Application of Crystallization Inhibitors to Chiral Separations. 1

UniVersity of Bradford, Bradford BD7 1DP, UK. ReceiVed August 24, 2006; ReVised Manuscript ReceiVed October 25, 2006. ABSTRACT: In the context of the ...
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CRYSTAL GROWTH & DESIGN 2007 VOL. 7, NO. 2 218-224

Articles Application of Crystallization Inhibitors to Chiral Separations. 1. Design of Additives to Discriminate between the Racemic Compound and the Pure Enantiomer of Mandelic Acid R. K. Mughal,† R. J. Davey,*,† and N. Blagden‡ The Molecular Materials Centre, School of Chemical Engineering and Analytical Sciences, UniVersity of Manchester, SackVille Street, Manchester M60 1QD, UK, and School of Pharmacy, UniVersity of Bradford, Bradford BD7 1DP, UK ReceiVed August 24, 2006; ReVised Manuscript ReceiVed October 25, 2006

ABSTRACT: In the context of the potential use of crystallization for spontaneous separation of racemates, it is well-known that the frequent appearance of racemic solid forms (racemic compounds) is a serious obstacle. Given previous studies on the selective inhibition of centric versus noncentric polymorphic structures, we report here the application of a similar rationale for the selective crystallization inhibition of a racemic compound compared to a pure enantiomer. We have chosen the system R,S-mandelic acid (MA) in water which, from racemic solutions, crystallizes as two polymorphs of a racemic compound. The selection of potential additives is described and from the effects of additive concentration on the crystallization of (R,S)-MA compared to (S)-MA, at various crystallization temperatures, it is shown that while none of the additives dramatically inhibit the crystallization of (S)-MA even at 3% they significantly inhibit the crystallization of the racemate. With 10% additive, the crystallization of (R,S)-MA is totally inhibited for over 4 months. This leads the way to a new potential crystallization process for chiral enrichment discussed in the accompanying paper in this issue. 1. Introduction Crystallization is widely used in the pharmaceutical and chemical industries for the preparation, purification, and separation of a range of active compounds. In the particular case of chiral molecules, the possible separation or enrichment of enantiomers through crystallization is of great academic and commercial interest, being one of the most economical methods by which high enantiomeric excesses might be obtained.1 The use of crystallization in this context is, however, limited by the observed behavior of chiral molecules isolated from racemic solutions. Three possible outcomes are possible during crystallization of a racemic solution: (i) a racemic compound is formed in which each crystal contains a 1:1 mixture of enantiomers, (ii) a conglomerate appears, which is a physical mixture of homochiral crystals; (iii) a solid solution is formed in which each crystal is heterochiral. According to available data,1,2 the formation of a racemic compound is the most common outcome. For this reason, resolution of enantiomers is normally carried out through the use of chiral acids or bases to form diastereomeric salts which may be separated by crystallization due to their different physical properties. The work reported here is the first part of a study in which we have considered possible crystallization processes occurring * To whom correspondence should be addressed. † University of Manchester. ‡ University of Bradford.

within different domains of the ternary system, R- and Smandelic acid and water. Even in such a system in which a racemic compound exists, there are certain regions of the phase diagram where the crystallized solid will be a mixture of pure enantiomer and racemic compound.1 We have been interested in the possibility of developing a crystallization process in this region such that the formation of the compound is selectively inhibited while allowing the pure enantiomer to form, hence yielding an enriched product. Thus, in this first part of the study, we have explored the design and assessment of “tailor-made” additives3 as a means of selectively delaying the nucleation of the compound compared to that of the pure enantiomer. In a second part, we will show how this information may be combined with the ternary phase data4 to develop a strategy for chiral enrichment. We have chosen mandelic acid (MA) (Scheme 1) as a model material largely because it is a commonly used resolving agent for isomer separation,5 and consequently much of the data we need are already available. Thus, the crystal structures of the pure enantiomer (no polymorphs known)6 and the polymorphs (R,S)-MA I (thermodynamically stable form) and (R,S)-MA II (monotropically related metastable form) of the racemic compound are known,7,8 and the ternary phase diagram of (R,S)MA and water has been previously published.4 Selection of Additives. The principle of additive selection used here for discrimination between the centrosymmetric (R,S)-

10.1021/cg0605638 CCC: $37.00 © 2007 American Chemical Society Published on Web 01/05/2007

Use of Crystallization Inhibitors in Chiral Separations

Figure 1. (a) (R,S)-MA I and (b) (S)-MA chains. Blue and pink circles indicate the similarities and differences along the fast growth axes.

Scheme 1. Molecular Structure of Mandelic Acid

MA I and the noncentric (S)-MA structures has been expounded and demonstrated before.9,10 Briefly, in a centric structure where the two ends of a fast growing crystal axis are related by symmetry, it is possible for a single additive to simultaneously block both ends of a crystal and arrest its growth. In a noncentric structure in which the ends of the fast growth direction are not symmetry related, while an additive can halt the growth of one end of a crystal it cannot inhibit both ends simultaneously. Thus, the growth of the noncentric form may proceed with only partial disruption. This principle has been amply verified in a number of cases, for example, for the centric and noncentric polymorphs of N-(2-acetamido-4- nitrophenyl) pyrolidene9 and dihydroxybenzoic acid,11 and perhaps most relevant to the current work, the racemic compound, histidine hydrochloride dihydrate and the conglomerate histidine hydrochloride monohydrate.12 In this latter case, the use of a specifically designed polymeric inhibitor effectively enabled the simultaneous inhibition of the compound and one of the enantiomers such that chiral enrichments of up to 100% were obtained, with concomitant yield losses. Previous studies of crystallization inhibition of MA have been restricted to an unsuccessful attempt to exploit symmetry reduction by incorporation of stereospecific additives in the {111} faces.13 Using the known structures of the pure enantiomer and of the racemic compound polymorphs,6-8 it is possible to analyze the main distinguishing features of the structures and the crystal morphologies. The stable form of the compound, (R,S)-MA I, is composed of C11(5) chains connected via R22(10) centrosymmetric dimers running along the c-axis, as seen in Figure 1a. Its crystal morphology is shown in Figure 2. Two habits commonly appear, one in which the c-axis is clearly the fast growth direction and one in which the {111} faces bound the fast growing edges of a platelike morphology. In both cases, the centric space group of the structure means that both ends of the fast growing directions are equivalent and expose both

Crystal Growth & Design, Vol. 7, No. 2, 2007 219

hydroxyl and carboxyl functionalities. A similar description applies to the metastable form II although not shown here. In the pure enantiomer (Figure 1b), the polar C11(5) chains are connected by noncentric carbonyl-hydroxyl interactions running along the b-axis, and the morphology is as seen in Figure 2. Again two habits are prevalentsone in which the b-axis is clearly the fast growth direction and the other a rhomb bounded by fast growing {110} and {111}. In both cases, the polar C11(5) chain is exposed at the growing surfaces such that one end is rich in hydroxyl functionality and the other rich in carboxyl groups. Thus, unlike the racemic compound, opposite ends of the fast growth direction are nonequivalent. This conclusion is reinforced by the red and blue circles seen in Figure 1, which highlight the carboxylic acid and hydroxyl functions, respectively. It is this inherent difference in chemistry between the surfaces of the two forms that provides the basis for the selection of additives to act as selective inhibitors of the compound while leaving the pure enantiomer to crystallize essentially as in pure solution. The additives chosen were very similar in molecular structure and functionality to MA itself but with specific modifications designed both to aid binding onto the growing crystal surface but also once bound to terminate the growing C11(5) chains. For example, molecules that lack either of the hydroxyl functionalities were selected. An additive molecule with no alcoholic -OH can continue to bind to both ends of (R,S)-MA through its carboxylic acid group, but this additive molecule will only be able to bind to one end of (S)-MA. This should result in selective inhibition of (R,S)-MA. Likewise, an additive with no acid hydroxyl will terminate the chains while continuing to bind to crystal surfaces through its alcoholic -OH, again inhibiting both ends of (R,S)-MA and only one end of (S)-MA. Table 1 lists all the additives selected, which include molecules lacking the alcoholic hydroxyl: phenylacetic acid (PAA), benzoylformic acid (BFA), 2-phenylpropionic acid (PPA), and 2-phenylglycine (PG) as well as those with the acid hydroxyl functionality missing: methyl mandelate (MM) and benzyl mandelate (BM). The crystal structures of PAA,14 BFA,15 and BM16 are known, and for these additives molecular conformations were extracted for visualization, in Cerius2.17 Additive molecules were docked visually onto MA sites to explore how the growth of (R,S)- and (S)-MA might be affected. As an example, Figure 3 illustrates the proposed mode of action of PAA. Along both the + and - c-axes, a PAA molecule (orange) can join a C11(5) chain of MA molecules via an H-bond through its acid -OH. However, once incorporated, because it does not bear a hydroxyl group (in contrast to MA), the next incoming MA molecule cannot join the chain. This effectively terminates the chain. Conversely in (S)-MA the additive molecule (orange) incorporates into the crystal along -b, terminating the bottom face, but due to the polar nature of the b-axis, the opposite end is nonequivalent and the additive cannot incorporate into the crystal from this end and hence the crystal can continue to grow along +b. In Figure 3, the green circles indicate hydrogen-bonding sites on an incoming MA molecule that cannot be satisfied due to the adsorption of the additive, while the blue circles show the location of the “missing” alcoholic -OH in the additives. In the case of chiral additive molecules, it was necessary to include both R- and S-isomers as additives in the crystallization of (R,S)-MA I to terminate both C11(5) chains. In the case of the pure (S)-mandelic acid, however, the S-isomer additives alone could be used if available. Thus, in the case of PPA and

220 Crystal Growth & Design, Vol. 7, No. 2, 2007

Mughal et al.

Figure 2. Experimental morphologies: schematic and microscope images of the (R,S)-MA I (above) and (S)-MA (below). Table 1. Additive Molecules Selected

PG, both the racemate and pure enantiomers were used, while for MM and BM the racemates were used in all experiments. Experimental Procedures To test the selectivity of the chosen additives, induction times were measured18 in experiments in which either the pure enantiomer was crystallized from a solution of S-mandelic acid in water or (R,S)-MA was crystallized from a racemic solution. The induction time, tind, is the time that elapses from the establishment of supersaturation to the appearance of a new crystalline phase, and it is often assumed to be inversely proportional to the nucleation rate. In this work, we have measured the induction time visually in a series of controlled temperature-jump cooling crystallization experiments carried out in stirred

Figure 3. (R,S)-MA I (left) and (S)-MA (right) with additive molecule PAA in orange. Blue circles show missing hydroxyls, and green circles show the potential hydrogen-bonding site on the incoming MA molecule.

(magnetic), stoppered, jacketed, glass crystallization vessels, (10-20 mL scale), whose temperature was controlled within ( 0.1 °C by a Haake circulating water bath. Saturated MA solutions were prepared on an 8 g of water scale and heated to 70 °C for approximately 1 h. Additives were dissolved in the saturated solutions, and compositions are given here as % additive based on the total mass of MA. The

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Crystal Growth & Design, Vol. 7, No. 2, 2007 221

Figure 4. The effect of 3% additive at 15 °C on (S)-MA crystallization. Repeat 10 is the average induction time.

Figure 5. The effect of 3% additive loading on crystallization of (R,S)-MA at 15 °C. Repeat 8 is the average induction time. Table 2. (a) Effects of Temperature, Supersaturation, and 3% Additives on Mean (S)-MA Induction Times and (b) Effects of Increasing Amounts of Additives on (S)-MA Induction Times (a) Effects of Temperature, Supersaturation, and 3% Additives on Mean (S)-MA Induction Times σT and % additive

PAA tind (mins)

BFA tind (mins)

PG tind (mins)

PPA tind (mins)

control tind (mins)

σ15°C ) 3.82, 3% σ20°C ) 3.73, 3%

9.3 12.0

5. 4 14.8

6.1 25.3

5.1 22.2

1.5 4.6

(b) Effects of Increasing Amounts of Additives on (S)-MA Induction Times tind (mins) σT and % additive

PAA

BFA

PPA

methyl mandelate

control

σ25°C ) 2.67, 6% σ25°C ) 2.67, 10% σ25°C ) 2.67, 12% yield (%)

50.38 83.0 96.5 49.7%

84.3 134.7 198.0 51.4%

38.0 50.2 43.0 57.8%

107.0 55.4 70.5 53.6%

76.0 64.1 103.8 67.6%

solutions were then “crash” cooled (4 months (R,S)-MA I

>4 months (R,S)-MA I

>4 months (R,S)-MA I

>22 h (R,S)-MA II

(b) Average Induction Times for the Effect of Combined Additives on (R,S)-MA Crystallization Av. tind (mins), σT and % additive σ15°C ) 3.82 σ20°C ) 3.2 % yield σ25°C ) 1.71 a

3% MM-

6% MM

3% MM + 3% PAA

6% MM + 3% PAA

control

518.7, (R,S)-MA II 487.3 (R,S)-MA I

1110.2 (R,S)-MA II 865.3 (R,S)-MA I

1146.3 (R,S)-MA II 36577 (R,S)-MA II

13.35 (R,S)-MA II 45 (R,S)-MA II

69.3% 750 (R,S)-MA I

64.5% 1211 (R,S)-MA I

986.4 (R,S)-MA II 27189 (R,S)-MA II 58.7% 58.7% 62347.3 (R,S)-MA II

54.6% 59024 (R,S)-MA II

73.2% 184.3 (R,S)-MA II

Experiment not performed due to limited solubility of additive.

Figure 6. (R,S)-MA inhibition with methyl mandelate and methyl mandelate and PAA at 20 °C. the equilibrium saturation concentration (gsolute/100 gsolvent) at the crystallization temperature. Solubility data were taken from refs 19 and 20. The induction times were determined for the nucleation of (R,S)MA and (S)-MA in the presence of the six different additives shown in Table 1. Two different additive concentration levels (3 and 6% based on total MA) have been thoroughly investigated, at 15, 20, and 25 °C. It is evident that, in comparing additive effectiveness, 3% of a chiral additive operating in the enantiomerically pure system is equivalent to 6% of the same additive present as a racemate and acting in the racemic system. A limited number of measurements were also made at 10 and 12% and as admixtures to test the ultimate potency of the additives. The solubility of 2-phenylglycine limited experiments with this additive to a maximum concentration of 3%. (R,S)-Mandelic acid (99%), S-(+)-

mandelic acid (99+%), and the additives (R,S)-benzyl mandelate, (R,S)ethyl mandelate (97%), (R,S)-methyl mandelate (97%), (R,S)-2phenylpropionic acid, (97%), (R,S)-2-phenylglycine (95%), (S)-2phenypropionic acid (97%), (S)-2-phenylglycine (99%), phenylacetic acid (99%), benzoylformic acid (97%) were all obtained from Sigma Aldrich and used as purchased. Distilled, deionized water was used throughout. The pKa of the acidic proton in mandelic acid is 3.4.21 The experimental pH was measured to be 1.2, indicating that crystallization took place from solutions in which the acid was fully protonated.

Results Effect of Additives on (S)-Mandelic Acid. The effect of 3% at 15 °C (σ15°C ) 3.82) and 20 °C (σ20°C ) 3.73), 6 and

Use of Crystallization Inhibitors in Chiral Separations

10% at 25 °C (σ25°C ) 2.67) of all additives tested on the induction time of (S)-MA was minimal; although some inhibition occurred, none of the additives were potent inhibitors. Table 2a,b gives some numerical data and Figure 4 shows the variation in tind of (S)-MA, crystallized at 15 °C with 3% additives. Both individual experiments and the average induction times are shown. The trend in induction times indicates that even in the best case (PAA) the inhibition only extends the average induction time from 2 min to about 10 min. Even with 10% additive loading at 25 °C, the average induction time for the control is 64 min, while the best inhibitor, BFA, only extends this to 135 min. Interestingly, PPA appears to consistently encourage crystallization (shorter induction times than the controls) with associated increase in yield in comparison to the other additives. Neglecting the impact of temperature, the effect of increasing supersaturation from 2.67 to 3.82 in pure solutions is, as expected, to reduce the induction time from 103.8 to 1.5 min. A combination of 3% methyl mandelate (lacking the carboxylic acid -OH) and 3% PAA (lacking the alcoholic -OH) were also used. It was expected that each additive would block the opposite ends of the fastest growing crystal face and hence totally inhibit the crystallization of (S)-MA. In fact, the combined additives had a relatively negligible effect on the inhibition of (S)-MA at 15 °C, with the average tind of 23 min compared to the control of tind ) 13 min. Effect of Additives on (R,S)-MA. The effect of 3 and 6% additives on the induction time of (R,S)-MA were studied at 15 (σ15°C ) 3.82), 20 (σ20°C ) 3.2), and 25 °C (σ25°C ) 1.71). In all cases, at the onset of crystallization and in the absence of additives, the metastable polymorph, (R,S)-MA II was the first phase to appear. As with (S)-MA, the induction times increase with decreasing supersaturation. With those additives in which the alcoholic hydroxyl is missing (Table 3a and Figure 5), the metastable polymorph continues to be the first phase to appear at 15 and 20 °C, but now with significant increases in induction time. At 25 °C and for 6 and 10% additive, the induction time increases dramatically (for example, to 20 days) and the first phase to appear is the stable (R,S)-MA I. For the mandelate additives (Table 3b and Figure 6), which lack the acid hydroxyl, the situation is slightly different with experiments at 15 °C giving the metastable form and those at 20 and 25 °C giving the stable form. Mixtures of methyl mandelate and PAA consistently give the metastable form. Given that both the stable and the metastable racemic compound polymorphs are centric structures based on an identical hydrogen-bonded chain, it is not surprising that the additives influence the crystallization of both. Notwithstanding this, it is clear that all the additives significantly inhibit the crystallization of (R,S)-MA solutions. PPA is the most effective, in not only delaying the appearance of crystals but also slowing down the subsequent crystal growth process. This was evidenced visually by the observation that this additive alone gave relatively few well-formed crystals, while other additives gave numerous small crystals (ie powders). When 6% of this additive is used at 15 °C, its effect is to extend the average induction from 9.5 h (control) to 121 h. The induction time for the crystallization at 25 °C with 6% was over 20 days, while in the presence of 10% additive no crystallization took place for over 4 months. In a pure racemic solution, the crystallized (R,S)-MA II was stable for 3 days before converting to the more stable (R,S)MA I, whereas 6% PPA extended this time to more than 7 days. The effect of a combination additives was also studied (Table 5) for the racemic compound to check for their expected

Crystal Growth & Design, Vol. 7, No. 2, 2007 223

complementary effect, by combining one lacking the alcoholic -OH with one the acid -OH. At 15, 20, and 25 °C, 3 and 6% methyl mandelate coupled with 3% PAA the inhibition in general is more dramatic. A comparison of 3 and 6% methyl mandelate and a combination of methyl mandelate and PAA at 20 °C is shown in Table 5 and Figure 6. Discussion The results of this study demonstrate the expected selectivity of the chosen additives for the racemic compound over the pure enantiomer. It is evident that, under the conditions chosen, the additives in racemic form are all effective inhibitors for the nucleation of the racemic compounds. This result follows the expectations of a nucleation process based on the self-assembly and growth of hydrogen-bonded chains in which the intermolecular -COO-H‚‚‚OH-R interactions may be terminated either by molecules lacking the alcoholic or acid hydroxyl group. With single molecule additives, the crystallization has been inhibited by up to 20 days, while with a combination of both additive types this time may be doubled. In the wider context of nucleation of a conglomerate versus a racemic compound, it is interesting to note that even from these racemic solutions that remain metastable for such long periods no conglomerate forms. This outcome strongly suggests that the nucleation behavior is dominated by statistics that favor R/S clusters. In contrast, the pure enantiomer is relatively unaffected even by a combination of additives with induction times extending at best to a few hours. For example, at a total of 10% loading of additives at 25 °C the maximum induction time found for the pure enantiomer was 135 min, while for the compound it was over 4 months. It is not clear why the pure enantiomer should be relatively unaffected by these combinations of additives, but the result is consistent with the fact that in none of the experiments with the pure enantiomer was the expected evolution of a polar morphology evident. This may be interpreted in one of two ways: either there is some unforeseen surface chemistry limitation that makes these additives ineffective (e.g., strongly bound solvent layer) or the assumption that the nuclei have the same structure as a mature crystal is incorrect in this case. While the latter might explain the lack of an effect on the induction times, it would not explain why a polar morphology does not develop. This would favor the former surface chemistry based explanation. References (1) Jacques, J.; Collett, A.; Wilsen, S. H. Enantiomers, Racemates and Resolutions; John Wiley and Sons Inc.: New York, 1994. (2) Brock, C. P.; Schweizer, W. B.; Dunitz, J. D. J. Am. Chem Soc. 1991, 113, 9811-9820. (3) Weissbuch, I.; Lahav, M.; Leiserowitz, L. Cryst. Growth Des., 2003, 3, 125-150. (4) Lorenz, H.; Seidel-Morgenstern, A. Thermochim. Acta 2002, 382, 129-142. (5) Larsen, S.; Dediego, H. L. ACH - Models Chem. 1995, 132, 441450. (6) Patil, A. O.; Pennington, W. T.; Paul, I. C.; Curtin, D. Y.; Dykstra, C. E. J. Amer. Chem. Soc. 1987, 109, 1529-1535. (7) Wei, K-T.; Ward, D. L. Acta Crystallogr. 1977, B33, 797-800. (8) Fischer, A.; Profir, V. M. Acta Crystallogr. 2003, E59, o1113-o111. (9) Staab, E.; Addadi, L.; Leiserowitz, L.; Lahav, M. AdV. Mater. 1990, 2, 40-43. (10) Weissbuch, I.; Popovitz-Biro, R.; Leiserowitz, L.; Lahav, M. The Lock and Key Principle; Behr, J.-P., Ed.; John Wiley and Sons: Chichester, 1994, Chapter 6, pp 222-226. (11) Davey, R. J.; Blagden, N.; Righini, S.; Alison, H.; Ferrari, E. S. J. Phys. Chem. B 2002, 106, 1954-1959. (12) Weissbuch, I.; Zbaida, D.; Addadi, L.; Leiserowitz, L.; Lahav, M. J. Amer. Chem. Soc. 1987, 109, 1869-1871. (13) Addadi, L.; Berkovitch-Yellin, Z.; Weissbuch, I.; Leiserowitz, L.; Lahav, M Top. Stereochem. 1986, 16, 1.

224 Crystal Growth & Design, Vol. 7, No. 2, 2007 (14) Hodgson, D. J.; Asplund, R. O. Acta Crystallogr. 1991, C47, 19861897. (15) Chen, C. D.; Brunskill, A. P. J.; Hall, S. S.; Lalancette, R. A.; Thompson, H. W. Acta Crystallogr. 2000, C56, 1148-1151. (16) Mughal, R. K.; Pritchard, R. G.; Davey, R. J. Acta Crystallogr. 2004, E60, 1984-1986. (17) Cerius2; Accelrys Software Inc.: San Diego, USA. (18) Pino-Garcia, O.; Rasmuson, Å.C. Ind. Eng. Chem. Res. 2003 42, 4899-4909. Davey, R. J.; Garside, J. From Molecules to Crystallizers - An Introduction to Crystallisation; Oxford University Press: Oxford, 2000.

Mughal et al. (19) Profir, V. M.; Furusjo, E.; Danielsson, L. G.; Rasmuson A. C. Cryst. Growth Des. 2002, 2, 273-279. (20) Mughal, R. K. Chiral Crystallisation - Additive Induced Crystallisation of Mandelic Acid, Ph.D. Thesis, University of Manchester, UK, 2005. (21) Brittain, H. G. Anal. Profiles Drug Subst. Excipients 2002, 29, 179.

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