting the crystal packing in (a) Form I and (b) 90InterB36, the second most stable crystal structure on the crystal energy landscape (Figure 3). Hydrogen bonds are shown (purple dotted line). ACS Paragon Plus Environment


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The other crystal structures that are calculated to be competitive in energy with Form I are very different. The second lowest energy structure (Figure 5b) has a totally different overall conformation, with the aromatic rings densely packed, and C11 (5) chains of intermolecular hydrogen bonds between the pyridine nitrogen and the hydroxyl group. Although the majority of the other lower energy structures were found in the searches with θCO=180°, there are marked differences in the θpyridine, θCPhCl and θPhF torsion angles, (SI Table 10) and hence in overall molecular shape. Many of the conformations in the crystal structures are quite close to the local conformational minima for the molecule in isolation, or as modeled in a polarizable continuum (SI Table 9). All three carboxylic acid proton configurations give rise to a range of hydrogen bonding motifs (SI Table 11). The alternative low energy crystal structures may have some more favorable intermolecular interactions, such as hydrogen bonding, but this has generally been balanced by being in an unfavorable conformation, albeit often close to a local minimum in the conformational energy surface. The covalent bonding constraints can restrict the ability of the molecule to close pack, giving less dense structures which reduces the stabilization from the intermolecular dispersion energy. Indeed, void space calculations showed that 8 of the calculated low energy structures (SI Table 11, structures below −173 kJ mol-1 on Figure 3) had voids that were large enough to accommodate a water molecule. The hydrogen bonding found in the low energy structures can be compared with the Logit Hydrogen Bond Propensity results (SI Section 5) which suggest two likely hydrogen bonding arrangements in the crystal structures of GSK269984B: the S11 (5) intramolecular hydrogen bonding present in the Form I crystal structure and all “Intra” crystal structures on Figure 3, and an intermolecular hydrogen bonding arrangement between the carboxylic groups. These hydrogen bonding pairs were observed on the crystal energy landscape as many of the structures with the InterA and InterB polar proton conformation adopt either the R22 (8) carboxylic acid dimer or the C11 (4) chain between carboxylic acid groups (SI Tables 11 and 12). This indicates that the hydrogen bonding motifs in the majority of the energetically favorable structures would also be expected based on their structural similarity to molecules in the CSD. However, in marked contrast, the second (Figure 5b) and fourth lowest energy structures have a carboxylic acid∙∙∙aromatic nitrogen intermolecular hydrogen bond that had a markedly lower propensity. There were also close contact “hydrogen-bonds” between the carboxylic acid proton and fluorine or chlorine atoms in some low energy


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structures. However, some of the low energy structures, including Form I, have no specific stabilizing interactions stronger than C-H close contacts to polar atoms, emphasizing the importance of the van der Waals forces. Thus the crystal energy landscape shows that a range of crystal structures are energetically plausible for GSK269984B, though these would probably be metastable polymorphs. However, all these structures require a significant change in the relative conformation of the aromatic rings (SI Table 10). Some structures could be stabilized by solvent in the void spaces. Hence, solvents that might cause a conformational change in solution, or crystallization experiments that could lead to novel forms via desolvation of an unstable solvate, appeared worth investigating in the search for polymorphs. Since many low energy structures have the carboxylic acid involved in intermolecular hydrogen bonding, crystallizations in solvents that would promote this were also considered.

Experimental Form Screening The input material to all experiments was the anhydrate, Form I. The solvent-mediated thermodynamic and metastable screens performed during this project led to the observation of six new forms, of which four could be characterized (a summary of results is presented in SI Tables 6 & 7). Form I was obtained from the majority of experiments including temperature cycling experiments in 42 out of 48 screening solvents and microemulsion crystallization; both techniques should favor crystallization of the thermodynamically stable form. Two solid-state forms of a dimethyl sulfoxide (DMSO) solvate (Form 2 and Form 6) were prepared by crystallization from that solvent. During screening, Form 2 was obtained by evaporation of a saturated solution, whereas Form 6 was obtained from undersaturated solutions using either rapid or slow cooling crystallizations. Although both forms were obtained from DMSO, the solid-state data clearly show that they are not the same form (SI Figures 1-3). Further characterization showed that Forms 2 and 6 differ in their solvation state and represent a mono- and di-solvate, respectively. An N-methylpyrrolidone (NMP) mono-solvate (Form 3) was obtained by temperature cycling a slurry of Form I in NMP for more than 48 hours. Forms 2, 3, and 6 all converted to Form I when stored at 40 °C / 75% RH for 70 hours, or by slurrying in water for more than 15 hours. A putative solvate with benzonitrile (Form 5) was also observed which had a needle habit when examined in situ, in contrast to the plate habit of Form I; however, on isolation this rapidly converted to Form I thus preventing any characterization. Evaporation of a nitromethane solution resulted in a small quantity of solid with a Raman spectrum different to that of Form I (SI Figure 1b); this material was tentatively assigned as Form 4 and considered to be unsolvated since no ACS Paragon Plus Environment


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bands attributable to solvent were observed in the spectrum. There was insufficient solid for further characterization and so attempts were made to reproduce this form; all experiments yielded Form I. Additionally re-analysis of the original sample was consistent with Form I however these spectra were acquired by Raman microscopy, which probes only a small area of the sample, so differences could arise from an inhomogeneous sample rather than form conversion. Based on the lack of characterizing data, Form 4 cannot be definitively assigned as a new polymorph. The data and observations indicate that it either represents a metastable form or a degradent which was inhomogeneous in the original sample.

Thermal analysis of Form I and solvates Form I and the solvates (Forms 2, 3 & 6) were heated to their melting points in an attempt to grow crystals on cooling. Irrespective of whether a fast (20 oC/min) or slow (2 oC/min) cooling rate was used these experiments yielded amorphous powder or gum. The amorphous material began converting to Form I within 30 hours at 40 °C / 75% RH. The solvates were further investigated to determine whether novel forms could be prepared via desolvation (SI Section 3.3.2). The 1:1 DMSO solvate (Form 2) and the NMP solvate (Form 3) were desolvated directly to Form I in variable temperature Raman microscopy investigations. When Form 6 (1:2 API:DMSO solvate) was heated to 60 °C, Raman analysis showed that a new form (Form 7) was prepared (SI Figure 4). After 24 hours at room temperature Form 7 was found to have converted to Form I. DSC/TGA indicated solvent was still associated with the sample after Form 7 had crystallized and so this form is thought to be a product of a partial desolvation of the 1:2 DMSO solvate (Form 6).

Single crystal X-ray studies on Form I and the solvates The single crystal structure of the anhydrate Form I, as shown in Figure 4 and Figure 5a, has an intramolecular O-H∙∙∙N hydrogen bond. There are no additional classical hydrogen bonds, although weaker C-H∙∙∙O and C-H∙∙∙Cl interactions are associated with the crystal packing. In contrast, the two solvate structures have an intermolecular hydrogen bond involving the solvent (Figure 6 & Figure 7). In the single crystal structure of the 1:1 NMP solvate (Form 3; Figure 6) this hydrogen bond links a GSK269984B and solvent molecule to give a discrete pair. In the single crystal structure of the 1:2 DMSO solvate (Form 6; Figure 7) one of the DMSO molecules and a GSK269984B molecule again form a discrete pair through hydrogen bonding of the carboxylic group and the arrangement can be ACS Paragon Plus Environment

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described as D11 (2) . A hydrogen atom from both methyl groups on this DMSO molecule act as weak hydrogen bond donors to the oxygen on the second DMSO, which in turn has a similar weak bifurcated interaction to the carbonyl oxygen of the API (Figure 7). Thus, the three crystalline forms of GSK269984B contain the three possible conformations of the carboxylic acid proton suggested by the computational work. However, the notable feature of all three single crystal structures of GSK269984B is the similarity of the molecular conformation (excluding the acidic proton) to that of the isolated molecule as determined by ab initio methods (Figure 8). Single crystal data on anhydrate Form I and the solvates, Form 3 and Form 6, show they have very similar conformations (RMSD1≤ 0.3 Å).

Figure 6. Unit cell of the NMP solvate (Form 3) of GSK269984B.

(a) (b) Figure 7. (a) The unit cell and (b) the close contacts in the (1:2) DMSO solvate (Form 6) of GSK269984B.



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Figure 8. Top - the overlay of the experimentally observed conformations of Form I (colored by element), NMP solvate (Form 3; blue) and DMSO solvate (Form 6; orange) of GSK269984B showing the different carboxylic acid conformations. The RMSD1 for Forms I and 3 is 0.29 Å; Forms I and 6 is 0.31Å; Forms 3 and 6 is 0.24 Å. Bottom – the overlay of each experimental conformation with the closest gas phase optimized minimum with the same carboxylic acid conformation: Form I (colored by element) with 180Intra (black); NMP solvate (Form 3; blue) with 180InterB (light blue); and DMSO solvate (Form 6; orange) with 180InterA (yellow). These figures were generated by overlaying the ring atoms of the chlorophenyl and the fluorochlorophenyl rings and the linking carbon and oxygen atoms. In contrast to the molecular conformations, the packing of the molecules within the crystal structures of the solvates is significantly different from Form I and the other low energy calculated structures due to the solvent being hydrogen bonded to the API. The closest match between a solvate and one of the calculated crystal structures was between the NMP solvate (Form 3) and structure 180InterA25, which had a lattice energy of -165.31 kJ mol-1, so is not among the low energy set reported in the SI. These two structures had 11 common molecules, with an RMSD11 of 0.80 Å. The 1:2 DMSO solvate crystal structure (Form 6, Figure 7) is clearly a channel solvate, indicating a potential mechanism for the partial desolvation of this form to the new transient Form 7 seen by variable-temperature Raman microscopy (SI Section 3.3.2). As the DMSO molecules are hydrogen-bonded in two ways in the structure (half are classically hydrogen-bonded to the drug and the others are held in the channels through weaker bifurcated short contacts), it is possible that as the temperature is increased the DMSO molecules that are held in place only by the weaker interactions move and are lost through the channels. The other solvent molecules would remain in place ACS Paragon Plus Environment

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until the temperature is increased further, at which point desolvation destabilizes the solid, with a polar proton shift leading to the material converting to Form I. The Raman spectra (SI Figures 1 & 4), and differing stability at ambient, show that Form 7 is distinct from the 1:1 solvate Form 2.

Discussion Summary and Discussion of Experimental Screening The thermodynamic screening and scale up activities indicated that the most stable form was the anhydrous form, Form I. Both kinetic and thermodynamic experiments gave Form I (SI Table 6). This indicates that Form I is the most stable form and that it is unlikely that any metastable anhydrates will form under normal crystallization conditions; these would have been expected to crystallize prior to Form I in the kinetic experiments. The only new forms obtained from thermodynamic and metastable screening were solvates (Figure 9).

Figure 9. Diagram showing how the different forms of GSK269984B were prepared and the relationships between them. The putative forms which could not be characterized are on a grey background. ACS Paragon Plus Environment


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The solvates (Figure 9) were obtained from polar solvents that are in the same quadrant of a principal component analysis (PCA) solvent model based on that of Carlson,25 namely benzonitrile, NMP and DMSO (SI Figure 5).The observation of DMSO solvates with two stoichiometries may arise from different supersaturation achieved during the different crystallization modes; this has been observed for another GSK molecule in development.26 It is worth noting that multiple solid-state forms of solvates are not often reported in polymorph screens of pharmaceutical molecules primarily because, unless they are solvated with a process solvent, the solvates are of little interest once it has been established that they do not provide a novel form following desolvation. In this study, the solvates were considered as a plausible route to generating diverse conformations in solution and solvates, but the characterization studies were only pursued as far as needed to establish that this was unsuccessful in targeting metastable polymorphs. The thermodynamic screen used both seeded (slurry) and non-seeded (evaporation, cooling, and vapor diffusion) crystallizations with the aim of identifying whether there are easily accessible metastable forms. Of particular value are the evaporations which typically produce more metastable forms than the other modes of crystallization in the thermodynamic screen; often they produce an amorphous solid, oil, or gum. However, for this compound, with the exception of DMSO and benzonitrile experiments, both the slow and fast evaporation experiments which gave sufficient material to analyze predominately generated crystalline Form I. This may be due to fast crystallization kinetics for Form I and/or the method used to set up the evaporation experiments causing unintentional seeding. The preparation of saturated solutions for the cooling crystallizations and evaporations in the thermodynamic screening included a filtration step. To the naked eye, this produced a visibly clear solution but it is unlikely that the pores in the filter (0.7μm) prevented all crystal growth nuclei from passing through into the saturated solution.27 This can be used to rationalize the formation of a new DMSO solvate (Form 6) from experiments where saturated solutions were undersaturated (by heating) prior to cooling. This may be due to the nuclei remaining after filtration being dissolved on heating, so no Form I template is available and other forms are able to crystallize. Thus, residual seeding of Form I and fast growth kinetics may explain why it was not possible to isolate novel polymorphs. An alternative reason to suggest that a new form could be prepared from pre-heated filtrate is that the heated molecules had greater energy to access a wider range of conformations in solution. If there are appropriate barriers in the conformational energy in the solvent, then, on cooling, molecules trapped in other conformational wells have ACS Paragon Plus Environment

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the opportunity to crystallize out as polymorphs with different conformations. No evidence of this was seen however.

Relationship between Experimental and Computational Screening Most stable form

The crystal energy landscape confirms the experimental conclusion that Form I is the most stable form. The screen incorporated a range of solvents and crystallization conditions that would be expected to yield thermodynamicallyand kinetically-favored forms. It was observed that Form I nucleated and grew rapidly which suggests that it is favored both kinetically and thermodynamically. Based on experimental results alone it would have been concluded that Form I is probably the most stable and would have presented a low risk of form change were the compound to have progressed through development. However, without the calculated crystal energy landscape, which also indicates Form I is probably the most stable form, there would have been some residual uncertainty in whether the fast kinetics of Form I was masking other forms. Combining the two approaches would have given additional confidence that there was a low risk of a new form appearing late in development or during commercial manufacture. This computed crystal energy landscape cannot be used as the only confirming evidence that Form I is the most thermodynamically stable, because the energy difference between the known and hypothetical structures is comparable with the uncertainty in the calculated crystal energies. For example, prior to estimating the effect of polarization by the crystalline environment by the PCM model, Form I was 5 kJ mol-1 above the global minimum (SI Table 13). More accurate models for the intermolecular and intramolecular forces or the inclusion of temperature (which will tend to favor the less dense structures over the dense known form) are likely to re-rank the lower energy structures. However, despite the uncertainty in the relative energies, it is clear that the structures that are competitive in energy with Form I have the molecule in very different, less stable molecular conformations. Both the experimental screening and the calculations indicate that Form I is probably the thermodynamically most stable form. Since the screening has not covered a full range of temperatures, pressures, templates or other conditions that have produced a novel polymorph for other similarly well-screened molecules,28 the failure to experimentally crystallize a more stable form is no proof that it does not exist. There are similar doubts for the computational work, since more space groups and more than one molecule in the asymmetric unit cell could have ACS Paragon Plus Environment


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been considered. For example, our search would not have found the most stable form of 7-fluoroisatin29 whose hydrogen bonding motif requires Z’=2. However, taken together, there is more confidence that Form I is the thermodynamically most stable form, which is further supported by the results of the microemulsion experiments. Accessing new polymorphs by conformational change

The crystal energy landscape showed the importance of conformation when searching for new forms and, in particular, the importance of the intramolecular hydrogen bonding in stabilizing Form I. The solvents for the metastable screen were therefore chosen to include solvents that encourage hydrogen bonding between drug and solvent, thereby avoiding the intramolecular bond and possibly allowing new forms to crystallize. The initial thermodynamic screen used solvents that were selected based on their diversity as judged by viewing their distribution in a PCA model (SI Figure 5), whereas the attempts to produce conformational change by varying the solvent have produced some metastable solvates of NMP and DMSO. Formation of solvates was shown to alter the position of the hydrogen bonding proton, since the carboxylic acid group formed a hydrogen bond to the solvent rather than intramolecularly. The potentiometric GLpKa measured pKa of this molecule is 1.04 (acid) and 5.5 (pyridine) and the sodium salt can be formed;9 therefore proton transfer between the oxygen atoms of the carboxylic acid would be expected to be relatively easy, possibly via a zwitterionic intermediate. Hence, experiment and computation agree that the intramolecular hydrogen bond in Form I of GSK269984B does not present a barrier to accessing alternative solid forms with different hydrogen bonding motifs. In contrast, it has not been possible to obtain a solid form with a significantly different gross conformation in the solid state from Form I. Desolvation causes a significant rearrangement of the packing of the molecules but hardly alters the molecular conformation, apart from the carboxylic acid proton. The other route to new forms suggested by the crystal energy landscape is to force a change in the relative orientation of the aromatic rings prior to crystallization. Simply heating or sonicating the solutions has the advantage of giving the molecule the energy to vibrate and potentially change conformation as well as ensuring that any nuclei dissolve; this proved ineffective in producing crystals containing very different conformations. NMR Nuclear Overhauser Effect (NOE) experiments (SI Section 7) in CDCl3 strongly suggest that the conformation in this solution is not solely that found in Form I. This implies that the barriers between the different conformations are sufficiently low in solution that there is always a fast kinetic route to the 180Intra conformation observed in Form I during crystallization. The computational conformational analysis of the molecule in isolation or a polarizable continuum (SI ACS Paragon Plus Environment

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Table 9) suggests that there are many conformational minima (probably with many more in solution than the 18 found for the isolated molecule) and that the barriers between conformations are quite low, with the possible exception of routes between the three different θCO regions (Figure 2 and SI Figures 7, 9, 10 and 11). However, these calculations have been carried out with an electronic structure theory which will underestimate the intramolecular dispersion but be subject to intramolecular basis set superposition errors.30 Hence establishing that there is no significant energy barrier between the conformations seen in the low energy crystal structures for even the isolated molecule would be a major undertaking. The conformational variability within any given solvent would depend on the specific solvation shell structure, which could increase the barriers to conformational change far more than suggested by approximating the solvent by a polarizable continuum. Hence, establishing that our, or any other experimental conditions, could not produce a sufficient population of the conformations needed to form the hypothetical low energy crystal structures is beyond the current capability to determine the range of conformations in solution and their effect on crystallization kinetics.31,32 The inability to crystallize metastable anhydrous forms may also have been due to residual seeds in experiments using saturated solutions. One approach to reducing this risk would be the use of amorphous input which would also allow access to higher supersaturation (with respect to the crystalline forms). However, although amorphous material does not show any long range order, it could be biased towards conformations closely related to those in the most stable form.33 Since this is likely if the barriers to conformational rearrangement are high, the crystal energy landscape therefore suggests that the conformational diversity in amorphous material should be assessed for its ability to allow access to metastable alternative forms. Small quantities of amorphous material were prepared by cooling from the melt and this material was found to be long-lived under ambient conditions. However, attempts to generate larger quantities of amorphous material suitable for use as input to the screen, e.g. using spray or freeze drying, were not attempted within this study. Why aren’t there more forms?

Pharmaceutical molecules which are polymorph screened at GSK exhibit a range of solubility in organic solvents and it is recognized that high-throughput screening has limited effectiveness at the extremes of this range. At very high solubility (>300 mg/ml) fewer hits from temperature cycling experiments are obtained since insufficient material is often available to maintain slurries, and consequently solutions are often too undersaturated to give hits from cooling and evaporation. If the material is very insoluble (

Evaluating a Crystal Energy Landscape in the ... - ACS Publications

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