Impact of Molecular Flexibility on Double Polymorphism, Solid

Article pubs.acs.org/molecularpharmaceutics

Impact of Molecular Flexibility on Double Polymorphism, Solid Solutions and Chiral Discrimination during Crystallization of Diprophylline Enantiomers Clément Brandel,† Youness Amharar,†,‡ Judith M. Rollinger,§ Ulrich J. Griesser,§ Yohann Cartigny,† Samuel Petit,*,† and Gérard Coquerel† †

Unité de Cristallogenèse, SMS, EA 3233, Université de Rouen, PRES Normandie Université, F-76821 Mont-Saint-Aignan Cedex, France § Institute of Pharmacy, University of Innsbruck, Innrain 52, 6020 Innsbruck, Austria S Supporting Information *

ABSTRACT: The polymorphic behavior of racemic and enantiopure diprophylline (DPL), a chiral derivative of theophylline marketed as a racemic solid, has been investigated by combining differential scanning calorimetry, powder X-ray diffraction, hot-stage microscopy and single-crystal X-ray experiments. The pure enantiomers were obtained by a chemical synthesis route, and additionally an enantioselective crystallization procedure was developed. The binary phase diagram between the DPL enantiomers was constructed and revealed a double polymorphism (i.e., polymorphism both of the racemic mixture and of the pure enantiomer). The study of the various equilibria in this highly unusual phase diagram revealed a complex situation since mixtures of DPL enantiomers can crystallize either as a stable racemic compound, a metastable conglomerate, or two distinct metastable solid solutions. Crystal structure analysis revealed that the DPL molecules adopt different conformations in the crystal forms suggesting that the conformational degrees of freedom of the substituent that carries the only two H-bond donor groups might be related to the versatile crystallization behavior of DPL. The control of these equilibria and the use of a suitable solvent allowed the design of an efficient protocol for the preparative resolution of racemic DPL via preferential crystallization. Therefore, the resolution of DPL enantiomers despite the existence of a racemic compound stable at any temperature demonstrates that the detection of a stable conglomerate is not mandatory for the implementation of preferential crystallization. KEYWORDS: chirality, binary phase diagram, conformational polymorphism, mixed crystals, preferential crystallization, nucleation inhibitions

1. INTRODUCTION Polymorphism, the ability of a chemical compound to exist in multiple crystalline forms, is an important issue in the pharmaceutical industry.1−3 Approximately 90% of marketed drug substances are small organic molecules, and it is estimated that at least half of them exhibit polymorphism.4 The necessity to select the right form for the development and manufacturing of a dosage form stems from the fact that each polymorph shows specific features in terms of stability, physical properties and processing behavior.5,6 These differences may result in an altered bioavailability and affect the biological activity and safety of a medication. In order to understand and control the nature of a polymorphic material it is important to establish the relative thermodynamic stability of each polymorph as function of temperature7 and to consider the role of kinetics for the phase stability and the formation of different forms. The crystallization process of polymorphs is governed by the nucleation and growth kinetics of each form, and kinetic factors often override thermodynamic considerations. Therefore the polymorph that crystallizes is often the fastest growing form © XXXX American Chemical Society

(kinetic form) and not the thermodynamically stable one. However, the nucleation and growth of an individual form is sensitive to a series of factors8 and can be strongly affected by impurities or additives. In the case of flexible molecules, a critical aspect for crystallization is the existence of multiple conformers in the crystallization medium, which may cause unusual crystallization behavior such as disappearing or concomitant polymorphs.9−12 The term used to highlight this situation is “conformational polymorphism”,13,14 which denotes the existence of distinct molecular conformations in the various crystal forms. Extensive structural systematic studies15 have revealed that the crystallization rate is reduced in molecules with high conformational flexibility, a consequence of the higher degree of freedom of the building blocks that may also result in Z′ > 1 structures.16 Received: May 23, 2013 Revised: August 21, 2013 Accepted: August 28, 2013

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dx.doi.org/10.1021/mp400308u | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

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behaviors and chiral discrimination in the solid state. Even if the solid phase landscape appeared highly unfavorable, the possibility of chiral resolution via preferential crystallization was envisaged.

In the case of chiral compounds, representing more than half of the marketed drugs, the situation may be even more complex, especially in racemic mixtures that consist of two molecular components of opposite absolute configuration (enantiomers).17 Today, the use of a single enantiomer is almost mandatory for new active pharmaceutical ingredients (API) since the opposite enantiomer often has significantly different pharmacological and toxicological effects.18 Crystallization is the most widely used purification method for pharmaceuticals and fine chemicals, and suitable crystallization routines for the separation of enantiomers can be developed with the knowledge of their equilibrium phase diagrams.19,20 In most cases (about 90%), a racemic mixture crystallizes as a racemic compound, where the two enantiomers form a welldefined arrangement in an equimolar ratio. Conglomerates can be regarded as physical mixtures of homochiral particles and account for about 5 to 10% of the reported crystalline racemic mixtures. The third and most rare case is the formation of a solid solution (mixed crystal or pseudoracemate), where the two enantiomers coexist in a random fashion within the macroscopically homogeneous crystal.19 The rare occurrence of solid solutions among chiral systems highlights the high stereoselectivity of crystallization processes.19,21−34 An overview of literature dealing with polymorphic chiral systems published during the past decades provides illustrative examples including polymorphic enantiomers35 and polymorphic racemic compound,36 as well as various stability relationships involving a stable racemic compound and a metastable conglomerate,37 or the reverse.38 Furthermore, a double polymorphism, i.e. the existence of several crystal forms for both the racemic compound and the enantiomers, has been reported for n-hexylsuccinic acid39 and the marketed drug modafinil.40,41 These phenomena were analyzed using a purely thermodynamic approach42 or on the basis of nucleation and growth mechanisms,43 whereas polymorphic transitions combined with solid solutions were shown to constitute key factors in the discovery of Preferential Enrichment.44 Diprophylline (Chart 1, DPL hereafter), also known as dyphylline, is a representative of a series of theophylline

2. EXPERIMENTAL SECTION 2.1. Preparation of Diprophylline Samples with a Defined Enantiomeric Composition. Racemic DPL was purchased from Sigma-Aldrich (USA, purity 99%) and was recrystallized from pure ethanol prior to further treatments. After drying at room temperature, solid samples were manually ground. All used solvents were of analytical grade and were purchased from Fisher Scientific (USA). Pure enantiomers (S)- and (R)-DPL were synthesized as follows: 1 equiv (6.94 g) of theophylline (Acros Organic, Belgium, purity 99%) and 1.15 equiv (2.48 g) of potassium hydroxide (Acros Organic, purity 85%) were dissolved at 70 °C in 50 mL of water. 1.15 equiv (4.90 g) of (R)- or (S)-3-chloro1,2-propanediol (Alpha Aesar, USA, purities 98%, 97% ee) mixed with 5 mL water was then added dropwise, and the reacting mixture was stirred during 24 h at 70 °C. The water was evaporated under reduced pressure, and the solid was dried in an oven at 50 °C before it was dissolved in 300 mL of ethanol. The system was heated at reflux for 3 h (i.e., DPL dissolves whereas potassium chloride remains in suspension) and filtered. (S)- or (R)-DPL crystallized in the filtrate upon cooling at room temperature. The harvested solid was slurried for 24 h in 100 mL of a 95:5 (v:v) mixture of ethanol/water under ambient conditions to eliminate any trace of KCl. After filtration, the enantiomerically pure product was dried in an oven at 50 °C and ground with a mortar and a pestle. Yield: 75%. Mp: 164 °C. [α 365 ] = 0.34° dm −1 L g −1 (in dimethylformamide at 25 °C). eeHPLC = 99.9%. Samples of intermediate enantiomeric compositions were prepared by mixing different amounts of accurately weighed (±0.05 mg) racemic DPL and enantiopure DPL. The physical mixtures were carefully homogenized by trituration with a pestle and a mortar before further treatments. Suitable single crystals of the two stable phases (racemic and enantiopure) were obtained by slow evaporation of either (±)-DPL or (+)-DPL saturated solutions at room temperature in a 95:5 (v:v) acetone:water mixture and seeded with RI or EI, respectively. In order to produce metastable RII single crystals suitable for X-ray diffraction analysis, 1.3 g of (±)-DPL was dissolved in 30 mL of isopropyl alcohol (IPA) at reflux (83 °C), and the solution was rapidly cooled down to room temperature to initiate nucleation under stagnant conditions. The system was stored at 35 °C, and crystal growth was allowed to proceed overnight. 2.2. Chiral HPLC Analysis (C-HPLC). The accurate ratio of R- and S-enantiomers in solid samples (powder or single crystals) was determined with chiral high performance liquid chromatography (C-HPLC) using a CHIRALPAK IC column (DAICEL group, Chiral Technologies Europe), 250 × 4.6 mm. The mobile phase was a heptane:ethanol mixture (7:3, v:v), and the flow rate was 1 mL/min. The wavelength for UV detection was 273 nm, and the temperature of the analysis was 25 °C. Under these conditions, retention times of 16.6 and 20.3 min were observed for (R)-DPL and (S)-DPL respectively. Analyses were performed using a Finnigan Surveyor apparatus (Fisher Thermoscientific). The resolution was greater than 1.5 for all separations. The enantiomeric excess (ee) values were obtained

Chart 1. Molecular Structure of Diprophylline (DPL)a

a

The theophylline moiety is displayed in blue.

derivatives that are used for the treatment of obstructive airway diseases such as bronchial asthma. This chiral drug is orally administered and is marketed as a racemic mixture. Like theophylline,45 and other theophylline derivatives such as etophylline46 and proxyphylline,47 DPL exhibits polymorphism,48 but its crystallization behavior has hardly been investigated. The present study deals with the polymorphic behavior of enantiopure and racemic DPL. From the elucidation of stability relationships through the determination of the binary phase diagram between DPL enantiomers combined with structure analysis, our objective is to clarify the impact of conformational variability on both crystallization B



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(Mettler Toledo RE50 apparatus) for global concentrations and polarimetry (Perkin-Elmer 341 apparatus) for the determination of enantiomeric excess values.

by integrating and comparing the peak areas of (R)-DPL and (S)-DPL, using the ChromQuest software. Due to the excellent reproducibility, the error bars of the sample compositions in the constructed phase diagrams are smaller than the plotted measuring points. 2.3. Differential Scanning Calorimetry (DSC). DSC experiments were performed using a Netzsch DSC 204 F1 apparatus equipped with an intracooler. Each DSC run was performed with 5 mg of a powdered sample (±0.05 mg) in aluminum pans with pierced lids at 1, 2, 5, and 10 K/min heating rates. The atmosphere of the analyses was regulated by a helium flux (40 mL/min). The Netzsch-TA Proteus Software v4.8.4 was used for data processing. Onset (respectively endset) temperatures are calculated from the intersection between the baseline and the slope of the first (respectively last) part of the endotherm. For the construction of phase diagrams, error bars on temperatures result from at least three measurements (±0.75 °C) from a single sample preparation. The reliability of these error bars was confirmed by using multiple sample preparations for selected compositions. Liquidus temperatures are obtained by extrapolation of the melting endsets at 0 K/min heating rate.22 2.4. X-ray Powder Diffraction (XRPD). XRPD analyses were performed using a D8 diffractometer (Bruker, Germany) equipped with a modified goniometer of reverse geometry (−θ/−θ) and a LynxEye detector (Bruker, Germany). Using Cu Kα (λ = 1.54059 Å) with a tube voltage and amperage set at 40 kV and 40 mA respectively, XRPD analyses were performed with a step of 0.04° (2θ), and a 4 s/step counting time from 3 to 30° (2θ). 2.5. Single Crystal X-ray Diffraction (SC-XRD). Single crystals were fixed on a glass fiber which was mounted on the full three circle goniometer of a Bruker SMART APEX diffractometer equipped with a CCD area detector (with Mo Kα1 = 0.71071 Å). The SMART49 software was used to determine the cell parameters and the orientation matrix for each crystal. Intensities were integrated and corrected for Lorentz polarization and adsorption effects using SAINT49 software. Crystal structures were solved by direct methods, using the SHELX-97 program,50 and anisotropic displacement parameters were refined for non-hydrogen atoms. All hydrogen atoms were located by Fourier difference synthesis and fixed geometrically according to their environment with a predefined isotropic thermal factor. 2.6. Hot-Stage Microscopy (HSM). Thin film samples were prepared by melting ca. 5 mg of powder in a quartz cell using a hot bench. The melt was covered with a glass slide before quenching. The quartz cell was placed in a THMS 600 hot-stage (Linkham) coupled with a Nikon Eclipse LV100 microscope (maximum magnification: ×1000) connected to a computer for image capture via a CCD camera. The temperature was regulated via the Linksys32 software. 2.7. Solubility Measurements and Monitoring of Preferential Crystallization. Dimethylformamide (DMF) was used as solvent for preferential crystallization experiments. The solubility curves in the ternary system ⟨DMF−(S)-DPL− (R)-DPL⟩ were estimated at 15 and 66 °C by gravimetric measurements starting with saturated DMF solutions of EI and RI or mixtures of the two. The identity of solid phase(s) in equilibrium with saturated solution was monitored by XRPD analysis. The enantiomeric composition of the dried solids was determined by C-HPLC analysis. Preferential crystallization experiments were monitored by a combination of refractometry

3. RESULTS 3.1. Preparation and Characterization of Racemic and Enantiopure DPL Phases. The previous results of Griesser and co-workers48 were confirmed for racemic DPL. Commercial batches consist of the stable form RI, melting at ca. 159 °C without detectable degradation or sublimation. Recrystallization from the supercooled melt (SCM hereafter) at 90 °C results in a second polymorph of the racemic compound, RII (mp ca. 148 °C). This form is thermodynamically less stable than RI but exhibits a rather high kinetic stability allowing its handling and storage for many months (Table 1). Figure 1 reports the Table 1. Thermochemical Data Obtained for the Different DPL Forms by DSC racemic DPL

enantiopure DPL

RIa

RIIa

SCb

MCc or SC (IPA)

SC

TS tconversione conditions

stable

stable

Tfusonsetf (°C) Tfuseqg (°C) ΔfusH (kJ/mol)

159.9 161.3 32.8

metastable 4 to 12 months 20 °C, open atmosphere 148.1 151.0 27.7

preparation d

EIa

164.4 165.7 31.7

EIIa MC (100 min at 90 °C) metastable 15 to 25 min 20 °C, open atmosphere 120.8 124.0 22.7

a

Crystal form. bSolvent crystallization. cMelt crystallization. dThermodynamic stability. eThe conversion time to the stable state was estimated from at least 3 experiments and monitored by XRPD. fOnset of melting measured at 5 K/min. gEquilibrium melting temperature obtained by extrapolating the endset of melting endotherms at 0 K/ min.

Figure 1. XRPD patterns of racemic (RI and RII) and enantiopure (EI and EII) DPL polymorphs.

distinct XRPD patterns of racemic DPL recrystallized from ethanol (stable form RI) and from the SCM (metastable form RII). Optical microscopy shows (Figure 2a) that RII particles recrystallize from the SCM as large (10−50 μm) birefringent spherulites consisting of single crystals with characteristic morphological features. Additionally, we were able to obtain RII by solvent crystallization. This was achieved by rapid cooling of a highly supersaturated (β > 3) isopropyl alcohol (IPA) solution of racemic DPL to room temperature under magnetic C



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However, numerous attempts to produce suitable single crystals for EII failed. Table 2 summarizes the main results and relevant parameters, indicating a satisfactory quality of these crystal Table 2. Crystallographic Data and Refinement Parameters at 298 K for EI(S), RI and RII EI(S)a crystal system space group (Z) a (Å) b (Å) c (Å) β (deg) vol (Å3) calcd density (g cm−3) measd/unique data obsd data (Fo > 4σ(Fo)) no. of restraints/ params goodness-of-fit on F2 R1/wR2 (Fo > 4σ(Fo)) R1 (all data) largest diff peak and hole (e− Å−3)

Figure 2. Polarized light microscopy images of the metastable forms of racemic (RII) and enantiopure (EII) DPL growing in the supercooled melt after annealing at 90 °C for 10 min: (a) RII, (b) EII.

stirring (β is the relative supersaturation, defined as C/Cs where C stands for the actual concentration and Cs for the solubility at the same temperature). The thermochemical data obtained by DSC for racemic DPL during this study (Table 1) are in good agreement with those found by Griesser et al.. The so-called “heat of fusion rule”7 confirms that the two polymorphs are monotropically related, which means that RII is the thermodynamically less stable form in the entire temperature range between 0 K and the melting point. The recrystallization of enantiopure DPL from commonly used solvents (e.g. alcohols, ketones, esters, toluene, apolar aprotic solvents, or more polar solvents, such as dimethylformamide, dimethyl sulfoxide and water) yields small acicular crystals of stable form EI (Figure 1c). Similar to the racemic mixture, a metastable form of the enantiopure DPL (labeled EII) can be produced by annealing the SCM for about 100 min at 90 °C. The XRPD of this form (Figure 1d) is well distinguishable from the stable form (EI) and from the polymorphs of the racemic mixture though the pattern exhibits a weak halo in the 2θ range 18°−30°, indicating some degree of disorder. Optical microscopy (Figure 2b) reveals that EII grows to spherical aggregates of crystals that developed from a single nucleus. In contrast to RII these spherulites consist of much smaller, acicular crystals, and it was noticed that, under similar experimental conditions, the crystal growth rate of EII is clearly lower than that observed for RII. Moreover EII is kinetically less stable than RII and undergoes the progressive and irreversible solid−solid transition to EI within about 20 min after its preparation (monitored by means of XRPD). The small size of the domains (less than 1 μm wide) may explain the observed background and the line broadening in the diffractogram EII. Thermochemical data for the enantiopure polymorphs are listed in Table 1. Similarly to the racemic composition, the “heat of fusion rule”7 can be used to confirm that EII is monotropically related to EI. The observation of two forms for the racemic compound as well as for the pure enantiomers (double polymorphism) now raises the question about the type of the phase relationship (racemic compound, conglomerate or solid solution). From the clear differences between the four diffractograms (Figure 1) and the different melting points (Table 1), it can be concluded that RI and RII are not racemic conglomerates of the corresponding enantiomers. A conglomerate would show the same pattern and a lower melting point than the pure enantiomer. To elucidate the structural difference of the four phases, single crystal structural analyses were performed. 3.2. Crystal Structures of the DPL Phases. Single crystals of sufficient quality for a structure analysis with conventional Xray diffraction methods were obtained for EI, RI and RII.

a

RIa

RIIa

monoclinic P21 (2) 4.5191(6) 14.1612(19) 8.9530(12) 99.129(2) 565.70 1.493

monoclinic P21/c (4) 4.5605(8) 12.8524(22) 19.1195(3) 92.029(3) 1119.96 1.508

monoclinic P21/c (4) 7.4520(9) 12.2227(15) 12.8962(16) 98.015(2) 1163.16 1.452

4566/2315 2178

6108/2285 1563

6557/2364 2082

0/167

0/167

1/175

0.833 0.0381/0.1032

1.054 0.0701/0.1626

1.031 0.0469/0.1332

0.0395 0.37, −0.24

0.1084 0.55, −0.22

0.0522 0.23, −0.17

Crystal form.

structure determinations. In the three crystal structures, all intramolecular bond lengths and angles are in the range of expected values and will not be further discussed hereafter. The molecular geometry of the methyl xanthine moiety (theophylline fragment) fits well with that of theophylline.51−53 Therefore, we will only discuss the conformational features of the propanediol substituent, intermolecular interaction motifs and molecular packings of the three DPL structures. In the following figures, (S)- and (R)-DPL enantiomers are represented with gray and green carbon atoms, respectively. Classical H-bonds are shown as red dashed lines, π stacking as purple dashed lines, and non H-bonding hydrogen atoms have been removed for clarity. All geometrical data of the most relevant intermolecular interactions in the three crystal structures are listed in Table 3, and the data for CH···O contacts are listed in Table S1 (Supporting Information). Figure 3 depicts the orientation of the propanediol substituent in the four conformers of a same (S) enantiomer in the three structures. Consistent with values given in Table 3 for the relevant torsion angles (C7−N4−C8-C9 and N4−C8− C9-C10), it can be seen that these conformers differ from one another. This conformational variability is closely related to the strongest intermolecular contacts since the propanediol moiety contains the two H-bond donors of the DPL molecule (O3H and O4H hydroxyl groups). The two stable crystal forms EI and RI consist of stackings of monolayers formed by H-bonded DPL molecules. In the EI crystal structure, the slices are held together by means of van der Waals and CH···O contacts. The theophylline fragments are almost perpendicular to the (001) planes, allowing π stacking that stabilizes the molecular slices (Figure 4). By contrast, the theophylline fragments in the RI structure are roughly oriented parallel to the slices, allowing the occurrence D



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Table 3. Hydrogen Bond Lengths and Angles (in Å and deg) and Selected Torsion Angles (in deg) of the Different (S)-DPL conformers in the EI, RI and RII structures torsion angle C7−N4−C8−C9 N4−C8−C9−C10 H-bond O4−H···N3 O3−H···O4 O3−H···O1

EI

D···A 2.850(2) 2.729(2)

92.27(21) 168.41(15) D−H···A 160.36 178.54

RI

D···A

−90.52(41) −68.82(47) D−H···A

RII-A

RII-B

96.71(24) −172.14(20) D···A D−H···A

−84.64(71) −176.03(66) D···A D−H···A

2.867(4)

164.75

2.854(2)

167.43

2.854(2)

167.43

2.892(3)

164.04

2.808(3)

168.48

2.750(7)

167.02

of π stacking between corrugated (100) molecular layers (Figure 5).

Figure 3. Molecular conformations of the (S) enantiomer in the three crystal forms of DPL. Thermal ellipsoids are displayed at the 50% probability level, and H atoms are shown as spheres with arbitrary radii. See text for further details about the two different conformations (“A” and “B”) in the RII crystal structure.

Figure 5. Projection of the RI crystal packing along the a axis (a) showing the heterochiral and corrugated (100) molecular slices and (b) projection along the c axis showing the stacking of these slices along a.

The structural analysis of RII revealed a more complex situation. A significant residual electronic density (roughly equivalent to 2 e−/Å3) was detected in the vicinity of the H atom bonded to the chiral C9 atom, indicating the possibility for a same crystallographic site to be occupied by the two opposite enantiomers with different conformations, hereafter referred to as “enantiomeric disorder”. The structural model could be further refined by simulating this enantiomeric disorder and led to occupancy factors close to 80% for one enantiomer and 20% for the opposite enantiomer in the asymmetric unit of the P21/c centrosymmetric space group. As a consequence of this statistical occupancy, each enantiomer in the RII structure can exhibit two distinct molecular conformations, labeled A and B, differing only by the orientation of the propanediol substituent (see Table 3 and Figure 3). Therefore, a given molecular site is occupied by the major enantiomer adopting conformation A, which is similar to

Figure 4. Projections of the EI crystal structure along the c axis (a) and along the a axis (b) showing the stacking along c of (001) slices.

E



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(but slightly different from) the conformation B adopted by the minor enantiomer. The RII crystal structure consists of bimolecular layers formed by centrosymmetric dimeric associations. Considering only the major enantiomer of each molecular site, the two heterochiral DPL molecules are H-bonded in a head-to-tail fashion. Each dimer is connected by means of (O4−H···N3) Hbonds to four neighboring dimers of the same (100) slice. The packing of these slices is stabilized by weak H-bonds as well as by π-stacking between theophylline fragments. It is remarkable that the presence of the minor conformer B on each molecular site does not alter the H-bond network since only the O3 atom is involved in the enantiomeric disorder (Figure 6).

Figure 7. Melting diagram between (R)-DPL and racemic DPL showing the theoretical curves for the four crystal forms and associated possible eutectic invariants (with corresponding temperatures and compositions).

are symmetrical with respect to the racemic composition, the study is restricted to the 0 < Xs < 0.5 section. The intersections between liquidus curves give rise to six predicted eutectic invariants whose occurrence has been assessed through the construction of the experimental binary phase diagram between DPL enantiomers. 3.3.1. Stable Equilibrium between EI and RI. The thermodynamically stable phase diagram between DPL enantiomers was derived from DSC analyses using physical mixtures of EI and RI (Figure 8). The experimental eutectic

Figure 6. Unit cell of the RII crystal structure showing the heterochiral dimers (a) and their stacking (b), the minor enantiomer being shown with light colors, and projection along the c axis (c). Molecules from a same dimer are drawn with the same color.

As an extension of this crystallographic study, the refinement of the RII crystal structure using the P21 space group with two independent molecules was attempted in order to obtain more precise information about the distribution of the enantiomers in the lattice. Although the statistical indicators were slightly improved, the use of a lower symmetry did not enhance the quality of the structural model. This observation is actually related to the difficulty, by means of X-ray diffraction methods, to differentiate between an ordered heterochiral packing and a semirandom distribution of the two enantiomers giving rise to an apparent but only statistical higher centrosymmetry.21,22,24,34,54 3.3. Binary Phase Diagram between DPL Enantiomers. Prior to the use of DSC, XRPD and HSM for the determination of stable and metastable equilibria, an estimate of this binary system was performed using the Shröder−Van Laar (for EI and EII) and Prigogine−Defay equations19 (for RI and RII) using data from Table 1. Assuming no miscibility at the solid state, the evolution of these theoretical liquidus curves versus the molar ratio of (S) enantiomer (Xs hereafter) is shown in Figure 7. Since phase diagrams between enantiomers

Figure 8. DSC melting endotherms obtained by heating 5 mg of EI + RI (a) or EI + RII (b) mixtures at 5 K/min. The compositions are indicated on the thermograms.

invariant temperature was found at 152 ± 0.75 °C, and the reasonable agreement with theoretical calculation (Tcalc = 153.2 °C) indicates the quasi-ideal behavior of these mixtures, with a eutectic composition found at Xs = 0.25 ± 0.02 (Figure 9a, green lines). The absence of miscibility in the solid state (no detectable domain of solid solution) is consistent with the crystallographic study of RI and EI. 3.3.2. Metastable Equilibrium between RII and EI. The right part of Figure 8 shows the DSC curves obtained for mixtures of EI and RII. For samples with 0 < Xs < 0.4, a eutectic invariant is clearly identified at 146 ± 0.75 °C and the eutectic composition was found at Xs = 0.35 ± 0.05. The corresponding equilibrium is shown as red lines in Figure 9a, F



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Figure 9. (a) Experimental phase diagram between (R)-DPL (EI) and racemic DPL (RI and RII). The dotted black lines correspond to two hypothetical curves. (b) Experimentally accessible part of the phase diagram between the metastable forms RII and EII showing two superimposed stability regions associated with the formation of two distinct solid solutions. Figure 11. (a) DSC curves (heating rate: 5 K/min) of the recrystallized SCM for samples with varying ratios of the enantiomers (Xs). (b, c) Enlargements for compositions Xs = 0.15 and Xs = 0.10.

and experimental results are also in good agreement with theoretical predictions (Ttheoretical = 147.3 °C). However, the occurrence of a eutectic melting for 0.4 < Xs < 0.5 samples cannot be clearly established: it can be suspected that the onset of melting endotherm continuously increases from Xs = 0.425 to Xs = 0.5. 3.3.3. Metastable Equilibria Involving RII and EII. This binary system was successfully studied via the in situ generation of RII and EII mixtures by recrystallization from the SCM. The resulting samples were gently ground using a mortar and a pestle prior to analysis. Figure 10 presents the XRPD patterns

endotherm with increasing Xs from 0 to 0.10. Additional XRPD recordings using an internal standard (data not shown) to investigate peak shifting did not reveal any detectable progressive change of lattice dimensions. Thermograms obtained for Xs = 0.15 and Xs = 0.10 compositions (Figure 11b,c) can be interpreted as the successive meltings of the two coexisting solid solutions, in consistency with XRPD analyses. Actually, the melting of ssEII starting for Xs = 0.15 (at 108.5 °C) is partially overlapped with the recrystallization of ssRII. The independent melting of the two solid solutions was confirmed by HSM experiments carried out with the Xs = 0.15 sample. As shown in Figure 12, one can

Figure 10. XRPD patterns of various compositions of RII and EII obtained by recrystallizing the SCM. The 10−11° 2θ scale region is enlarged in the right plot.

at room temperature of these recrystallized samples. For samples in the range Xs = 0.2 to 0.5, the diffractograms are similar to that of RII, indicating crystallization of a single phase isomorphous to RII. Besides, samples with Xs = 0, 0.05, and 0.10 consist of phases isomorphous to EII since no additional peak is detected. For Xs = 0.15, XRPD indicates that phases isomorphous to RII and EII have crystallized concomitantly. Each of these mixtures was analyzed by means of DSC (Figure 11) revealing the absence of invariant eutectic melting. Instead, a progressive depression of the onset of melting and a splitting of the melting endotherms are observed from Xs = 0.2 to 0.5, consistent with the presence of a biphasic domain liquid + solid in the phase diagram,55 and confirming the existence of a metastable solid solution (type II in the Roozeboom classification56), labeled ssRII hereafter. A Roozeboom type III solid solution (labeled ssEII) can be postulated from the XRPD results and the continuous decrease of the melting

Figure 12. Polarized light microscopy images of the recrystallized SCM (Xs = 0.15, thin film preparation) at different temperatures (see text for details).

successively observe the progressive recrystallization of ssEII (A, B), the melting of ssEII and the simultaneous recrystallization of ssRII (C), the growth of ssRII particles (D, E) and finally the melting of ssRII (F). Thus, XRPD, DSC and HSM experiments provide consistent evidence for the existence of two distinct solid solutions that can nucleate from the SCM. The crystallization of ssEII is kinetically favored only for compositions of Xs < 0.20. Figure 9b displays the metastable equilibria derived from these experiments. The plot shows the superimposition of two distinct stability domains of ssRII and ssEII solid solutions in blue and magenta, respectively. G



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In order to study the metastable equilibrium between the pure enantiomers, a DSC analysis was conducted on a 50/50 physical mixture of EI(R) and EI(S). The recorded DSC curve (Figure S2 in the Supporting Information) shows a broad endothermic event starting at Tonset = 137.0 °C (predicted at Tcalc = 133.3 °C). HSM observations of the same sample confirm the occurrence of a metastable conglomerate between EI(R) and EI(S), shown with gray dashed lines in Figure 13. However, in the absence of a solvent, the physical mixture readily converts to RI as soon as manual grinding is applied.

Figure 14. Isothermal sections at 66 and 15 °C in the DMF-rich part of the ternary solubility diagram ⟨DMF−(R)-DPL−(S)-DPL⟩. Dashed lines correspond to metastable solubility lines. Error bars are smaller than the dots representing experimental data points.

compound RI and the metastable form RII under these conditions. This favorable situation motivated us to apply the preferential crystallization (PC) technique to the racemic mixture in order to separate the DPL enantiomers. This method and its variants57 involves the enantioselective crystallization in a nearly racemic supersaturated solution as a result of the kinetic advantage provided by seeding with the desired enantiomer. The implementation of PC requires the existence of a conglomerate of sufficient stability.58 The efficiency of the process can be limited by the presence of a metastable racemic compound,59,60 by multiepitaxy during crystal growth61,62 or by enantiomorphous solid solutions.63,64 The possibility to perform preferential crystallization implemented via a metastable conglomerate has been discussed by Levilain et al.65 Hereafter, all enantiomeric compositions are expressed as enantiomeric excess (ee). Table 4 summarizes the

Figure 13. Global Xs−T phase diagram between DPL enantiomers. Stable equilibria are shown by solid lines whereas dashed lines indicate metastable equilibria.

The global phase diagram between DPL enantiomers resulting from the above investigations is shown in Figure 13. The diagram shows two predicted invariants (eutectic behavior at 152 and 146 °C, see Figure 7) and two solid solutions ssEII and ssRII. The solvus in the (RII−EI) system (red dotted line in Figure 13) is not experimentally accessible but is indicated by the existence of ssRII.20,55 From a thermodynamic point of view, the existence of two additional crystal forms can thus be inferred from this phase diagram since the extension of ssEII and ssRII solid solutions indicates the existence of RIII and EIII (corresponding to the extremity of ssEII at Xs = 0.5 and ssRII at Xs = 0). However, because of the very low stability of these phases it was not possible to obtain them by a seeding crystallization procedure from solvents or from the SCM.22 It should be stressed that, to our knowledge, the chiral binary system of DPL is the first example that exhibits all three phase types, namely, racemic compound, conglomerate without partial solid solutions and complete solid solutions. 3.4. Crystallization in Solution and Preparative Chiral Resolution of Racemic DPL. In order to further characterize the conglomerate between EI(R) and EI(S), a solvent screening was carried out and the isotherms at 15 and 66 °C were determined in dimethylformamide (DMF). Reliable solubility data could be obtained for the two solid forms RI, EI and their mixtures. This was confirmed by XRPD analysis of the solid phase(s) of the suspensions. Moreover, we found that a DMF solution saturated with RI at 66 °C (solubility S(RI) = 22.2% w/ w) and rapidly cooled under magnetic stirring to 15 °C (solubility S(RI) = 7.8% w/w, i.e. β = 2.8 with respect to RI) remains supersaturated for at least 3 h before spontaneous crystallization of the RI crystal form (the green zone in Figure 14 refers to the stability domain of RI). The persistence of the out-of-equilibrium state is highly reproducible and illustrates the difficult primary nucleation of both the stable racemic

Table 4. Starting Conditions for the Implementation of Preferential Crystallization Process preferential crystallization mode

SIPC

mass of DMF (g) mass of (±)-DPL (g) concentration (g g−1) mass of seeds (R) (mg) stirring mode stirring speed (rpm)

15.00 4.436 0.228 230 magnetic 700

starting conditions for the PC experiments with DPL in DMF. We selected the SIPC method (Seeded Isothermal Preferential Crystallization), comprehensively described elsewhere.57 The principle of SIPC can be explained via the red lines in Figure 14 depicting the composition of the mother liquor during a PC cycle. The starting situation is point A: the solution is saturated at 66 °C with RI (i.e., the ee of the solution is 0%). After rapid cooling down to 15 °C, the racemic solution is seeded with pure EI(R) (eeC‑HPLC = 99.9%) inducing the stereoselective crystallization of this enantiomer in the highly supersaturated solution. When the composition of the mother liquor reaches point B, the crude crops are collected by filtration and the system moves to point C after compensation (i.e., addition of racemic compound RI and solvent). The homogeneous solution obtained by heating at 70 °C is enriched with (S)-DPL. Cooling to 15 °C and seeding with this H



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counter enantiomer cannot be incorporated in the crystal lattice). Consistently, samples of high ee (>99.5% HPLC) could be obtained by a single recrystallization.

enantiomer facilitates its crystallization, and the composition of the solution moves from C to D. At D filtration and compensation are performed in order to reach E, which may be the starting point of a new cycle. It can be seen in Figure 15

4. DISCUSSION The experimental determination of the binary phase diagram between DPL enantiomers clearly revealed that the two racemic compounds are thermodynamically more stable than the conglomerate. Considering the common assumption that a racemic mixture can be resolved by PC only if it crystallizes as a stable conglomerate, one would not have attempted to apply this resolution method to this system. This situation underlines that the applicability of PC could be actually determined by other parameters, such as the kinetic competition between the nucleation rates of the various solid forms. Davey et al.66 recently highlighted that nucleation mechanisms are still not well understood and that one cannot directly estimate the nature of the solvated state (in particular the predominant conformation) from structural data in the solid state. Nevertheless, in the DPL system, it can be postulated that the presence of different conformations in the three crystal structures is involved in the unusual nucleation behavior of DPL in DMF solutions, which is characterized by a rather wide metastable zone. The assumption that the difficult nucleation of DPL might be related to its conformational diversity is reinforced by the presence of two H-bond donor groups (i.e., main way of bonding) on the flexible substituent of the DPL molecule. Postulating the coexistence of several solvated conformers in dynamic equilibrium separated from each other by a significant energy barrier, it can be supposed that the formation of molecular clusters of critical size (required for nucleation of any crystal form) in solution is hindered by the difficult interconversion between conformers. In this respect, the successful implementation of PC appears to be the consequence of seeding, which means that the nucleation step is skipped, and that the major part of the excess enantiomer in the supersaturated solution is rapidly assembled in the growing seed crystals. This point highlights a second decisive criterion for the applicability of PC, namely, the highly stereoselective character of crystallization during the crystal growth of the pure enantiomer EI. Further investigations, using mainly modeling tools, are required in order to account for chiral discrimination in the crystal lattice of EI. However, it is rather surprising that this crystal form presents a high tendency to discriminate the enantiomers during crystal growth (i.e., stereoselectivity)43 whereas RII and EII forms were shown to consist of solid solutions in which both enantiomers can be incorporated in the crystal lattice.

Figure 15. Monitoring of the preferential crystallization in SIPC mode starting from racemic conditions: ee of the mother liquor (triangles) and ee in collected crystals (circles).

that, moving from A to B, the final ee (eef‑motherliquor) is reached after ca. 300 min. However, the evolution of ee in collected crystals versus time shows that the optical purity decreases significantly after ca. 200 min because of the heterogeneous nucleation of RI (checked by XRPD, data not shown). The filtration window was therefore fixed close to 200 min. The kinetics of the entrainment was found to be similar when the starting point was a racemic or a quasi racemic solution (i.e., t(AtoB) is equivalent to t(CtoD) in Figure 14, so the filtration window was unchanged). Six consecutive preferential crystallizations were performed, and the results are presented in Table 5, indicating that PC in this case is mainly based on the crystal growth of seeds (large amount of seeds versus the mass of collected crystals) rather than the secondary nucleation. This statement was confirmed by optical microscopy, which shows that the crystal growth of the EI(R) seed crystals predominates (see Figure S4 in Supporting Information) until the secondary nucleation of RI starts. The formation of RI occurs when the crystallization process is allowed to proceed for more than three hours. The average final ee of the mother liquor is 7.0%, a result which is slightly below the value expected for robust PC processing (

Impact of Molecular Flexibility on Double Polymorphism, Solid

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