CRYSTAL GROWTH & DESIGN
Concomitant Crystallization of ROY on Patterned Substrates: Using a High Throughput Method to Improve the Chances of Crystallization of Different Polymorphs
2009 VOL. 9, NO. 2 1182–1185
Aniruddh Singh, In Sung Lee, and Allan S. Myerson* Department of Chemical and Biological Engineering, Illinois Institute of Technology, Chicago, Illinois 60616 ReceiVed September 19, 2008
ABSTRACT: In this study, we demonstrate how increasing the number of crystallization trials can help crystallize polymorphs which may not be obtained in a fewer number of trials due to statistical reasons. Crystallization experiments were conducted using patterned substrates of self-assembled monolayers (SAMs) with solutions of 5-methyl-2-[(2-nitrophenyl)amino]-3-thiophenecarbonitrile (known as ROY for its red, orange, and yellow crystals) in dimethylsulfoxide (DMSO). The patterned bifunctional surface was immersed and slowly withdrawn from undersaturated solutions. The solution preferentially wetted the metallic islands, and as the solvent evaporated, ROY crystals exclusively nucleated on the lyophilic metallic islands. Raman microscopy was utilized to characterize the crystalline form on each metallic island. In one of the experiments, over 10 000 islands were analyzed, and we calculated the probability of crystallizing a particular polymorph on an island. We were able to crystallize six of the seven stable polymorphs of ROY using this method, including form YT04, which to the best of our knowledge, has never been obtained from solution crystallization. Introduction The ability of a compound to exist in more than one crystal structure is known as polymorphism. Polymorphism is a result of differences in the molecular packing arrangement and/or molecular conformation.1 Different polymorphs exhibit different physical and chemical properties such as solubility, dissolution rate, density, heat capacity, melting point, and particle morphology. Different physical and chemical properties of polymorphs affect the process acceptability and bioavailability of drug substances.2 Thus, polymorph screening is an important part of the drug development process. 5-Methyl-2-[(2-nitrophenyl)amino]-3-thiophenecarbonitrile, commonly known as ROY for its red, orange, and yellow crystals, is a precursor to the antipsychotic agent olanzapine.3 ROY is currently the most polymorphic system of known structures.4 It has 10 known polymorphs, seven of them with solved structures. The existence of a high number of wellcharacterized polymorphs makes ROY a good model compound to evaluate polymorph screening methods and to study concomitant nucleation. High throughput polymorph screening methods attempt to counter the stochastic nature of crystallization from solution with thousands of parallel experiments. Large sample sizes allow a higher probability for detection of a polymorphic form having a small Pi, where Pi is the probability of yielding an i form crystal in a crystallization trial.5 Recently, our group has developed patterned substrates of self-assembled monolayers (SAMs), which consist of lyophilic gold islands surrounded by a lyophobic domain.6-8 Arrays of small solution droplets are generated using these patterned SAMs, and solvent evaporation from the droplets leads to crystallization. This method allows us to carry out a large number of independent crystallization trials with a minimal amount of material. It is also possible to achieve statistical accuracy of polymorph distribution by analysis * To whom correspondence should be addressed. Mail: Philip Danforth Armour Professor of Engineering, Department of Chemical and Biological Engineering, Illinois Institute of Technology, 10 W. 33rd St., Chicago, IL 60616. Phone: 312-567-3101. Fax: 312-567-8856. E-mail:
[email protected].
of a large number of islands. We have previously used this method to demonstrate concomitant nucleation of multiple forms under the same conditions with glycine, mefenamic acid, and sulfathiazole6-8 and have studied the effect of island size, solution concentration, and solution pH on polymorphic outcome.5-8 In this work, crystallization experiments were carried out using patterned substrates of SAMs and solutions of ROY in dimethylsulfoxide (DMSO) to study concomitant nucleation of ROY polymorphs. In one of the experiments, more than 10 000 islands were analyzed to achieve statistical accuracy of polymorph distribution while using only ∼1 mL of solution. We were able to show that increasing the number of crystallization trials could lead to nucleation of polymorphs that have escaped solution crystallization experiments using conventional methods. Experimental Methods Materials. ROY was a gift from Eli Lilly & Company and was used without further purification. Titanium (99.995%) and gold pellets (99.999%) were purchased from Kurt J. Lesker Company. 4-Mercaptobenzoic acid (4-MBA) and octadecyltrichlorosilane (OTS) were obtained from TCI America and Acros Organics, respectively. Anhydrous ethanol (200 proof) and DMSO were acquired from Decon Laboratories Inc. and Pharmco Products respectively. 3-Mercaptopropionic acid, anhydrous toluene (99.8%), and N-methylpyrrolidone (NMP) were purchased from Sigma-Aldrich. Gold Islands and SAMs Preparation. The methods used to prepare the gold islands have been described previously.6-8 Metallic gold islands were prepared by evaporation of titanium through a mesh onto glass slides, followed by evaporation of gold. The dimensions and Table 1. Initial ROY Polymorph Screening Experiments with DMSO as Solvent and 725 µm Islands experiment concentration no. of crystallization no. (M) islands time monolayer 1
0.386
1500
∼4 days
4-MBA
2
0.386
800
∼1 week
3-MPA
3 4
0.386 0.771
800 1000
∼2 weeks ∼3 weeks
3-MPA 3-MPA
10.1021/cg801055x CCC: $40.75 2009 American Chemical Society Published on Web 12/02/2008
forms obtained ON, R, Y, YN, YT04, R05/ ON, R, Y, YN,R05/ ON, R, Y, YN ON, R, Y, YN
Concomitant Crystallization of ROY on Patterned Substrates
Crystal Growth & Design, Vol. 9, No. 2, 2009 1183
Table 2. ROY Polymorph Screening Experiments with DMSO as Solvent and 500 and 250 µm islands experiment no.
island size (µm)
no. of islands
concentration (M)
5 6
500 250
3000 900
0.386 0.386
monolayer 3-MPA 3-MPA
forms obtained ON (95%), R (0.03%), Y (1.45%), YN (0.35%), YT04 (0.03%) ON (80%), R (2.92%), Y (10.67%)
Table 3. Results of ROY Polymorph Screening Experiments with DMSO as Solvent and 725 µm Islands experiment no.
island size
no. of islands
concentration (M)
monolayer
forms obtained
7
725 µm
11556
0.386
3-MPA
Y (37.73%), ON (26.59%), R (14.75%), YT04 (0.35%), YN (0.14%), ORP (0.09%)
Table 4. Estimated Probabilities of Yielding Particular ROY Polymorphs on an Island (Pi) and the Probability of Failure to Crystallize Particular Polymorphs for 100 and 1000 Trials polymorph
Pi
Y ON R YT04 YN ORP
0.3773 0.2659 0.1475 0.0035 0.0014 0.0009
P (T ) 100, P (T ) 1000, Pi range (99% confidence level) Si ) 0, Pi ) k) Si ) 0, Pi ) k) (0.3657, 0.3889) (0.2553, 0.2765) (0.1390, 0.1560) (0.0021, 0.0049) (0.0005, 0.0023) (0.0002, 0.0016)
2.6785 × 10-21 3.7629 × 10-14 1.1734 × 10 -7 0.7043 0.8693 0.9139
1.9005 × 10-206 5.6915 × 10-135 4.9475 × 10-70 0.0300 0.2464 0.4064
patterns of the islands depend on the size and shape of the holes in the mesh. Typically, square-shaped islands have been prepared. 4-MBA or 3-mercaptopropionic acid (3-MPA) was used as the thiol monolayer that self-assembled onto the gold surface, while octadecyltrichlorosilane (OTS) was used to backfill the glass-exposed substrate. The SAMs were formed on the gold islands by immersing the substrates overnight in 10 mM solutions of 4-MBA or 3-MPA in ethanol followed by immersing them into a 2 mM solution of OTS in toluene for 1 h. After removal from solution, the substrates were rinsed with toluene and ethanol and blow dried with nitrogen. This process generated patterned substrates with a bifunctional surface. Solution Preparation, Crystallization and Characterization. The patterned bifunctional surface was immersed and slowly withdrawn from undersaturated solutions of ROY in DMSO to prepare arrays of small solution droplets. The number of islands analyzed in each experiment varied from ∼800 to more than 10 000. The evaporation rate of solvent from the droplets was controlled by placing the substrate in a plastic petridish, covering it with parafilm, and puncturing holes in the parafilm (in all experiments except Experiments 1 and 6). ROY crystals nucleated and were attached to the gold islands. In Experiment 1, a desiccator was saturated with DMSO vapors by placing a beaker containing pure DMSO in it for ∼5 days. On the sixth day the beaker was removed and the patterned SAMs substrate with an array of solution droplets generated on it was placed in the desiccator. In Experiment 6, the patterned SAMs substrate was stuck on the bottom of a small glass jar. The jar was filled with a solution of ROY in DMSO and sealed. A syringe was then used to drain out the solution through a small hole in the jar cap, leaving an array of droplets on the patterned SAMs. The solution droplets were then allowed to evaporate inside the jar. Raman spectroscopy was employed to identify the polymorphic forms of ROY that had nucleated on the gold islands. The Raman spectra were obtained using a Raman Microprobe from Kaiser Optical Systems, Inc., equipped with a 450 mW external cavity stabilized diode laser as the excitation source, operating at 785 nm. The Raman spectra of the polymorphs of ROY are different from each other and have been used previously in the literature to distinguish between polymorphs. In particular, the peak positions in the nitrile stretch (2200-2250 cm-1) have been used to identify ROY polymorphs.3,4,9
Results and Discussion Polymorph Screening Experiments. ROY has 10 known polymorphs, Yellow Prism (Y), Red Prism (R), Orange Needle (ON), Orange Plate (OP), Yellow Needle (YN), Orange Red Plate (ORP), Red Plate (RPL), Yellow 2004 (Y04), Y04 transformed (YT04), and red 2005 (R05), including seven with solved structures. Form Y is the most stable and the structures of forms RPL, Y04, and R05 are yet to be solved.4 Polymorph screening experiments using patterned SAMs with island sizes 250, 500, and 725 µm were carried out as the first
part of this study. Undersaturated solutions of ROY in DMSO with concentrations 0.386 and 0.771 M were used. In our initial experiments, evaporation under ambient conditions led to a high percentage of oiling out for all island sizes. For island sizes of 250 and 500 µm almost all the islands on the substrate oiled out. The evaporation rate was slowed down by placing the substrates with droplets in a desiccator saturated with DMSO vapors or in a plastic petridish covered with parafilm with holes punctured in it. As smaller islands are more likely to result in oiling out due to the higher rate of evaporation of solvent from the smaller droplets formed on them, 725 µm islands with different evaporation rates and different concentrations were used. The results of the initial polymorph screening experiments are shown in Table 1. We were able to obtain five of the seven stable forms of ROY in our initial experiments. It is interesting to note that, to the best of our knowledge, there is no other work in the literature where form YT04 has been obtained from solution crystallization. YT04 was discovered by melt crystallization and subsequently a seed based crystallization method was developed to grow single crystals.10 This result shows how increasing the number of crystallization trials by using high throughput methods could lead to nucleation of forms that are difficult to obtain from solution crystallization using conventional methods. Slow evaporation rates and higher solution concentration led to higher percentages of form Y (most stable) being obtained. Although exact percentages of the various forms were not calculated for these initial experiments, the percentage of form Y crystals increased from ∼30% in Experiment 1 to ∼70% in Experiment 4. These results are consistent with the results of Lee et al.8 who observed that the polymorphic distribution on the islands became biased toward the stable form of sulfathiazole, as the rate of evaporation was reduced. The amount of ROY used was approximately 15 mg, 8 mg, 8 mg, and 20 mg for Experiments 1, 2, 3, and 4, respectively. Red colored plate-like crystals of ROY (R05/) were obtained in Experiments 1 and 2. Their Raman spectra showed a positive shift from the Raman spectrum of the form R05 discovered by Chen et al.4 Also, in Experiment 1, while forms Y, R, and ON crystallized on hundreds of islands, form YT04 crystallized on one island only (∼1500 islands analyzed). On the basis of these results, we carried out 10 000 crystallization trials to see if increasing the number of experiments could lead to obtaining any polymorphs that we had missed in our initial experiments due to statistical reasons. Improving Polymorph Selectivity through Kinetic Means. The existence of the polymorphs of ROY in a small free-energy range contributes to poor thermodynamic polymorph selectivity. It has been previously reported that fast crystallization favors ON and YN.11 In experiments using patterned SAMs, as the island size decreases, the size of the solution droplet also decreases leading to faster evaporation times and a higher rate of supersaturation generation. This results in higher percentages of metastable forms being obtained.6,8
1184 Crystal Growth & Design, Vol. 9, No. 2, 2009
Singh et al.
Figure 1. (a) A microscope image showing an array of gold islands with a dimension of 725 µm with ROY crystals on them and (b) different forms of ROY crystallized on 725 µm islands.
Figure 2. Raman spectra of the red plate-like crystals obtained in our experiments R05/ (red spectrum) and R05 (blue spectrum).
Initial attempts to crystallize ROY on 500 µm islands led to oiling-out. However, we were subsequently able to slow down the evaporation rate by placing the patterned SAMs in a petri dish covered with a parafilm immediately after generating the droplets. ON, R, Y, YN, and YT04 were the five polymorphs of ROY obtained on the 500 µm islands as shown in Table 2. Forms YT04 and R crystallized on only one island each out of ∼3000 islands. We calculated the percentages of the different polymorphs of ROY that had crystallized on the 500 µm islands and found that more than 95% of the crystals were ON. More than 3000 islands were analyzed to achieve statistical accuracy. This result shows how for a system such as ROY, which shows poor thermodynamic selectivity of polymorphs, polymorph selectivity can be improved through kinetic means. Solution droplets generated on the 250 µm islands had an even greater tendency to oil-out. Our efforts to slow down the evaporation rate through the methods used for 725 and 500 µm islands were unsuccessful as 100% of the droplets on the islands
Figure 3. The nitrile stretch (2200-2250 cm-1) of different Raman spectra obtained from ROY crystals on the islands (peaks from left to right: R, ORP, R05/, YN, ON, YT04, and Y).
oiled out. To solve this problem, the patterned SAMs substrate was stuck on the bottom of a small glass jar. The jar was filled with a solution of ROY in DMSO and sealed. A syringe was then used to drain out the solution through a small hole in the jar cap, leaving an array of droplets on the patterned SAMs. The solution droplets were then allowed to evaporate inside the jar. ON, R, and Y crystals were obtained on the 250 µm islands with 80% of the crystals being ON. The amount of ROY used was less than 30 and 8 mg for Experiments 5 and 6, respectively. 10000 Crystallization Trials. Because of the stochastic nature of crystallization from solution, a high number of experiments need to be carried out to achieve statistical accuracy of polymorph distribution. Polymorphs YT04 and YN (in some experiments) were obtained in very low percentages in our initial polymorph screening experiments. On the basis of these results, we carried out an experiment in which we analyzed more than
Concomitant Crystallization of ROY on Patterned Substrates
Crystal Growth & Design, Vol. 9, No. 2, 2009 1185
10 000 islands to achieve statistical accuracy and to see if increasing the number of experiments could lead to crystallization of a polymorph(s) that we had missed in our initial polymorph screening experiments. 11 556 islands were analyzed and 9616 crystals were obtained and characterized. The amount of ROY used for this experiment was approximately 115 mg. As shown in Table 3, six out of the seven stable polymorphs of ROY nucleated concomitantly under seemingly identical conditions. The existence of polymorphs within a small freeenergy range and a broad conformational distribution in solution contribute to the poor polymorph selectivity and concomitant polymorphism of ROY.11 Interestingly, form ORP, which had not been obtained in any of our previous experiments crystallized on 10 islands out of 11556. Chen et al.4 have previously reported that ORP crystals are difficult to obtain without seeds. Three out of the six forms of ROY obtained during the experiment, YT04, YN, and ORP, were obtained on less than 1% (total) of the islands. If we assume that each island is an independent trial of crystallization, the probability of obtaining a particular form in an experiment can be obtained through the formula for the binomial probability given as12
P(T,Si, k) )
T! (k)Si(1 - k)T-Si (T - Si) ! · (Si)!
(1)
where, T is the total number of islands, Si is the number of islands that produce the i form, and k is the probability of success in each trial. The parameter k is unknown; however, if Pi is the probability of yielding an i form crystal on the surface of a gold island, k ) Pi can be estimated from the experimental data acquired from a large number of samples. Using the data acquired from the 11 556 samples, we calculated the probability of yielding a particular polymorph on an island under the given set of conditions. Table 4 shows the estimated values of Pi for different polymorphs of ROY and the probabilities of failure to crystallize a particular form when the number of crystallization trials is 100 and 1000. From Table 4, we can see that for forms having high values of Pi (Y, ON, and R) even a relatively low number of crystallization trials (100) should be enough to obtain them. However, if we had carried out only 100 trials, there is a 70% chance that we would not have obtained YT04 crystals. This figure rises to 87% for YN and 91% for ORP. The extremely low value of Pi for form ORP means that even if 1000 trials are carried out, there is a 40% chance of ORP crystals not being obtained. In our initial experiments under various conditions, the number of crystallization trials was between 800 and 3000 and we failed to obtain ORP crystals in any of these experiments. The appearance of YT04 crystals in solution crystallization experiments, which to the best of our knowledge has never been observed before, could also be because we carried out a very high number of independent crystallization trials. Conclusion Multiple polymorphs of ROY nucleated on patterned metallic gold islands. We were able to obtain six out of the seven stable
polymorphs of ROY under seemingly identical conditions. Form YT04 crystals, previously prepared by melt crystallization and seeding methods, were obtained in very low percentages in our experiments from solution crystallization. This result shows how high throughput methods could lead to crystallization from solution of polymorphs that are difficult to obtain using conventional solution crystallization methods. Over 95% of the crystals obtained using 500 µm islands were ON, due to the faster rate of supersaturation generation in smaller size solution droplets formed on them. Reducing the rate of supersaturation generation and increasing concentration of the solution favored the most stable polymorph, Y. Thus, kinetic effects can be used to improve polymorph selectivity for compounds such as ROY that show poor thermodynamic selectivity of polymorphs. Form ORP crystals were obtained in very low percentages only when we carried out more than 10000 trials. The low probability of obtaining crystals of forms YN and ORP on an island means that they could be missed even if 1000 trials are carried out. In this study, approximately 204 mg of ROY was used for 19 556 independent crystallization trials. Our method enables us to carry out a high number of independent crystallization trials and can also be used to study concomitant nucleation and the effect of monolayer, solvent etc. on polymorph selectivity, while using a minimal amount of material. Acknowledgment. Financial support from the Office of Naval Research (N00173-06-1-G007) is gratefully acknowledged. We thank Dr. Lian Yu of the University of Wisconsin-Madison for providing reference Raman spectra of ROY polymorphs.
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CG801055X
