Racemic Compound, Conglomerate, or Solid Solution: Phase

Publication Date (Web): March 2, 2010. Copyright © 2010 American Chemical Society. *To whom correspondence should be addressed. Telephone: +31 (0)15 ...
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DOI: 10.1021/cg901483v

Racemic Compound, Conglomerate, or Solid Solution: Phase Diagram Screening of Chiral Compounds

2010, Vol. 10 1808–1812

Sukanya Srisanga and Joop H. ter Horst* Intensified Reaction and Separation Systems, Process & Energy Laboratory, Delft University of Technology, Leeghwaterstraat 44, 2628CA Delft, The Netherlands Received November 27, 2009; Revised Manuscript Received February 19, 2010

ABSTRACT: Crystallization in racemic solutions usually results in a racemic compound, a conglomerate or a solid solution. Resolution of a chiral compound strongly depends on the kind of solid state that it forms. Solid-liquid equilibria are related to the prevailing solid state of the chiral compound. A phase diagram screening of enantiomer mixtures in solution can therefore give important indications on the possibility of chiral resolution through crystallization. We describe a new simple and straightforward phase diagram screening method for chiral compounds in solution. Rather than determining the saturation concentration at a certain temperature, we more conveniently determined the saturation temperature of a certain solution composition. A number of chiral compounds were tested with the new method. Asparagine in water behaves as a conglomerate system, ibuprofen in hexane shows racemic compound/enantiopure compound formation, and atenolol shows solid solution behavior, consistent with observations elsewhere. Additionally, the method results in the ternary phase diagram of both enantiomers in a solution which may enable separation methods to be identified and designed. 1. Introduction Enantiomers of chiral drugs may differ drastically in their pharmacological and toxicological effects: the R-enantiomer of thalidomide has a sedative effect on pregnant women while the S-enantiomer causes birth defects.1 In the absence of in vivo racemization, resolution (separation of enantiomers) of racemic mixtures or impure enantiomers would prevent such disasters. Resolution is therefore of paramount importance in the pharmaceutical industry. One of the techniques that can be employed for chiral resolution is crystallization.2 The solid phase formed from a racemic solution can be a racemic compound, a conglomerate, or a solid solution.2 A crystal of a racemic compound consists of an even ratio of both enantiomers in a regularly structured array. A conglomerate is a physical mixture of pure enantiomer crystals, with the overall mixture being essentially racemic. A solid solution or a pseudoracemate forms when both enantiomers compete for the same position in the crystal structure. While the formation of a solid solution is rare, the formation of a racemic compound occurs most often. In only about 10% of the cases, a stable conglomerate forms.2 The kind of solid state of a chiral compound has important implications on the ability for resolution through crystallization. A prerequisite for direct resolution through preferential crystallization is the ability of enantiomer mixtures to form a conglomerate.3 When enantiomer mixtures form a racemic compound, an enantiopure compound can only be crystallized when one of the enantiomers is present in a sufficient excess.4 For solid solutions, several crystallization steps may lead to enantiomer enrichment (fractional crystallization).2 The prevailing phase diagram is related to the kind of solid state of a chiral compound. The phase diagram thus can give important information on the possibility of chiral resolution *To whom correspondence should be addressed. Telephone: þ31 (0)15 278 6661. Fax: þ 31 (0)15 27 82460. E-mail: [email protected]. Web: http://www.pe.tudelft.nl/. pubs.acs.org/crystal

Published on Web 03/02/2010

through crystallization. Furthermore, the phase diagram gives quantitative data that may enable chiral separation methods to be identified and designed. It would therefore be beneficial to have a method for the fast and straightforward determination of the ternary phase diagram of both enantiomers of a chiral compound in solution. This paper proposes such a phase diagram screening method based on saturation temperature measurements of mixed compositions. The proposed method can be used to identify opportunity systems and would precede an exhaustive phase diagram and solid state study. The method is tested on three chiral compounds of which enantiomer mixtures were known to form a racemic compound, a conglomerate, or a solid solution. 2. Theory Upon crystallization, enantiomer mixtures form a racemic compound, an enantiopure compound, or a solid solution. Which of these solid states is stable at ambient pressure depends on the chiral compound, the system composition, and the temperature. Common temperature-composition binary phase diagrams are shown in Figure 1. The upper lines in Figure 1a and b indicate the melting temperature for a certain R-enantiomer fraction yR. The lower horizontal line in Figure 1a and b indicates the eutectic temperature. Figure 1a shows a conglomerate. This is the case when the enantiopure crystalline compounds R and S are the stable compounds in the entire compositional range 0 e yR e 1 below the melting temperature. At the outer sides, for yR =0 and 1, the melting temperatures are those of the pure enantiomer crystalline compounds S and R, which are equal. Decreasing enantiomer purity toward a racemic mixture shows a decrease of the melting temperature of either the solid enantiopure R or S phase down to the eutectic temperature. Below the eutectic a mixture of enantiopure crystals R and S exists. Figure 1b shows a racemic compound. At the outer sides, for yR = 0 and 1, the melting temperature is that of the pure enantiomer crystalline compounds S and R. Slightly decreasing enantiomer purity decreases the melting temperature. r 2010 American Chemical Society

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the value of x=1, while for ternary phase diagrams, in which a certain fraction of solvent is present, 0 < x < 1. For a constant value of the total enantiomer fraction x, the solvent excluded R-enantiomer ratio yR can be varied xR xR ð2Þ ¼ yR ¼ xR þ xS x

Figure 1. Binary phase diagram of enantiomer mixtures forming (a) a conglomerate, (b) a racemic compound, and (c) a solid solution.2 R, S, RS, and L represent respectively solid R-enantiomer phase, solid S-enantiomer phase, racemic compound RS, or solid solution RS and liquid phase.

However, for compositions around yR =0.5 the racemic compound RS rather than the enantiopure compound R or S is the more stable compound, which causes the melting temperature to rise toward yR = 0.5. Figure 1c shows a binary phase diagram of an ideal solid solution. Both enantiomers compete for the same lattice position in the solid phase. In this case of a chiral compound of Roozeboom type I the melting temperature is constant and not depending on the enantiomer fraction yR. The behavior of solid solutions can be more complex than shown here.2,3 Differential scanning calorimetry (DSC) is often used to determine binary phase diagrams. However, many organic compounds decompose before melting. Furthermore, industrial crystallizations are performed well below the melting temperature. The binary phase diagram then may not be representative for the system behavior at lower temperatures. The stability of a racemic compound and its conglomerate counterpart may switch at a certain transition temperature lower than the melting temperature. It is reported that at room temperature the racemic compound of the hydrochloric salt of histidine is the stable form in racemic solutions in water while at elevated temperatures the conglomerate is more stable.2 It is thus important to know phase behavior in solutions at industrial preferential crystallization conditions. Generally, the solubility of a compound is measured isothermally upon equilibration of a crystal suspension. This is a time-consuming method for which a concentration measurement is required (gravimetry, acid-base titration, HPLC, GC, etc.) and, in the case of enantiomer mixtures, a specific calibrated concentration measurement that can make a distinction between the enantiomers (e.g., chiral-HPLC). Rather than determining the saturation concentration at a certain temperature, we more conveniently determine the saturation temperature of a certain solution composition. This can be performed much faster. Increasing the temperature of a suspension with a sufficiently slow heating rate will result in a temperature at which the slurry becomes a clear liquid and all crystals are dissolved, the clear point. At the clear point, the solution composition is known. If the crystals dissolve sufficiently fast, the clear point can be taken as the thermodynamic saturation temperature. Adapted from a phase diagram screening method for cocrystals,5 we developed a phase diagram screening method for the ternary phase diagram of enantiomers in a solvent. Similar to the determination of a binary phase diagram, the total amount of enantiomer was taken at a constant value x x ¼ xR þ xS

ð1Þ

where xR and xS are the mole fractions of the R and S enantiomers, respectively. In the case of a binary phase diagram,

By determining the saturation temperature along the enantiomer fraction yR at a constant total amount x of enantiomer, effectively a pseudobinary phase diagram is determined: the determined saturation temperatures then are equivalent to the melting temperatures in Figure 1. Similar figures as in Figure 1 would thus be obtained, showing the solid state characteristics at lower temperatures in solutions. The contour of the measured saturation temperatures in the pseudobinary phase diagram then identifies the kind of solid state of the chiral compound. This is exactly what is done for the model compounds asparagine, ibuprofen, and atenolol. 3. Experimental Section R-Atenolol (99%), RS-atenolol (98%), L-asparagine (minimum 98% TLC), DL-asparagine monohydrate (minimum 98% TLC), and S-Ibuprofen (99%) were purchased from Sigma-Aldrich. The RSIbuprofen (>99.9%) used was produced by Albemarle. Chemicals were used without further purification. Ethanol (absolute, Merck), n-hexane (95% for HPLC, J.T. Baker), and double distilled deionized water (ultrapure water) prepared in the laboratory were used as solvents. The Crystal16 equipment of Avantium Technologies was used to determine the saturation temperatures of the mixtures of enantiomers in different solvents. In the Crystal16 equipment, the clear and cloud points of sixteen 1-mL solution aliquots can be measured in parallel and automatically, based on turbidity. A physical mixture of the enantiopure and racemic crystalline phase was dissolved in the appropriate amount of solvent at a sufficiently high temperature. Upon cooling the clear solution with 0.5 °C per minute to 0 °C and maintaining this temperature for 5 h, recrystallization occurred. The recrystallized solid was slowly dissolved again upon increasing temperature with a rate of 0.5 °C per minute. The temperature at the point the suspension becomes a clear solution upon heating was taken as the saturation temperature of the measured sample, of which the composition was established beforehand. Samples were stirred at 700 rpm. Enantiomers have identical chemical and physical properties except for their ability to rotate plane-polarized light by equal amounts but in opposite directions. The binary phase diagrams of chiral compounds are therefore symmetrical around yR = 0.5. Determination of only one side of the binary or ternary diagram is sufficient to know the full phase diagram.

4. Results Asparagine, ibuprofen, and atenolol (Figure 2) were chosen as model chiral compounds. For all these compounds saturation temperatures were determined of samples with varying solvent excluded enantiomer fraction yR but a constant total molar fraction x of the chiral compound. It was investigated whether the measured pseudobinary phase diagram of saturation temperature against solvent excluded enantiomer fraction yR reflects the solid state characteristics of the chiral compound. The phase diagram screens of asparagine, ibuprofen, and atenolol are shown from top to bottom in Figure 3. The sample compositions of mixed enantiomers with constant total amount x = xR þ xS of chiral compound and varying solvent excluded R-enantiomer fraction yR =xR/(xR þ xS) are shown in the left graph. The right graph shows the measured saturation temperature as a function of the enantiomer fraction yR.

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Figure 2. Molecular structure of the model compounds used in the phase diagram screening: (a) asparagine; (b) ibuprofen; (c) atenolol. The chiral center is indicated with an asterisk.

Asparagine. Asparagine (Figure 2a) is an amino acid that is soluble in water. Based on preliminary saturation temperature measurements, two total asparagine hydrate fractions, x = 15.2 and 25.7 mmol/mol, were chosen for the phase diagram screening, taking into account that asparagine forms a monohydrate solid state in water. At these total asparagine fractions, several solvent excluded R-enantiomer fractions yR were chosen. The sample compositions of these are shown in Figure 3a. The sample compositions for a constant total asparagine fraction x are positioned on a straight line from xR =x to xS =x in the xR-xS diagram (Figure 3a). At these constant total asparagine fractions, the solvent excluded R-enantiomer fraction yR is varied. The measured saturation temperatures of these samples are shown in Figure 3b as a function of the solvent excluded R-enantiomer fraction yR. In Figure 3b at yR = 1, only the R-enantiomer is present in water, at yR = 0.5 a racemic mixture is present in water. At yR =1 the saturation temperature is measured to be Ts = 60.8 °C (x = 15.2 mmol/mol) and 73.1 °C (x = 25.7 mmol/mol). When the solvent excluded R-enantiomer fraction is decreased toward yR = 0.5, a decrease in saturation temperature is observed down to Ts = 44.5 °C (x=15.2 mmol/mol) and 56.8 °C (x=25.7 mmol/mol). The result is a pseudobinary phase diagram for the asparagine enantiomers in water that can be compared to Figure 1 in order to suggest the kind of solid state of asparagine. Figure 3b shows a single eutectic system which can be identified in Figure 1a as a conglomerate. Asparagine in water therefore behaves as a conglomerate. The asparagine solid state in water enables the separation of the enantiomers using preferential crystallization.6 The line for 0.5 e yR e 1 in Figure 3b represent the expected saturation temperatures based on the R-enantiomer solubility in the solvent (in the absence of the other enantiomer). The theoretical and experimental saturation temperatures are in close agreement. This indicates that the presence of one enantiomer hardly affects the solubility of the other enantiomer. This valuable thermodynamic information from the pseudobinary phase diagram could further be used in crystallization process design. Ibuprofen. Ibuprofen (2-[4-isobutylphenyl]propanoic acid, Figure 2b) contains a chiral carbon in the 2-position of the carboxylic acid. Although it was found that only (S)-ibuprofen is active,7 ibuprofen is marketed in its racemic form because there is a metabolic stereoisomeric inversion of ibuprofen in mammals.8 The solubility of (RS)-ibuprofen in hexane was found to be 175 mmol/mol at 44.5 °C, which was chosen as the total ibuprofen fraction of x=175 mmol/mol. Figure 3c shows the sample compositions used at this total ibuprofen fraction. The saturation temperatures for a total ibuprofen fraction of x=175 mmol/mol are shown in Figure 3d as a function of the solvent excluded mole fraction yR. A racemic mixture (yR = 0.5) in hexane has a saturation temperature of 44.5 °C while the enantiopure ibuprofen (yR = 0) in hexane has a

saturation temperature of 18.1 °C. Starting from enantiopure ibuprofen in hexane and moving toward the racemic mixture (increasing yR from 0 to 0.5) shows first a decrease down to 15.7 and then an increase to 44.5 °C at yR =0.5 in the measured saturation temperatures. The pseudobinary diagram for ibuprofen in hexane shows two eutectic points situated at around a solvent excluded R-enantiomer fraction of yR =0.03 and yR =0.97. Comparing the pseudobinary phase diagram of Figure 3d to Figure 1b shows that ibuprofen forms a racemic compound over a large range of yR-values. This is consistent with the previously reported binary phase diagram of ibuprofen.9 In this case the racemic compound is very stable compared to enantiopure ibuprofen, with the eutectic points at an R-enantiomer fraction of about yR = 0.18 and 0.82. To crystallize enantiopure ibuprofen, a large excess of one of the enantiomers in the solution is needed. Atenolol. The molecular structure of atenolol, 2-{4-[2hydroxy-3-(propan-2-ylamino)propoxy]phenyl}acetamide, is shown in Figure 2c. Atenolol is a β1 receptor selective antagonist, used primarily in cardiovascular diseases.10 It was reported that administering (S)-atenolol instead of the marketed (RS)-atenolol avoids the side effect of an excessively lowered heart rate sometimes encountered with (RS)atenolol.11 A chiral separation of the enantiomers would therefore be beneficial for atenolol activity. Figure 3e shows the composition of the atenolol samples studied. A total atenolol fraction of x=10 and 25 mmol/mol in ethanol was chosen. Figure 3f shows the measured saturation temperatures as a function of the solvent excluded R-enantiomer mole fraction yR. Over the whole range of fractions yR the measured saturation temperature remains nearly constant. Comparing the pseudobinary phase diagram Figure 3f to the binary phase diagram Figure 1c suggests atenolol to be a solid solution. RS-AT has previously been identified as a solid solution using DSC12 and single crystal structure determination.13 This agrees with our results. In order to obtain enantiopure atenolol by crystallization, the solid state of atenolol should be altered by for instance diastereomeric salt formation. 5. Discussion The aim of the fast and straightforward phase diagram screening method proposed here is to obtain a first indication of the kind of solid state and the effect of solution composition on its solubility. In order to validate the observed behavior, more extensive phase diagram and solid state studies as well as other analytical tools such as XRPD are needed. Additionally, more complex solid state behavior is possible, which will be discussed below. Our methodology is targeted so that the probability for the formation of the thermodynamically most stable phase is high. However, formation of metastable phases is possible

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Figure 3. Phase diagram screens of asparagine in water (a, b), ibuprofen in hexane (c, d), and atenolol in ethanol (e, f). The left graphs show the used compositions in the screen in a xR-xS graph with the mole fractions expressed in mmol/mol. The right graphs show the measured saturation temperatures Ts as a function of the solvent excluded R-enantiomer fraction yR of these samples. The lines in part b indicate the pure enantiomer solubility. Other lines are drawn as a guide to the eye. Comparison of Figure 1 and the figures on the right shows that asparagine is a conglomerate system, ibuprofen forms a racemic compound, and atenolol is a solid solution.

and should be considered to play a role. For instance, when the stable racemic compound does not recrystallize, but rather the metastable conglomerate is formed previous to the saturation temperature measurement, the system would be erroneously identified as a conglomerate system. The phase diagram screening assumes the formation of either a racemic compound, an enantiopure compound, or an ideal solid solution. The majority of chiral compounds forms a racemic compound from a racemic solution, in only about 10% of the cases a conglomerate forms, while solid solutions are relatively rare. However, the possibility of solid solution formation cannot be ruled out, as the case of atenolol shows. Atenolol is an ideal solid solution where the satura-

tion temperature is not affected by the solvent excluded R-enantiomer fraction yR in the sample but only by the total fraction x=xR þxS of atenolol. Other compounds might show nonideal solid solution behavior, which would result in more complex behavior of the saturation temperature as a function of the solvent excluded R-enantiomer ratio yR. Additional analysis techniques then might be needed to distinguish between solid states. The formation of solvates, such as in the case of the asparagine monohydrate, has to be accounted for when constructing the isothermal phase diagram from the phase diagram screen. Even more complex related behavior, such as partial solid solutions, was observed.14,15 The phase diagram screening

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method is not aiming to identify such complex behavior. These exceptional cases need a further and more extensive study of the solid state and phase diagram to correctly identify the complex behavior. The proposed phase diagram screening method with its assumptions and limitations gives an indication on the solid state behavior and ternary phase diagram of chiral compounds that sets the basis for an extensive further study. 6. Conclusions We have proposed a new phase diagram screening method to identify the solid state behavior of chiral compounds. Asparagine, ibuprofen, and atenolol were identified respectively as a conglomerate system, a racemic compound, and a solid solution. This is consistent with previous findings. Furthermore, the additional quantitative data from the phase diagram screen is essential information in identifying and designing chiral separation processes based on crystallization. The phase diagram screening method is therefore an interesting new and fast way to obtain an indication of the solid state behavior and the phase diagram of chiral compounds. Acknowledgment. The authors thank Somnath Kadam for providing the (RS)-ibuprofen raw materials and (RS)-ibuprofen solubility data in hexane.

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