Article pubs.acs.org/crystal
Polymorphism of Mechanochemically Synthesized Cocrystals: A Case Study Franziska Fischer,†,‡ Adrian Heidrich,† Sebastian Greiser,†,‡ Sigrid Benemann,† Klaus Rademann,‡ and Franziska Emmerling*,† †
BAM Federal Institute for Materials Research and Testing, R.-Willstätter-Strasse 11, 12489 Berlin, Germany Department of Chemistry, Humboldt-Universität zu Berlin, B.-Taylor-Strasse 2, 12489 Berlin, Germany
‡
S Supporting Information *
ABSTRACT: The liquid-assisted grinding cocrystallization of theophylline with benzamide leading to polymorphic compounds was investigated. A solvent screening with 17 different solvents was performed. The dipole moment of the solvent used in the synthesis determines the structure of the polymorphic product. A detailed investigation leads to the determination of the kinetically and thermodynamically favored product. In situ observations of the formation pathway during the grinding process of both polymorphs show that the thermodynamically favored cocrystal is formed in a two-step mechanism with the kinetic cocrystal as an intermediate.
■
INTRODUCTION In organic solid state chemistry, the terms “polymorphism” and “cocrystals” are nowadays inherently connected to the research of active pharmaceutical ingredients (APIs). Polymorphs are compounds having the same chemical composition but solidify in different crystal structures.1,2 These differences in the molecular arrangement of these compounds are connected to altered therapeutic effects and physicochemical properties including water solubility, bioavailability, and storage behavior.3−6 Therefore, various crystallization methods have been established to trigger the formation of distinct polymorphic forms.7−13 Despite the intensive research on pharmaceutical polymorphs, the crystallization of pure polymorphs is still challenging.14−17 Because of their similar crystal energies, polymorphs can convert spontaneously into a more stable, sometimes undesired, form. Cocrystallization was once supposed to circumvent the polymorphism issues of any given API.18 Cocrystals are defined as stoichiometric crystalline two- or more-component systems consisting of neutral molecules stabilized via noncovalent intermolecular forces such as hydrogen bonds, π−π-stacking, or halogen bonds.19,20 The cocrystallization of an API renders its physicochemical properties without altering its pharmaceutical activity. Pharmaceutical cocrystals consist of at least one API and an appropriate coformer.21−25 With the increasing number of synthesized cocrystals, various polymorphic forms were identified based on different crystallization methods.26−34 It was described that the addition of a distinct solvent by grinding can control the polymorphic outcome.26 In the last few decades, the mechanochemical approach for synthesizing cocrystals has © 2016 American Chemical Society
become an established, efficient route for the screening of cocrystals and their polymorphs.30,35 Although a lot of work was focused on the fields of cocrystals and mechanochemistry, the underlying formation pathways are not completely understood. To get first insights into the mechanisms, ex situ investigations were conducted. Here, the grinding process was stopped after short milling periods, and small amounts of sample were extracted for further investigations. On the basis of the results of these ex situ experiments, first reaction pathways including stepwise formation processes were obtained.30,36,37 The interruption in the ex situ investigations can lead to a different product compared to the continuous reaction.38 To circumvent this problem, in situ setups for milling syntheses using powder X-ray diffraction (PXRD) and Raman spectroscopy were recently introduced. These studies reveal stepwise formation processes as well as unexpected, quick conversions after a long induction time and reaction rates.39−43 So far, no cocrystallizations with polymorphic outcomes were investigated. Especially for pharmaceutical applications, it is important to know all the polymorphic forms of a given compound. Only a complete characterization including stability, thermodynamic, and kinetic behavior can help to understand the solid-state chemistry of the polymorphs. On the basis of this understanding, precautions can be taken in order to prevent unwanted conversions during the storage. In this study, we Received: December 15, 2015 Revised: January 27, 2016 Published: January 27, 2016 1701
DOI: 10.1021/acs.cgd.5b01776 Cryst. Growth Des. 2016, 16, 1701−1707
Crystal Growth & Design
Article
the figure-of-merit factor. The crystal structure of the cocrystal was solved by the simulated annealing procedure on a standard personal computer within 12 h finding the deepest minimum of the cost function several times during the procedure. To complete the structure determination, the structural solution obtained from Monte Carlo/ simulated annealing was subsequently subjected to a Rietveld refinement employing the TOPAS software.47 CCDC-1425986 (tp:ba II) contains the supplementary crystallographic data for polymorph II. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.cdc.cam.ac.uk/ data_request/cif. In Situ Synchrotron XRD. The in situ X-ray diffraction experiments were performed at the μSpot beamline (BESSY II, Helmholtz Centre Berlin for Materials and Energy, Germany) in a ball mill Pulverisette 23 (Fritsch, Germany) in a Macrolon jar for 25 min at 50 Hz. For the experiments a beam diameter of 100 μm at a photon flux of 1 × 109 s−1 at a ring current of 100 mA was used. The experiments were performed with a wavelength of 1.000 Å using a double crystal monochromator Si (111). The spot size on the sample was 200 μm. Scattered intensities were collected with a twodimensional X-ray detector (MarMosaic, CCD 3072 × 3072 pixels, pixel size 73 μm).48 Measurements were carried out every 30 s with a delay time of 3 or 4 s between two measurements. For clarity the reaction times were given as full 30 s in the text of the results. The occasionally observed doubling of reflections is caused by diffraction of two different areas in the reaction jar, due to differences in the distance between detector and sample. The obtained scattering images were processed employing an algorithm of the computer program FIT2D.49 For the graphical representations, q values were transformed to the diffraction angle 2θ (Cu) to provide a direct comparison to results obtained by XRD experiments performed with Cu radiation. The resulting patterns (2θ angle vs intensity) were analyzed and plotted using the evaluation software EVA.50 The XRD plots are background corrected. Raman Spectroscopy. Raman measurements were performed using a Raman RXN1 Analyzer (Kaiser Optical Systems, France). The spectra were collected using a laser with a wavelength of λ = 785 nm and a contactless probe head (working distance 1.5 cm, spot size 1.0 mm). Raman spectra were recorded with an acquisition time of 5 s and 5 accumulations. NIR excitation radiation at λ = 785 nm and an irradiation of 6.6 W/cm2 were performed. ssNMR Spectroscopy. 1H NMR measurements were performed on a Bruker AVANCE 600 spectrometer using a 2.5 mm doublebearing magic angle spinning (MAS) probe (Bruker Biospin) and applying a spinning speed of 25 kHz. The spectra were recorded with a π/2 pulse length of 2.75 μs, a recycle delay of 300 s, and an accumulation number of 32. The EASY experiment was used to suppress existent rotor and probe background signals.51,52 Adamantane served as secondary field standard with a chemical shift of 1.78 ppm. SEM. SEM images were obtained using a scanning electron microscope ZEISS SUPRA 40 equipped with a thermal field emission cathode (Schottky-emitter, ZrO/W-cathode). The acceleration voltage was set to 10 kV, and the working distance was between 6.0 mm and 6.1 mm. The images were adapted with an In-lens secondary electron detector, a SE2 secondary electron detector, and a QBSD backscatter detector. In addition, the scanning electron microscope is equipped with the energy dispersive X-ray spectrometers Thermo NSS (SiLi 5665) and Bruker X-Flash 5010 3403, Quantax 400. DSC-TG Analysis. Differential scanning calorimetry and thermogravimetric analysis (DSC-TG) measurements were conducted using an EXSTAR DSC-7020 (Seiko, Japan). The measurements were performed in an open Pt jar in a N2 flow with a heating rate of 10 K/min. No cycle measurements were performed. BFDH Morphology. The BFDH morphologies of the crystallites of tp, ba, tp:ba Form I, and Form II shown in Figure 7 were calculated with the standard procedure of the program Mercury 3.5.1.53
present the thermodynamics and kinetics of the polymorphic cocrystallization including the API theophylline (tp) and the coformer benzamide (ba). The structure of Form I (tp:ba I) was described in a previous publication.44 Here, we present the structure of Form II (tp:ba II) based on high-resolution PXRD patterns. The two polymorphs can be obtained quantitatively by changing the added solvent in the liquid-assisted grinding (LAG) strategy. Raman spectroscopy, solid-state NMR (ssNMR) spectroscopy, and differential scanning calorimetry (DSC) with coupled thermogravimetric (TG) analysis lead to a complete characterization of the cocrystal forms. A solventscreening of the LAG syntheses and slurry experiments corroborate the determination of the thermodynamic and the kinetic product. In situ investigations using PXRD coupled with Raman spectroscopy permit the evaluation of the formation pathways of the cocrystal forms. A reaction mechanism could be derived supporting the prediction of kinetics and thermodynamics.
■
EXPERIMENTAL SECTION
Materials. Theophylline, C7H8N4O2 (99%, Sigma-Aldrich, Germany), and benzamide, C7H7NO (≥99.5%, Sigma-Aldrich, Germany), were purchased commercially and were used without further purification. The solvents were purchased from Acros Organics (Belgium), Fisher Chemical (USA), Merck (Germany), and Th.Geyer (Germany) in analytical grade or higher purity. Milling Synthesis. After a 10 mL steel vessel with two steel balls of 10 mm diameter and 4 g in mass was prepared, the reactants were accurately weighed in a stoichiometric ratio of 1:1 into the vessel for a total load of 1 g (0.5980 g of theophylline and 0.4020 g of benzamide). For LAG experiments 250 μL of the chosen solvent was added, and the vessel was immediately closed by screwing. In the case of neat grinding, the vessel was directly sealed before milling. Grinding was performed for all reactions (LAG and neat grinding) at 30 Hz for 25 min in a milling mill MM400 (Retsch, Germany). After the milling process, the grinding jar was opened, and the products were dried overnight at room temperature. Seeding Experiments. After a 10 mL steel vessel with two steel balls (10 mm diameter, 4 g) was prepared, the reactants were accurately weighed in a stoichiometric ratio of 1:1 into the vessel for a total load of 1 g (0.5980 g of theophylline and 0.4020 g of benzamide). After a small amount of Form II seeds was added, the vessel was immediately closed by screwing. Grinding was performed as described for the previous reactions at 30 Hz for 25 min in a milling mill MM400 (Retsch, Germany). After the milling process, the grinding jar was opened, and the products were investigated via PXRD. Slurry Experiments. Slurry experiments are commonly performed to identify the thermodynamically stable polymorph. Aqueous slurry experiments were conducted in order to determine the stability against hydrate formation and to gain the thermodynamically stable form of the cocrystal at ambient conditions. 250 mg of one cocrystal form or a physical, equimolar mixture (0.1495 g theophylline and 0.1005 g benzamide) of the reactants were slurried in 1 mL of water for 4 days. The solid phase was separated by filtration and analyzed by PXRD. PXRD Measurements. The products were investigated by PXRD. The obtained powder patterns of the pure cocrystals did not show any residues of the reactants. The final PXRD patterns were compared to the theoretical patterns of tp:ba I44 and tp:ba II. All PXRD experiments were carried out using a D8 diffractometer (Bruker AXS, Karlsruhe, Germany) in transmission geometry (Cu−Kα1 radiation, λ = 1.54056 Å). The crystal structure of tp:ba II was solved based on the PXRD pattern using the open source program FOX for indexing and structure solution.45 The program CHEKCELL was used to confirm the unit cell and the space group.46 FOX uses globaloptimization algorithms to solve the structure by performing trials in direct space. This search algorithm uses random sampling coupled with simulated temperature annealing to locate the global minimum of 1702
DOI: 10.1021/acs.cgd.5b01776 Cryst. Growth Des. 2016, 16, 1701−1707
Crystal Growth & Design
■
Article
RESULTS AND DISCUSSION Characterization of Cocrystal Polymorphs. Both polymorphic forms of the tp:ba cocrystal were obtained by neat or liquid-assisted grinding of an equimolar ratio of tp and ba (Scheme 1). The corresponding powder X-ray diffraction Scheme 1. Molecular Structure of Theophylline and Benzamide
Figure 2. Molecular motifs of the tp:ba cocrystal Form I (left)44 and Form II (right).
amino group with an amide group of a ba molecule, whereas in Form I only every second tp molecule shows this interaction. The main difference between the polymorphic forms is the arrangement of the plane of the R22(9) heterodimers in the unitcell. In tp:ba I these dimers are twisted about approximately 80° toward each other leading to four sheets in the unit cell. Within each sheet the molecules are arranged in a parallel order, and π−π-stacking can be observed. The heterodimers in Form II are arranged in parallel planes stabilized via π−π-stacking (Figure S1). Although polymorph I has a higher density, the cocrystal Form II exhibits more intermolecular interactions including hydrogen bonds and π−π-stacking. The differences in the crystallite morphologies are reflected in the SEM images in Figure 3b. The images indicate similar
(PXRD) patterns are depicted in Figure 1. For both polymorphs, the XRD data show that all reflections of the reagents disappeared completely.
Figure 1. Measured PXRD patterns of tp:ba Form I,44 Form II, and the corresponding reactants.
On the basis of the powder patterns, a structure determination of both polymorphs followed by a Rietveld refinement (Table S1 and Figure S2) was possible.44 Form I crystallizes in the space group P41. The unit-cell contains two crystallographically independent tp molecules and two independent ba molecules. Each tp molecule forms a R22(9) dimer with another ba molecule including the carbonyl group and the secondary amino group of tp and the amide group of ba (Figure 2, left). The heterodimers are stabilized via further hydrogen bonds between each other. The crystal structure of polymorph Form II of the tp:ba cocrystal was solved in the space group P21/n. As depicted in Figure 2 each tp molecule interacts in the cocrystal with two different ba molecules. One hydrogen bond is formed between the amino group of the ba molecules and a carbonyl group of tp. Another interaction between the acidic imidazolic nitrogen atom of tp and the carbonyl oxygen atom of ba results in the formation of a R22(9) dimer. The third hydrogen bond is based on the interaction of the tertiary amine of tp and the amide group of another ba molecule leading to a chain-structure. Both polymorphic forms reveal similar hydrogen-bond motifs. In Form II every tp molecule interacts via the tertiary
Figure 3. (a, b) SEM images of both polymorph materials in different magnitudes.54−56
particle sizes for both mechanochemically obtained compounds. A preferred formation based on different dimensions can be excluded. Compared to Form I, the crystallites of Form II agglomerate to thinner rods. Both cocrystals were further characterized to understand the stability of the polymorphic forms. Raman measurements and ssNMR spectroscopy allow determining salt formation during cocrystallization. The Raman spectra (Figure 4) of the polymorphs are very similar, which can be attributed to the same hydrogen bonding pattern in both structures. Since the carbonyl stretching absorption bands of tp at 1665 and 1707 cm−1 shift to 1643 and 1690 cm−1 in the cocrystals, salt formation can be excluded. A complete proton transfer would lead to a shift of these absorption bands by 30−40 cm−1 to lower frequencies.57 Additionally, the stretching band of the 1703
DOI: 10.1021/acs.cgd.5b01776 Cryst. Growth Des. 2016, 16, 1701−1707
Crystal Growth & Design
Article
evoke a ssNMR signal at 3.2 ppm and the proton at the tertiary carbon at 7.6 ppm.60 In the spectra of the cocrystals, these signals shift only slightly due to the new chemical environment. For the determination of a salt formation, the secondary amine of tp with a resonance frequency of 14.5 ppm has to be considered.60 In Form I this signal shifts to 13.0 ppm, whereas it can be observed at 13.3 ppm in Form II. On the basis of these shifts it can be supposed that in Form II the acidic proton of tp is bridged slightly stronger than in Form II without forming a salt. This result is in accordance to the investigation of the crystal structure. In order to determine the stability against humidity and to assess the thermodynamically favored polymorphic form, aqueous slurry experiments were conducted. Here, the cocrystals were slurried in water for 4 days. The powder patterns of the dried products show only the appearance of Form II independent of the cocrystal form used as starting material for slurrying. One can conclude that Form II of the tp:ba cocrystal is thermodynamically more stable than Form I. This assumption is supported by DSC-TG analysis: the melting point of polymorph I is 18 K below the melting point of the second polymorphic form (Figures S3−S4). Structurally, the differences in the stability can be assigned to the higher number of intermolecular interactions in the structure of Form II. Additionally, tp hydrate was slurried in water in an equimolar ratio of ba. The dried product shows also only the reflections of the tp:ba cocrystal Form II suggesting that this polymorphic form is stable against hydrate formation at high humidity. Solvent-Screening in LAG Experiments. Liquid-assisted grinding conditions were investigated by adding different solvents to the reaction mixture before starting milling. Typically, 1 g of the reaction mixture was ground with 250 μL of the solvent at 30 Hz for 25 min with two steel balls. On the basis of the powder patterns of the final products a polymorph determination was possible. In most of the experiments the thermodynamically favored Form II was formed. Only under neat grinding conditions or in the presence of a nonpolar solvent, the metastable cocrystal Form I is built. Prolonged milling time led to the same product. It can be assumed that the formation of tp:ba I proceeds without any interaction between the solvent and the reactant molecules. Form II is gained when a polar is solvent added to the reaction mixture. It seems likely that the crystallization of Form I has a lower activation energy than Form II and can be regarded as the kinetic product of the cocrystallization. Most probably the
Figure 4. Raman spectra of the tp:ba cocrystal Form I, Form II, and the respective reactants.
secondary amine (3123 cm−1) would vanish in the case of salt formation, and a new absorption band of an ammonium group between 2000 and 2200 cm−1 could be observed. In both cocrystal forms the band of the secondary amine of tp shifts only slightly by a few wavenumbers.58,59 The ssNMR measurements support the results of the Raman investigations. As illustrated in Figure 5 the methyl groups of tp
Figure 5. Solid-state NMR spectra of the tp:ba cocrystal Form I, Form II, and the respective reactants.
Table 1. Solubility of tp and ba in Water, Ethanol, and Diethyl Ether at Room Temperature and Solvents Added in the LAG Syntheses and the Resulting Polymorph of the tp:ba Cocrystal62−65 solubility [mg·mL−1] in compound benzamide solvent cyclohexane heptane pentane 1,4-dioxane 1-butanol 1-hexanol 1-propanol acetone acetonitrile
water
EtOH
Solubility [mg·mL−1] in Et2O
compound
13.5 166.7
