Deracemization of NMPA via Temperature Cycles - Crystal Growth

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Deracemisation of NMPA via temperature cycles Francesca Breveglieri, Giovanni Maria Maggioni, and Marco Mazzotti Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.7b01746 • Publication Date (Web): 19 Jan 2018 Downloaded from http://pubs.acs.org on January 24, 2018

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Crystal Growth & Design

Deracemisation of NMPA via temperature cycles Francesca Breveglieri, Giovanni Maria Maggioni, and Marco Mazzotti∗ Separation Processes Laboratory, ETH Zurich, Zurich E-mail: [email protected] Phone: +41 44 632 24 56. Fax: +41 44 632 11 41

Abstract 15th January 2018 Recent studies have shown that total deracemisation of a racemic suspension of a conglomerate forming compound can be attained in the presence of a racemising agent through either attrition enhanced deracemisation or temperature cycles. We experimentally investigate the deracemisation of N-(2-methylbenzylidene)-phenylglycine amide, in the presence of DBU as racemising agent in a mixture of isopropanol and acetonitrile (95/5 w/w), at several different operating conditions. Based on several experiments, we determine how the operating parameters influence the temperature cycles, by varying the initial enantiomeric excess, the cooling rate, the operating temperature range, and the system volume. We examine how each parameter affects the phenomena characterising the temperature cycles, e.g. total process time or total number of cycles to attain deracemisation. Finally, we discuss in general how to improve the performance of the process.

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Introduction

Chiral molecules represent a significant fraction of active pharmaceutical ingredients, whose purity requirements are becoming more and more stringent, since enantiomers have typically different physiological effects 1 . Therefore, scientific and technological efforts are devoted to how to produce selectively the target enantiomer. Traditional approaches cover a broad range of techniques. One of them is the production of the desired enantiomer only via asymmetric synthesis, which often requires the use of complex catalysts and conditions, proceeds through multiple steps, and frequently has low final yields 2 . Another approach is based on the physical separation of the target enantiomer from its counterpart after the synthesis, for instance through chiral preparative chromatography 3,4 or preferential crystallisation 5,6 . The former is a powerful and widely applicable technique, but the purified product needs to be crystallised in a further step to be separated from the liquid phase. The latter leads to a pure crystalline product, but it can be performed only with substances crystallising as conglomerates, i.e. a mechanical mixture of pure crystals of the two enantiomers 7 , and exhibits some operational challenges, such as the possible nucleation of the undesired enantiomer, which reduces the chiral purity of the final product. Furthermore, all separation techniques have a maximum theoretical yield of 50%, which can be improved e.g. by adding one more step after the separation: a racemisation unit to convert the undesired enantiomer into the desired one, followed by a recycle of the racemate to the separation unit 8 . Viedma first, and other researchers later, have demonstrated that it is possible to attain complete deracemisation of conglomerate crystals by combining solute racemisation, crystallisation, and grinding in a single step, thus realising the process known as ”attrition enhanced deracemisation” or ”Viedma Ripening” 9–12 . In this process, the key phenomena enabling deracemisation are the size-dependent solubility of crystals and the enantioselective agglomeration of crystals of different sizes 13–15 . The former is responsible for Ostwald Ripening and leads to the growth of the largest particles at the expense of the smallest ones. 2


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The latter promotes the formation of larger particles, which cannot dissolve, thus introducing an asymmetry in the system. As suggested by its descriptive name Viedma Ripening is enhanced by attrition, which breaks the particles and creates fragments small enough to dissolve because of their size dependent solubility. In his original experiment, Viedma used glass beads to promote attrition, but he needed to separate the final crystalline product from the glass beads once deracemisation was attained. To avoid this further step, alternative strategies based on similar concepts have been explored: for example, reducing the particles size by milling 16 , or by sonication 11,17,18 , or by using high pressure homogenisation 19 . Attrition can be obtained through several different techniques. For example, total resolution of the intermediate compound in the synthesis of the marketed drug Clopidogrel is attained after 17 hours, when 300 mL of the slurry are ground with glass beads 20 , but after only 45 minutes, if an industrial bead mill is used as generator of attrition in the process scale up (see Figure 3, in Noorduin et al. 2010) 16 . Hence, a higher attrition intensity leads to a faster process, thanks to larger particles comminution. However, a more intense attrition could also be responsible for a significant local temperature variation, further contributing to the deracemisation. From these observations Suwannasang et al. first 21,22 , and others later, 23 studied a different approach, consisting in periodically programmed temperature swings (temperature cycles). Such approach is particularly convenient thanks to the simple experimental set-up needed and to the several degrees of freedom that one can exploit to operate and to optimise the process. However, temperature cycles are still operated in a rather heuristic way, since the phenomena on which they rely have not yet been fully understood and an accurate model has not yet been developed. Thus the potential of this technique is underexploited. The experimental investigations in this work focus on examining the influence of the main operating parameters on the temperature cycles, in order to understand the role of the phenomena occurring during the deracemisation and their relative importance. In this paper, we first characterise the investigated system and illustrate the experimental

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protocol, describing also how the periodical temperature swings are believed to influence the system (Section 2). Then, we present and discuss the experimental results obtained by investigating the effect of the initial enantiomeric excess, the cooling rate, the operating temperature range, and the system volume. For each series of experiments, we comment on the mechanisms driving the enantiomeric deracemisation and on the way the operating conditions could be affecting them (Section 3). Finally, we summarise the effects of the investigated operating conditions on the deracemisation process and we discuss how one could take advantage of these conditions to optimise the process in view of an industrial application (Section 4).

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Materials and Methods

2.1

Materials and Equipment

The experiments were performed with N-(2-methylbenzylidene)-phenylglycine amide (NMPA), synthesised and used in previous investigations 19 , which racemises in solution in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (Figure 1). The solvent was a 95/5 (w/w) mixture of 2-propanol (IPA) and acetonitrile (ACN). The racemising agent and the solvents were purchased from Sigma-Aldrich with a purity of 99%, and used as received. The solid for the analysis was collected by vacuum filtration with a B¨ uchner funnel and a MS PTFE Membrane Filter 0.45 µm. The experiments were performed in a customised version of Crystal16 (Technobis): the device consists of 16 independent vials of ca. 1.8 mL, each of them equipped with a thermocouple to monitor the temperature. Rare Earth cylindrical PTFE stirring bars (8×3 mm) were used to stir the suspension at 1250 rpm. The vials used as crystallisers were 32×10 mm standard glass chromatographic vials. Only the experiments designated as 1a-1b in Table 2 were performed in an Easymax (Mettler Toledo), with a glass cylindrical crystalliser (2×10 cm); the temperature in the Easymax was monitored using a thermocouple and the 4


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suspension was stirred at 1000 rpm with a magnetic PTFE stirring bar.

Figure 1: Racemisation reaction of N-(2-methylbenzylidene)-phenylglycine amide (NMPA) in the IPA:ACN mixture (95/5 w/w), where 1,5-diazabicyclo[5.4.0]undec-5-ene (DBU) is used as racemising agent.

The solubility, expressed as gram of solute per kilograms of solvent, was measured gravimetrically at different temperatures with three independent experiments, whose average value and standard deviation are reported in Table 1. Table 1: The solubility data of NMPA in the IPA:ACN mixture (95/5 w/w), measured at different temperatures. These values are plotted in Figure 3.

Temperature [ ◦ C] Solubility [g/kgs ] 20 11.9±0.2 25 12.8±0.4 30 16.1±0.1 35 19±1 41 23±1

2.2

Experimental Procedure

We developed an experimental protocol to prepare a homogeneous powder with the desired initial enantiomeric excess in order to minimise the variability between two different repetitions. In fact, previous works reported that deracemisation experiments via attrition enhanced deracemisation 24–28 , as well as via temperature cycles 22 , are sensitive to variability in the initial conditions. First, we wet-ground a solid racemic mixture of NMPA powder 5



with the necessary amount of powder of the desired enantiomer and few µL of pure acetonitrile, which then evaporated. In all experiments, (S)-NMPA was used to produce the initial enantiomeric excess. The desired amount of DBU (3.85 µL/gs ) was added to a filtered saturated solution of racemic NMPA in the specified solvent mixture IPA:ACN. Finally, the vials containing the solids were filled with the saturated solution, to produce a suspension with a solid density, ρ, of 2.5% (gsolid /gs ). The suspension was stirred at the lowest temperature (see Section 2.3) for two minutes, then the first sample was taken and the temperature cycles started. The experiments ran until the enantiomeric excess was at least 98%. The final product was collected by vacuum filtration and washed with n-hexane, to remove the residual solution. Each experiment at a given set of operating conditions was repeated three times. Note that for Exps. 1a-1b the three repetitions have been sampled at different times.

2.3

The Temperature Cycles

Each experiment consisted in a series of cycles, during which the temperature went from the lowest value, Tmin , to the highest, TMAX , following a four-stage profile: an initial heating ramp, a high-temperature isothermal period, a cooling ramp, and a final low-temperature isothermal period. Once the last stage was completed, a new cycle started. Figure 2 illustrates conceptually the mechanisms thought to occur during the different stages. Upon heating the scalemic suspension (enriched in (S)-enantiomer), an equal amount of (R)- and (S)-solids, which is determined by the temperature- and size-dependent solubility, dissolves, thus yielding a racemic solution of the two enantiomers in contact with their crystalline scalemic physical mixture. The composition of the solid phase remains enriched in the (S)-enantiomer, hence more (S)-surface is available to incorporate molecules of this handedness. As a consequence, during the cooling period one expects that the supersaturation of (S)-NMPA is consumed faster than that of the (R)-enantiomer, and that (S)-crystals grow and agglomerate more than (R)-crystals. While crystals grow, the racemisation reaction in solution converts the (R)-enantiomer into the (S)-enantiomer, to compensate for the larger 6


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amount of (S)-enantiomer that crystallises. Note that in principle also breakage occurs, although there is evidence that it plays no key role in our specific system (see Exp. 2a reported Figure 6, Section 3.2). At the end of each cycle, the fraction of the desired enantiomer in the solid phase has increased, and a new cycle starts. The operating temperature range, ∆T = TMAX − Tmin , is associated to a maximum concentration change, ∆c, determined by the solubility, c∗ , represented for NMPA as a solid black line in Figure 3. ∆T and ∆c, together, define the attainable region, i.e. the region in the (T, c)-plane that the system can explore during every cycle. A typical cycle starts from point A and moves towards TMAX during the heating stage, when the solubility increases. A fraction of the solids dissolves to compensate the difference between the actual solute concentration and the solubility, thus the operating line bends upwards along the c-axis, towards point C. Note that point C can be attained or not during the dissolution step, depending on the kinetics of the system: the actual value of the concentration at the final temperature may also be lower. When the cooling starts, the operating point moves away from the segment BC towards the segment DA: the decrease in solubility creates supersaturation, which is predominantly consumed by crystal growth. Consequently, the solute concentration decreases, reaching the value in point A (or stopping on another point along DA, depending, once again, on the actual kinetics). The rectangle A-B-C-D in Figure 3 thus defines the attainable region. It is clear that the operating line, irrespective of the actual kinetics of the investigated system, depends mainly on the heating and cooling rates: the slower the rates, the closer the concentration profile is to the solubility line; the faster the rates, the further away it is. The sides of the rectangle correspond to the operating lines obtained with infinitely fast heating and cooling (red and blue arrows, respectively). On the contrary, for an infinitely slow heating and cooling rate the operating line would correspond to the solubility line.

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Figure 2: A conceptual representation of deracemisation via temperature cycles. Upon heating and due to size-dependent solubility, (R)-crystals (orange squares) and (S)-crystals (blue squares) dissolve, while upon cooling they grow. At the same time, the racemisation reaction ideally maintains the solution at the racemic composition. Agglomeration among crystals of the same handedness enables deracemisation that is enhanced by breakage.

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Figure 3: The solubility of NMPA in IPA:ACN (95:5 w/w). Symbols and error bars are the experimental values from Table 1, while the solid black curve represents the solubility line fitted to the data, with a = 4.73 × 10−4 g/kgs and b = 0.0344 1/K. The red and blue paths represent the changes in temperature during a typical cycle and the corresponding expected change in concentration for heating and cooling periods, respectively. The coloured areas represent the attainable regions during heating (red) and cooling (blue), as explained in the main text.

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Experimental Results

In this work, we investigate the following operating parameters: the initial enantiomeric excess, ee0 , the cooling rate, Rc , the operating temperature range, ∆T , i.e. the difference ∆T = TMAX − Tmin , in which the cycles work, and the system volume, VS . In all experiments, we kept constant the concentration of DBU, the solid density of the suspension at Tmin , ρ, and the amount of crystals dissolved at TMAX , ∆c. Note that, by fixing Tmin and keeping ∆c constant, TMAX is not a degree of freedom, but it is determined by Tmin and c∗ , through: ∆c = c∗ (TMAX ) − c∗ (Tmin )

(1)

In the following, we analyse the influence of each operating parameter separately. We report in Table 2 a summary of all experiments with the values of their operating parameters, the total process time, ttot , and the total number of cycles, ntot , performed to attain complete deracemisation. We used the conditions of Exp. 2 as reference scenario.

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Table 2: Summary of all performed experiments and the values of the investigated operating parameters: ee0 is the initial enantiomeric excess; Rc the cooling rate; Tmin and TMAX the minimum and the maximum temperature in a cycle, respectively; VS the volume of the crystalliser; ttot the duration of the process and ntot the total number of cycles. The gray-shaded cells highlight the specific parameter investigated for each set of experiments. The following parameters are kept constant in all the experiments: solid density, ρ (2.5% g/gs ), amount of DBU (3.85 µL/gs ), heating rate (1.3 ◦ C/min) and amount of compound dissolved every cycle (30.4% of the suspended solid).

Exp. 1 2 3 2a 2b 2c 2d 2− 2+ 2d− 2d+ 1a 1b

3.1

ee0 [%] 7 18 32 18 18 18 18 18 18 18 18 7 7

Rc [◦ C/min] Tmin [◦ C] TMAX [◦ C] VS [mL] 0.22 25.0 38.0 1.8 0.22 25.0 38.0 1.8 0.22 25.0 38.0 1.8 25.0 25.0 1.8 0.14 25.0 38.0 1.8 0.43 25.0 38.0 1.8 1.30 25.0 38.0 1.8 0.22 20.0 34.9 1.8 0.22 30.0 41.3 1.8 1.30 20.0 34.9 1.8 1.30 30.0 41.3 1.8 0.22 25.0 38.0 4 0.22 25.0 38.0 8

ttot [h] ntot [#] 16-18 11-12 9 6 6 4 12 6 8 8 7 11 13 8 7 5 8-10 12-14 4 7 16-18 11-12 16-18 11-12

Initial Enantiomeric Excess

As mentioned in Section 2, previous experimental studies have shown that an increase of the initial enantiomeric excess accelerates the deracemisation process and improves the experimental reproducibility 10,17,19,23 . This effect of the ee0 has also been demonstrated for attrition enhanced deracemisation in a comprehensive series of simulations elsewhere 24 . In light of these studies, we have performed experiments at three different values of ee0 , namely 7%, 18%, and 32%, in Exps. 1, 2, and 3, respectively, whose results are illustrated in Figure 4. Exp. 2 has attained resolution after 9 hours, while Exp. 3 after 6; since during Exp. 1 no samples could be taken between 12 and 22 hours, one could not determine exactly the time when the process had ended. However, the results of Exps. 1a-1b, conducted at similar

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conditions and reported for the sake of comparison in Figure 4 as green circles (see Figure 10 and Section 3.4), suggest that Exp. 1 should have been completed after 16-18 hours. In any case, the samples collected indicate that deracemisation has certainly been attained after 24 hours. Figure 4 also shows that ee increases more rapidly for higher values of ee0 (Exp. 3 > Exp. 2 > Exp. 1), even though the rate of variation of ee in time (at each value of ee) does not differ appreciably in the three experiments.

Figure 4: The evolution of the ee over time at different ee0 . The results are represented by green squares (Exp. 1, ee0 = 7%), orange diamonds (Exp. 2, ee0 = 18%), and red triangles (Exp. 3, ee0 = 32%). The filled, edged, and open symbols with dot represent the data points of the three repetitions conducted for every experiment. The green dots correspond to Exp. 1b, conducted at the same conditions as Exp. 1, but with VS = 8 mL.

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3.2

Cooling Rate

Previous works have reported contradicting results about the effect of the cooling rate Rc on the deracemisation process 21,23 . Suwannasang et al. found that the total process time did not change with the cooling rate, but that more cycles were required, if the operating conditions favoured secondary nucleation of the undesired enantiomer, for example by operating with fast cooling 21 . On the contrary, Li et al. reported that neither the total process time nor the number of cycles were significantly influenced by the cooling rate 23 . In order to better understand the effect of the cooling rate, we have performed experiments at four different values of Rc : 0.14, 0.22, 0.43, and 1.30 ◦ C/min, in Exps. 2b, 2, 2c, and 2d, respectively. Note that each value of Rc corresponds to a partially different temperature profile, thus to a different duration of the cycle (Figure 5). We have also investigated the limit case Rc = 0 (Exp. 2a), where we have operated the system at constant temperature (25 ◦

C) to assess if deracemisation could occur in a comparable time due to attrition only. As

one can see in Figure 6 (pink triangles), at these conditions deracemisation never occurred.

Figure 5: The temperature profiles applied in the experiments. Tmin and TMAX were the same in all the tests of this series, but different cooling rates have been tested: 0.14 ◦ C/min (Exp. 2b, green dashed line), 0.22 ◦ C/min (Exp. 2, orange dotted line), 0.43 ◦ C/min (Exp. 2c, blue dash-dotted line), and 1.30 ◦ C/min (Exp. 2d, red solid line).

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The results of the experiments in terms of evolution of ee at different cooling rates are reported in Figure 6a as function of time and in Figure 6b as function of the number of cycles. Exp. 2b is clearly the slowest one, taking about 12 hours to attain complete deracemisation, while Exps. 2, 2c, and 2d are faster, attaining deracemisation after 9, 8, and 7 hours, respectively. In spite of their rather similar behaviour, one can still observe a clear trend for Exps. 2, 2c, and 2d, with the total process time reducing for increasing values of Rc (Figure 6a). Contrary to the total process time, ttot , the total number of cycles, ntot , required by the deracemisation process increases with Rc : total resolution is attained in 8 and 11 cycles in Exp. 2c and 2d, respectively, while it occurs in 6 cycles for both Exps. 2 and 2b (Figure 6b). This behaviour is not consistent with the observations of Li et al. 23 , but it does not contradict those of Suwannasang et al. 21 , i.e. that secondary nucleation and growth of the undesired enantiomer may occur for high values of Rc , thus slowing the deracemisation process. Note also that since the cycles are shorter when Rc is higher, the total time can still decrease, despite the increase of total number of cycles. Finally, the similarity of Exps. 2, 2c, and 2d suggests that the system attains asymptotic behaviour for an infinitely fast cooling. This would be consistent with the mechanism of the temperature cycles discussed in Section 2.3, since, during a single cycle, a finite amount of material can be dissolved, hence converted from one enantiomer to the other. On the contrary, when Rc decreases, the number of cycles should reduce, but eventually attain a constant value, while the total process time increases, given that the individual cycle becomes longer.

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(a)

(b)

Figure 6: The evolution of enantiomeric excess at different cooling rates, Rc , (a) ee as a function of time and (b) as a function of the number of cycles performed. The results are represented by green triangles (Exp. 2b, 0.14 ◦ C/min), orange diamonds (Exp. 2, 0.22 ◦ C/min), blue squares (Exp. 2c, 0.43 ◦ C/min), and red circles (Exp. 2d, 1.30 ◦ C/min). Exp. 2a, performed as control experiment at 25 ◦ C, is reported in pink triangles. The filled, edged, and open symbols with dot represent the data points of the three repetitions conducted for every experiment.

3.3

Operating Temperature Range

Li et al. studied the influence of the operating temperature range, ∆T , by operating at constant Tmin , thus changing the fraction of solid dissolved in every cycle, for each different ∆T investigated 23 . We have performed experiments at different ∆T , but keeping ∆c constant, hence being able to directly correlate the effect of temperature on the total process time, since the amount of solid dissolved was the same at each ∆T considered. We have investigated three values of Tmin and their associated values of TMAX (see Eq. 1), at two different cooling rates, i.e. Rc = 0.22 ◦ C/min and Tmin = 20, 25, and 30 ◦ C (the experiments are labelled 2− , 2 and 2+ , respectively), and Rc = 1.30 ◦ C/min and again Tmin = 20, 25, and 30 ◦ C (the experiments are labelled 2d− , 2d and 2d+ , respectively). Figure 7a shows the attainable regions in the (T, c)-plane, corresponding to Tmin = 20 ◦ C, and 30 ◦ C (heating and cooling 15



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stages are indicated by red and blue arrows respectively; light and dark shades correspond to Exps. 2− -2d− and Exps. 2+ -2d+ , respectively), together with that for Tmin = 25 ◦ C (red and blue areas for heating and cooling stages, respectively). Since the solubility increases non-linearly with the temperature, ∆T is smaller for higher Tmin and the cycles are shorter at higher Tmin , because the cooling rate is constant (Figure 7b).

(a)

(b)

Figure 7: (a) The attainable regions. As in Figure 3 we report the solubility curve of NMPA in IPA:ACN (95:5 w/w) and the attainable regions at Tmin = 25 ◦ C (coloured areas). Red and blue shaded arrows, for the heating and the cooling period, respectively, represent the attainable region at Tmin : 20 ◦ C (orange and light blue), 25 ◦ C (red and blue), and 30 ◦ C(dark red and blue). (b) The temperature profiles defined at the three Tmin : 20◦ C (Exps. 2− - 2d− , blu lines), 25◦ C (Exps. 2 - 2d, orange lines), 30◦ C (Exps. 2+ - 2d+ , red lines), and with two cooling rates: 0.22 ◦ C/min (Exps. 2− , 2, and 2+ , dotted lines) and 1.30 ◦ C/min (Exps. 2d− , 2d, and 2d+ , solid lines).

Let us consider in more detail the experiments, starting first with those at Rc = 0.22 ◦

C/min, in Figure 8a. The process is completed in 13, 9, and 7 hours for Exps. 2− , 2, and

2+ , respectively; similar results are also obtained in Exps. 2d− , 2d, and 2d+ with Rc = 1.30

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◦

C/min (Figure 9a), where the resolution time decreases for increasing Tmin , i.e. Exp. 2d− >

Exp. 2d > Exp. 2d+ . Considering all investigated values of Tmin , the process is faster at higher temperatures; additionally, deracemisation is faster also for higher Rc , in agreement with the observations of Section 3.2. Similarly, for a constant value of Rc , more cycles have to be performed when Tmin is lower, hence the number of cycles increases with the total process time (Figures 8b and 9b). Interestingly, the experiments seem more reproducible at higher Tmin , likely due to a different dependence on the temperature of the several phenomena active in the process, for example, the racemisation reaction and the crystal growth. Since all experiments in this work start with an enantiomeric excess of the (S)-enantiomer in the solid phase, during the cooling stage (S)-crystals consume more solute from the solution than (R)-crystals. The concentration of the (S)-enantiomer in solution, initially with a 1:1 ratio, decreases faster than that of the (R)-enantiomer: this variation should be compensated by the racemisation reaction. Nevertheless, if racemisation is not fast enough, the concentration of the (R)-enantiomer (hence, its supersaturation) would become larger than that of the (S)-enantiomer, then promoting growth and possibly secondary nucleation of the (R)-enantiomer. The occurrence of these phenomena could lead not only to a higher process variability, but also to a greater number of cycles required to attain complete resolution (see Section 3.2). Preliminary studies of the racemisation kinetics suggest that such a reaction is in this system indeed sensitive to the temperature, so that, racemisation may be the phenomenon affecting most the process reproducibility when operating in different temperature ranges.

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(a)

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(b)

Figure 8: The evolution of ee at different temperature intervals, but at constant Rc = 0.22 C/min. (a) ee as a function of time and (b) as a function of the number of cycles performed. The results are represented by red triangles (Exp. 2+ , Tmin = 30◦ C), orange diamonds (Exp. 2, Tmin = 25◦ C), and blue circles (Exp. 2− , Tmin = 20◦ C). The filled, edged, and open symbols with dot represent the data points of the three repetitions conducted for every experiment. ◦

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(a)

(b)

Figure 9: The evolution of ee at different temperature intervals, but at constant Rc = 1.30 C/min. (a) ee as a function of time and (b) as a function of the number of cycles performed. The results are represented by red triangles (Exp. 2d+ , Tmin = 30◦ C), orange diamonds (Exp. 2d, Tmin = 25◦ C), and blue circles (Exp. 2d− , Tmin = 20◦ C). The filled, edged, and open symbols with dot represent the data points of the three repetitions conducted for every experiment. ◦

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3.4

System Volume and Scalability

The mathematical models describing attrition enhanced deracemisation 13,26,29–36 and temperature cycles 37–39 do not depend explicitly on the size of the system, but only on its intensive properties. Nevertheless, additional factors not accounted for in such models could influence the process performance when the system is scaled up, e.g. the fluid dynamic regime. To verify this possibility, we have performed a series of experiments varying the total volume of the system from 1.8, to 4.0, and to 8.0 mL (Exps. 1, 1a, and 1b in Table 2, respectively). Note that the larger volumes were operated at similar, but not identical, mixing conditions, since the geometry of the Easymax vessel is not the same as that of Crystal16. Figure 10 shows that deracemisation is completed after 16-18 hours in Exps. 1a-1b. For Exp. 1 it was not possible to collect data at this time, but one can hypothesise that total deracemisation was attained after the same time or possibly slightly earlier, since the ee evolution overlaps in the three cases during the first 12 hours and also afterwards for Exps. 1a and 1b. One can see that the tests conducted in the three volumes are not significantly different from each other. Therefore, the system volume does not seem to affect the process time, as long as the system is well-mixed and the fluid dynamic conditions (which influence particles attrition, secondary nucleation, etc.) do not appreciably change, in agreement with the results for attrition enhanced deracemisation.

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Figure 10: The evolution of ee over time with cooling rate 0.22 ◦ C/min and ee0 7% for experiments performed in reactor of volume: 1.8 mL (Exp. 1, green squares), 4.0 mL (Exp. 1a, blue circles), and 8.0 mL (Exp. 1b, orange triangle).

4

Discussion and Conclusions

In this work, we have investigated the influence of several operating parameters on the deracemisation of NMPA via temperature cycles, aiming at understanding better the features of this technique. The experimental results suggest that the operating conditions influence not only the total time required by the process, but also the process reproducibility. Thereafter the relevant observations are as follows: • The higher ee0 , the faster the system attains total deracemisation and also the more reproducible the process is. • Faster cooling rates correspond to shorter process times; the cooling rate does not seem to affect the process reproducibility in the investigated system. This result could be due to the relatively high solid density in the suspension, which could favour the

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growth of the crystals already present rather than the formation of new ones. Further investigations are required to assess if a fast Rc lowers the reproducibility in case of low solid density, a condition at which nucleation could become more significant; • When the amount of crystals dissolved during each cycle is kept constant, performing experiments at a higher operating temperature, Tmin , reduces the process time as well as the number of cycles required to attain deracemisation. Even though both growth and racemisation kinetics likely accelerate at higher temperatures, preliminary studies suggest that racemisation should be the phenomenon most sensitive to the choice of Tmin , in the system considered here. • The volume of the system does not influence the behaviour of the deracemisation process, at least for crystallisers with volume in the range 1 to 10 millilitre. Whether this volume invariance also applies at larger scales has to be checked, particularly because different mixing conditions might affect the process significantly. Let us now discuss how the operating conditions can be tuned to improve the process performance in terms of productivity, P . This is defined as the ratio between the total mass of target enantiomer produced during the process per unit mass of solvent (which is computed as the product of the solid suspension density, ρ, multiplied by half of the difference between the final, eef , and the initial, ee0 , enantiomeric excess) and per unit process time, ttot (which is calculated as the total time to reach complete deracemisation): ρ P = ttot (ee0 , Rc , Tmin , ρ)

eef − ee0 2

(2)

Clearly, the productivity increases when the amount of target enantiomer converted increases, and reduces when the process time increases. The numerator in Eq. 2 can be incremented by operating with a lower ee0 or with a larger ρ. The denominator can be reduced by operating at high values of ee0 , of the cooling rate, or of Tmin . Note that ee0 and ρ play an opposite role in the numerator and in the denominator. As a consequence, a 22


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maximum value of the productivity as a function of ee0 and ρ could exist, even though it may not be experimentally detectable. Figure 11 shows how the productivity changes with ee0 , Rc , and Tmin . In Figure 11a, P keeps increasing with ee0 and no maximum value seems to occur for the values investigated. In Figure 11b, the productivity seems to be approaching an asymptotic value for increasing cooling rates, while Figure 11c shows that P increases monotonically with Tmin . Note that also in this case, for a fixed temperature range, the productivity is higher when the cooling rate is faster. Even though one might be tempted to consider as a general rule that high values of Rc and Tmin are better, it is important to recall that the choice of the operating conditions depends also on additional constraints, such as the solvent evaporation, the energy consumption, and the specific chemistry of the system. For instance, high temperatures accelerate the racemisation reaction, but may be detrimental with thermolabile compounds, or in the presence of undesired reactions forming side products.

(a)

(b)

(c)

Figure 11: The effect of the investigated operating conditions on the productivity. (a) The productivity as a function of the ee0 : 7% (Exp. 1, green squares), 18% (Exp. 2, orange diamonds), and 32% (Exp. 3, red triangles). (b) The productivity as a function of the cooling rates perfomed: 0.14 ◦ C/min (Exp. 2b, green triangles), 0.22 ◦ C/min (Exp. 2, orange diamonds), 0.43 ◦ C/min (Exp. 2c, blue squares), and 1.30 ◦ C/min (Exp. 2d, red circles). (c) For two different cooling rates, the productivity as a function of the temperature range, defined by the minimum temperature: 20 ◦ C (Exp. 2− and 2d− , blue circles), 25 ◦ C (Exp. 2 and 2d, orange diamonds), 30 ◦ C (Exp. 2+ and 2d+ , red triangles). The lines are guides to the eye.

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It is clear that adopting an empirical approach for the proper selection of the optimal operating conditions requires a large number of experiments. On the contrary, the development of a mathematical model would not only greatly reduce the experimental effort needed for determining the optimal conditions, but also improve the understanding of the fundamental phenomena characterising the process. Thus concluding, the production of enantiopure compounds is an important goal of the pharmaceutical industry and deracemisation via temperature cycles is a promising technique. The experiments show that the deracemisation of a scalemic suspension of a conglomerate forming compound can be successfully and reproducibly attained via temperature cycles. Our robust experimental procedure has allowed to investigate the effect of the chosen operating conditions on the process and on its productivity. The results reported here represent the basis for a further development and optimisation of deracemisation via temperature cycles to an industrially relevant scale.

Acknowledgements This research received funding as part of the CORE project (October 2016-September 2020) from the European Unions Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 722456 CORE ITN.

Supporting information Supporting information is available free of charge on the ACS Publication website. It reports a preliminary evaluation of the racemisation reaction and its features, including a description of the experimental protocol and of the analytical method (chiral HPLC) used to measure the enantiomeric excess in the samples.

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For Table of Contents Use Only Deracemisation of NMPA via temperature cycles Francesca Breveglieri, Giovanni Maria Maggioni, Marco Mazzotti

In this paper, we experimentally investigate the deracemisation of the conglomerate forming compound NMPA via temperature cycles in the presence of a racemising agent. We examine how different operating parameters affect the deracemisation process time and the total number of cycles to attain deracemisation in order to discuss how to improve the process performance.

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Deracemization of NMPA via Temperature Cycles - Crystal Growth

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