A Biocatalytic Route to the Novel Antiepileptic Drug Brivaracetam

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A bio-catalytic route to the novel anti-epileptic drug Brivaracetam Arnaud Schule, Alain Merschaert, Christophe Szczepaniak, Christophe Marechal, Nicolas Carly, John O'Rourke, and Celal Ates Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.6b00094 • Publication Date (Web): 24 Aug 2016 Downloaded from http://pubs.acs.org on August 25, 2016

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Organic Process Research & Development

A bio-catalytic route to the novel anti-epileptic drug Brivaracetam Arnaud Schülé*, Alain Merschaert, Christophe Szczepaniak, Christophe Maréchal, Nicolas Carly, John O’Rourke and Célal Ates. UCB Pharma S.A., Chemical Process Research and Development, Pharma Sciences, Chemin du Foriest, 1420 Braine l’Alleud, Belgium.

ACS Paragon Plus Environment

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Table of Contents

Enzymatic resolution 42% yield 97.1% ee 4

6

Brivaracetam 1


2

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Abstract

An asymmetric synthesis of the novel antiepileptic drug Brivaracetam 1 is described. The stereochemistry of the 4-n-propyl substituent is introduced by a bio-catalytic resolution of (rac)methyl 2-propylsuccinate 4-tert-butyl ester 4. The selection of the resolution substrate and the screening of enzymes were carried out from our in-house screening platform. The development and scale-up of the best conditions including solvent media, pH control, work-up and enzyme supply led up to a successful demonstration conducted at 1 kg scale in a 10 L vessel. The chiral intermediate (R)-2-propylsuccinic acid 4-tert-butyl ester 6 was reproducibly obtained in 42% yield and 97% ee all along the development. The control of the stereochemistry via the biocatalytic resolution allowed the production of Brivaracetam 1 within the required commercial quality specifications.

Keywords: enzymatic resolution, scale-up, process chemistry, Brivaracetam


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Introduction to Brivaracetam and control of the stereochemistry Brivaracetam 1, (2S)-2-[(4R)-2-oxo-4-propyltetrahydro-1H-pyrrol-1-yl]butanamide also referred to as ucb 34714, is a novel antiepileptic drug (AED) with a high-affinity for synaptic vesicle protein 2A ligand1 (SV2A), that successfully went through clinical Phase III2. Brivaracetam 1 was recently approved in Europe and United States as adjunctive therapy in the treatment of partial onset seizures in patients of 16 years age and older with epilepsy under the trade name of Briviact®. The control of the stereochemistry of the 4-n-propyl moiety is the major challenge from a synthetic perspective. The stereochemistry of the (2S) center might be accessed and controlled by the widely available (S)-2-aminobutanamide 2 building block. In our continuous efforts to find new and potentially useful synthetic approaches that could be applied on large scale, the transformations on the succinate derivatives 3-6 were selected for further investigation (scheme 1). Scheme 1. Succinate intermediates towards Brivaracetam

2

3, R=Et, 4, R=Me 5, R=H

6

Brivaracetam 1, ucb 34714

ucb 34713

The transformation from the racemic series 3-5 into the chiral intermediate 6 was envisaged by both an enzymatic resolution3 (3-4→6) and a salt resolution (5→6). The synthesis of the three resolution substrates 3, 4 and 5 at laboratory scale is described in the Supporting Information.


4

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Enzymatic resolution approach The evaluation of the bio-catalytic resolution of substrates 3 and 4 was performed with the screening of four families of enzymes4: lipases (30 enzymes), acylase (1 enzyme), protease (15 enzymes) and esterase (30 enzymes). The objective was to be able to perform readily a demonstration at scale if some hits were identified in the perspective of a potential industrialization. Scheme 2. Bio-catalytic resolution of (rac)-ethyl ester 3 and (rac)-methyl ester 4

Enzymatic resolution

3, R=Et 4, R=Me

6

7, R=Et 8, R=Me

The screening of enzymes was performed in the following conditions: 20 mg of substrate, ca. 50% w/w of enzyme load, a phosphate buffer (KH2PO4, K2HPO4, 0.1 M, pH 7.5, 90 mL/g), methyl tert-butyl ether (MTBE) as co-solvent (12.5 mL/g), 27.5 °C during ca. 16 hours on a shaking plate at 250 rpm5. The full screening of the enzymes was performed on the ethyl ester substrate 3. No conversion was observed with the acylase and esterase families. The best results were obtained with lipase and protease families, especially alkaline protease B and protease C from Bacillus subtilis with 97-98% ee. The results are summarized in the table 1. Table 1. Hydrolysis results on (rac)-ethyl 2-propylsuccinate 4-tert-butyl ester 3


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Sub-class of the hydrolase Lipase Lipase Protease Protease

Strain of the hydrolase

Conversion (%)

A from Rhizomucor miehei C from Rhizomucor miehei Alkaline B C from Bacillus subtilis

13 52 40 14

ee of (S)ethyl ester 7 (%) 11 69 64 15

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ee of (R)succinic acid 6 (%) 75 63 97 98

E value6 8 9 148 96

The influence of alternative co-solvents and the absence of a co-solvent were also evaluated on the four identified hits by testing cyclohexane, dimethyl sulfoxide, acetone and toluene, all other parameters were kept unchanged. The results are reported in the table 2. Table 2. Influence of alternative co-solvents and absence of a co-solvent

Sub-class of the hydrolase Protease Protease Lipase Lipase Protease Protease Lipase Lipase Protease Protease Lipase Lipase Protease Protease Lipase Lipase Protease Protease Lipase Lipase


Cosolvent

Conv. (%)

Alkaline B C from Bacillus subtilis A from Rhizomucor miehei C from Rhizomucor miehei Alkaline B C from Bacillus subtilis A from Rhizomucor miehei C from Rhizomucor miehei Alkaline B C from Bacillus subtilis A from Rhizomucor miehei C from Rhizomucor miehei Alkaline B C from Bacillus subtilis A from Rhizomucor miehei C from Rhizomucor miehei Alkaline B C from Bacillus subtilis A from Rhizomucor miehei C from Rhizomucor miehei

54 40 No 58 100 33 0 Cyclo60 hexane 71 55 Dimethyl 51 sulfoxide 53 100 56 51 Acetone 41 77 ≤ 15 0 Toluene ≤ 15 ≤ 15

ee of (S)ethyl ester 7 (%) 99 64 82 47 99 99 99 99 72 99 99 48 99 -

ee of (R)succinic acid 6 (%) 85 98 59 98 65 40 81 94 65 77 95 70 30 -

E value 65 190 9 176 23 10 48 167 10 38 210 9 8 -


6

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In general, the results using the protease hits were better than the ones using the lipase hits: a conversion close to 50%, a high ee for the desired product 6 and also a higher E value. Dimethyl sulfoxide and acetone emerged as alternative co-solvents to methyl tert-butyl ether used in the screening. The absence of a co-solvent also led to similar good results. The lipase hits appeared to be less stereoselective with higher conversions, sometimes nearly complete. Based on the resolution results obtained with the ethyl substrate 3, the methyl derivative 4 was tested with the four enzymes hits only in order to optimize the screening duration. The screening conditions were the same than the ones used for the ethyl derivative 3, expending the screening to the best co-solvents identified with the ethyl substrate 3. The results are reported in the table 3. Table 3. Hydrolysis results on (rac)-methyl 2-propylsuccinate 4-tert-butyl ester 4 Sub-class of the hydrolase Protease Protease Lipase Lipase Protease Protease Lipase Lipase Protease Protease Lipase Lipase Protease Protease Lipase Lipase Protease Protease Lipase Lipase


Cosolvent

Conv. (%)

Alkaline protease B C from Bacillus subtilis A from Rhizomucor miehei C from Rhizomucor miehei Alkaline protease B C from Bacillus subtilis A from Rhizomucor miehei C from Rhizomucor miehei Alkaline protease B C from Bacillus subtilis A from Rhizomucor miehei C from Rhizomucor miehei Alkaline protease B C from Bacillus subtilis A from Rhizomucor miehei C from Rhizomucor miehei Alkaline protease B C from Bacillus subtilis A from Rhizomucor miehei C from Rhizomucor miehei

57 52 No 61 100 50 ≤ 10 Cyclo62 hexane ~75 ~74 Dimethyl 55 sulfoxide 52 ~98 64 53 Acetone ~24 55 51 ~33 MTBE ≤ 10 ~28

ee of (S)methyl ester 8 (%) 99 99 95 96 99 99 77 99 99 82 99 -

ee of (R)succinic acid 6 (%) 76 91 60 96 62 83 72 57 89 68 94 -

E value

37 115 14 180 21 54 14 17 93 13 162 -


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The trends obtained previously with the ethyl substrate 3 were also observed with the methyl derivative 4. Proteases were again more efficient than the lipases that appeared significantly less stereoselective. At this stage of the screening, with the results in hands and insight on enzymes prices at larger scale7, the two proteases hits were selected to progress further in the development of the enzymatic resolution. Both ethyl and methyl substrates 3 and 4 were similarly attractive at this stage of the evaluation. In order to differentiate the two substrates, parameters of the screening conditions were challenged to reflect more realistic industrial resolution conditions: 10%-w of enzyme load (vs. 50% w/w), 25 mL/g of phosphate buffer/pH 7.5 (vs. 90 vol), 2 mL/g of co-solvent (vs. 12.5 mL/g) and 30 °C for the temperature. The tests were performed on 100 mg of substrates 3 and 4. The results are reported in the table 4. Table 4. Hydrolysis results on 3 and 4 with more realistic operating conditions

Substrate


Co-solvent

4, R=Me 4, R=Me 3, R=Et 3, R=Et 4, R=Me 4, R=Me 4, R=Me 3, R=Et 3, R=Et 4, R=Me 4, R=Me 3, R=Et 3, R=Et 4, R=Me 3, R=Et

Alkaline B C from Bacillus subtilis Alkaline B C from Bacillus subtilis Alkaline B Alkaline B C from Bacillus subtilis Alkaline B C from Bacillus subtilis Alkaline B C from Bacillus subtilis Alkaline B C from Bacillus subtilis Alkaline B Alkaline B

No Cyclohexane Dimethyl sulfoxide

Acetone

MTBE

Conv. (%) 43 28 31 6 33 45 36 38 22 46 34 36 20 43 25

ee of (S)derivative 7 or 8 (%) 75 39 44 6 48 80 57 59 27 82 50 55 25 72 32

ee of (R)succinic acid 6 (%) 97 98 98 99 98 97 98 98 98 97 98 98 99 97 98

E value 149 126 154 211 135 162 170 182 156 168 139 182 169 143 175


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In these resolution conditions, the methyl ester derivative 4 emerged as the best resolution substrate with both proteases. Additionally, the methyl ester derivative 4 was also more attractive from a synthesis perspective thanks to the efficient one-pot dealkoxycarbonylation transformation (scheme 3). The alkaline protease B gave also better results than the protease C from Bacillus subtilis allowing to nominate a lead enzyme and a back-up one if necessary. It was worthwhile to note that the absence of a co-solvent gave similar results than the resolution using a co-solvent. This option without a co-solvent could be attractive to simplify the work-up of the reaction. The temperature influence was also evaluated (35 °C vs. 30 °C) without significant impact on the outcome. In a similar way, the quality of the substrate – a polishing distillation vs. a chromatography – did not reveal any resolution difference. Work-up of the enzymatic resolution The opportunity to conduct the resolution without any co-solvent was prioritized to simplify the work-up. Once the resolution completed (pH 7.5), the unreacted (S)-methyl ester derivative 8 was extracted with cyclohexane. The analysis of the discarded organic layer showed that ca. 12% of the succinic acid derivative 6 was also eliminated. To circumvent that issue the pH was adjusted at 9 before the extraction. In these conditions, the product 6 was totally ionized into the succinate form and remained in the aqueous phase without any racemization identified. The pH was then adjusted to pH 1 in order to extract the product 6 with isopropyl acetate. During both liquid-liquid separations at pH 9 and pH 1, some foaming was observed probably due to protein material but also due to amphiphilic properties of the product 6. Moreover, at pH 1, the precipitation of the denatured enzyme was observed. After some tests, the most appropriate


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solution found was to pre-filter the biphasic mixture prior to separation with addition of diatomaceous earth (Celite®) which enhances the filtration by adsorbing the protein material. The final work-up procedure was conducted in the following conditions: the unreacted ester 8 is extracted in cyclohexane (1 x 2 mL/g) at pH 9 (adjustment with NaOH 0.1 M solution, ca. 0.2 mL/g). The pH is then adjusted to pH=1 with HCl 3 M (1 mL/g). Isopropyl acetate (3 mL/g) and Celite (17% w/w) are added then the slurry is filtered. The cake is washed with isopropyl acetate (1 mL/g) and the liquid-liquid separation is performed. The organic layer is finally evaporated to give 6 as an oil8. Supply of the enzyme at scale At the time of performing the initial scale-up, the most promising enzymes – protease alkaline B and protease C from Bacillus subtilis – could unfortunately not be provided immediately by the supplier at 2 kg scale. In agreement with the supplier, alternative enzymes from the same strains available from the supplier’s stock were therefore quickly evaluated. The back-up protease C from Bacillus subtilis was replaced by two similar strains referenced as type 1 and type 2 respectively. The lead enzyme protease alkaline B was replaced by alkaline B n°2 whose formulation was described as different. The enzymatic resolution was tested on the methyl ester substrate 4 in the second screening conditions: 100 mg of substrate, 10%w/w of enzyme load, 25 mL/g of phosphate buffer (pH 7.5), 2 mL/g of co-solvent (or not) and 30 °C for the temperature. The results are reported in the table 5. Table 5. Hydrolysis results on 4 with new operating conditions


10

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Co-solvent

Strain of the protease

Alkaline B n°2 Protease C from Bacillus subtilis (type 1) No Protease C from Bacillus subtilis (type 2) Alkaline B n°2 Cyclohexane Protease C from Bacillus subtilis (type 1) Protease C from Bacillus subtilis (type 2) Alkaline B n°2 Protease C from Bacillus subtilis (type 1) DMSO Protease C from Bacillus subtilis (type 2) Alkaline B n°2 Protease C from Bacillus subtilis (type 1) Acetone Protease C from Bacillus subtilis (type 2) Alkaline B n°2 Protease C from Bacillus subtilis (type 1) MTBE Protease C from Bacillus subtilis (type 2)

Conv. (%) 17 16 33 11 6 20 9 15 36 26 15 36 20 2 27

ee of (S)methyl ester 8 (%) 20 19 48 11 6 25 9 18 54 34 17 56 23 2 35

ee of (R)succinic acid 6 (%) 97 99 97 95 99 97 96 98 97 97 98 97 96 99 98

E value 117 240 105 43 211 83 56 121 116 90 144 138 69 203 114

The back-up from the lead enzyme alkaline B n°2 and the protease C from Bacillus subtilis (type 1) gave poor results in the conditions tested. Protease C from Bacillus subtilis (type 2) gave acceptable results whereas poorer than the best results obtained during the screening phases especially regarding the conversion (33-36% vs. 43-45% with the former lead enzyme). The influence of the pH and the enzyme loading was tested in order to evaluate the scope of potential improvements for the resolution. A pH 8.0 was tested and the enzyme loading was fixed between 10% w/w and 20% w/w. All other parameters being unchanged compared to the second screening conditions. Two enzymes were tested: the best from each back-up strain provided. The results are reported in the table 6.


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Table 6. Influence of pH and enzyme load on the hydrolysis of 4

Enzyme load (%-w)

pH

Strain of the protease

10 15 20 15 10 15 20 15 10 15 20 15 10 15 20 15 10 15 20 15

8.0 8.0 8.0 8.0 8.0 8.0 8.0 8.0 8.0 8.0 8.0 8.0 8.0 8.0 8.0 8.0 8.0 8.0 8.0 8.0

C from Bacillus subtilis (type 2) C from Bacillus subtilis (type 2) C from Bacillus subtilis (type 2) Alkaline B n°2 C from Bacillus subtilis (type 2) C from Bacillus subtilis (type 2) C from Bacillus subtilis (type 2) Alkaline B n°2 C from Bacillus subtilis (type 2) C from Bacillus subtilis (type 2) C from Bacillus subtilis (type 2) Alkaline B n°2 C from Bacillus subtilis (type 2) C from Bacillus subtilis (type 2) C from Bacillus subtilis (type 2) Alkaline B n°2 C from Bacillus subtilis (type 2) C from Bacillus subtilis (type 2) C from Bacillus subtilis (type 2) Alkaline B n°2

Cosolvent

No

Cyclohexane

Dimethyl sulfoxide

Acetone

MTBE

Conv. (%) 36.6 39.5 41.2 33.8 22.0 26.9 33.8 21.2 41.7 42.4 44.2 39.3 38.2 42.0 43.2 38.0 28.1 32.0 34.0 26.9

ee of (S)methyl ester 8 (%) 55.7 63.0 67.8 49.6 27.4 35.7 49.6 25.7 68.7 70.8 75.9 62.2 60.1 69.8 73.3 59.0 38.1 45.9 50.0 35.4

ee of (R)E succinic value acid 6 (%) 96.6 96.4 96.9 97.0 97.3 97.1 97.2 95.6 96.2 96.3 95.8 96.1 97.0 96.4 96.2 96.3 97.8 97.6 97.3 96.3

101 104 130 107 95 96 115 58 107 111 106 96 123 114 114 95 131 128 119 74

A positive effect of the pH on the conversion was observed for both enzymes, maintaining similar ee (96-97%) for the product 6. This was particularly true for the alkaline B n°2 with a conversion improvement from 9-26% at pH 7.5 (table 5) to 21-38% at pH 8.0 (Table 6). Nevertheless, the best enzyme remained the protease C from Bacillus subtilis (type 2) either without co-solvent or with dimethyl sulfoxide and acetone as co-solvent. A 10-15% w/w of enzyme loading might appeared as an acceptable compromise between conversion and enzyme price at this stage of the study. The new lead enzyme was supplied at kg scale without any issue during the development of the project9.


12

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Scale-up preparation for the enzymatic resolution The switch from the phosphate buffer, used in the screenings, to aqueous media with pH control in order to limit the overall volume of reaction for industrial perspectives was identified as a key factor to ensure the successful enzymatic scale-up from the screening conditions to standard agitated vessels. The phosphate buffer solution (pH 7.5 at 0.1 M) was replaced by water with a pH stat Metrohm® Titrando 902 using a NaOH solution in order to control the pH and to limit the overall dilution. Controlling the pH of the reaction mixture during the resolution by adding a NaOH solution might lead to the ester hydrolysis as a side-reaction in case of pH overshoot. This was confirmed by a stress test with the substrate 4 in NaOH 1M at 30 °C overnight. As expected, a significant amount of chemical hydrolysis was observed (ca. 12%). Hence, it was crucial to perform the reaction and the scale-up in agitated vessels with the appropriate agitation design and the appropriate positioning of the pH probe and the dosing pipe (NaOH). The set-up of the resolution at laboratory scale was done in a 250 mL double-jacketed reactor equipped with a multistage mechanical stirrer and the pH stat Metrohm® Titrando 902. Some preliminary blank tests were run by simulating the bio-resolution to determine the appropriate i) dilution of the NaOH solution, ii) positioning of probes/pipes and iii) agitation conditions in order to achieve an smooth pH control10. A first bio-catalytic resolution in the 250 mL reactor set was run in the following conditions: 30 g of the methyl ester substrate 4, 10% w/w of enzyme (protease C from Bacillus subtilis, type 2), 6 mL/g of water, no co-solvent, pH 8.0, 30 °C and a 2 M NaOH solution to control the pH along the resolution. An in-process control after 18.5 hours showed 43% conversion and 97% ee.


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The volume of 2 M NaOH required to control the pH during the resolution was about 1 mL/g. The work-up developed was applied and the (R)-succinic acid derivative 6 was isolated in 33% yield, 95% purity and 97% ee. The modest isolated yield was explained by the number of manipulations in order to recover the final product in this first attempt. The reaction was repeated – same scale and conditions – to evaluate the robustness of the resolution and to assess a more representative yield. The in-process control after 20 hours showed again 43% conversion and 97% ee. After work-up, the (R)-succinic acid derivative 6 was isolated in 42% yield, 95% purity and 97% ee. These results confirmed the bio-resolution route as a valuable stereo-controlling step. When the resolution was conducted at pH 8.5, the yield (42%) and enantiomeric excess (97%) were reproducible in a shorter time without any chemical hydrolysis observed. The usual conversion (ca. 43%) was obtained after ca. 17 hours at pH 8.5 compared to ca. 20 hours at pH 8.0. This result was useful in the perspective of the definition of an acceptable pH range to ensure robustness of the process. The resolution was also tested with 15% w/w of enzyme loading. As expected, the initial rate was higher compared to 10% w/w. But since the kinetic trend is asymptotic, the increase of the average rate was more modest. These observations were confirmed, at the same reaction time, by a yield slightly higher (95% 1H NMR

O

16

6

HBr 33% w/w in AcOH (4 eq) 80 °C, 2 h 30

O

O

Br 89% 93.3% HPLC 93.0% GC

EtO 14

O 3- IPAC (1 L/kg)

O

N

NH 2

32%, 97.1% w/w 95.9% ee

O 1

Figure 1. (a) bio-resolution in a 10 L vessel with pH-stat control equipment. (b) pH monitoring


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The reduction-deprotection transformation of 6 into the propyllactone 11 (5 batches, 2.9 kg, nearly quantitative yield, 91% purity, 95.9% ee21) and the two-step sequence into the bromoester 14 (2 x 3 batches, 3.04 kg, 70% overall yield, 92.5% purity) were achieved without any technical issue at the scale studied. As expected, the final condensation remained the less optimal step from the demonstration with a 32% overall step yield. The condensation was done in 3 batches (2.47 kg, 99% yield, 74% w/w, 85.4%, 1.94% ucb 34713), the pre-treatment in methyl tert-butyl ether was performed in 2 batches but finally appeared as useless with respect to the purity (2.06 kg, 82% yield, 64% w/w, 86.9%, 2.52% ucb 34713). A single batch crystallization afforded the API 1 (0.5 kg, 39% yield) with a purity of 97.1% w/w and 2.03% ucb 34713. The assessment of the potential genotoxic compounds and impurities (GTIs) associated to the synthetic route unsurprisingly showed alerting structures for the tert-butyl 2-bromoacetate and the intermediates 13 and 14 by in-silico Derek analysis22. The GLP Ames testing showed that the compounds tert-butyl 2-bromoacetate and 13 were not mutagenic23.


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Conclusion The control of the stereochemistry of the 4-n-propyl substituent borne by the novel AED Brivaracetam 1 was efficiently achieved by the bio-catalytic resolution of (rac)-methyl 2propylsuccinate 4-tert-butyl ester 4. The selection of the best substrate and the protease C from Bacillus subtilis (type 2) was carried out from our in-house screening platform. The transformation was then optimized in 250 mL laboratory scale reactors with a final demonstration at 10 L scale showing reproducible and consistent results all along the development. The chiral intermediate (R)-2-propylsuccinic acid 4-tert-butyl ester 6 was obtained in 42% yield and 97% ee. A fit-to-purpose development of upstream and downstream chemistry towards Brivaracetam 1 allowed to produce the API within the required specifications. Furthermore, the racemization24 of the unwanted enantiomer 8 was demonstrated offering a recycling opportunity to maximize the overall yield of the resolution. The selection of the biocatalytic route over the salt resolution one also gave the chance to identify an important impurity control point with the isolation of a (S)-phenylethylamine salt of 6. The process mass intensity (PMI) calculated with the kilo-scale demonstration data was 1428 kg/kg API hampered by the weak final step. Indeed, the work-up, the methyl tert-butyl ether treatment and the isolation of the final step represented 68% of the overall PMI. A recalculated metric taken account of the racemization and recycling of 8 and without the useless methyl tert-butyl ether treatment gave a PMI estimation of 785 kg/kg API showing there was still room for improvement. Finally, an optimization of the final step minimizing the suspected substrate degradation and improving the isolation of the API would be necessary to consider this bio-catalytic route to Brivaracetam 1 as a viable commercial route.


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Experimental section Analytical methods for the determination of chemical purity by HPLC and GC: HPLC analysis were run on a Water Alliance 2695 apparatus, column Xbridge C18 (3.5 µm, 4.6 x 50mm), 35 °C, H2O with H3PO4 1%/acetonitrile from 90/10 to 10/90 v/v in 5 min, 3 mL/min, UV detection at 210 nm. GC analysis were run on an Agilent 6890, column VF-5ms (15 m x 0.25 mm x 1µm), 25 min run (50 °C/5 min, 50 °C to 280 °C/10 min, 280 °C/10 min), helium, FID detection at 300 °C. The chemical purity of 1 was monitored on HPLC Waters Alliance 2695, column Ascentis Express C18 (2.7 µm, 100 x 4.6 mm), 40 °C, (A) water/phosphoric acid 1000/1 (v/v) and (B) acetonitrile gradient (t = 0 min 95/5 A/B, t = 25 min 64/36 A/B, t = 26 min 95/5 A/B, t = 28 min 95/5 A/B), 1.5 mL/min, UV detection at 205 nm. Analytical methods for the determination of chiral purity: the chiral purity of 6 (enzymatic and salt resolutions) was monitored on an HPLC Water Alliance 2695 apparatus, column AS-H (5 µm, 4.6 x 250mm), 25 °C, IPA 3%/n-heptane 97%/TFA 0.01% v/v, 1 mL/min, UV detection at 210 nm. The chiral purity of 20 was monitored on a GC Agilent 6890, column Macherey – Nagel Lipodex E 25 m x 250 µm x 1 µm, isotherm 130 °C, helium (1 mL/min), FID detection at 220 °C. The chiral purity of 1 was monitored on HPLC, column Chiralpak AD (10 µm, 4.6 x 250 mm), 20 °C, 80/20 (v/v) n-hexane/ethanol, 1 mL/min, UV detection at 205 nm. NMR spectra were recorded on a Bruker Avance 400 MHz instrument. Elemental analyses were performed on an Elementar Vario EL3 apparatus.


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(R)-2-propylsuccinic acid 4-tert-butyl ester.(S)-phenylethylamine salt 9: A 100 mL bottom flask equipped with refrigerant and mechanical agitation is charged with (rac)2-propylsuccinic acid 4-tert-butyl ester 5 (5 g, 23.12 mmol, 1.0 eq), ethyl acetate (30 mL, 6 vol) and (S)-phenylethylamine (2.80 g, 23.12 mmol, 1.0 eq) at room temperature. The salt precipitates within 10 min. The suspension is heated to gentle reflux (80-85°C, oil bath) until the salt has entirely dissolved. The reflux is maintained for 20 min then cooled to room temperature in ca. 1 h 30 min. The salt precipitates at room temperature and is agitated an extra 1 h 30 min at room temperature. The suspension is then cooled to 0-5 °C and filtered. The cake is washed with cold ethyl acetate (10 mL) and dried under vacuum oven at 40 °C to give 9 (3.35 g, 9.94 mmol, 43%) with a chiral purity of 64% de. A 100 mL bottom flask equipped with refrigerant and mechanical agitation is charged with 9 (3.28 g, 9.7 mmol) and ethyl acetate (33 mL) at room temperature. The suspension is heated to gentle reflux (85-90 °C, oil bath) until the salt has entirely dissolved. The reflux is maintained for 20 min then slowly cooled to room temperature in ca. 2 h 30 min. The salt precipitates at ca. 30 °C. The suspension is agitated at room temperature for an extra 1 h 30 min then cooled to 0-5 °C and filtered. The salt is dried under vacuum oven at 40 °C to give 9 (2.01 g, 61%) with a chiral purity of 95.2% de. O1-Tert-butyl O2,O2-dimethyl pentane-1,2,2-tricarboxylate 16: A 60 L double jacketed reactor equipped with a mechanical stirrer and a condenser is charged with dimethyl n-propylmalonate 15 (3000 g, 17.22 mol, 1.0 eq) and THF (6 L, 2 L/kg) under a nitrogen atmosphere. The solution is cooled to -5.0 °C. NaHMDS 40.9% w/w solution in THF (8140 g, 18.08 mol, 1.05 eq) is added maintaining the temperature between -2.8 °C and -0.3 °C


23


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over a period of 1 h 10 min. The addition funnel is washed with THF (0.3 L, 0.1 L/kg). The solution is agitated at -5 °C for 1 hour. Tert-butyl 2-bromoacetate (3529 g, 18.096 mol, 1.05 eq) is added maintaining the temperature between -8.0 °C and -0.3 °C over a period of 1 hour. The addition funnel is washed with THF (0.3 L, 0.1 L/kg). The reaction mixture is warmed to 17 °C in 1 h 15 min and agitated at 20 °C for an additional 1 hour. Water (3 L, 1 L/kg), saturated ammonium chloride solution (3 L, 1 L/kg) and ethyl acetate (9 L, 3 L/kg) are successively added at 20 °C. After decantation the aqueous layer is separated and discarded. The organic layer is washed with 50% saturated ammonium chloride solution (2 x 6 L, 2 x 2 L/kg). After decantation and phase separation, the organic layer is evaporated to give 13 as an oil (5154 g, 17.87 mol, 104%) with a purity of 95.9% GC and 91.8% HPLC. 1H NMR (CDCl3, 400 MHz) 3.67 (s, 6 H) 2.82 (s, 2 H) 1.90-1.86 (m, 2 H) 1.36 (s, 9 H) 1.23-1.12 (m, 2 H) 0.86 (t, J = 7.3 Hz, 3 H) ppm; 13

C NMR (CDCl3, 100 MHz) 171.0, 169.4, 81.1, 55.7, 52.5, 38.9, 35.5, 27.9, 17.7, 14.2 ppm.

Anal. Calcd for C14H24O6: C, 58.32; H, 8.39. Found: C, 57.75; H, 8.49. (rac)-Methyl 2-propylsuccinate 4-tert-butyl ester 4: A 60 L double jacketed reactor equipped with a mechanical stirrer and a condenser is charged with lithium bromide (985 g, 11.34 mol, 1.0 eq), intermediate 16 (3272 g, 11.35 mol, 1.0 eq), water (0.409 L, 22.72 mol, 2.0 eq) and DMF (16.4 L, 5 L/kg) under a nitrogen atmosphere. The reaction mixture is heated to 139 °C for 5 hours. The reaction mixture is cooled to 20 °C in 1 h 05 min. Diisopropyl ether (13.2 L, 4 L/kg), water (6.6 L, 2 L/kg) and saturated ammonium chloride solution (6.6 L, 2 L/kg) are added. After decantation, an additional portion of saturated ammonium chloride solution (2.5 L, 0.76 L/kg) is added in order to have a clear phase separation. After phase separation, the organic layer is washed with water (2 x 10 L, 2 x 3.05 L/kg) then evaporated to give 4 as a crude oil (2467 g, 10.71 mol, 94%) with a purity of


24

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95.9% GC and 89.8% HPLC. 1H NMR (CDCl3, 400 MHz) 3.54 (s, 3 H) 2.70-2.60 (m, 1 H) 2.46 (dd, J = 16.3, 9.4 Hz, 1 H) 2.20 (dd, J = 16.3, 5.2 Hz, 1 H) 1.52-1.40 (m, 1 H) 1.35-1.25 (m, 1 H) 1.28 (s, 9 H) 1.22-1.10 (m, 2 H) 0.76 (t, J = 7.3 Hz, 3 H) ppm;

13

C NMR (CDCl3, 100 MHz)

175.6, 171.1, 80.6, 51.5, 41.2, 37.4, 34.0, 28.0, 20.2, 13.8 ppm. Anal. Calcd for C12H22O4: C, 62.58; H, 9.63. Found: C, 61.64; H, 9.53. (R)-2-propylsuccinic acid 4-tert-butyl ester 6: A 10 L double jacketed reactor equipped with a mechanical stirrer and a condenser is charged with protease C from Bacillus subtilis type 2 (100 g, 10% w/w) and water (4.5 L, 4.5 L/kg). NaOH 0.1 M (30 mL, 0.03 L/kg) is added so as to reach an initial pH of 8.55. Then the racemic substrate 4 (1000 g, 4.34 g, 1.0 eq) is added one pot. The reaction mixture is stirred (200 rpm) at 30 °C maintaining the internal pH at 8.55 with a 2 M NaOH solution (pH-stat) for 18.7 hours (NB: 930 mL, 0.93 L/kg of 2 M NaOH is added overall). The unreacted ester is extracted with cyclohexane (2 L, 2 L/kg) at pH 9 (0.1 M NaOH, 60 mL, 0.06 L/kg). The pH is then adjusted to 2 with 3 M HCl (640 mL, 0.64 L/kg) and isopropyl acetate (3 L, 3 L/kg) is added to extract the product. Celite (167 g, 16.7% w/w) is added into the reactor to facilitate the filtration of the slurry (NB: removal of a part of the denatured enzyme). The cake is washed with isopropyl acetate (0.5 L, 0.5 L/kg). The layers are separated and the organic phase (emulsion) is filtered through the pad of Celite previously obtained. The cake is washed with isopropyl acetate (5 L, 0.5 L/kg). The final organic layer is evaporated (45 °C, 3 mbar) to give 6 (394.4 g, 1.82 mol, 42.0%) with a purity of 95.1% GC, 92.4% HPLC and 97.1% ee. 1H NMR (CDCl3, 400 MHz) 2.88-2.78 (m, 1 H) 2.63 (dd, J = 16.4, 9.3 Hz, 1 H) 2.39 (dd, J = 16.4, 5.2 Hz, 1 H) 1.72-1.60 (m, 1 H), 1.55-1.48 (m, 1 H) 1.45 (s, 9 H) 1.45-1.35 (m, 2 H) 0.94 (t, J = 7.3 Hz, 3 H) ppm ;

13

C


25


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NMR (CDCl3, 100 MHz) 181.5, 171.1, 80.9, 41.2, 37.1, 33.8, 28.0, 20.1, 13.8 ppm; HRMS calculated for [M-H] C11H19O4 215.1283, found 215.1328. (R)-3-n-Propyllactone 11: A 20 L double jacketed reactor equipped with a mechanical stirrer and a condenser is charged with 6 (1000.6 g, 4.63 mol, 1.0 eq) and toluene (8.5 L, 8.5 L/kg). The solution is cooled to -5.1 °C under nitrogen flow. Triethylamine (561.5 g, 5.55 mol, 1.2 eq) is added maintaining the temperature between -6.0 °C and -5.1 °C over a period of 35 min. Ethyl chloroformate (551.9 g, 5.09 mol, 1.1 eq) is added maintaining the temperature between -6.6 °C and -3.4 °C over a period of 38 min. The heterogeneous reaction mixture is stirred for 1 hour at -5 °C. Then the reaction mixture is warmed to 20 °C and filtered to remove the triethylammonium chloride salt. The cake is washed with toluene (1.5 L, 1.5 L/kg) at 20 °C. The filtrate is transferred into the reactor and the solution is cooled to -18.7 °C. Under nitrogen flow, powdered sodium borohydride (349.8 g, 9.25 mol, 2.0 eq) is added one pot at -18.5 °C. Then methanol (2 L, 2 L/kg) is transferred dropwise in order to maintain the temperature below -18 °C and to limit the formation of foaming (08 h 40 min addition time between -21.4 °C and -19.1 °C). The reaction mixture is stirred for minimum 1 hour at -20 °C (15 h 15 min, overnight). At -21.4 °C, under nitrogen flow, HCl 4 M (2 L, 2 L/kg) is added dropwise in order to maintain the temperature below -18 °C and to limit the formation of foaming (05 h 20 min addition time between -21.9 °C and -21.5 °C). Then the reaction mixture is warmed to 20 °C. Water (5 L, 5 L/kg) is added until dissolution of salts and the aqueous layer is discarded. An additional water wash (4 L, 4 L/kg) is done. At 25 °C, trifluoroacetic acid 95% v/v (0.1 L, 0.1 L/kg, ~0.27 eq) is added one pot and the reaction mixture is stirred until the completion of the reaction (~1 h). The organic layer is washed with water (2 x 3 L, 2 x 3 L/kg) then evaporated to give 11 (608.9 g, 4.75 mol, 103%)


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with a purity of 93.2% GC and 95.7% ee GC. 1H NMR (CDCl3, 400 MHz) 4.42 (tapp, J = 8.0 Hz, 1 H) 3.93 (tapp, J = 8.0 Hz, 1 H) 2.65-2.54 (m, 2 H) 2.19 (dd, J = 16.3, 7.3 Hz, 1 H) 1.481.44 (m, 2 H) 1.40-1.30 (m, 2 H) 0.95 (t, J = 7.1 Hz, 3 H) ppm ; 13C NMR (CDCl3, 100 MHz) 177.3, 73.4, 35.4, 35.2, 34.5, 20.5, 13.9 ppm; HRMS calculated for [M+H] C7H13O2 129.0916, found 129.0916. Non isolated intermediate 10 (tert-butyl 3-hydroxymethyl hexanoate): 1H NMR (CDCl3, 400 MHz) 4.42 (dd, J = 9.0, 7.4 Hz, 1 H) 3.92 (dd, J = 9.0, 7.1 Hz, 1 H) 2.65-2.50 (m, 2 H) 2.18 (dd, J = 16.4, 7.4 Hz, 1 H) 1.50-1.44 (m, 2 H) 1.40-1.30 (m, 2 H) 0.94 (t, J = 7.2, 3 H) ppm;

13

C

NMR (CDCl3, 100 MHz) 177.4, 73.4, 69.1, 35.4, 35.2, 34.5, 31.1, 20.5, 13.9 ppm. Anal. Calcd for C11H22O3: C, 65.31; H, 10.96. Found: C, 62.65; H, 10.44. (R)-γ-bromo-3-propylbutyric acid 13: A 10 L double jacketed reactor equipped with a mechanical stirrer, a condenser and a sodium hydroxide scrubber is charged under a nitrogen flow with HBr 33% w/w in acetic acid (4.4 L, 25.49 mol, 4 eq). The solution is cooled to 5.9 °C and a solution of 11 (816.7 g, 6.37 mol, 1.0 eq) in acetic acid (0.4 L, 0.5 L/kg) is added slowly maintaining the temperature at ca. 5 °C over a period of 40 min. The solution is warmed to room temperature and then heated to 80 °C for 2 h 30 min. The reaction mixture is cooled to 20 °C, diluted with water (4.1 L, 5 L/kg) and extracted with dichloromethane (4.1 L, 5 L/kg then 2 L, 2.5 L/kg). The combined organic phases are washed with water (3 x 2.4 L, 3 x 3 L/kg), dried over anhydrous sodium sulfate and evaporated to 13 as a pale yellow oil (1075.9 g, 5.15 mol, 81%) with a purity ≥ 95% by 1H NMR. 1H NMR (CDCl3, 400 MHz) 11.0-10.0 (s, 1 H) 3.60 (dd, J = 10.3, 4.1 Hz, 1 H) 3.51 (dd, J = 10.3, 5.2 Hz, 1 H) 2.59 (dd, J = 16.4, 7.3 Hz, 1 H) 2.44 (dd, J = 16.4, 5.9 Hz, 1 H) 2.28-2.18


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(m, 1 H) 1.50-1.30 (m, 4 H) 0.96 (t, J = 7.0) ppm ;

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13

C NMR (CDCl3, 100 MHz) 179.2, 38.2,

37.4, 36.0, 34.7, 19.7, 13.9 ppm; HRMS calculated for [M+H] C7H14O2Br 209.0177, found 209.0182. Ethyl (R)-γ-bromo-3-propylbutyrate 14: A 10 L double jacketed reactor equipped with a mechanical stirrer and a condenser is charged under a nitrogen atmosphere with 13 (1014.7 g, 4.85 mol, 1.0 eq) and ethanol (4.04 L, 4 L/kg) at 20 °C. HCl 37% (120 g, 100 mL, 1.21 mol, 0.25 eq) is added and the reaction mixture is heated to 40.6 °C for 19 h 05 min. Ethanol is removed by evaporation and the residue is diluted in ethyl acetate (4.04 L, 4 L/kg). The solution is successively washed with aqueous sodium hydroxide (97 g, 2.42 mol, 0.5 eq in 1.52 L, 1.5 L/kg of water) and water (1.52 L, 1.5 L/kg). The organic phase is then evaporated to give 14 as a pale yellow oil (1025 g, 4.32 mol, 89%) with a purity of 93.0% GC and 93.3% HPLC. 1H NMR (CDCl3, 400 MHz) 4.16 (q, J = 7.2, 2 H) 3.57 (dd, J = 10.2, 4.2 Hz, 1 H) 3.51 (dd, J = 10.2, 5.1 Hz, 1 H) 2.50 (dd, J = 16.0, 7.3 Hz, 1 H) 2.35 (dd, J = 16.0, 6.0 Hz, 1 H) 2.25-2.15 (m, 1 H) 1.50-1.30 (m, 4 H) 1.28 (t, J = 7.2, 3 H) 0.94 (t, J = 7.1, 3 H) ppm ; 13C NMR (CDCl3, 100 MHz) 172.3, 60.4, 38.5, 37.6, 36.2, 34.7, 19.7, 14.2, 14.0 ppm; HRMS calculated for [M+H] C9H18O2Br 237.0490, found 237.0493. (2S)-2-[(4R)-2-oxo-4-propyltetrahydro-1H-pyrrol-1-yl]butanamide 1: A 10 L double jacketed reactor equipped with a mechanical stirrer and a condenser is charged under a nitrogen atmosphere with 14 (1041.8 g, 4.39 mol, 1.0 eq), (S)-2-aminobutanamide 2 (673.3 g, 6.60 mol, 1.5 eq), tetrabutylammonium iodide (487 g, 1.32 mol, 0.3 eq), sodium carbonate (932 g, 8.79 mol, 2.0 eq) and isopropyl acetate (5.2 L, 5 L/kg). The suspension is heated to reflux (Tmass = 88.8 °C) for 28 hours then cooled to 15.7 °C. The suspension is filtered


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at 15 °C and the cake is washed with isopropyl acetate (1 L, 1 L/kg). The filtrate is charged into the reactor, diluted to ca. 9-10 vol overall with isopropyl acetate (+4 L, +4 L/kg) and then heated to 60.3 °C. Acetic acid (197.9 g, 3.30 mol, 0.75 eq) is added slowly over a period of 40 min then the reaction mixture is agitated at 60 °C for 1 h 30 min. The suspension is cooled to 25.1 °C and filtered. The cake is washed with isopropyl acetate (1 L, 1 L/kg). Water (1 L, 1 L/kg) is added and sodium bicarbonate (185 g) is added until pH 7 is reached in the aqueous layer then the phases are separated. An additional wash with water (1 L, 1 L/kg) is done. The organic phase is evaporated to give crude product (892 g, 95.7%) as a yellow oily solid. A blended crude product (1560 g) is partially dissolved in methyl tert-butylether (15.6 L) into a 20 L reactor and agitated at 20 °C for 22 h 30 under a nitrogen atmosphere. The insoluble solid is eliminated by filtration and the filtrate is evaporated to a crude 1 (1283 g, 82%) as a yellow oily solid. After complete dissolution in isopropyl acetate (500 mL), the mass temperature is set at 36 °C and seeded with 1 (8 g). After 30 min aging time at the seeding temperature, the suspension is cooled down to 23 °C in 2 hours then to 15 °C in 1 hour with a post stirring time of 30 min at 15 °C. The product is isolated by filtration. The cake is washed with isopropyl acetate (350 mL and 150 mL). The product is dried 40 °C under vacuum to afford Brivaracetam 1 (500 g, 32% overall step yield) with a purity 97.1% w/w assay and 2.03% of ucb 34713. 1H NMR (CDCl3, 400 MHz) 6.51 (s, 1 H) 5.93 (s, 1 H) 4.46 (dd, J = 8.9, 7.9, 1 H) 3.47 (dd, J = 9.8, 7.8 Hz, 1 H) 3.05 (dd, J = 9.8, 7.1 Hz, 1 H) 2.54 (dd, J = 16.7, 8.6, 1 H) 2.39-2.23 (m, 1 H) 2.06 (dd, J = 16.7, 8.1, 1 H) 1.99-1.85 (m, 1 H) 1.70-1.62 (m, 1 H) 1.45-1.37 (m, 2 H) 1.37-1.25 (m, 2 H) 0.94-0.84 (m, 6 H) ppm ; 13C NMR (CDCl3, 100 MHz) 175.4, 172.6, 55.7, 49.4, 37.8, 36.4, 31.9, 21.2, 20.5, 13.9, 10.4 ppm; HRMS calculated for [M+H] C11H21O2N2 213.1603, found 213.1607.


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Supporting Information. Synthesis of the racemic substrates 3, 4 and 5 and improvement of the synthesis of 4. Racemization of the unwanted enantiomer 8. List of enzymes and chiral bases tested in the enzymatic and salt resolutions. 1H, 13C NMR, HPLC and/or GC chromatograms for compounds 1, 3, 4, 6, 10, 11, 13, 14, 15 and 16. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author *E-mail: [email protected]; Tel: +32.2.386.6208 Funding Sources We thank the Wallonia Region DGO 6 (direction générale opérationnelle de l'Économie, de l'Emploi et de la Recherche) for financial support. Acknowledgements We also thank David Vasselin and Paul Deutsch for fruitful discussion during the preparation of this manuscript.


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Reference section 1

(a) Kenda, B.M.; Matagne, A.C.; Talaga, P.E.; Pasau, P.M.; Differding, E.; Lallemand, B.I.; Frycia, A.M.; Moureau, F.G.; Klitgaard, H.V.; Gillard, M.R.; Fuks, B.; Michel, P. J. Med. Chem. 2004, 47(3), 530–549. (b) Meir Bialer, M.; Johannessen, S.I.; Levyc, R.H.; Peruccad, E.; Tomsone, T.; White, H.S. Epilepsy Res. 2013, 103(1), 2– 30. 2 (a) Klein, P.; Schiemann, J.; Sperling, M.R.; Whitesides, J.; Liang, W.; Stalvey, T. Brandt, C.; Kwan, P. Epilepsia 2015, **(*), 1-9 [doi: 10.1111/epi.13212]. (b) Yates, S.L.; Fakhoury, T.; Lianga, W.; Eckhardt, K.; Borghs, S.; D'Souza, J. Epilepsy & Behavior 2015, 52, 165–168. 3 Bio-catalytic resolution on similar substrates have been described. (a) Beaulieu, P.L.; Gillard, J.; Bailey, M.; Beaulieu, C.; Duceppe, J-S.; Lavallée, P.; Wernic, D. J. Org. Chem. 1999, 64, 6622-6634. (b) Bailey, M.D.; Halmos, T.; Adamson, D.; Bordeleau, J.; Grand-Maître, C. Tetrahedron: Asymmetry 1999, 10, 3285–3295. 4 The whole list of enzymes tested are available in the Supporting Information. 5 The screening conditions (buffer and co-solvent types) were established as a common denominator based on enzymes data provided by the suppliers. 6 Enantiomeric ratio E was calculated from enantiomeric excess of ester substrate (S) and acid product P: E=ln[eeP*(1-eeS)/(eeP+eeS)]/ln[eeP*(1+eeS)/(eeP+eeS)]. 7 Protease alkaline B was quoted at 72 €/kg at 500 kg scale. 8 The representative crude yield of 6 was ca. 42% in the optimal resolution conditions described later. 9 2 kg of protease C from Bacillus subtilis (type 2) was provided. The enzyme was quoted at 130 €/kg at 500 kg scale. 10 The bio-resolution was simulated by the controlled addition of acetic acid with syringe pump miming the formation of the succinic acid product 6 in the reaction mixture. 11 The solvents used in the screening were ethanol, ethyl acetate, tetrahydrofuran (THF) and methyl isobutylketone (MIBK). 12 The complete list can be found in the Supporting Information. 13 10-20 €/kg at multi-ton scale. 14 The crystallization and elimination of the wrong enantiomer with the cheaper L-phenylalaninol was not envisaged. 15 The proof of concept was demonstrated in the laboratory with the formation of the (S)-phenylethylamine salt of 6 in isopropyl acetate at the end of the bio-catalytic step in 85% yield. The chemical purity was improved from 95% up to >98% along with a chiral upgrade up to >99%. The salt release was then carried out quantitatively with an aqueous citric acid solution in toluene to telescope the next step. 16 Davis, R.E.; Gottbrath, J. A. J. Am. Chem. Soc. 1962, 84, 895-898. 17 Snider, B.; Lu, Q. J. Org. Chem. 1996, 61, 2839-2844. 18 HPLC and GC techniques appeared to be not suitable to monitor the ring opening of 20 in the short time allocated to process research. 19 Brivaracetam has a solubility of ca. 0.84g/mL of water. 20 The high numbers of batches run were due to the size limitation of the reactor with the suitable pH-stat monitoring available in the laboratory. This was also the opportunity to assess the repeatability and robustness of the biotransformation by changing parameters in order to generate data on both the reaction conditions and the work-up. 21 The difference of chiral purity between 6 and 19 was assigned to the early qualification status of the analytical methods used and the lack of equivalence tested at this stage of the project (chiral HPLC for 6 and chiral GC for 19). No racemization was expected in the reaction conditions used to perform the reduction/cyclization transformation. Similarly, no racemization of the n-propyl moiety was expected in the downstream chemistry. The chiral purity was further controlled at the API step only. 22 A single in-silico methodology was enough to evaluate potential genotoxicity at the time of the study. Two (Q)SAR prediction methodologies that complement each other should now be applied (ruled-based and statisticalbased methodologies) according to ICH M7 “Assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk”, June 2014. 23 The testing of 23 was initially scheduled later but was finally not performed when the development of this route was stopped. 24 The racemization of the unwanted enantiomer 17 is detailed in the Supporting Information.


31

A Biocatalytic Route to the Novel Antiepileptic Drug Brivaracetam

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