Preferential crystallization of a racemic compound via its conglomerate co-crystals

Master Thesis

Oscar F. Villamil R

August 24th 2016

Faculty of 3ME

Department: Process & Energy

Section: Intensified Reaction & Separation Systems

Graduation Committee

Ir. W. Li PDeng

Dr. ir. H.J.M Kramer

Dr. ir. H.W.Nugteren

Dr. ir. A. van der Heijden

1 Abstract

Preferential crystallization, as a powerful chiral resolution technique, is intrinsically limited to chiral molecules that crystallize as conglomerates. Many studies have been conducted on using chemical reactions to convert the target molecules, which originally form racemic compounds, into conglomerate-forming derivatives salts or by creating solvate, for the application of preferential crystallization. Up to this date conglomerate co-crystals of racemic compounds have never been applied as the intermediate for chiral resolution.

In this study, preferential crystallization of the model compound Ibuprofen (IBU), originally a racemic compound, was carried out via its conglomerate co-crystal with 2,4-bipyridine ethylene (BPE) in heptane. Suitable operation conditions were selected based on pseudo- binary phase diagram of the model compound system constructed under different IBU-BPE ratio. A unique measurement method combining polarimeter and Nuclear Magnetic Resonance (NMR) measurements was developed to identify the enantiopurity and the yield of the final product, which was a mixture of racemic IBU and IBU-BPE co-crystals, a likely result from this complex system.

With respect to the results, preferential crystallization of IBU was successfully performed by slowly cooling down a saturated solution of racemic IBU-BPE, initially at T=57.5°C, after seeding it with S-IBU/BPE crystals to T=53°C with a cooling rate of 0.3°C/min. The recovered crystalline product contained pure IBU and a mixture of R-co-crystals and S-co-crystals with a yield of 44%, with the amount of S-co-crystals recovered four times higher than the amount of R-co-crystals present in the final product. The existence of R-IBU/BPE indicates that the primary nucleation of the undesired enantiomer still took place. This can be minimized by performing the experiment at bigger scale, where samples of the mother liquor can be taken during the process in order to monitor the evolution of the enantiomeric excess enabling the defining of an optimum filtration time. The crystallization of racemic IBU along with the co- crystals lowered the purity of S-IBU. By using new ratios of IBU/BPE close to the stoichiometric co-crystal ratio and with IBU in excess, this impurity can be diminished. Additionally a comprehensive study of the Metastable Zone Width (MSZW) in a bigger volume and the exploration of mixture of solvents can improve the definition of the final temperature in order to avoid the presence of racemic IBU and R-co-crystals in the crystals produced.

Key words: conglomerate co-crystal, preferential crystallization, chiral resolution

2 3 Acknowledgements

Firstly thank you to the Process and Energy Department of TU Delft for giving me the scholarship that allowed me to pursue my master of science in Sustainable Process and Energy Technologies.

Thanks to the Intensified Reactions and Separation (IRS) group for its support during the development of my project, especially to my daily supervisor, Weiwei Li, for guiding me during the experiments and for having a positive and enthusiastic attitude regarding my work, making always a space on his agenda for my project, even when he was out of the country. I would also like to thank Marloes Reus for dedicating me time, explaining me good practice in the laboratory and for organizing the group meetings and sharing her knowledge with nice tutorials. I want to thank Debby den Besten (DDB) for motivating me with her hard work attitude, for explaining me the equipment and procedures and for the countless technical discussions. Herman Kramer, my supervisor, I want to thank you for introducing me into the crystallization world initially through your lectures during the first year and afterwards with your advices, guidelines and feedback during the execution of my project, thank you for your positive attitude and for being always willing to help me when I needed it. Stephen Eustace, thank you for all your help with the NMR measurements. Moj and Priya, thank you for all the emotional support and for making the office a nice place to be. I would also like to thank Henk Nugteren and Antoine van der Heijden for being part of my thesis committee.

I also want to thank Silvia for bringing me all the joy from Colombia especially in the most hectic period of my project. Dennis, thank you for encouraging me and for being literally always there in the long days of work. Yeyo, thank you because without all your help I would have never been able to succeed during my master.

Oscar Villamil

4 Contents

Abstract ...... 2 Acknowledgements ...... 4 1. Abbreviations ...... 9 2. Introduction...... 10 3. Theory...... 12 3.1 Chirality...... 12 3.2 Crystallization ...... 13 3.2.1 Nucleation ...... 14 3.2.2 Metastable zone (MSZ) ...... 16 3.2.3 Crystal growth ...... 18 3.3 Preferential crystallization ...... 18 3.3.1 Enantiopurification from a conglomerate forming system...... 21 3.4 co-crystals...... 23 3.5 Screening of conglomerates...... 25 3.5.1 Specific Optical rotation and its use for the determination of the enantiomeric excess 27 4. Materials and methods ...... 28 4.1 Materials...... 28 4.2 Methods ...... 28 4.2.1 Solubility determination...... 28 4.3 Seeds synthesis...... 29 4.3.1 XRPD patterns...... 29 4.4 Purity measurement...... 29 4.4.1 Determination of the ratio IBU/BPE with the NMR ...... 30 4.4.2 Specific Optical rotation and its use for the determination of the enantiomeric excess 30 4.5 Preferential crystallization ...... 31 5. Results and discussion...... 33 5.1 Initial system...... 33 5.2 Influence of the ratio IBU/BPE in the system...... 36 5.3 New solvent selection ...... 38 5.4 Phase diagram of IBU/BPE...... 40 5.5 MSZW (Metastable Zone Width) definition ...... 42

5 5.6 Calibration lines for specific optical rotation measurement...... 44 5.7 The preferential crystallization in different batches...... 45 6. Conclusions...... 55 7. Recommendations...... 56 8. Bibliography...... 59 9. Appendix...... 62 9.1 Specifications of the solubility measurement experiments...... 62 9.2 Relaxation time for compounds in NMR tests ...... 63 9.3 Single crystals of seeds...... 67 9.4 Preferential crystallization ...... 69 9.5 XRPD Patterns IBU/BPE in different ratios in Heptane ...... 71

Figures

Figure 1 Evolution of the PC ...... 10 Figure 2 Routes to access pure enantiomers ...... 12 Figure 3 Representation of the supersaturation creation. Left, cooling crystallization without solvent lost. Right. Evaporative crystallization at constant temperature...... 14 Figure 4 Types of nucleation ...... 14 Figure 5 Primary nucleation. (A) Homogeneous nucleation. (B) Heterogeneous nucleation...... 15 Figure 6 Secondary nucleation classifications as applicable to an industrial crystallizer ...... 16 Figure 7 Representation of the MSZ. The dotted line represents the limit of the MSZW ...... 17 Figure 8 Representation of polythermal method for the MSZW determination...... 17 Figure 9 Block diagram of the conventional PC (*) the enantiomer used as seed depends on the cycle and changes its sign after each crops recovery). The solution is stirred continuously...... 19 Figure 10 Block diagram of the AS3PC method (*) the enantiomer used as seed depends on the cycle and changes its sign after each crops recovery). The solution is stirred continuously...... 20 Figure 11 Ternary phase diagram observed for a conglomerate forming system...... 21

Figure 12 Ternary phase diagrams observed for a conglomerate-forming system at T1 (Blue) and T2 (Red) ...... 22 Figure 13 Principle of crystallization by entrainment ...... 22 Figure 14 Schematic representation of three possible arrangements of chiral molecules in a crystal structure...... 23 Figure 15 Schematic representation of pharmaceutically relevant multicomponent crystalline solids. (a) Solvate; (b) co-crystal; (c) salt. API, active pharmaceutical ingredient...... 24 Figure 16 Representation of the formation of a conglomerate in the case of the IBU-BPE system..... 24 Figure 17 Screening of conglomerates techniques ...... 25 Figure 18 XRPD diffractograms of the ethanolamine mandelates: racemic mixture and pure enantiomer...... 25

6 Figure 19 PXRD diffractograms for (a) pure indomethacin, (b) pure saccharin, and (c) IND-SAC co- crystals produced using the solvent evaporation method. The arrows indicate the characteristic peaks of IND-SAC co-crystals...... 26 Figure 20 Binary phase diagrams (a) conglomerate (b) racemic compound...... 26 Figure 21 Purity measurement method ...... 31 Figure 22 Phase diagram screens of ibuprofen in hexane ...... 33 Figure 23 Solubility of S-IBU and RS-IBU in Hexane ...... 33 Figure 24 Views of ((±)-ibu).(BPE) highlighting: (a) two component assembly and (b) extended packing ...... 34 Figure 25 the non-coformer and coformer phase diagram for RS-IBU...... 35 Figure 26 Phase diagram of IBU/BPE in Hexane concentration c =180[mg/ml] ...... 35 Figure 27 Influence of BPE on the saturation temperature of the system (a) Racemic, (b) S- Enantiopure. The solid lines are guide to the eyes...... 36 Figure 28 XRPD patterns for the system IBU/BPE in different ratios of IBU/BPE [mg/mg] ...... 37 Figure 29 (a) Solubility of RS-IBU in different solvents ...... 39 Figure 30 Influence of BPE on the system saturation temperature in Heptane. (a) Comparison in Heptane and Hexane. (b) Different cycles for Heptane ...... 39 Figure 31 (a) Binary phase diagram IBU/BPE in Heptane, with a ratio of 180/40 [mg/mg]. (b) no- coformer and coformer phase diagram ...... 40 Figure 32 XRPD patterns IBU/BPE in Heptane...... 41 Figure 33 Saturation temperature and cloud point for IBU/BPE in Heptane. Figure in the left shows the saturation temperature when the solution is completely clear. Pictures in the right show the appearance of the first crystals, indicating the cloud point...... 42 Figure 34 Ratio IBU/BPE vs. Filtration temperature...... 43 Figure 35 Calibration line for optical rotation of IBU based on e.e in Ethanol. c=6 mg/100cm3 and T=20°C ...... 44 Figure 36 Calibration line for optical rotation of IBU/BPE 1:1 molar ratio based on e.e in Ethanol. c=11 mg/100cm3 and T=20°C...... 44 Figure 37 Calibration line for optical rotation of IBU/BPE in different ratios. Solvent: Ethanol, c=11 mg/100cm3 and T=20°C...... 45 Figure 38 XRPD patterns of co-crystals of IBU/BPE...... 46 Figure 39 Steps of the PC...... 47 Figure 40 PC monitoring Batch 1. On top, the temperature (black line) is monitored together with the transmissivity (blue line) of the solution. Below, pictures at the moment of total dissolution, seeding, stabilization of the seeds and crops formation...... 47 Figure 41 spectrum of a mixture of crops of IBU/BPE...... 48 Figure 42 Distribution of compounds inside the crops ...... 49 Figure 43 XRPD PC Batch #1 ratio 180/40 [mg/mg] ...... 50 Figure 44 Mass distribution of IBU in the crops Batch 1 (a) Distribution of the total amount of IBU in either co-crystal or pure racemic form. (b) Distribution of the enantiomers in the co-crystals and racemic form. (c) Discrimination of the S enantiomer recovered...... 51 Figure 45 Solubility of mixtures in the 1:1 molar ratio IBU/BPE in Heptane ...... 52 Figure 46 Mass distribution of IBU in the crops Batch 1 Initial solution in the 1:1 mole ratio IBU/BPE (a) Distribution of the total amount of IBU in either co-crystal or pure racemic form. (b) Distribution

7 of the enantiomers in the co-crystals and racemic form. (c) Discrimination of the S enantiomer recovered...... 53 Figure 47 XRPD PC Batch #1 ratio 20/18 [mg/mg] ...... 53 Figure 48 Left. Initial solution IBU/BPE [20/18] [mg/mg]. Right, Clear initial solution for the system IBU/BPE 180/40 [mg/mg]...... 54 Figure 49 Influence of the final temperature on the solution e.e. during the crystallization (AS3PC mode) ...... 56

Figure A. 1 Relaxation time for IBU ...... 63 Figure A. 2 Relaxation time for BPE...... 63 Figure A. 3 NMR spectrum for IBU ...... 64 Figure A. 4 NMR spectrum for BPE...... 65 Figure A. 5 Experiment for accuracy of NMR in application for IBU/BPE ratio measurement ...... 65 Figure A. 6 crystallization during the NMR test...... 66 Figure A. 7 co-crystals of S-IBU/BPE...... 67 Figure A. 8 Microscopic view of the co-crystals of S-IBU/BPE...... 67 Figure A. 9 XRPD analysis of the single crystal obtained for one batch of seeds production ...... 68 Figure A. 10 Seeds grinded...... 69 Figure A. 11 Illustration of the seeding mechanism...... 70 Figure A. 12 XRPD Patterns IBU/BPE in different ratios in Heptane ...... 71

Tables

Table 1 Experimental conditions for the PC system 180/40 [mg/mg] IBU/BPE...... 32 Table 2 Solvent substitution...... 38 Table 3 Yield measurement in the different batches...... 49 Table 4 Experimental conditions for the PC system 20/18 [mg/mg] IBU/BPE...... 52 Table 5 Solubility measured using Crystal 16 ™ ...... 62

Equations

Equation 1 Enantiomeric excess...... 27 Equation 2 S-Enantiomer present in crops...... 27

8 1. Abbreviations

PC: Preferential Crystallization

S: Supersaturation c: Concentration

MSZW: Metastable Zone Width

AS3PC: Auto-Seeded Polythermic Programmed Preferential Crystallization

ASPreCISE: Auto-Seeded Preferential Crystallization Induced by Solvent Evaporation

SOAT: Second-Order Asymmetric Transformation

XRPD: X-Ray Powder Diffraction

NMR: Nuclear Magnetic Resonance

IBU: Ibuprofen

BPE: 2,4-bipyridine ethylene e.e: Enantiomeric excess

HPLC: High Performance Liquid Chromatography

IR: Infra-red

9 2. Introduction

Chiral resolution or enantiomeric separation is vitally important to pharmaceutical and food industries, where enantiopure products are required. Many techniques have been developed in response to the increasing demand from the industry. Among them, Preferential Crystallization (PC) was mentioned for the first time in 1866 by Gernez[1], but it was around 68 years later when it was started to be considered in industrial applications after the resolution by entrainment of Histidine Mononohydrochloride performed by Duschinsky (Figure 1). Recent studies have developed techniques such as the Auto Seeded Programmed Polythermic Preferential Crystallization [2] that continue contributing to the understanding of this powerful technique.

Resolution by preferential crystallization is only possible when the racemic mixture forms a conglomerate. This means that the two enantiomers crystallize as distinct enantiopure solid phases when both enantiomers are present in the mother liquor[3]. However, conglomerates present in nature represent just 5 to 10% of the racemic species [1] which seems to be a limitation for the application of PC.

Figure 1 Evolution of the PC

In order to broaden the spectrum of the application of the PC, many studies were conducted on the use of chemical reactions to convert the target molecules, which originally form racemic compounds, into conglomerate-forming derivative salts or by the formation of solvates. Up to this date, conglomerate co-crystals of racemic compounds have never been applied as the intermediate for chiral resolution.

In recent years co-crystal formation has emerged as a viable strategy towards improving the solubility and bioavailability of poorly soluble drugs[4] and, as an additional consequence of the co-crystallization process, it has been proved the possibility of forming conglomerate systems. [5][6] Ibuprofen (2-(4-isobutyl-phenyl) propionic acid) is a common analgesic pharmaceutical [7] that crystallizes as a racemic compound [8]. In the work of E. Elacqua et al. [6] it was reported the formation of a stable conglomerate co-crystal, using 2,4-bipyridine ethylene (BPE) as a coformer. This system was chosen as the model compound for the application of PC.

10 During this research, the study of the application of PC on the model conglomerate co- crystal system is performed, which composes three parts: 1) The identification of suitable operation conditions for PC, based on pseudo-binary phase diagrams, including solvent, composition and temperature profile etc. 2) The development of a measurement method, using Nuclear Magnetic Resonance (NMR) and Polarimeter, for the final product characterization, e.g. the co-crystal (enantio-)purity and yield and 3) The chiral resolution of model compound ibuprofen via its conglomerate co-crystals by preferential crystallization technique.

11 3. Theory

3.1 Chirality

The phenomenon attributed to the fact that an object is not superimposable onto its mirror image is known as chirality [11]. A chiral object may be present in two enantiomorphic forms [1] or enantiomers. The two enantiomers have identical chemical and physical properties, except for the specific optical rotation, that is equal in value, but opposite in sign. In a racemic mixture, both enantiomers are present in an equimolar ratio.

In industrial applications chiral substances can be commercialized in its racemic form or enantiopure[12]. International entities such as the FDA (US Food and Drug Administration) leaves the decision of producing racemic or enantiopure medicines to the developers, but always justifying the choice between enantiopure or racemic form[13].

When a medicine is supplied in therapy as a racemic compound, one of the enantiomers may display different chemical and pharmacologic behaviour than the other enantiomer [13] , because the human body is itself chiral. In some cases, both enantiomers have the same activity, or one is active while the other is inactive, when the activity differs, some side effects can be induced. As an example is the case of the Ethambutol, where the S- enantiomer is used in tuberculosis treatment and the R-enantiomer causes blindness[14]. Therefore, in some cases, obtaining an enantiopure compound is mandatory.

Noordium [15] identified three routes for the pure enantiomers production or chiral resolution.

Figure 2 Routes to access pure enantiomers1

Further information regarding these techniques can be found in the work of J.Jacques[1]. This research is mainly focused in one particular application of the enantiomeric resolution, named Preferential Crystallization (PC) or resolution by Entrainment. Hence, in order to understand the PC it is necessary to review the basic concepts involved in a crystallization process.

1 Reprinted from [15] 12 3.2 Crystallization

Crystallization is a separation technique that involves the creation of a solid phase [16], where mass is transferred from the liquid phase to the crystalline phase. It is present in almost all processes developed in the chemical industry, where is essentially used for production, purification or recovery of a solid material[17]. The applicability of crystallization also includes the production of medicines, especially in the interest of producing highly pure compounds with specific properties[4].

When at one specific temperature the solution is in thermodynamic equilibrium with the solid phase, it is said that the solution is saturated [17]. In order to initiate a crystallization process, the solution should be shifted to a supersaturated state by means of an external action. Supersaturation implies that the concentration of the dissolved compound is higher than the concentration at the solid liquid equilibrium or saturation concentration. Depending on the method to generate the supersaturation the crystallization method can be classified as:

 Crystallization from solution

• Evaporative crystallization

• Cooling crystallization

• Anti-solvent crystallization

• Precipitation

 Melt crystallization

Cooling and evaporative crystallization are the methods employed during this research. These processes are represented in Figure 3. In the case of evaporative crystallization, the concentration is increased by evaporation of the solvent. For cooling crystallization, the solubility decreases by decreasing the temperature. The supersaturation S induced can be quantified as the ratio of the final concentration c with respect to the concentration in 2 equilibrium ceq

2 The concentration in equilibrium is the value that lies in the solid line. 13 Figure 3 Representation of the supersaturation creation. Left, cooling crystallization without solvent lost. Right. Evaporative crystallization at constant temperature

In the Figure 3, when a solution is on the blue line, it is saturated and cannot hold anymore solute. On one hand, below the line, the solution is unsaturated and can hold more solute. On the other hand, above the line, it is in an unstable condition named supersaturation, holding more solute than it should.

3.2.1 Nucleation

Reaching a supersaturated state is not enough for the initiation of crystallization from a solution, it is necessary to have centres of crystallization or nuclei before the crystals can develop. [17] The classification of the nucleation is presented in the Figure 4.

Figure 4 Types of nucleation3

3 Refer to [1] 14 When the spontaneous formation of crystals take place in a solution that does not contain crystalline matter of the compound to crystalize, the formation is attributed to primary nucleation. If the primary nucleation takes place in a spontaneous fashion, it is named Homogenous. On the other hand, if the presence of solid impurities at the beginning of the process enhances the nucleation, it is named Heterogeneous.

Figure 5 Primary nucleation. (A) Homogeneous nucleation. (B) Heterogeneous nucleation

In the secondary nucleation, it is proposed in the literature that the nuclei originate either from a parental crystal or are formed from the solution layer adjacent to the surface[18]. Usually the fragmentation of the parental crystal is due to attrition by means of an impeller or collisions with the wall of the reactor. The stages of the secondary nucleation can be dived as:

• Generation of attrition fragments

• Removal of fragments from parent crystal

• Survival and growth of the fragments

Figure 6 shows the classification of secondary nucleation proposed by S. G. Agrawal & A. H. J. Paterson [18]. Two major phenomena are identified as responsible of the secondary nucleation, collision, and fluid shear. These mechanisms can produce secondary nuclei by either attrition or solute layer removal.

15 Figure 6 Secondary nucleation classifications as applicable to an industrial crystallizer4

3.2.2 Metastable zone (MSZ)

“The MSZ is an area between concentration of solubility (It is a thermodynamic equilibrium between a solid phase and a liquid phase) and concentration of detection the first nuclei (supersolubility)”[19]

Herein, it is important to define the next concepts, before analysing the methods for the MSZ characterization:

Clear point: Upon heating, there is a temperature where a suspension turns into a clear solution

Cloud point: Upon cooling, a solution there is a temperature that crystals will be detected

Metastable Zone Width: The difference between the saturation temperature (Clear point) and cloud point is the

In the Figure 7, the solubility curve (solid blue line) is well defined and determined via experiments, indicating the maximum amount of solid contained in a saturated solution[19]. The metastable zone width is defined as the zone between the saturation temperature (clear points) and the metastable limit curve (cloud points).

4 Reprinted from [18] 16 Figure 7 Representation of the MSZ. The dotted line represents the limit of the MSZW

The MSZW can be determined via Isothermal and polythermal methods[19].

Isothermal methods

• Attaining the supersaturation as fast as possible

• measurement of the time lag for the appearance of the first detectable nuclei

Polythermal methods (Figure 8)

• Start from a saturated solution at the point i

• Using a constant cooling rate, the solution is cooled down from a temperature slightly higher than the saturation temperature (Point 1) until the first crystals are detected in solution (Point 2)

Figure 8 Representation of polythermal method for the MSZW determination

17 The MSZW is not a fixed quantity, and its value can depend on different variables such as[20][19]:

 Cooling rate  Impurities  Solution thermal history  Fluid dynamics  Volume of solution

3.2.3 Crystal growth

When a stable centre of crystallization is formed, it can start to grow and form a visible crystal[17]. This growth can occur in a smooth way, layer growth or rough growth. Rough growth is where growth units attach anywhere to the rough crystal surface.

3.3 Preferential crystallization

The Pasteurian resolution is the most classical and well known method for the use of crystallization in enantiomeric purification [3], for which a chiral resolving agent is used to obtain the crystallization of diastereoisomers [11]. Nevertheless, it was Gernez in 1866, one of the students of Pasteur, whom observed the early indication of the preferential crystallization process as an alternative for the enantiomeric resolution. Gernez observed that if a saturated solution of one enantiomer is seeded by the same enantiomer, enantiomerically pure crystals are formed; on the other hand, if the solution was seeded with the opposite enantiomer, no crystallization was observed.

In 1934, Duschinsky performed the first efficient resolution via PC, applying this technique to the purification of histidine monohydrochloride [1]. The main advantages of its use with respect to the Pasteurian method is that no resolving agent is needed [2]. Although, the application of the PC is limited to conglomerate5 systems.

Based on the description of the PC developed by J.Jacques [1] , the main steps can be described as:

The racemic mixture with an excess of the desired enantiomer is heated up and stirred until total dissolution is obtained. Subsequently it is cooled down and seeded with the same enantiomer in the initial excess, and then the stirred solution is allowed to crystallize for a certain period. “Selective growth of one enantiomorphous crystal phase is achieved because

5 A conglomerate is the physical mixture of the two enantiomers [44] 18 the energy barrier for the spontaneous nucleation of the nonseeded enantiomer is higher than the energy required to grow the crystals present from the seed”[3].

Before the primary nucleation of the non-seeded enantiomer starts, the solution is filtered and the crops are recovered. The crops are defined as the crystals filtered from the solution. Because of the subtraction of the enantiopure crops, the mother liquid is enriched in the non-seeded enantiomer. Subsequently, the same mass of crops recovered is added in form of racemic mixture. Now the process can start again, but in this case, the opposite enantiomer will be crystallized.

The procedure is represented in the Figure 9, after each cycle, the sign of the enantiomer recovered changes.

Figure 9 Block diagram of the conventional PC (*) the enantiomer used as seed depends on the cycle and changes its sign after each crops recovery). The solution is stirred continuously.

Additional variations and improvements in the PC have been introduced; some of the most popular are:

AS3PC (Auto-Seeded Polythermic Programmed Preferential Crystallization): proposed by Aubin et al. [2] includes the addition of a controlled cooling step to a saturated solution.

19 Figure 10 Block diagram of the AS3PC method (*) the enantiomer used as seed depends on the cycle and changes its sign after each crops recovery). The solution is stirred continuously.

ASPreCISE (Auto-Seeded Preferential Crystallization Induced by Solvent Evaporation)[3]: in this case, controlled solvent loss is performed, inducing supersaturation, subsequently promoting the crystallization.

SOAT (Second-Order Asymmetric Transformation) [3] :This can be applied when the compound can be easily racemized in solution. Thus, the depletion of one of the enantiomers during the process is rapidly equilibrated and the solution remains in its racemic form.

Successful examples of the application of these techniques include the resolution of Ethanolamine salt of Mandelic Acid [2], Aspartic acid and Modafine Acid [3]

In 1882, Jungfleish described the supersaturation as the key factor [1] for the PC. He identified that if the initial solution is not strongly supersaturated, the yield of the process will be low, but the nucleation of the undesired enantiomer is easier to control. Finding high yields implies strong supersaturation, but the process should be operated inside the limit where the primary nucleation of the nonseeded enantiomer does not take place.

20 3.3.1 Enantiopurification from a conglomerate forming system

The preferential crystallization process based on the AS3PC method can be represented in a ternary phase diagram, where two of the components are the enantiomers and the third one corresponds to the solvent.[21]

Starting from a mixture of enantiomers with composition M with an enantiomeric excess (e.e) in S, the exact amount of solvent needed in order to reach the point K (Located in the tie line that divides the regions 1 and 2 ) situated in the tie line is added.(Figure 11)

Figure 11 Ternary phase diagram observed for a conglomerate forming system6

The suspension is heated up to a temperature slightly higher than T1, where just S enantiomer is present in the solid phase. (Figure 12)

6 Reprinted from [11] 21 7 Figure 12 Ternary phase diagrams observed for a conglomerate-forming system at T1 (Blue) and T2 (Red)

The solution is then cooled down to the saturation T0. Under these conditions, the point K lies on the three-phase domain and as long as the crystallization is stereoselective (occurs for the desired enantiomer), the liquid phase is shifted from the point I1 to the point Z0. This point Z0, is located on the metastable zone of S at T0, thus the liquid phase is enriched in R, resulting on entrainment. (Figure 13)

Figure 13 Principle of crystallization by entrainment8

The mentioned above corresponds to one cycle of the PC, if a new amount of racemic compound is added, the process can start again, obtaining the opposite enantiomer as a final product. For more details about the representation of this in the phase diagram, refer to the work of A. Collet. [22]

7 Reprinted from [11] 8 Reprinted from [11] 22 So far, the PC has not been performed in conglomerate co-crystals. In order to study the suitable conditions for this operation in this particular case, it is necessary to understand the principles of the co-crystallization and the conformation of a conglomerate system in this type of compounds.

3.4co-crystals

Co-crystals are defined as “crystalline materials in which two or more components are neutral molecules and solids at room temperature”[23]. The preparation of co-crystals is mainly achieved by solution crystallization; including solvent evaporation, temperature gradient, antisolvent addition etc.[24]. Preparation of co-crystals of racemic IBU and 2- aminopyrimidine by means of evaporative crystallization of a stoichiometric solution9, has been reported by Solhe F. Alshahateet[25]

As an alternative , the Mechanochemistry can be used in the co-crystal synthesis, where the cocrystallization is mainly produced in the solid-state and/or using liquid assisted grinding[6]. In this research the co-crystallization is going to be focused on crystallization process.

Most application of co-crystals takes place in the pharmaceutical industry, were the cocrystallization offers an alternative to improve the physical properties, solubility, and consequent bioavailability of poor water-soluble drugs [6] [4]. One additional application that can be derived from the cocrystallization is the transformation of a racemic compound in a conglomerate system, creating the possibility of resolution by PC. Is on this application where the main aim of this research relies.

Racemic mixtures can be present in the solid state in three different ways: as racemic compounds, racemic conglomerates and solid solutions[15]

Figure 14 Schematic representation of three possible arrangements of chiral molecules in a crystal structure10.

From the previous classification, only racemic conglomerates are suitable for a separation of the enantiomers into enantiopure solid phases [15] , due to the fact that the enantiomers can crystallize separately. Nevertheless, conglomerates present in nature represent only 5 to

9 Stoichiometric in the co-crystal formation 10 Reprinted from [15] 23 10% of the racemic species[1], which seems to be a limitation for the use of crystallization to obtain substances on its enantiopure form.

In order to broaden the spectrum of the application of resolution based on crystallization, many studies have been conducted on using chemical reactions to convert the target molecules, which originally form racemic compounds, into conglomerate-forming derivatives salts or solvate. This is not always possible and it is here where the formation of co-crystals that are substances based on noncovalent interactions such as hydrogen bonding, π-π stacking[15] can offer an alternative tool for the conformation of a conglomerate system.

Figure 15 Schematic representation of pharmaceutically relevant multicomponent crystalline solids. (a) Solvate; (b) co- crystal; (c) salt. API, active pharmaceutical ingredient.11

Ibuprofen (2-(4-isobutyl-phenyl) propionic acid) is a common analgesic pharmaceutical [7] that crystallizes as a racemic compound [8]. In the work of E. Elacqua et al. [6] it was reported the formation of a stable conglomerate co-crystal, using 2,4-bipyridine ethylene (BPE) as a coformer.

Figure 16 Representation of the formation of a conglomerate in the case of the IBU-BPE system12

The outcome of co-crystallization of a chiral compound is not always a conglomerate system[6], thus the identification of techniques that can screen for the presence of conglomerate should be introduced.

11 Reprinted from[45] 12 The 3D figures were taken from [6] 24 3.5Screening of conglomerates

In the work of V. Dupray et al. [11] different techniques are proposed for the screening of conglomerates. The Figure 17 summarizes the most commonly used. In the case of the comparison of patterns, the pattern of the enantiopure and racemic mixture should look alike in the case of conglomerate formation.

Figure 17 Screening of conglomerates techniques

Using XRPD (X-Ray Powder Diffraction) has been proven to be an easy non-destructive test that can give a fast indication of the formation of a conglomerate, just by the comparison of the patterns of the enantiopure and racemic mixture (Figure 18).

Figure 18 XRPD diffractograms of the ethanolamine mandelates: racemic mixture and pure enantiomer13

13 Reprinted from [2]. Here both patters look the same, indicating the formation of a conglomerate. 25 The use of XRPD cannot only give information of the formation of a conglomerate system, but also indicate the formation of a co-crystal. When the diffractogram of the “co-crystal” is compared with the patterns of the pure constituent substances and new peaks appear in positions where the pure substances have not resulted in a response, the new peaks indicate the formation of a new crystalline structure (Figure 19).

Figure 19 PXRD diffractograms for (a) pure indomethacin, (b) pure saccharin, and (c) IND-SAC co-crystals produced using the solvent evaporation method. The arrows indicate the characteristic peaks of IND-SAC co-crystals.14

One additional indication for a conglomerate besides the XRPD is the comparison of the binary phase diagram of the racemic mixture and the “conglomerate” (Figure 20). A conglomerate melts as if it were a pure substance and thus fits the definition of a eutectic[1] . The Figure 20 represents the most common type of phase diagram of enantiomers forming a racemic compound. This shape can present variations, depending if the racemic compound melting point is greater, lower or equal to that of the enantiomers [1]. An extensive list of examples of these phase diagrams can be found in the work of J. Sanger [26].

Figure 20 Binary phase diagrams (a) conglomerate (b) racemic compound.15

In the case of the IBU, it has been found that the racemic compound has a greater melting point than the enantiomers.[9][8]

14 Reprinted from [35] 15 Reprinted from [9]

26 3.5.1 Specific Optical rotation and its use for the determination of the enantiomeric excess

Optical activity is one of the manifestation of chirality. The fact that two enantiomers of a given compound have rotatory powers of equal absolute value but of opposite sign[1], can be used as a tool in order to determine the e.e of a mixture of crystals.

Using the Biot’s law, the optical rotation of a substance can be related with its e.e by means of its deviation from the enantiopure value.

 c l     T 100 c=Concentration [g/100cm3]

     T =Specific Rotation 3  dm g  cm 

I= Optical path length (Sample cell length [dm]) T=Temperature Λ=Wavelength

The e.e is defined as[27], where R and S represent the mass of each enantiomer .

RS  e. e  100  RS 

Equation 1 Enantiomeric excess

16 With IBUcrops as the total amount of IBU present in the crops, it is possible to express the amount of S-enantiomer as:

100 e . e     IBU crops 100 e . e S    100 e . e  1   100 e . e 

Equation 2 S-Enantiomer present in crops

16 This can be obtained straight after the NMR measurement 27 4. Materials and methods

4.1 Materials

Racemic Ibuprofen [[2-(4-isobutyl-phenyl) propionic acid] and BPE [trans-1-(2-pyridyl)-2-(4- pyridyl)-ethylene] were obtained from Santa Cruz Biotechnology. Enantiopure S-Ibuprofen (99% purity), Hexane (95% purity), Heptane (99% purity) and Ethanol absolute (99.5% purity) were supplied by Sigma Aldrich. For the NMR assays, Methanol D4 (water <0.03%) was purchased from Eurosi-top and Maleic Acid (99%) was obtained from Aldrich-Chemie. All chemicals were used without further purification.

4.2 Methods

4.2.1 Solubility determination

For the solubility measurement, the multireactor Crystal 16 ™ of Technobis was used. The samples were placed in Standard 1.8[ml] flat-bottomed HPLC vials and magnetic stirrers were employed for the solution mixing. The procedure as described in the work of Vellema et al.[28] is followed.

The analysis of the data obtained out of the measurements, allows the identification of the:

Clear point: Upon heating, there is a temperature where a suspension turns into a clear solution

Cloud point: Upon cooling, a solution there is a temperature that crystals will be detected

Metastable Zone Width: The difference between the saturation temperature (Clear point) and cloud point is the

As an additional tool, the Crystalline ™ of Avantium was used for the MSZW measurements. The equipment combines the same principle than the Crystal 16 ™ with real time particle viewers. It allows the visualization of the complete crystallization process through the temperature cycle, recording pictures based on the timing set, together with information about the particle size.

28 For the case of the binary phase diagram construction, a fix concentration was used. In this case 180[mg/ml] of IBU and 40[mg/ml] of BPE. The amount of BPE is maintained constant in all the samples. On the other hand, the first vial contains pure RS-IBU, and from the second onwards; the amount of RS-IBU was decreased and replaced by pure S-IBU. Finally, in the last vial, just 100% S-IBU is present in combination with the BPE.

One millilitre of solvent was added on each vial, and the saturation temperatures were measured based on the procedure described in 4.2.1

4.3Seeds synthesis

The seeds are co-crystals consisting of pure S-IBU and BPE. For the production of the seeds, solution crystallization can be used [4]. A 1:1 molar ratio solution of IBU/BPE with a concentration of 3 [mol/ml] in Ethanol is evaporated at a constant temperature of 50°C during 3 days. Afterwards, the slurry is left at room conditions for one day in order to evaporate the remaining solvent. These procedure has been described by Elacqua [6] for the production of IBU-BPE co-crystals.

4.3.1 XRPD patterns

The XRPD patterns were obtained using a Bruker AXS-D5005 diffractometer with a Bragg- Brentano focussing geometry and a CuKa1 radiation (λ = 1.54056 Å). The diffractograms are measured in the range 2θ [4°, 50°] with an increment of 0.006047°. Data interpretation is performed using the software DIFFRAC.EVA provided by Bruker. As a reference for the co- crystal formation, the XRPD patterns obtained are compared with a single crystal diffractograms supplied by the University of Nijmegen and the patters of the pure compounds.

4.4Purity measurement

For the application of the PC, it is required to have a conglomerate system. When the molecule to resolve contains an acidic or a basic structure, the formation of a salt as an intermediate compound is technique commonly used [29]. In this case, the ratio of the compounds in the crops is always the same and the analyses of the e.e via optical rotation can be executed. In the case of the PC for co-crystals, the initial solution contains an excess of IBU, which can contribute to the presence of pure racemic IBU in the crops, therefore the measurement of this ratio it is necessary before the e.e is evaluated.

29 4.4.1 Determination of the ratio IBU/BPE with the NMR

In order to obtain the ratio of IBU/BPE present in the crops, assay tests using the Agilent Direct Drive 400 MHz NMR with a 5 mm OneNMR™ Probe, based on Proton Magnetic Resonance Spectrometry [30] was performed. The samples preparation and the test execution are based the NMR Spectroscopy, User Guide[31]. The interpretation and analysis of the data takes as a reference the Quantitative NMR spectroscopy Relative method proposed by Bernd W.K Diehl et al [10].

Maleic acid is used as a reference in a concentration of 30 [mg/mL]. The crops are added in a concentration of 30[mg/ml], using Methanol D4[32] as a solvent.

The spectrum of the pure components was analysed and the relaxation time was defined as 40[s], based on the results depicted in Figure A. 1 and Figure A. 2. The number of scans was defined as 16 with a pulse angle of 45 degrees. Additionally, taking as a reference the spectrum of the pure compounds, the characteristic peaks for each one were defined (Figure A. 3 and Figure A. 4).

4.4.2 Specific Optical rotation and its use for the determination of the enantiomeric excess

The optical rotation of the pure compounds and crops is determined using the Polarimeter MCP 500 of Anton Paar. The protocol followed is the one defined for solid substances in the procedure of the vendor[33]. Samples with concentrations of minimum 6 [mg/100 cm3] are used for the different measurements. Ethanol absolute was chosen as solvent; the optical path length was defined as I= 1[dm], the measurement was recorded at a temperature of T= 20°C, using a wave length of λ= 589 [nm]

The method proposed for the purity measurements is summarized in the Figure 21.

30 Figure 21 Purity measurement method

4.5Preferential crystallization

The procedure followed for the PC performance, is in principle similar to the AS3PC, with the adaptation that the initial temperature corresponds to the saturation temperature of the system and not to a higher value.

Using the Crystalline™, an initial solution of 3[ml] of Heptane with concentrations of 180[mg/ml] of RS-IBU and 40[mg/mL] was heated up and stabilized to Ts= 57.5 °C, which corresponds to its saturation temperature17. The temperature and transmissivity were monitored with the camera, pictures were taken each 30 seconds and the total dissolution before seeding was verified. (Figure 40)

With the saturation temperature fixed and the solution stabilized, the seeds were added. For this, the vial was removed from the reactor and the seeds were added using a paper cone. (Figure A. 11). The total time estimated for the experiment is 120 minutes. At the end, the slurry was filtered using a vacuum pump.

17 The definition of this temperatures and the final temperature Tc is developed in the chapter 5.5 MSZW (Metastable Zone Width) definition 31 The experiment was performed with five different batches. The Table 1 summarizes the experimental conditions and the e.e measured, using the procedure described in 4.4 Purity measurement.

Th: Saturation temperature of the system, the system was seeded at this point.

Tc: Final temperature after the cooling step

Concen. Concen. Volume initial e.e Mass Cooling Th Tc Batch RS-IBU BPE Heptane purity seeds rate [°C] [°C] [mg/mL] [mg/mL] [mL] [%] [mg] [°C/min] 1 182 40.5 3.0 -6.0 64.6 57.5 53.0 0.30 2 183 40.7 3.0 -6.1 66.0 57.5 53.0 0.30 3 182 40.6 3.0 -6.1 66.0 57.5 53.0 0.30 4 182 40.4 3.0 -6.1 65.8 57.5 53.0 0.40 5 182 40.7 3.0 -6.1 65.8 57.5 53.0 0.50

Table 1 Experimental conditions for the PC system 180/40 [mg/mg] IBU/BPE

The next chapter describes the steps followed for solvent selection, concentration and operative window definition. Also the analysis of the results and the graphic representations of the system will be discussed.

32 5. Results and discussion

5.1 Initial system

Ibuprofen forms a Racemic compound in hexane[9], as seen in Figure 22.

Figure 22 Phase diagram screens of ibuprofen in hexane18

Firstly, the solubility of enantiopure and racemic IBU were determined using the TV Method (Figure 23). A concentration of 180 [mg/mL] of IBU was taken as an initial point since the saturation temperatures for the enantiopure and racemic compound ( around 12° C and 40°C respectively) are lower than the boiling point of the solvent (68.75°C).

Figure 23 Solubility of S-IBU and RS-IBU in Hexane

18 Reprinted from[9] .The diagram was built for a concentration of 175 [mmol/mol], which is equivalent to 274.4 [mg/mL] 33 In the studies of E. Elacqua [6], the formation of conglomerate co-crystals between IBU and 2-4 BPE in the 1:1 molecular ratio was investigated.

Figure 24 Views of ((±)-ibu).(BPE) highlighting: (a) two component assembly and (b) extended packing19

The PC starts with solution based on a racemic mixture[2]. For this system 159 [mg] of 2-4 BPE per 180 [mg] of IBU is required in order to have a stoichiometric solution. The PC is not going to start from a stoichiometric solution, because the region of the phase diagram most favourable to co-crystal formation may be missed.[23][34]

In this research, 10 [mg] of 2-4 BPE per 180 [mg] of IBU was taken as a starting point, in pursuance of studying its influence on the saturation temperature of the system. Once the influence on the saturation temperature was elucidated, the initial temperature was set in such a way that the entire compound was dissolved before the seeding.

The first part of this research consisted of the characterization of the saturation temperatures of the system and the verification of its applicability for the PC performance.

Using the temperature variation method (TV), the phase diagram of the system was constructed and compared with the non-coformer phase diagram (Figure 25)

19 Reprinted from [6] 34 Figure 25 the non-coformer and coformer phase diagram for RS-IBU20

The values shown in the Figure 25 correspond to the average values of the different sub- cycles during the TV method.

The phase diagram for the system IBU/BPE (Figure 26) presented two different trends; sometimes a shape indicating conglomerate system (red line) and sometimes the formation of a eutectic point was observed, indicating the dominant behaviour of the pure IBU in the system. The two trends were obtained based on experiments with the same concentrations, and temperature cycle. The latter suggests that the ratio of IBU/BPE chosen, can promote the formation of pure RS-IBU instead of co-crystals. From this, it was concluded that new ratios of IBU/BPE needed to be investigated.

Figure 26 Phase diagram of IBU/BPE in Hexane concentration c =180[mg/ml]

20 The blue dots represent the pure IBU in a concentration of 180 [mg/mL] in Hexane, the red squares represent the system IBU/BPE in the ratio 180/140 [mg/mg] in Hexane 35 In the next chapter, the results of experiments using higher concentrations of BPE in the system are evaluated and the verification of a conglomerate system will be evaluated via phase diagram and XRPD measurements.

5.2Influence of the ratio IBU/BPE in the system

As can be seen in the Figure 27, in a range between 10 and 40 [mg/ml] of BPE per 180 [mg/ml] of IBU, the saturation temperature is increased in an exponential fashion. In this case, while the system is getting closer to the 1:1 mole ratio, the solubility of the co-crystal is decreased with respect to the pure IBU. (See experiment #3)

Figure 27 Influence of BPE on the saturation temperature of the system (a) Racemic, (b) S-Enantiopure. The solid lines are guide to the eyes.

XRPD can be used for the characterization of the crystallographic structure of polycrystalline, as each crystalline phase shows a different XRPD pattern [35]. The diffractograms of the crystals formed at different ratios were analysed and are depicted in the Figure 28. The diffractograms show a mixture of crystalline structures. The appearance of new peaks at a higher BPE concentration is clearly visible at 2θ = 5 and 2θ = 25, indicating a new crystalline structure that corresponds to co-crystals. Qualitatively the intensity of the peaks corresponding to co-crystals increases while the amount of BPE increases. Nevertheless, the pattern associated to pure RS-IBU is predominant. The peaks corresponding to BPE are not identified, which can be an indication that the coformer is present in the new crystalline structure that corresponds to co-crystals.

36 Figure 28 XRPD patterns for the system IBU/BPE in different ratios of IBU/BPE [mg/mg]

Based on the trend exhibited in the Figure 28, it seems that reaching the 1:1 mole ratio (180/159 mg/mg) of IBU/BPE enables the presence of co-crystals and reduces the presence of pure RS-IBU. However reaching this 1:1 ratio may cause the saturation temperature to increase beyond the boiling point of the solvent (68.75 ˚C for Hexane)21. A new solvent was required that would enable the use of higher concentrations of BPE. Ideally, the new solvent would also have a bigger temperature difference between the phase diagram of pure IBU and IBU-BPE, which reduces the possibility of the formation of pure IBU in the final crops and creates a wider operative window.

It was also taken into account that Hexane is not recommended for pharmaceutical applications. Safety reasons encourage the search of a new solvent if this process wants to be scaled up for production conditions.

21 Taken from [46] 37 5.3New solvent selection

The U.S. Department of Health and Human Services Food and Drug Administration catalogues Hexane as a class 2 Pharmaceutical solvent. This implies that its use should be limited in pharmaceutical products because of its inherent toxicity [36].

Kim Alfonsi proposes Heptane as an alternative to the use of Hexane. The new solvent selected should such as the viscosity of the slurry remains low enough for an easy and fast filtration [2]. Additionally it is required that the saturation temperature of the system is lower than the boiling point of the solvent. As can be seen in Table 2, the preferred solvent is the Acetone

Table 2 Solvent substitution22

Ibuprofen is very soluble in Acetone[37] and almost insoluble in water[38]. Based on Figure 29 (a), the solubility obtained with Acetone (light green line) is much higher than the one obtained with Hexane. Acetone thus was discarded as an alternative. As a usable solvent, Heptane is proposed in the Table 2, additionally it has been used successfully as a substitute of Hexane in industrial applications[39].

The solubility of IBU in Heptane was measured using the TV method and is depicted in the Figure 29 (b). As can be seen, the performance is very similar to the one obtained in Hexane and additionally offers a higher boiling point (98.42°C)23.

22 Reprinted from [47] 23 Taken from [48] 38 Figure 29 (a) Solubility of RS-IBU in different solvents24

Additional solubility measurements were carried out at higher temperatures (78°C) and it was not possible to obtain results. It was found that IBU thermally decomposes at 76-77 °C [40]. Therefore the maximum temperature for solubility measurement of the system, was set to 70°C.

The influence of the amount of 2, 4-BPE on the saturation temperature of the system was evaluated using the new solvent. Similar values than with Hexane for the saturation temperatures of the system were found, however larger amounts of BPE could be added. This was due to the higher boiling point of the solvent (Figure 30)

Figure 30 Influence of BPE on the system saturation temperature in Heptane. (a) Comparison in Heptane and Hexane. (b) Different cycles for Heptane

Initially, the system was defined as 180/40 [mg/mg] of IBU/BPE. This results in a temperature around 57 ˚C. Higher temperatures were avoided due to health and safety reasons. as the seeding step was performed manually.

24 Ethanol, Acetone and 2-Methyl-1-propanol are taken from [37]; Hexane is taken from results using TV method. (b) Comparison between solubility of RS-IBU in Hexane and Heptane, data obtained from TV method. 39 The system is represented graphically in a binary phase diagram, combined with the characterization of its MTZ. Based on these graphs, a suitable cooling temperature was identified.

5.4Phase diagram of IBU/BPE

The binary phase diagram for the coformer and no-coformer system was plotted. In the Figure 31 (a) can be seen that the conglomerate system was present in all the different temperature cycles and no eutectic point were detected. This indicates that the presence of pure IBU close to the saturation temperature of the system was reduced compared to the case of the mixture 180/10 [mg/mg] in Hexane (Figure 26)

Figure 31 (a) Binary phase diagram IBU/BPE in Heptane, with a ratio of 180/40 [mg/mg]. (b) no-coformer and coformer phase diagram

XRPD measurements were taken in the points A, B, C, D and E at room conditions; with the purpose of verifying the crystalline structures (Figure 32). In all the 5 points, the presence of new peaks, indicating co-crystals, can be seen.

It was expected that the XRPD pattern would be the same in all the points of the diagram when a conglomerate was formed [2]. Nevertheless, in this case, the patterns were not the same as the samples used corresponded to a mixture of co-crystals and pure IBU. However, the characteristic peaks for co-crystals were present in all the points, indicating the formation of a conglomerate system.

Later it will be shown how the solution was filtered close to the saturation temperature, where, qualitatively, the predominant structure were co-crystals.

40 Figure 32 XRPD patterns IBU/BPE in Heptane25

Based on the binary phase diagram for the coformer system (Figure 31), the initial solution had to be heated up until 57.5°C in order to get total dissolution before the seeding.

The next step is to define the final temperature of the cooling step after seeding. If the interaction of BPE on the saturation temperature of IBU is neglected, pure IBU is expected at temperatures below the 39°C. Verification of this interaction was not done in this study and will need to be studied via a quaternary phase diagram in future research.

The solubility of BPE using the TV method was not possible, due to high dispersion in the data obtained. Thus the construction of a quaternary phase diagram was not possible. As an alternative for the operative temperature determination, a different approach was proposed.

The approach uses different samples with the same concentration filtered at different temperatures. The purpose is determining the evolution of the ratio IBU/BPE in the solid phase. Qualitatively this was done using XRPD, quantitatively by NMR. This approach will be discussed further in the MTZ definition chapter.

25 Characteristic peaks for co-crystals are present in all the samples, indicating the formation of a conglomerate system. 41 5.5MSZW (Metastable Zone Width) definition

The measurement of the saturation temperature and cloud point for the solution was performed. In this case, the process was monitored with the Crystalline ™ camera.

Figure 33 Saturation temperature and cloud point for IBU/BPE in Heptane. Figure in the left shows the saturation temperature when the solution is completely clear. Pictures in the right show the appearance of the first crystals, indicating the cloud point.

The average value for the saturation temperature measured with the Crystalline ™ was 57.5°C and is exactly the same as obtained from the Crystal 16 ™ measurement. The average value for the cloud point was 48.6°C and it represents the theoretical minimum value for the cooling temperature.

In the PC, the resolution was obtained via secondary nucleation and crystal growth of the desired enantiomer [2] around the seeds. The primary nucleation of the undesired enantiomer should be avoided. The above mentioned implies that the operative window for the PC should be located between 57.5°C and 48.6 °C.

After letting the solution equilibrate at different temperatures, it was filtered and the solid phase recovered was analysed in the NMR via an Assay. The results are depicted in Figure 34, it shows how before the cloud point (48.6°C) at 0°, 15°and 30°C, the ratio IBU/BPE is almost constant and close to 3.5, indicating the excess of IBU in the crystals. Once the filtration temperature gets closer to the clear point temperature (Ts= 57.5°C), the ratio is almost 1, indicating a predominant co-crystal structure in the solid phase.

42 Figure 34 Ratio IBU/BPE vs. Filtration temperature

During the PC, once the solution is seeded, its temperature is progressively decreased in order to induce an enantioselective secondary nucleation and crystal growth of the enantiomer seeded. Thus, the final temperature of the cooling step, should be closer to the saturation temperature, but low enough in order to allow for the secondary nucleation of S- co-crystals.

As long as the system remains above the solubility of the pure IBU, its crystallization will not take place, but, if the system is cooled below this point, crystallization of IBU may occur.

Based on the above, a final cooling temperature of 53°C was proposed (Tc).

43 5.6Calibration lines for specific optical rotation measurement

The calibration line for pure IBU was determined and the specific optical rotation was found to be 53.5 [◦/dm.g.cm-3].In the Figure 35 it can be seen that the correlation performs according to a linear trend.

Figure 35 Calibration line for optical rotation of IBU based on e.e in Ethanol. c=6 mg/100cm3 and T=20°C

In the case of the analysis of the crops, the presence of BPE can influence the specific optical rotation of the sample. The specific optical rotation of BPE was measured and its value of 0.048 [◦/dm.g.cm-3], is almost negligible when compared to IBU. For the ideal case of just co- crystal formation, a calibration line was built and is depicted in the Figure 35.

Figure 36 Calibration line for optical rotation of IBU/BPE 1:1 molar ratio based on e.e in Ethanol. c=11 mg/100cm3 and T=20°C

44 Due to the possibilities of having pure racemic IBU in the crops, a new calibration line was built. In this case, the effect of different amounts of BPE on the specific optical rotation of pure IBU was studied (Figure 37).

Figure 37 Calibration line for optical rotation of IBU/BPE in different ratios. Solvent: Ethanol, c=11 mg/100cm3 and T=20°C

5.7 The preferential crystallization in different batches

One batch of seeds was produced and the diffractograms were used in order to prove the formation of co-crystals. The XRPD patterns in the Figure 38 show that the seeds obtained (orange line) have no presence of pure IBU, because there are no representative peaks for it, indicating a pure co-crystal structure.

The XRPD patterns enclosed in the blue dotted rectangle in the Figure 38 show the same structure in all the measurements. Additionally it confirms the formation of a conglomerate system, as the patterns for racemic and enantiopure co-crystals are the same. These seeds were grinded (Figure A. 10), stored and subsequently used for the PC experiment.

45 Figure 38 XRPD patterns of co-crystals of IBU/BPE

In one of the experiments, the solution was evaporated at room conditions and a mixture of single crystals was produced after four days (see Figure A. 7 & Figure A. 9 in the appendix).

46 Figure 39 Steps of the PC

Figure 40 PC monitoring Batch 1. On top, the temperature (black line) is monitored together with the transmissivity (blue line) of the solution. Below, pictures at the moment of total dissolution, seeding, stabilization of the seeds and crops formation.

As can be seen in the Figure 40, once the solution starts to cool down around the minute 92, the transmissivity decreases, indicating the formation of crystals. Once the solution reaches the final temperature Tc=53°C, it is stabilized for a couple of minutes and subsequently filtered, using a vacuum pump.

For the yield measurement, the ratio IBU/BPE in the crops obtained after the different batches of the PC was determined using the NMR. The applicability of this method was verified in an experiment here known amounts of IBU and BPE were mixed and subsequently analysed in the NMR. The ratio of IBU/BPE measure through NMR was compared with the known initial conditions, it was noticed that NMR on average had a 2.6% deviation from the

47 known conditions. Therefore NMR can be considered to be accurate enough to measure IBU/BPE ratios. (Figure A. 5)

Figure 41 spectrum of a mixture of crops of IBU/BPE

The Figure 41 shows the final spectrum of a mixture of IBU/BPE, where the characteristics peaks for IBU and BPE are highlighted. The characteristic peak for BPE overlaps with the signals of the benzene ring of the IBU. In order to obtain the integrals of BPE, the aromatics corresponding to IBU were obtained, based on the ratio with the peak around 3.7 ppm and the signal on the left was assigned to BPE.

Because of the presence of some crystals during the NMR test (Figure A. 6), the measurement was run with an assisted temperature control at 50°C, guarantying the total dissolution of the samples.

The determination of the ratio consists on relating the value of the integrals for both compounds with the concentrations used in the test.

The yield was defined in the terms of additional S enantiomer obtained in the enantiopure co-crystals form, compared with the amount of S enantiomer added in the seeds.

S Yield  IBU S IBUseeds

48 26 SIBU  Additional SIBU recovered in form of co-crystals

It was measured on each batch and is depicted in the Table 3.

Ratio e.e Yield Batch IBU/BPE purity [%] mol/mol [%] 1 1.34 -63.5 44% 2 1.45 -55.2 14% 3 1.58 -53.8 8% 4 1.48 -58.7 16% 5 1.52 -61.7 35%

Table 3 Yield measurement in the different batches

The Batch 1 provided the highest yield of S enantiomer (44%). In this, the IBU present in the crops can be as part of the co-crystal structure or in pure racemic form.

Figure 42 Distribution of compounds inside the crops

The above mentioned was confirmed in the Figure 43, where the XRPD pattern of the crops showed characteristic peaks for pure IBU and co-crystals. Of particular note was the fact that peaks indicating pure BPE were not present, which implies that all the BPE were in the form of co-crystals.

26 The SIBU obtained in the form of pure racemic compound is not taken into account 49 Figure 43 XRPD PC Batch #1 ratio 180/40 [mg/mg]

The primary nucleation of pure racemic IBU and co-crystals can be due to variations in the MSZW. For the initial solution, the MSZW was defined between 57.5°C-48.6°C and the final temperature after the cooling was set in 53°C. This was done to avoid the lower limit of the MSZW and the start of the primary nucleation of the undesired enantiomer and racemic IBU. The characterization of the MSZW was carried out with a volume of 2 [ml] and the PC was performed with volumes of 3[ml]. In the research of S.Kadam et. al.[20], “It was experimentally observed that the MSZW is not a reproducible point at small volumes but a spread which increases roughly inversely proportional to the volume”.

Eventually, at 53°C, the solution is reaching a point where the primary nucleation can take place. Besides this, the MSZW was measured without the presence of crystals and during the PC; the presence of the seeds might influence the final measurement.

The seeds used were obtained via evaporative crystallization and even though the initial solution is stoichiometric in the co-crystal ratio, it might happen that some pure S-IBU was present as an impurity and during the process was converted in RS-IBU.

One additional mechanism that may contribute to the presence of racemic co-crystals and racemic IBU was the fact that the crops were not being washed and dried out after the

50 filtering in the vacuum pump. Once the mother liquid has evaporated, left over compounds can provide additional compound in the form of racemic co-crystals and racemic IBU.

In the Figure 44 the distribution of the different enantiomers is represented.

Figure 44 Mass distribution of IBU in the crops Batch 1 (a) Distribution of the total amount of IBU in either co-crystal or pure racemic form. (b) Distribution of the enantiomers in the co-crystals and racemic form. (c) Discrimination of the S enantiomer recovered.

The batches 2, 3 and 4 show low values of yield due to that part of the seeds attached to the wall of vial without being in contact with the solution. Thus the vial was agitated manually in order to incorporate the seeds. It is assumed that this agitation created a sudden temperature drop that propitiated the nucleation of RS-co-crystals before the programed cooling step.

In order to reduce the amount of racemic IBU present in the crops, a new initial solution with a concentration in the 1:1 mole ratio for IBU/BPE was proposed with the hypothesis that all the BPE was going to be associated to IBU in a co-crystal structure. Different concentrations were evaluated in the Crystal 16™ and the results are depicted in the Figure 45. Herein a stability in the saturation temperature of the system was observed around 69- 70°C, for concentrations higher than 100 [mg/mL].

51 Figure 45 Solubility of mixtures in the 1:1 molar ratio IBU/BPE in Heptane

A new concentration of 38 [mg/mL], constituted by 20 [mg] of RS-IBU and 18[mg] of BPE was selected as a new starting solution. The saturation temperature in this case was 58.9°C and the average value for the cloud point was found to be around 52.4°C. The final cooling temperature was set in 53°C and the cooling rate was 0.3°C/min.

The Table 4 summarizes the experimental conditions for the PC in the system starting with 1:1 mole ratio

Concen. Concen. Volume initial e.e Mass Cooling Mass Ratio e.e Th Tc Batch RS-IBU BPE Heptane purity seeds rate crops IBU/BPE purity [°C] [°C] [mg/mL] [mg/mL] [mL] [%] [mg] [°C/min] [mg] mol/mol [%] 1 20 17.8 5.9 -10.0 25.0 58.9 53.0 0.30 79.7 1.09 -32.9

Table 4 Experimental conditions for the PC system 20/18 [mg/mg] IBU/BPE

In a similar fashion for the previous system, the yield was measured, with a value of just 9%. The presence of pure racemic IBU (Figure 46) can be explained by the fact that in the initial solution, the molar ratio of IBU/BPE was slightly higher than one (it was 1.1). This means that a small amount of IBU was present in excess at the beginning. Due to the scale of the experiment (vials of 5mL), ensuring that the amount of compound added are exactly in a 1:1 molar ratio is complicated. By setting the experiments in larger scales, the errors associated to weighing can be reduced. Nevertheless, it was proved that the amount of pure racemic IBU in the crops could be reduced by starting solutions close to the 1:1 molar ratio. The final amount in this case was 8,2 % and in the previous system it was 25.4%.

52 Figure 46 Mass distribution of IBU in the crops Batch 1 Initial solution in the 1:1 mole ratio IBU/BPE (a) Distribution of the total amount of IBU in either co-crystal or pure racemic form. (b) Distribution of the enantiomers in the co-crystals and racemic form. (c) Discrimination of the S enantiomer recovered.

When the XRPD pattern of the crops is analysed (Figure 47), the presence of pure racemic IBU is qualitatively imperceptible and the predominant structure is pure co-crystal.

Figure 47 XRPD PC Batch #1 ratio 20/18 [mg/mg]

The main reason for the poor yield was the fact that at the saturation temperature set, it was not possible to obtain a clear solution before seeding (Figure 48). This means that the undesired enantiomer (R) was present in the solid phase. During the slow cooling of the seeded solution, secondary nucleation and crystal growth of the seeds took place, together with the nucleation of R- co-crystals.

53 Figure 48 Left. Initial solution IBU/BPE [20/18] [mg/mg]27. Right, Clear initial solution for the system IBU/BPE 180/40 [mg/mg]

The fact that it was not possible to get a total dissolution of the racemic solution at the predicted saturation temperature can be explained by the fact that in the temperature variation method, the laser beam [41] is measuring a 100% transmissivity in the solution in the Figure 48 . Once the solution was analysed under the microscope camera, it is possible to find small crystals undissolved which were not detected by the laser beam. The saturation temperature28 set was 58.9°C. In an additional experiment the solution with the same composition was heated up to 70°C and the same pictures of the Figure 48 were obtained.

The undissolved crystals correspond to BPE. Even though it was not possible to build a solubility line for BPE, there were some indications of its poor solubility in Heptane compared with IBU.

With the system of IBU/BPE 180/40 [mg/mg] (which corresponds to 3.97 molar ratio), total dissolution at the beginning was achieved. This can have an explanation on the positive effect of the IBU on the solubility of BPE.

The solubility of BPE at 57.5°C is unknown. In one experiment, one vial with 40 [mg] of BPE per [mL] of heptane was heated up and held at 57.5°C. In this case, it was not possible to obtain dissolution of the powder, indicating that the solubility of BPE at the set temperature is less than 40[mg/mL]. Nevertheless, when BPE is interacting with IBU in Heptane, its solubility is increased enabling the total dissolution in the mixture 180/40 [mg/mg].

In the second solution with 20/18 [mg/mg], the amount of IBU is not enough in order to enable the total dissolution of BPE. This performance can be an explanation for the stability of the saturation temperature in the experiment of the solubility of the mixture in the 1:1 molar ratio depicted in the Figure 45.

27 The laser beam detects this as 100% transmissivity, but with the microscope of the camera, it can be seen that there is no total dissolution. 28 Based on the Crystal 16 ™ measurement 54 6. Conclusions

 Heptane appears to be a suitable solvent for the PC, offering a reasonable solubility of IBU and a high boiling point (98.42°C) that allows exploring different ratios of the mixture IBU/BPE. The limitation of using mixtures with high concentrations of BPE is the need of reach saturation temperatures beyond the thermal stability of the IBU at 76 ˚C. Additionally Heptane is a safer alternative than the initial option of Hexane.

 The binary phase diagram for the system in heptane, using concentrations of IBU/BPE 180/40 [mg/mg] was successfully determined, describing the formation of a conglomerate system.

 The formation of co-crystals was verified using the phase diagram method and the XRPD measurements. The apparition of crystals of pure IBU in the final product of the PC, made necessary to implement NMR measurements in order to determine their composition.

 PC for the system IBU/BPE was performed with a maximum yield of 44%, starting from a solution with excess of IBU instead of a stoichiometric one. Nevertheless, pure racemic IBU and co-crystals of both enantiomers were present in the crops.

 The applicability of the NMR for measuring the ratio IBU/BPE in the final crops was successfully verified and subsequently applied to the process.

 The influence of the BPE in the specific optical rotation of the co-crystal was found to be linear, thus it was possible to implement polarimeter measurements in order to determine the enantiomeric excess of the final product, once the composition of the final crops was determined using NMR measurements.

 Starting with solutions in the stoichiometric co-crystal ratio does not allow performing PC, due to even for small initial concentrations of IBU/BPE. Close to the 1:1 molar ratio, is not possible to get total dissolution of the BPE without going beyond temperatures that compromise the thermal stability of the IBU.

55 7. Recommendations

 The formation of co-crystals for the system IBU/BPE either using evaporative crystallization or cooling crystallization was proved. It is suggested as an alternative, to produce a batch of seeds using cooling crystallization, pursuing to get higher purities.

 It is possible to obtain wrong solubility measurements out of laser beam based equipment, probably because the size of the crystals is not detectable for the laser beam or the refractive index of the particle is similar to the one of the solution. Using additional methods such as gravimetric or monitoring of the measurements with a microscope can reduce the chances of a wrong value.

 Obtain the solubility line of BPE by means of an alternative method such as gravimetric measurements, following procedures like the one described by D. Harvey[42]. This can be used for the construction of ternary and quaternary phase diagrams, where the operative windows for the PC can be delimited with more accuracy.

 Evaluate the effect of the final temperature on the purity of the crops. A procedure described by G. Levilain et al. in [29] can be taken as a reference. Essentially the PC should be performed in different batches, under the same initial conditions, but changing the set of the final temperature. It is expected that higher temperature drops produce more crops, but on the other hand the purity is decreased (Figure 49)

Figure 49 Influence of the final temperature on the solution e.e. during the crystallization (AS3PC mode)29

With this analysis, a better definition of the operative window can be achieved, by finding an optimal final temperature, with a good balance between purity and amount of crystals recovered.

29 Reprinted from [29] 56  Look for new ratios of IBU/BPE close to the stoichiometric co-crystal ratio, but always with an excess of IBU. In this way, the chances of total dissolution of the BPE could be increased and the possibilities of nucleation of racemic IBU are reduced. One experiment, measuring the saturation temperature of different mixtures can be carried out. These can be done by taking as reference the system of 180/40 [mg/mg]; the amount of BPE can be fixed in and the amount of IBU can be decreased, monitoring with the crystalline that in the transmissivity of 100% the totality of crystals are dissolved.

 Look for the application of mixtures of solvents that can allow the total dissolution of the compounds in starting solutions in the 1:1 molar ratio. A mixture of Acetone where IBU is highly soluble (Figure 29) with water, where the IBU is poorly soluble can end up in an intermediate solubility that can allow the total dissolution of the compounds without reaching extremely high concentrations.

 Investigate possible solvents that can be used for washing the crops. In this way, the precipitation of solute contained in the mother liquid after the filtering can be lowered.

 Perform the PC at a bigger scale. By doing this, the mother liquid could be monitored by taking samples and analysing them in the polarimeter.[43] This could provide a better understanding of the process and give a better definition of the filtration time.

 Additional studies of the effect of the cooling rate can be performed. In the PC with the first system it was tried to be analysed but due to problems in the seeding it was not possible. Performing the experiment with faster and lower cooling rates than the initially used (0.3°C/min) could provide a better understanding of the process kinetics.

 It is recommended to perform the PC following the method described in the Figure 9, which implies the use of a constant temperature, starting from a solution saturated in the desired enantiomer. In this case the steps proposed are as follow: a) Determine the clear point for a solution of IBU/BPE in the mixture 180/40 [mg/mg] with different e.e (see Figure 4 in the reference [2]) b) Choose a suitable initial temperature, where total dissolution with an initial e.e is achieved c) Heat up the solution and add the seeds d) Maintain the temperature fixed and filter the crops after certain time (initially it can be 20 minutes) and measure the purity and e.e of the mother liquid e) Repeat the step (d) with different times and analyse the evolution of the e.e.

57 With this experiment, a better understanding of the process kinetics can be gained, and a better definition of the filtration time before the primary nucleation of the undesired enantiomer can be set.

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61 9. Appendix

9.1Specifications of the solubility measurement experiments

Heating Stirring Experiment Solvent Cooling Rate speed Specification [°C/min] [rpm] Solubility racemic and Hexane 0.3 700 Evaluated in the range 50- S-enantiomer IBU 300[mg/ml]

Solubility IBU/BPE in Hexane 0.3 700 Concentration of IBU fixed in different ratios of BPE 180[mg/mL], BPE ranged from 10-40[mg/ml] Binary phase diagram Hexane 0.3 700 Concentrations of IBU IBU/BPE =180[mg/mL] BPE=10[mg/ml] Solubility racemic and Heptane 0.3 700 Evaluated in the range 50- S-enantiomer IBU 300[mg/ml]

Solubility IBU/BPE in Heptane 0.3 700 Concentration of IBU fixed in different ratios of BPE 180[mg/mL], BPE ranged from 10-124[mg/ml] Binary phase diagram Heptane 0.3 700 Concentrations of IBU IBU/BPE =180[mg/ml] BPE=40[mg/ml]

Table 5 Solubility measured using Crystal 16 ™

62 9.2Relaxation time for compounds in NMR tests

Figure A. 1 Relaxation time for IBU

Figure A. 2 Relaxation time for BPE

63 Figure A. 3 NMR spectrum for IBU

64 Figure A. 4 NMR spectrum for BPE

Figure A. 5 Experiment for accuracy of NMR in application for IBU/BPE ratio measurement

65 Figure A. 6 crystallization during the NMR test

66 9.3Single crystals of seeds

Figure A. 7 co-crystals of S-IBU/BPE

Figure A. 8 Microscopic view of the co-crystals of S-IBU/BPE

67 Figure A. 9 XRPD analysis of the single crystal obtained for one batch of seeds production

68 9.4Preferential crystallization

Figure A. 10 Seeds grinded

69 Figure A. 11 Illustration of the seeding mechanism

70 9.5 XRPD Patterns IBU/BPE in different ratios in Heptane

Figure A. 12 XRPD Patterns IBU/BPE in different ratios in Heptane

71