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Enzymatic process for N-substituted (3S)- and (3R)-3-hydroxypyrrolidin-2-ones Amarjit Singh, James Falabella, Thomas LaPorte, and Animesh Goswami Org. Process Res. Dev., Just Accepted Manuscript • Publication Date (Web): 24 Jun 2015 Downloaded from http://pubs.acs.org on June 24, 2015

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Organic Process Research & Development

Enzymatic process for N-substituted (3S)- and (3R)-3hydroxypyrrolidin-2-ones Amarjit Singh*, James Falabella, Thomas L LaPorte and Animesh Goswami

Chemical Development, Bristol-Myers Squibb, One Squibb Drive, New Brunswick, NJ 08903, USA *Corresponding author. Tel.: +1 732 227 5457; Fax: +1 732 227 3994. E-mail: [email protected]

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Figure for Table of Contents

Lipase PS 30 from Pseudomonas cepacia R3

O F N

O

Succinic anhydride 2-Methyltetrahydrofuran

R1 N

HO

X

I

RS-1 R2

RS-1. R1 = F, R2 = I, R3 = OH, X = C RS-4a. R1 = CH3, R2 = Br, R3 = OAC, X = N

ee >99.8% HO

RS-4a

O CH3

Novozym 435 Acetonitrile, water

N

N Br

ee >99.4%

2


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Abstract

Lipase PS 30 (immobilized on polypropylene) from Pseudomonas cepacia was used

for

enantioselective

esterification

of

(RS)-1-(2-fluoro-4-iodophenyl)-3-

hydroxypyrrolidin-2-one by using succinic anhydride and 2-methyltetrahydrofuran at 4 °C. The isolation of desired alcohol avoided use of column chromatography, a simple solvent extraction of undesired (R)-4-((1-(2-fluoro-4-iodophenyl)-2-oxopyrrolidin-3yl)oxy)-4-oxobutanoic acid into 5% potassium bicarbonate solution separated pure desired

(S)-1-(2-fluoro-4-iodophenyl)-3-hydroxypyrrolidin-2-one

into

the

2-

methyltetrahydrofuran solution. The reaction conditions were optimized and (S)-1-(2fluoro-4-iodophenyl)-3-hydroxypyrrolidin-2-one was prepared in high enantiomeric excess >99% and yield ~40% (theoretically possible yield 50%). Novozym 435 (Candida antarctica lipase B) was found to be a suitable biocatalyst for the resolution of (RS)-1(6-bromo-2-methylpyridin-3-yl)-2-oxopyrrolidin-3-yl acetate to form the undesired Sacetate and the desired R-alcohol. The optimized reaction conditions gave (R)-1-(6bromo-2-methylpyridin-3-yl)-3-hydroxypyrrolidin-2-one in ~37% isolated yield (maximum possible yield 50%) and high enantiomeric excess (ee >99.4%). The enzymatic resolution of (RS)-1-(6-bromo-2-methylpyridin-3-yl)-2-oxopyrrolidin-3-yl acetate followed by chromatography was successfully implemented to deliver material for two successive (4.1 Kg, ee >99.4% and 5.5 Kg, ee >99.5%) campaigns. The undesired S-alcohol was recycled back to the desired R-alcohol using a Mitsunobu inversion of stereochemistry in gram scale. An increase in the chain length from acetate to hexanoate improved the selectivity and subsequent optimization decreased the enzyme loading and enhanced the substrate input. Separation of the desired (R)-1-(6-bromo-2-methylpyridin-3-yl)-33



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hydroxypyrrolidin-2-one from (S)-1-(6-bromo-2-methylpyrrolidin-3-yl)-2-oxopyrrolidin-3-yl hexanoate was achieved using a solvent extraction. The process for the preparation of (S)-1-(2-fluoro-4-iodophenyl)-3-hydroxypyrrolidin-2-one

and

(R)-1-(6-bromo-2-

methylpyridin-3-yl)-3-hydroxypyrrolidin-2-one is scalable, economical, highly efficient, and avoids chromatography.

Key Words

Enzymatic succinylation, enzymatic hydrolysis, inversion of stereochemistry by Mitsunobu reaction, 3-hydroxypyrrolidin-2-one

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Introduction The projected increase in the global population with type 2 diabetes mellitus is a cause for concern. It is estimated that from 2010 to 2030, 54% more people will have type 2 diabetes mellitus which represents 7.7% of the total adult population of the world.1 A number of therapeutics are available for diabetes.2,3 G Protein-coupled receptor 119 (GPR119) agonists are an attractive target because they may: (i) improve glucose control with low added risk of hypoglycemia (ii) slow diabetes progression (iii) reduce food intake and body weight (iv) constitute small synthetic molecules which could be taken orally unlike GLP-1 analogs which require parenteral administration and (v) provide greater benefits when used in combination with a DPP4 inhibitor.3-4 More than 20 pharmaceutical companies have entered into GPR119 agonist programs since their discovery between 2000-2003.5 Bristol-Myers Squibb Company has disclosed a series of pyridone, pyridazone, benzothiazole, dihydrobenzofuran, bicyclic pyrimidines and piperidinyl sulfone analogs as GPR119 agonists.6 (S)-1-(2-Fluoro-4-iodophenyl)-3hydroxypyrrolidin-2-one S-1, and (R)-1-(6-bromo-2-methylpyridin-3-yl)-3hydroxypyrrolidin-2-one R-5 are key chiral intermediates for GPR119 agonist compounds.7 Synthesis of optically pure 3-hydroxypyrrolidin-2-one is needed while using them for drug development.8 Construction of optically pure 3-hydroxypyrrolidin-2one requires a multi-step synthesis where chiral template such as S-malic acid is used as precursor.9 Lactate dehydrogenases-catalyzed chemo-enzymatic approach has been reported to prepare (S)- and (R)-3-hydroxypyrrolidin-2-ones where chirality is introduced in the penultimate step and then (S)- or (R)-2-hydroxy acids are transformed into homochiral 3-hydroxypyrrolidin-2-ones.10 The reactions require expensive co-factors

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which could be recycled by using another enzyme to reduce the cost. Recently, enzymatic kinetic resolution approach was employed to prepare N-methyl,-ethyl and benzyl substituted 3-hydroxypyrrolidin-2-one and 3-hydroxypiperidin-2-ones from milligram to gram scale.11 Here we report lipase PS 30 (immobilized on polypropylenes) from Pseudomonas cepacia and Novozym 435 (Candida antarctica) catalyzed scalable processes for two new 3-hydroxypyrrolidin-2-ones, (S)-1-(2-Fluoro-4-iodophenyl)-3hydroxypyrrolidin-2-one S-1, (R)-1-(6-bromo-2-methylpyridin-3-yl)-3-hydroxypyrrolidin-2one R-5 (Scheme 1 and 3) which are needed in kilogram scale to supply respective APIs for drug development studies.7

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Results and discussion The enzymatic kinetic approach is commonly used to prepare chiral intermediates. We explored enzymatic kinetic resolution in an aqueous medium for the hydrolysis of 2 to form S-1 (Scheme 1). Many enzymes gave poor selectivity. Enzymatic acetylation of RS-1 also failed to form the desired compound S-1 in good yield. Among the different kinds of kinetic resolutions, enzymatic succinylation12 is preferred to develop as a process because the separation of product hemisuccinate from the opposite enantiomer of the unreacted alcohol is accomplished relatively easy by simple solvent extraction. Enzymatic succinylation screening experiments identified lipase PS 30 isolated from Pseuodomonas cepacia (immobilized on polypropylene beads) with good selectivity. Nine enzymes isolated from Pseudomonas/Burkhoderia cepacia were available in our inventory from different vendors13 formed desired compound, with best results of solution yield 30-40%, ee >99% in 18 h when lipases PSIM, sprin lipo, sprin epobond, lipase SL and lipase P1 were used. Although the succinylation reaction with enzymes from Pseudomonas cepacia showed good selectivity, we found that hydrolysis of racemic acetate 2 by the same enzymes gave poor ee for both alcohol (ee 99% was achieved in 6-8 h. However further reaction ageing to 24 h reduces the ee of the alcohol probably due to hydrolysis of the succinate derivative. In order to supply material quickly the approach was scaled up with minimum optimization work. The improved conditions (i) substrate input 50 g/L (ii) enzyme to substrate ratio 1:20 (iii) succinic anhydride 0.8 molar equivalent to substrate and (iv) 2-methyltetrahydrofuran as solvent were implemented and five lab batches (Scheme 2, Table S1) were carried out in 5.5 – 8.5 h at 4 °C to prepare the desired alcohol S-1 in yields of 35-43% (maximum possible yield 50%) and ee 99.2 to >99.9%.

Intermediates S-1 and R-5 have similarity in the core structure and only differed in substitution pattern in the aromatic ring. We thought that a similar enzymatic succinylation approach could be used for R-5, but our intuition turned out to be incorrect. About ~200 enzymes including lipase PS 30 (immobilized on polypropylene) were screened for succinylation reactions of RS-5 in different solvents but gave unacceptable ee for process development. We took the reverse approach and synthesized succinate derivative of alcohol RS-4e followed by enzymatic desuccinylation reactions. The desuccinylation reaction gave ee ~95% which could be optimized to ee >99%. However, due to poor stability of the succinate derivative in an

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extractive workup, the preparation of alcohol R-5 with ee >99% was not feasible. The desuccinylation route was ruled out because >99% ee of the chiral intermediate R-5 is necessary for drug development studies. Even 1% degradation of the homochiral succinate derivative could reduce the ee of R-5 by 2%. Most of the enzymes gave poor selectivity in acylation reactions with vinyl acetate or trifluoroethylbutyrate in MIBK and 2-methylTHF. After no success in enzymatic acylation reactions, enzymes were screened for hydrolytic reactions.

Conventionally, hydrolytic reactions are screened in aqueous medium. We followed literature screening precedence and many enzymes were found active with low selectivity. Some enzymes showed good selectivity but they were not available in large scale and/or reasonable cost to move forward with the process development. Novozym 435 (lipase from Candida antarctica) catalyzed the resolution of the racemic acetate RS-4a to form the R-alcohol R-5 and the S-acetate S-4a but the reaction was slow in water (Scheme 3, Figure S1a, S1b and 1, conversion ~24% in 50 h), nevertheless, the ee of the alcohol R-5 was >97%. Novozym 435 is a robust, well characterized, commercially available immobilized enzyme, tolerant to wide range of reaction conditions like high temperature, organic solvents, in-situ racemization, packed bed reactor conditions, etc.14 Immobilized Novozym 435 can easily be removed from the reaction mixture for reuse by a simple filtration.

A screen of aqueous organic solvent combinations (2-methylTHF, MTBE, acetonitrile, toluene and heptanes) yielded better conversion (>48%) and ee (>99%) for

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the alcohol R-5 in aqueous acetonitrile. Several parameters like solubility of alcohol RS5 (45 mg/mL) and acetate RS-4a (>100 mg/mL) in acetonitrile, water miscibility in acetonitrile, solubility of the byproduct acetic acid, stability of the S-acetate S-4a, and enzyme selectivity were taken into consideration while selecting aqueous acetonitrile as reaction media for Novozym 435 catalyzed reactions. Screening studies at 1.0 mL scale formed the alcohol R-5 with an ee >99% in 24 h without any temperature adjustment when acetonitrile 90% and water 10% were used with an enzyme to substrate ratio of 1:4. A significant difference in the alcohol RS-5 and acetate RS-4a mobility during thin layer chromatography further encouraged us to improve the reaction conditions and develop a scalable process where the desired alcohol R-5 could be separated from the undesired acetate S-4a by using chromatography.

The impact of temperature on the resolution of acetate RS-4a (Figure S2) was recorded at 10 °C to 50 °C for reactions run at 1.0 g scale (acetonitrile 90%, water 10%, E:S ratio 1:4 at 20 g/L concentration). In the linear range (0 to 10 h), the rate of the reaction increased as the temperature was increased from 10 °C to 40 °C and no significant difference was observed from 40 °C to 50 °C. However ~48% conversion was achieved for reactions run between 25 °C to 50 °C at around 24 hours. Even though the rate of the reaction increased with the increase in temperature (25-50 °C) at early stages of the reaction there was no significant benefit in reaction time to reach ~48% conversion, thus the temperature 25 °C was preferred for a greener and low energy intensive process.

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One of the byproducts of the enzymatic hydrolysis of acetate RS-4a is acetic acid (~0.5 molar equivalent). When 1.0 g scale reactions (20 g/L, E:S 1:4, water 10%, acetonitirle 90%) were done at 25 °C in the presence of 0.0 molar equivalent, 0.5 molar equivalent and 1.0 molar equivalent of acetic acid, minimum effect was observed on the yield, ee of alcohol R-5 and ee of acetate S-4a (Figure S3). The presence of acetic acid apparently imparted stability to the acetate RS-4a by keeping the pH below 7.0. Stability studies of acetate RS-4a have shown attrition of acetate in sodium phosphate buffer solution with time and, as expected, non-enzymatic hydrolysis increases with the increase in pH.

The separation of the R-alcohol R-5 from the S-acetate S-4a was only feasible using chromatography and storage stability of the reaction mixture prior to cumbersome chromatography was essential for the scale up. RS-acetate RS-4a (20 g/L) was hydrolyzed using Novozym 435 (E:S ratio 1:4) in 10% water and 90% acetonitrile at 30 °C. The reaction was stopped after 27 h (solution yield 49.5%, ee acetate S-4a 97.4%, ee alcohol R-5 99.3%) and the enzyme was filtered. The filtrate was divided into two portions with one part stored at room temperature and the other at 4 °C. HPLC analysis of the filtrates over a period of 14 days suggested no change in conversion, ee of alcohol R-5 and ee of acetate S-4a.

At this point, the process was finalized for the first scale up campaign. The process steps included: (i) Novozym 435 catalyzed hydrolysis of acetate RS-4a at 20 g/L concentration in acetonitrile-water (90:10) mixture, a reaction temperature of 25 °C,

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and an enzyme to substrate ratio of 1:4 (ii) filtration of enzyme after the reaction and (iii) separation of the desired alcohol R-5 from the undesired acetate S-4a by chromatography. A 50 g batch was run in the lab using the above conditions achieving 48.3% conversion in 28 h with an ee of alcohol R-5 of 99.4% and an ee of acetate S-4a of 99.2% (Figure S4). Alcohol R-5 and acetate S-4a were separated using silica gel chromatography with a gradient of heptane and ethyl acetate as eluents. The R-alcohol R-5 was isolated as an off-white solid 16.04 g, yield 37.1% (theoretical possible yield 50%), ee 99.9%, AP 99.4. S-Acetate S-4a was isolated as a white solid 19.04 g, yield 38.1% (theoretical possible yield 50%), ee 99.8%, AP 99.5.

For the first scale up campaign, 13 Kg of racemic acetate RS-4a was hydrolyzed in the pilot plant in two batches (6.5 Kg each) to supply the chiral intermediate R-alcohol R-5 for API synthesis. The conversion of the acetate RS-4a into the alcohol R-5 can be seen from the plots in Figure 2a and 2b. The process samples taken at reaction times of 1, 3, 5, 7 and 22 hours were analyzed by HPLC for conversion, ee of alcohol R-5 and ee of acetate S-4a. The reactions were completed in 20-22 hrs. The enzyme was filtered, filtrate from the two batches was mixed and the R-alcohol R-5 was separated from the S-acetate S-4a by silica gel chromatography on a 20 Kg pre-packed column (Biotage 400M). Elution by 50% ethyl acetate-50% heptane removed the acetate S-4a. The alcohol R-5 was flushed out of the column using ethyl acetate as eluent. From 13 Kg of racemic acetate RS-4a input, 4.1 Kg, yield 36.5% (theoretical yield 5.627 Kg), ee 99.4% of dry crystallized alcohol R-5 was isolated.

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In the first campaign, the enzymatic hydrolysis was carried out in 10% water and 90% acetonitrile. For the subsequent column chromatography step, most of the water was removed as an azeotropic mixture with acetonitrile and the acetonitrile was exchanged with dichloromethane. The water content was reduced to 1% in a solvent swap with acetonitrile and dichloromethane. We decided to improve the process, with an aim to develop a resolution condition that would require minimum solvent exchange reducing the pilot plant operation time and overall cost of the process.

We looked into the water content in detail and ran a series of 1.0 g scale (20 g/L) reactions with an enzyme to substrate ratio of 1:4 by varying the water percentage in anhydrous acetonitrile. The results (Figure 1, S1a and S1b) showed that, though a small amount of water is required, only a very small amount of water is best. The reaction rate decreased with increasing amount of water above the optimum; 0.5% ≅ 1% (optimum amount) > 0.2% > 3% > 5% ≅ 10% > 20% > 50% > 100% water > 100% acetonitrile. Notably, a positive impact on the process was evident as ≥2 fold increase in reaction rate was seen just by reducing water from 10% to 1%. The lower amount of water (1%) would not only increase the reaction rate but it can also substantially reduce the solvent waste and the solvent swap time in the pilot plant.

Under the modified conditions (1% water in acetonitrile and an enzyme to substrate ratio of 1:4) >45% conversion was achieved in ~5 h suggesting that still lower enzyme load could be sufficient to complete (>48%) the enzymatic hydrolysis in 24 h. We undertook studies of reducing the enzyme loading for our process. A series of 1.0 g

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scale (20 g/L) reactions were executed to explore the impact of enzyme loading at 25 °C in 1% water in acetonitrile. The analysis of reaction plot (Figure S5) reveals ~48% conversion can be achieved in 24 h under two different reaction conditions. One experiment utilized 10% water and an E:S of 1:4 (25% enzyme loading) while the other utilized 1% water and an E:S of 1:25 (4% enzyme loading). Essentially enzyme loading was reduced by six fold when water concentration was reduced from 10% to 1%.

Driven by the desire to improve the volumetric productivity of the process, we examined different substrate concentrations under optimum water conditions. Under new conditions (water 1%, acetonitrile 99%, temperature 25 °C) 1-5 gram scale reactions were run at 20 g/L, 40 g/L, 60 g/L, 80 g/L and 100 g/L concentrations and the results (Figure S6-S11) showed water 1% is optimum for acetate RS-4a hydrolysis for substrate input from 20 g/L to 100 g/L.

The improved conditions were implemented in the second pilot plant campaign and 17 Kg of racemic acetate RS-4a was processed at 60 g/L concentration. The reaction performed as expected and the alcohol R-5 was separated from acetate S-4a using chromatography as described above. In the second campaign 1% water was used and the solvent swap which was taking ~16 h of pilot plant time was eliminated. The enzyme loading was reduced from 250 g/Kg of ester to 40 g/Kg of ester. R-alcohol R-5 5.5 Kg (theoretical yield 7.35 Kg), yield 37.4% (theoretical possible yield 50%), ee >99.5% was isolated as white solid for API synthesis.

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The enzymatic hydrolysis process from acetate requires chromatography for the separation of the desired alcohol R-5 from the acetate S-4a which reduces throughput and increases the cost of the overall process. We reasoned that it might be possible to separate longer chain esters from the alcohol by solvent extraction. We synthesized propionate RS-4b, butyrate RS-4c and hexanoate RS-4d and studied Novozym 435 catalyzed resolution. We found that by increasing the chain length from acetate RS-4a to hexanoate RS-4d (Scheme 3, Table S2, and Figure S12), the rate of the reaction increased and the hexanoate RS-4d showed highest reaction rate >48% conversion in 99.9% ee of the desired alcohol R-5. We also observed that the reaction almost stalled at 47% conversion when concentration of water was 1% however the reaction proceeded beyond 48% conversion with the increase in water concentration to 1.5% and 2%. A series of batches were run by varying the enzyme loading, water concentration and substrate input at different temperatures (Table S2). The optimized reaction conditions for enantioselective RS-hexanoate RS-4d resolution were established as follows: substrate input 100 g/L, acetonitrile 98%, water 2%, enzyme to substrate ratio 1:200 at 45 °C (the reaction was complete in ~15 h). To demonstrate the process, a 5.0 g batch was run and after the reaction was complete (conversion >48%) the enzyme was filtered out and the solvent was removed. The residue was dissolved in water and toluene extraction was implemented to remove the undesired S-hexanoate S-4d from the reaction mixture. The R-alcohol R-5 was extracted into dichloromethane from the aqueous layer and pure alcohol R-5 was isolated as a white solid yield 38.9% (theoretical possible yield 50%), AP 98.9, ee >99.9%.

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The enzymatic hydrolysis process from hexanoate does not require column chromatography and simple solvent extraction was sufficient to separate the desired enantiomer from the undesired enantiomer. The enzyme loading was reduced from 250 g/kg of substrate in the first campaign to 40 g/Kg in the second campaign to 5.0 g/Kg of RS-ester in the hexanoate process (Table 1). Most importantly use of low concentration of water (1-2%), eliminated the solvent swap step, which lowered solvent use, pilot plant time and resources. Novozym 435 catalyzed resolution of RS-4a was also successfully implemented in packed bed reactor.15

The enzymatic resolution reaction formed a chiral alcohol R-5 and chiral acetate S-4a. To determine the absolute stereochemistry, S-acetate S-4a was hydrolyzed into alcohol S-5 by using potassium carbonate. Both enantiomers of the alcohol, R-5 and S5, were derivatized with (+)- and (-)-MPTA in pyridine-d5 in an NMR tube. From NMR analysis the chemical shift for -CH2 group next to chiral center of the alcohol R-5 was upfield when the alcohol R-5 was derivatized with R-MTPA-Cl (δ 2.75, 2.48) as compared to when the alcohol was derivatized with S-MTPA-Cl (δ 2.72, 2.29). According to Mosher’s rule, the results are in agreement with R-absolute stereochemistry for the alcohol R-5. Similarly, from NMR analysis the chemical shift for the -CH2 group next to the chiral center in the alcohol S-5 was upfield when the alcohol S-5 was derivatized with S-MTPA-Cl (δ 2.74, 2.47) as compared to when derivatized with R-MTPA-Cl (δ 2.72, 2.29). According to Mosher’s rule, the results are in agreement with S-absolute stereochemistry for the alcohol S-5. NMR analysis confirmed that the Ralcohol R-5 is formed after enzymatic hydrolysis. The R-Alcohol R-5 was derivatized

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with 1-(S)-camphanic acid chloride and the product was crystallized. The single crystal X-ray crystallography of 6 assigned absolute stereochemistry as R for the new chiral center (Figure 4).

A major disadvantage of an enzyme catalyzed kinetic resolution reaction is that theoretically 50% of the material is the desired compound and the remaining 50% is waste. S-Acetate S-4a was hydrolyzed using potassium carbonate in methanol in 88.8% isolated yield, ee >99.9%. The S-alcohol S-5 was transformed into p-nitrobenzoate derivative using the Mitsunobu reaction. A total inversion of stereochemistry was observed during the Mitsunobu reaction and the product 7 was isolated in 88.2% yield, ee 99.3%. The subsequent hydrolysis of the p-nitrobenzoate derivative 7 formed the desired R-alcohol R-5. By using the Mitsunobu reaction all of the undesired enantiomer S-5 was converted into the desired alcohol R-5.

Finally, after a successful inversion of stereochemistry, (Scheme-3) we envisioned enzymatic acylation and an inversion of stereochemistry in one pot. One pot transformation is only feasible if the enzymatic acylation reaction would form both alcohol S-5 and acetate R-4a in high ee (>99%). A list of solvents; acetonitrile, toluene, 2-methylTHF, dichloromethane, ethanol, heptanes, acetone, tetrahydrofuran, MTBE, 1,4-dioxane, 2-ethoxyethanol, 1-propanol, 2-methyl 1-propanol and 1,2-propanediol were screened to engineer the reaction conditions. Acetonitrile, 2methyltetrahydrofuran, dichloromethane, and tetrahydrofuran formed the acetate R-4a in good ee >96%. Tetrahydrofuran was a solvent of choice for one pot transformation

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because the inversion of stereochemistry was successfully done in tetrahydrofuran. After a minimal amount of optimization, a 1.0 g batch of RS-alcohol RS-5 was acetylated with vinyl acetate (2 molar equivalent) and Novozym 435 100 mg (enzyme to substrate ratio 1:10) in 50 mL of tetrahydrofuran at room temperature. The reaction was over in 3 h (conversion 49%, ee of desired acetate 99%, ee of alcohol 95%), however, a couple of attempts to implement the in-situ Mitsunobu reaction could not exceed >80% conversion. Further work is necessary to develop an in-situ enzymatic acetylation coupled with the Mitsunobu reaction to provide the desired chiral alcohol in both good yield and ee.

Conclusions

A preparative scale process for chiral intermediates S-1 and R-5 was essential to drive the synthesis of the respective APIs.7 A process was developed at 50 g/L concentration where the reaction was over within 8.5 h, immobilized enzyme was removed by simple filtration and intermediate S-1 was isolated in ~40% yield and ee >99%. 2-Methyltetrahydrofuran served as a reaction medium and a solvent to extract the desired compound from the reaction mixture eliminating the use of chromatography. A scalable enzymatic process for R-5 was possible only after extensive solvent screen and water dependency of hydrolytic reactions. Traditional screening experiments in aqueous media could have easily missed Novozym 435 as a lead biocatalyst, thus, it is recommended to study different solvents and water concentrations 18


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while dealing with immobilized enzymes specifically Novozym 435. The current process is industrially relevant and a major disadvantage of kinetic resolution of losing half of the material was overcome by recycling the undesired enantiomer into the desired enantiomer via an inversion of stereochemistry, which was demonstrated at small scale. After a list of changes, (Table 1) a highly economical process was developed to supply the chiral intermediate R-5 at kilogram scale.

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Experimental Materials

Chemicals were purchased from Sigma-Aldrich chemical company. NMR spectra were recorded in CDCl3 (except as indicated) on a BRUKER-500 MHz or Jeol 400 MHz spectrometer. LCMS data were recorded on a Shimadzu LCMS system with positive ion electrospray (ES+) or negative ion electrospray (ES-) methods. (RS)-1-(6-bromo-2methylpyridin-3-yl)-3-hydroxypyrrolidin-2-one and (RS)-1-(2-Fluoro-4-iodophenyl)hydroxypyrrolidin-2-one were prepared as described elsewhere.7

HPLC methods

a.

Achiral HPLC methods for racemic alcohol RS-1, racemic acetate 2 and

racemic hemisuccinate RS-3

Method -1: Samples were analyzed on a Phenomenex Gemini 5 µM 4.6 x 50 mm HPLC column by using solvent A (0.05% TFA in water-acetonitrile, 95:5) and solvent B (0.05% TFA in water – acetonitrile, 5:95) with a gradient of 20% to 70% of B in 6 min at a flow rate of 2.0 mL/min. The detection was done by UV at 240 and 254 nm and the detection at 240 nm was used for calculations. The retention times are as follow: racemic alcohol RS-1 2.18 min, racemic acetate 2 3.64 min, and racemic hemisuccinate RS-3 3.21 min. Method-2: Samples were analyzed on a Waters XTerra RP18 3.5 µM 4.6 x 50 mm HPLC column by using solvent A (0.05% TFA in water-acetonitrile, 95:5) and solvent B (0.05% TFA in water – acetonitrile, 5:95) with a gradient of 10% to 70% of B in 6 min at a flow rate of 2.0 mL/min. The detection was done by UV at 240 and 254 nm and the 20


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detection at 240 nm was used for calculations. The retention times are as follow: racemic alcohol RS-1 2.20 min, and racemic acetate 2 3.35 min.

b.

Chiral HPLC methods for racemic alcohol RS-1, racemic acetate 2 and

racemic hemisuccinate RS-3 Chiral HPLC method (normal phase method)- Samples were analyzed on a ChiralPak AD-H 5 µM 4.6 x 250 mm HPLC column by using solvent A (heptane) and solvent B (heptane : IPA, 1:1) with a gradient of 20% to 70% of B in 32 min at a flow rate of 1.0 mL/min. The detection was done by UV at 240 and 254 nm and the detection at 240 nm was used for calculations. The retention times are as follow: S-alcohol S-1 14.88 min, R-alcohol R-1 17.12 min, S-acetate S-2 22.63 min, R-acetate R-2 27.25 min, R-hemisuccinate R-3 26.3 min, and S-hemisuccinate S-3 28.8 min.

Chiral HPLC method (reverse phase method)- Samples were analyzed on a Chiralpak IC 5 µM 4.6 x 150 mm HPLC column by using solvent A (0.01 M NH4OAc in water-MeOH, 80:20) and solvent B (0.01M NH4OAc in water-MeOH–CH3CN, 5:20:75) with a gradient of 25% of B for 24 min, 25% to 40% of B for 1 min, 40% to 40% of B for 25 min. at a flow rate of 0.5 mL/min, stop time 50 min. The detection was done by UV at 240 and 254 nm and the detection at 240 nm was used for calculations. The retention times are as follow: S-alcohol S-1 18.55 min, R-alcohol R-1 21.44 min, R-acetate R-2 42.46 min, and S-acetate S-2 45.02 min.

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c.

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Achiral HPLC methods for alcohol RS-5, acetate RS-4a, propionate RS-4b ,

butyrate RS-4c, hexanoate RS-4d and succinate RS-4e: Samples were analyzed on a Waters SunFire C18 3.5 µm 4.6 x 150 mm HPLC column by using solvent A (0.05% TFA in water-acetonitrile, 95:5) and solvent B (0.05% TFA in water – acetonitrile, 5:95) with a gradient of 40% to 100% of B in 5 min, 100% B to 100% of B in 10.0 min at a flow rate of 1.0 mL/min. The detection was done by UV at 220 and 275 nm and the detection at 220 nm was used for calculations. During the course of the work it was observed that UV signal is slightly higher at 226 nm as compared to 220 nm. The retention times are as follow: RS-alcohol RS-5 2.02 min, RS-acetate RS-4a 3.68 min, RS-propionate RS-4b 4.74 min, RS-butyrate RS-4c 5.59 min, and RS-hexanoate RS-4d 6.95 min. Hemisuccinate of alcohol RS-4e was analyzed on a Waters XTerra RP18 3.5 µm 4.6 x 150 mm HPLC column by using solvent A (0.05% TFA in water-acetonitrile, 95:5) and solvent B (0.05% TFA in water – acetonitrile, 5:95) with a gradient of 10% to 50% in 8 min at a flow rate of 1.0 mL/min. The detection was done by UV at 220 and 275 nm and the detection at 220 nm was used for calculations. The hemisuccinate of alcohol RS-4e appears at retention time 5.87 min.

d.

Chiral HPLC method for RS-alcohol RS-5 and RS-acetate RS-4a: Samples

were analyzed on a Chiralpak AD-H 5 µM 4.6 x 250 mm HPLC column by using isocratic solvent system of 100% B (solvent A, methanol and solvent B, acetonitrile) in 25 min at a flow rate of 0.5 mL/min. The detection was done by UV at 220 and 275 nm and the detection at 220 nm (in some cases detection at 226 nm was used which produced minimum effect on conversion) was used for calculations. The retention times

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are as follow: R-acetate R-4a 7.72 min, S-acetate S-4a 9.16 min, S-alcohol S-5 11.41 min, and R-alcohol R-5 14.63 min. Samples were analyzed on a Chiralpak AD-3R 3 µM 4.6 x 150 mm HPLC column by using isocratic solvent system of 100% B (solvent A, methanol and solvent B, acetonitrile) in 15 min at a flow rate of 0.5 mL/min. The detection was done by UV at 220 and 275 nm and the detection at 220 nm was used for calculations. The retention times are as follow: R-acetate R-4a 4.52 min, S-acetate S-4a 5.22 min, S-alcohol S-5 6.41 min, and R-alcohol R-5 7.69 min.

e.

Reverse phase Chiral HPLC method for acetate RS-4a: Samples were

analyzed on a Chiralpak AD-RH 5 µM 4.6 x 150 mm HPLC column by using solvent A (0.01M ammonium acetate in water-acetonitrile, 95:5), and solvent B (0.01M ammonium acetate in water-acetonitrile, 5:95) with a gradient of 10% to 70% of B in 30 min at a flow rate of 0.5 mL/min. The detection was done by UV at 220 and 275 nm and the detection at 220 nm was used for calculations. The retention times are as follow: Racetate R-4a 15.52 min, and S-acetate S-4a 19.97 min.

f.

Chiral HPLC method for propionate RS-4b: Samples were analyzed on a

ChiralPak AS-RH, 5 µm 4.6 x 150 mm HPLC column by using solvent A (0.01M NH4OAc in water-methanol, 80:20) and solvent B (0.01M NH4OAc in water-methanolacetonitrile, 5:20:75) with a gradient of 20% to 70% of B in 30 min at a flow rate of 0.5 mL/min. The detection was done by UV at 220 and 275 nm and the detection at 220 nm was used for calculations. The retention times are as follow: (R)-1-(6-bromo-2-

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methylpyridin-3-yl)-2-oxopyrrolidin-3-yl propionate R-4b 14.88 min, and (S)-1-(6-bromo2-methylpyridin-3-yl)-2-oxopyrrolidin-3-yl propionate S-4b 16.88 min.

g.

Chiral HPLC method for butyrate RS-4c: Samples were analyzed on a

ChiralPak AS-RH, 5 µm 4.6 x 150 mm HPLC column by using solvent A (0.01M NH4OAc in water-methanol, 80:20) and solvent B (0.01M NH4OAc in water-methanolacetonitrile, 5:20:75) with a gradient of 30% to 70% of B in 30 min at a flow rate of 0.5 mL/min. The detection was done by UV at 220 and 275 nm and the detection at 220 nm was used for calculations. The retention times are as follow: (R)-1-(6-bromo-2methylpyridin-3-yl)-2-oxopyrrolidin-3-yl butyrate R-4c 14.40 min, and (S)-1-(6-bromo-2methylpyridin-3-yl)-2-oxopyrrolidin-3-yl butyrate S-4c 17.02 min.

h.

Chiral HPLC method for hexanoate RS-4d: Samples were analyzed on a

ChiralPak AS-RH, 5 µm 4.6 x 150 mm HPLC column by using solvent A (0.01M NH4OAc in water-methanol, 80:20) and solvent B (0.01M NH4OAc in water-methanolacetonitrile, 5:20:75) with a gradient of 40% to 80% of B in 30 min at a flow rate of 0.5 mL/min. The detection was done by UV at 220 and 275 nm and the detection at 220 nm was used for calculations. The retention times are as follow: (R)-1-(6-bromo-2methylpyridin-3-yl)-2-oxopyrrolidin-3-yl hexanoate R-4d 15.87 min, and (S)-1-(6-bromo2-methylpyridin-3-yl)-2-oxopyrrolidin-3-yl hexanoate S-4d 18.66 min.

i.

Chiral HPLC methods for hemisuccinate derivative of alcohol RS-4e:

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Method 1-Samples were analyzed on a Chiralpak IC 5 µM 4.6 x 150 mm HPLC column by using solvent A (0.05% TFA in water-methanol, 8:2), and solvent B (0.05% TFA in acetonitrile–water, 8:2) with a gradient of 20% to 50% of B in 20 min at a flow rate of 0.5 mL/min. The detection was done by UV at 220 and 275 nm and the detection at 220 nm was used for calculations. The retention times are as follow: S-hemisuccinate S-4e 9.93 min, and R-hemisuccinate R-4e 12.05 min. Method 2-Samples were analyzed on a Chiralpak IC 5 µM 4.6 x 150 mm HPLC column by using solvent A (0.05% TFA in watermethanol, 8:2), and solvent B (0.05% TFA in acetonitrile –water, 8:2) with 20% of B isocratic solvent at a flow rate of 0.5 mL/min for 30 min. The detection was done by UV at 220 and 275 nm and the detection at 220 nm was used for calculations. The retention times are as follow: S-hemisuccinate S-4e 12.21 min, and R-hemisuccinate R-4e 17.50 min.

j.

Achiral HPLC analysis of 1-(6-bromo-2-methylpyridin-3-yl)-2-oxopyrrolidin-

3-yl 4-nitrobenzoate RS-7: Samples were analyzed on a Waters SunFire C18 3.5 µm 4.6 x 150 mm HPLC column by using solvent A (0.05% TFA in water-acetonitrile, 95:5) and solvent B (0.05% TFA in water – acetonitrile, 5:95) with a gradient of 40% to 100% of B in 10 min, 100% of B to 100% of B for 15 min at a flow rate of 1.0 mL/min. The detection was done by UV at 220 and 260 nm and the detection at 260 nm was used for calculations. The retention time for (R, S)-1-(6-bromo-2-methylpyridin-3-yl)-2oxopyrrolidin-3-yl 4-nitrobenzoate RS-7 is 7.62 min.

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k.

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Chiral HPLC analysis of 1-(6-bromo-2-methylpyridin-3-yl)-2-oxopyrrolidin-3-

yl 4-nitrobenzoate RS-7: Samples were analyzed on a Chiracel OJ-RH 5 µM 4.6 x 150 mm HPLC column by using solvent A (0.01M NH4OAc in water-methanol, 80:20) and solvent B (0.01M NH4OAc in water-methanol-acetonitrile, 5:20:75) with a gradient of 50% to 70% of B in 30 min at a flow rate of 0.5 mL/min. The detection was done by UV at 220 and 260 nm and the detection at 260 nm was used for calculations. The retention times are as follow: (R)-1-(6-bromo-2-methylpyridin-3-yl)-2-oxopyrrolidin-3-yl 4nitrobenzoate R-7 11.48 min, and (S)-1-(6-bromo-2-methylpyridin-3-yl)-2-oxopyrrolidin3-yl 4-nitrobenzoate S-7 16.82 min.

General method of enzymatic esterification and isolation of (S)-1-(2-Fluoro-4iodophenyl)-3-hydroxypyrrolidin-2-one, S-1: Racemic alcohol RS-1 (75 g, 234 mmol, 50 g/L) and 2-methyltetrahydrofuran (1.5 L) were added to a 2.0 L jacketed glass reactor with continuous stirring of the reaction mixture by using a magnetic stirrer. The temperature was adjusted to 4 °C by using a water circulator. Succinic anhydride (18.7 g, 187 mmol, 0.8 molar equivalent to racemic alcohol, RS-1) followed by lipase “Amano” PS-30 (immobilized on polypropylene, 3.75 g, enzyme to substrate ratio 1:20) were added to the same reaction mixture. The racemic alcohol RS-1 was not completely soluble in 2-methyltetrahydrofuran in the beginning of the reaction however with the progress of the reaction it most likely became completely soluble. Samples (10 µL) were taken out at different times, solvent was removed by flushing nitrogen stream, the residue was dissolved in 1.0 ml of methanol, filtered through 0.2 µM filter and analyzed by using HPLC. The reaction was stopped after 5.5 h (ee ≥99%) and filtered. The residue was washed with 2-methyltetrahydrofuran and 2-methyltetrahydrofuran solution 26


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was pooled. The filtrate was extracted with 5% potassium hydrogen carbonate (1 x 1.5 liter, 2 x 1.0 liter) until no hemisuccinate derivative (99.9%. LCMS shows a peak with M/Z of the desired S-alcohol. Compound S-5: 1H NMR (500 MHz, DMSO-d6): δ 7.65 (d, J = 8.2 Hz, 1H), 7.55 (d, J = 8.2 Hz, 1H), 5.76 (d, J = 6.1 Hz, 1H), 4.36 - 4.26 (m, 1H), 3.69 - 3.54 (m, 2H), 2.48 2.37 (m, 1H), 2.31 (s, 3H), 2.01 - 1.89 (m, 1H). 13C NMR (125 MHz, DMSO-d6): δ 173.6, 157.0, 138.2, 137.7, 133.9, 126.1, 68.8, 45.6, 29.1, 20.6. HRMS [M + H]+ calculated for C10H11BrN2O2, 271.00767; found, 271.00757.

Derivatization of (R)- and (S)-alcohol with (R)- MTPA-Cl and (S)-MTPA-Cl:

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The Novozym 435 catalyzed hydrolysis of RS-4a produced alcohol R-5 and acetate S4a. Alcohol R-5 was derivatized separately with (R)-3,3,3-trifluoro-2-methoxy-2phenylpropanoyl chloride (R-MTPA-Cl) and (S)-3,3,3-trifluoro-2-methoxy-2phenylpropanoyl chloride (S-MTPA-Cl). The reactions were carried out in pyridine-d5 in an NMR tube. Similarly, S-acetate S-4a formed after the enzymatic reaction was hydrolyzed with potassium carbonate to form the alcohol S-5. The alcohol S-5 was derivatized separately with R-MTPA-Cl and S-MTPA-Cl. The reactions were carried out in pyridine-d5 in an NMR tube.

Derivatization of (R)-1-(6-bromo-2-methylpyridin-3-yl)-3-hydroxypyrrolidin-2-one R-5 with S-camphanic chloride and x-ray crystallography (6)

The alcohol R-5 (500 mg, 1.84 mmol), (1S)-(-)-camphanic chloride (519 mg, 2.40 mmol) and 3.0 mL tetrahydrofuran were added to a three neck flask. The reaction mixture was stirred in an ice bath for 15 min. Pyridine (2.0 mL, d= 0.978, 24.73 mmol) was added to the reaction mixture, and it was stirred for 10 min using an ice bath and then raised to room temperature. The reaction was over in 3 h as evidenced by HPLC analysis of the reaction mixture. The tetrahydrofuran was removed under vacuum and the residue was diluted with 50 mL of ethyl acetate. The ethyl acetate solution was washed with dilute hydrochloric acid (1N HCl, 1 x 30 mL), water (2 x 25 mL), 10% sodium chloride solution (1 x 30 mL) and with water again (2 x 25 mL). The ethyl acetate layer was separated, dried over anhydrous sodium sulfate, filtered and the solvent was removed under vacuum. (1S,4S)-(R)-1-(6-bromo-2-methylpyridin-3-yl)-2-oxopyrrolidin-3-yl 4,7,7trimethyl-3-oxo-2-oxabicyclo[2.2.1]heptane-1-carboxylate 6 was isolated as a white 31



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solid 826 mg, AP 99.3%, yield 99.3%. The compound 6 was crystallized out from two solvent mixtures: (1) 550 mg was dissolved in a 50 mL solution of 25% methanol-75% ethyl acetate, warmed, the flask was closed with a cotton plug, and the solution was stored in the hood for six days with slow evaporation. Needle shaped crystals (280 mg) were isolated by filtration. (2) 100 mg of compound 6 (native) was dissolved in 10 mL of acetone, the flask was closed with a cotton plug and stored in the hood for two days with slow evaporation. Needle shaped crystals 51 mg were filtered out. The crystals obtained from acetone crystallization were analyzed by X-ray crystallography. A single crystal X-ray crystallography confirmed that absolute stereochemistry for the alcohol R5 formed after enzymatic hydrolysis was R. Compound 6 : 1H NMR (400 MHz, CDCl3): δ 7.40 (d, J = 8.3 Hz, 1H), 7.36 (d, J = 8.3 Hz, 1H), 5.68 (t, J = 8.3 Hz, 1H), 3.82 - 3.70 (m, 2H), 2.73 (dddd, J = 13.2, 8.9, 6.3, 4.0 Hz, 1H), 2.59 – 2.36 (m, 5H), 2.08 (ddd, J = 13.5, 9.2, 4.5 Hz, 1H), 1.96 (ddd, J = 13.2, 10.8, 4.5 Hz, 1H), 1.71 (ddd, J = 13.3, 9.2, 4.3 Hz, 1H), 1.13 (s, 3H), 1.09 (s, 3H), 1.04 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 178.1, 168.6, 166.8, 157.3, 140.1, 136.8, 132.3, 126.3, 90.8, 71.3, 54.9, 54.8, 46.5, 30.5, 28.8, 25.9, 20.9, 16.6, 16.5, 9.7. HRMS [M + H]+ calculated for C20H23BrN2O5, 451.08631; found, 451.08635.

Synthesis of 1-(6-bromo-2-methylpyridin-3-yl)-2-oxopyrrolidin-3-yl propionate RS4b

Alcohol RS-5 (5.0 g, 18.44 mmol) and pyridine (2.98 mL, d=0.978, 36.89 mmol, 2 molar equivalent) were added to a three neck round bottom flask and the temperature of the reaction mixture was lowered using an ice bath. Propionic anhydride (4.73 mL, d= 32


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1.015, 36.89 mmol) was added drop wise into the reaction. The ice bath was removed and the reaction mixture was stirred overnight (16 h) at room temperature. The residue was suspended in 200 mL of ice cold water and stirred. The solid was filtered and the precipitates were thoroughly washed with water, 2N HCl solution and water. 1-(6bromo-2-methylpyridin-3-yl)-2-oxopyrrolidin-3-yl propionate RS-4b was isolated as a light brownish solid 4.5 g, yield 75.25%, AP 99.2. Compound RS-4b: 1H NMR (500 MHz, CDCl3): δ 7.40 (d, J = 8.2 Hz, 1H), 7.37 (d, J = 8.2 Hz, 1H), 5.44 (t, J = 8.0 Hz, 1H), 3.81 - 3.60 (m, 2H), 2.74 (m, 1H), 2.58 - 2.35 (m, 5H), 2.31 - 2.13 (m, 1H), 1.20 (t, J = 7.4 Hz, 3H). 13C NMR (125 MHz, CDCl3): δ 173.7, 169.6, 157.3, 140.0, 136.8, 132.5, 126.3, 70.5, 46.6, 27.3, 26.7, 21.0, 8.9. HRMS [M + H]+ calculated for C13H15BrN2O3, 327.03388; found, 327.03378.

Synthesis of 1-(6-bromo-2-methylpyridin-3-yl)-2-oxopyrrolidin-3-yl butyrate RS-4c

Alcohol RS-5 (5.0 g, 18.44 mmol) and pyridine (2.98 mL, d=0.978, 36.89 mmol, 2 molar equivalent) were added to a three neck round bottom flask and the temperature of the reaction mixture was lowered by using an ice bath. Butyric anhydride (6.035 mL, d=0.967, 36.89 mmol) was added drop wise into the reaction mixture. The ice bath was removed and the reaction mixture was stirred overnight (14 h) at room temperature. The residue was suspended in ice cold water and stirred. The solid was filtered and the precipitates were washed thoroughly with water, 2N HCl and again with water. The solid was dried and 1-(6-bromo-2-methylpyridin-3-yl)-2-oxopyrrolidin-3-yl butyrate RS-4c was isolated as an off-white solid 5.7 g, yield 90.6%, AP99.4.

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Compound RS-4c: 1H NMR (500 MHz, CDCl3): δ 7.40 (d, J = 8.2 Hz, 1H), 7.37 (d, J = 8.2 Hz, 1H), 5.44 (t, J = 8.2 Hz, 1H), 3.78 - 3.65 (m, 2H), 2.80 - 2.69 (m, 1H), 2.52 - 2.36 (m, 5H), 2.30 - 2.17 (m, 1H), 1.74 - 1.67 (m, 2H), 0.99 (t, J = 7.3 Hz, 3H). 13C NMR (125 MHz, CDCl3): δ 172.8, 169.6, 157.3, 140.0, 136.8, 132.5, 126.3, 70.5, 46.6, 35.8, 26.7, 21.0, 18.3, 13.5. HRMS [M + H]+ calculated for C14H17BrN2O3, 341.04953; found, 341.04953.

Synthesis of 1-(6-bromo-2-methylpyridin-3-yl)-2-oxopyrrolidin-3-yl hexanoate, RS4d

Alcohol RS-5 (10.0 g, 36.89 mmol) and pyridine (5.967 mL, d=0.978, 73.77 mmol, 2 molar equivalent) were added to a round bottom flask and the temperature of the reaction mixture was lowered using an ice bath. Hexanoic anhydride (17.04 mL, d= 0.928, 73.78 mmol) was added drop wise into the reaction mixture. In the beginning, the reaction mixture was heterogeneous. After 4 hours, the reaction mixture became homogeneous and after some time precipitates were formed. The ice bath was removed and the reaction mixture was stirred overnight (15 h) at room temperature. The residue was suspended in 100 mL of ice cooled water, stirred, and filtered. The precipitates were thoroughly washed with 100 mL ice cooled water, 100 mL 2N HCl solution and 150 mL cold water. The precipitates were suspended in 300 mL heptanes, stirred vigorously for 30 min, filtered and the precipitates were washed with another 200 mL of heptane. 1-(6-bromo-2-methylpyridin-3-yl)-2-oxopyrrolidin-3-yl hexanoate RS-4d was isolated as off-white solid 13.05 g, yield 95.8%, AP 98.

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Compound RS-4d: 1H NMR (500 MHz, DMSO-d6): δ 7.68 (d, J = 8.2 Hz, 1H), 7.58 (d, J = 8.2 Hz, 1H), 5.50 (t, J = 8.5 Hz, 1H), 3.80 - 3.70 (m, 1H), 3.70 - 3.62 (m, 1H), 2.65 2.54 (m, 1H), 2.38 (t, J = 7.3 Hz, 2H), 2.33 (s, 3H), 2.22 - 2.07 (m, 1H), 1.56 (br. s., 2H), 1.28 (br. s., 4H), 0.86 (br. s., 3H). 13C NMR (125 MHz, CDCl3): δ 173.1, 169.7, 157.4, 140.1, 136.8, 132.6, 126.3, 70.5, 46.6, 34.0, 31.2, 26.7, 24.5, 22.3, 21.0, 13.9. HRMS [M + H]+ calculated for C16H21BrN2O3, 369.08083; found, 369.08075.

Enzymatic hydrolysis of hexanoate RS-4d

Acetonitrile (49 mL) and water (1.0 mL, 2%) were added to a jacketed glass reactor and the reaction temperature was maintained at 45 °C by using a water circulator. RSHexanoate RS-4d (5.0 g, 13.54 mmol, concentration 100 g/L) was added to the reaction mixture with continuous stirring followed by addition of Novozym 435 (25 mg, enzyme to substrate ratio of 1:200). The reaction progress was monitored by HPLC, 20 µL samples were taken out, diluted with 1.980 mL of acetonitrile, vortexed, filtered through 0.2 µM filter and analyzed by achiral and chiral HPLC. The reaction conversion of 49% was achieved in 25 h (ee of R-alcohol R-5 >99.9%, Table S2). The enzyme was filtered out and the residue was washed with 10 mL of acetonitrile. The solvent was removed under reduced pressure in a rotatory evaporator and the residue was suspended in 70 mL of water. The solution was extracted with toluene (2 x 25 mL) and a nice separation of aqueous layer from organic layer was observed. The undesired hexanoate S-4d was extracted into toluene and the desired R-5 remained in the aqueous layer. The aqueous layer was diluted with 30 mL of saturated (~35%) sodium chloride solution. The aqueous layer was extracted with dichloromethane (3 x 50 mL) and complete extraction 35



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of alcohol R-5 in to the dichloromethane was observed. The combined dichloromethane extracts were washed with water (50 mL). The dichloromethane solution was dried over anhydrous sodium sulfate, filtered, and the solvent was removed. The residue was stirred with heptanes (200 mL) and filtered. The white solid R-alcohol R-5 was dried in the vacuum oven, 1.42 g, yield 77.8%, AP 98.9, ee >99.9%.

Enzymatic enantioselective acylation of racemic alcohol RS-5 in different solvents Multiple solvents were screened including acetonitrile, toluene, 2-methyltetrahydrofuran, dichloromethane, ethanol, heptanes, acetone, tetrahydrofuran, methyl tertiary butyl ether, 1, 4-dioxane, 2-ethoxyethanol, 1-propanol, 2-methyl 1-propanol and 1,2propanediol. A stock solution of racemic alcohol RS-5 (1.0 mg/10 µL) was prepared in DMSO and each vial was charged with ~ 10 mg of Novozym 435, 100 µL of vinyl acetate, 10 µL of stock solution of racemic alcohol RS-5 and the vials were shaken in a Thermomixer R at 30 °C and 500 RPM. The reactions were stopped after 3 h, the solvent was removed, the residue was dissolved in 1.0 mL acetonitrile, vortexed and analyzed by HPLC. Acetonitrile (conversion 46.1%, ee of S-alcohol S-5 83.5%, ee of Racetate R-4a 97.8%), 2-methyltetrahydrofuran (conversion 38%, ee of S-alcohol S-5 56.8%, ee of R-acetate R-4a 92.8%), dichloromethane (conversion 45.6%, ee of Salcohol S-5 81.4%, ee of R-acetate R-4a 96.9%), and tetrahydrofuran (conversion 48.2%, ee of S-alcohol S-5 90% and ee of R-acetate R-4a 96.7%) gave good selectively; with a better combination of ee and conversion in tetrahydrofuran than other solvents. The Mitsunobu inversion of stereochemistry was successfully implemented in

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tetrahydrofuran and the tetrahydrofuran was selected for further studies. In follow up studies the acylation of the racemic alcohol RS-5 with vinyl hexanoate (n-caproic acid vinyl ester) in different solvents (2-methyltetrahydrofuran, tetrahydrofuran, acetonitrile, toluene, dichloromethane, acetone, MTBE, and MIBK) under similar conditions showed 2-methyltetrahydrofuran (conversion 50.2%, ee of R-hexanoate R-4d 99.1%, ee of Salcohol S-5 >99.9%) is a the better solvent.

Racemic alcohol RS-5 (1.0 g, 3.69 mmol), vinyl acetate (680 µL, d=0.934, 7.38 mmol), Novozym 435 (100 mg, enzyme to substrate ratio 1:10) and tetrahydrofuran (50 ml) were added to a round bottom flask . The reaction progress was monitored by HPLC and the reaction was over in 3 h (conversion 48.9%). R-acetate R-4a and S-alcohol S-5 were formed with an ee of 99.1% and 94.7%, respectively. The enzyme was filtered out, the solvent was removed and the residue was used for the inversion of stereochemistry without any purification as follows. After the reaction, the expected S-alcohol S-5 in the reaction mixture was 433 mg (1.59 mmol). To the residue added 25 mL tetrahydrofuran, triphenyl phosphine 628 mg, 2.40 mmol, p-nitrobenzoic acid 400 mg, 2.40 mmol and the reaction mixture was stirred in an ice bath. To the reaction mixture added diisopropyl azodicarboxylate 472 µL, 2.39 mmol. Unlike the reaction done with pure S-alcohol S-5 (experimental section), the reaction with crude material (in the presence of R-acetate R4a and other likely impurities from the enzyme) did not go to completion (maximum conversion ~80%).

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Synthesis of (RS)-1-(6-bromo-2-methylpyridin-3-yl)-2-oxopyrrolidin-3-yl 4nitrobenzoate RS-7

Racemic alcohol RS-5 (2.0 g, 7.38 mmol), triphenylphosphine (2.902 g, 11.07 mmol, 1.5 molar equivalent), p-nitro benzoic acid (1.849 g, 11.07 mmol, 1.5 molar equivalent) and 50 mL anhydrous tetrahydrofuran were added to a round bottom flask. The reaction mixture was cooled to ~0 °C using an ice bath. Diisopropyl azodicarboxylate (2.179 mL, d=1.027, 11.07 mmol, 1.5 molar equivalent) was added drop wise into the reaction mixture. The reaction was stirred for 30 min using an ice bath. The ice bath was removed and the reaction mixture was stirred overnight (16 h) at room temperature. The reaction progress was assessed by HPLC, 25 µL samples were taken out, solvent was removed and the residue was dissolved in 1.0 mL of acetonitrile, filtered and analyzed by HPLC. The reaction was over in 3-4 h as evident from HPLC analysis and no significant change in peak area percentage was observed over 16 h. The solvent was removed under reduced pressure and the residue was purified with silica gel column chromatography using 50% ethyl acetate and heptanes (Rf-0.25). The pure fractions were pooled and the white solid product, (RS)-1-(6-bromo-2-methylpyridin-3yl)-2-oxopyrrolidin-3-yl 4-nitrobenzoate 2.849 g, AP 98.7, yield 91.93% was isolated. Compound RS-7: 1H NMR (500 MHz, DMSO-d6): δ 8.39 (d, J = 8.5 Hz, 2H), 8.25 (d, J = 8.5 Hz, 2H), 7.73 (d, J = 8.2 Hz, 1H), 7.60 (d, J = 8.2 Hz, 1H), 5.83 (t, J = 8.5 Hz, 1H), 3.91 - 3.70 (m, 2H), 2.80 - 2.67 (m, 1H), 2.46 - 2.30 (m, 4H). 13C NMR (125 MHz, CDCl3): δ 168.9, 163.9, 157.3, 150.8, 140.2, 136.8, 134.5, 132.3, 131.1, 126.3, 123.6,

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71.9, 46.5, 26.5, 21.0. HRMS [M + H]+ calculated for C17H14BrN3O5, 420.01896; found, 420.01865.

Synthesis of (R)-1-(6-bromo-2-methylpyridin-3-yl)-2-oxopyrrolidin-3-yl 4nitrobenzoate R-7

(S)-1-(6-bromo-2-methylpyridin-3-yl)-3-hydroxypyrrolidin-2-one S-5 (3.0 g, 11.07 mmol), triphenylphosphine (4.354 g, 16.60 mmol, 1.5 molar equivalent), p-nitro benzoic acid (2.774 g, 16.60 mmol, 1.5 molar equivalent) and 75 mL anhydrous tetrahydrofuran were added to a round bottom flask. The reaction mixture was cooled to 0 °C using an ice bath. Diisopropyl azodicarboxylate (3.268 mL, 16.60 mmol, 1.5 molar equivalent) was added drop wise into the reaction mixture. The reaction mixture was stirred for 30 min, the ice bath was removed and the reaction mixture was stirred at room temperature for 3 h. Samples (10 µL) were taken out, solvent was removed, the residue was dissolved in 1.0 mL of acetonitrile, filtered and analyzed by HPLC. The reaction is fast and minimum to no change in peak area percentage was observed for samples analyzed at 30 min, 2 h and 3 h. With the addition of DIAD, yellow color appears which disappears instantaneously. In the end of the DIAD addition yellow color persists, this could be an indicator of reaction completion. The reaction was stopped after 3 h and the solvent was removed under reduced pressure. The residue was purified with silica gel column chromatography using a gradient of 40% ethyl acetate-heptane and 50% ethyl acetate heptanes (Rf-0.25). The pure fractions were pooled and the white solid product, (R)-1-

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(6-bromo-2-methylpyridin-3-yl)-2-oxopyrrolidin-3-yl 4-nitrobenzoate R-7, 4.1 g, AP 99.5, yield 88.17%, ee 99.3% was isolated. Compound R-7: 1H NMR (500 MHz, DMSO-d6): δ 8.39 (d, J = 8.9 Hz, 2H), 8.25 (d, J = 8.5 Hz, 2H), 7.74 (d, J = 8.2 Hz, 1H), 7.60 (d, J = 8.2 Hz, 1H), 5.83 (t, J = 8.5 Hz, 1H), 3.89 - 3.71 (m, 2H), 2.79 - 2.68 (m, 1H), 2.45 - 2.32 (m, 4H). 13C NMR (125 MHz, CDCl3): δ 168.6, 163.6, 157.0, 150.5, 138.7, 137.8, 134.4, 133.2, 130.9, 126.2, 124.0, 72.2, 46.0, 25.9, 20.6. HRMS [M + H]+ calculated for C17H14BrN2O5, 420.01896; found, 420.01880.

Hydrolysis of (R)-1-(6-bromo-2-methylpyridin-3-yl)-2-oxopyrrolidin-3-yl 4nitrobenzoate R-7 to R-alcohol R-5

(R)-1-(6-bromo-2-methylpyridin-3-yl)-2-oxopyrrolidin-3-yl 4-nitrobenzoate (500 mg, 1.19 mmol), potassium carbonate (164 mg 1.19 mmol) and 25 mL methanol were added to a round bottom flask and the reaction mixture was stirred at room temperature for 30 min. The reaction progress was assessed by using HPLC. Samples (50 µL) were taken out, diluted with 950 µL of acetonitrile and analyzed using HPLC. Hydrolysis of (R)-1-(6bromo-2-methylpyridin-3-yl)-2-oxopyrrolidin-3-yl 4-nitrobenzoate R-7 was fast and almost all the material was hydrolyzed in 10 min. The reaction was stopped after 30 min and methanol was removed under vacuum. No racemization of (R)-1-(6-bromo-2methylpyridin-3-yl)-2-oxopyrrolidin-3-yl 4-nitrobenzoate R-7 or R-alcohol R-5 was observed during hydrolysis. The residue was dissolved in 50 mL ethyl acetate, extracted with 25 mL water, and 25 mL of 5% sodium bicarbonate. The ethyl acetate layer was washed with 25 mL of 20% sodium chloride and 25 mL water. The aqueous layer 40


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contains a part of desired alcohol R-5 which was back extracted with ethyl acetate (3 x 75 mL). The combined ethyl acetate solution was dried over anhydrous sodium sulfate, filtered and the solvent was removed. The residue was crystallized from 50% ethyl acetate-heptane. The pure R-alcohol R-5 was isolated as a white solid 240 mg, yield 74.3%, AP 98.9, ee >99.9%.

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Acknowledgements We thank our ABD colleagues Drs Charles Pathirana for NMR analysis, Michael Peddicord for HRMS analysis and Qi Gao for X-ray crystallography. Dr Nicolas Cuniere is acknowledged for helpful discussions. Authors are thankful for Dr. David Kronenthal for reviewing the manuscript and making helpful suggestions.

Supporting Information Additional figures and tables are in the Supporting Information. This Information is available free of charge via the internet at http://pubs.acs.org/.

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Chen, L.; Magliano, D. J.; Zimmet, P. Z. Nat. Rev. Endocrinol. 2012, 8, 228236.

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Inagaki, N.; Watada, H.; Murai, M.; Kagimura, T.; Gong, Y.; Patel, S.; Woerle, H. J. Diabetes, Obes. Metab. 2013, 15, 833-843 and references cited therein.

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Juillerat-Jeanneret, L. J. Med. Chem. 2014, 57, 2197-2212

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(a). Ohishi, T.; Yoshida, S. Expert Opin. Investig. Drugs 2012, 21, 321-328. (b) Darout, E.; Robinson, R. P.; McClure, K. F.; Corbett, M.; Li, Bryan.; Shavnya, A.; Andrews, M. P.; Jones, C. S.; Li, Q.; Minich, M. L.; Mascitti, V.; Guimaraes, C. R. W.; Munchhof, M. J.; Bahnck, K. B.; Cai, C.; Price, D. A.; Liras, S.; Bonin,

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P. D.; Cornelius, P.; Wang, R.; Bagdasarian, V.; Sobota, C. P.; Hornby, S.; Masteron, V. M.; Joseph, R. M.; Kalgutkar, A. S.; Chen, Y. J. Med. Chem. 2013, 56, 301-319. 5.

(a). Overton, H.; Fyfe, M.; Reynet, C. Br. J. Pharmacol. 2008, 153, S76-S81 (b). Buzard, D. J.; Lehmann, J.; Han, S.; Jones, R. M. Pharm. Pat. Analyst 2012, 1, 285-299.

6.

(a). Wacker, D. A.; Wang, Y.; Broekema, M.; Rossi, K.; O’Connor, S.; Hong, Z.; Wu, G.; Malmstrom, S. E.; Hung, C.; LaMarre, L.; Chimalakonda, A.; Zhang, L.; Xin, L.; Cai, H.; Chu, C.; Boehm, S.; Zalaznick, J.; Ponticiello, R.; Sereda, L.; Han, S.; Zebo, R.; Zinker, B.; Luk, C. E.; Wong, R.; Everlof, G.; Li, Y.; Wu, C. K.; Lee, M.; Griffen, S.; Miller, K. J.; Krupinski, J.; Robl, J. A. J. Med. Chem. 2014, 57, 7499-7508. (b) Ye, X.; Morales, C. L.; Wang, Y.; Rossi, K. A.; Malmstrom, S. E.; Abousleiman, M.; Sereda, L.; Apedo, A.; Robl, J. A.; Miller, K. J.; Krupinski, J.; Wacker, D. A. Bioorg. Med. Chem. Lett. 2014, 24, 25392545.

7.

Pyrimidinylpiperidinyloxypyridone analogues as GPR119 modulators. Broekema, M.; Wu, G.; Wacker, D. A. PCT Int. Appl. WO 2013173198A1 20131121, 2013.

8.

FDA’s Policy Statement for the development of new stereoisomeric drugs. Chirality, 1992, 4, 338-340.

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Pires, R.; Burger, K. Tetrahedron 1997, 53, 9213-9218.

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(a). Bentley, J. M.; Wadsworth, H. J.; Willis, C. L. J. Chem. Soc., Chem. Commun. 1995, 231-232. (b). Gibbs, G.; Hateley, M. J.; McLaren, L.; Welham, M.; Willis, C. L. Tetrahedron Lett. 1999, 40, 1069-1072.

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Kamal, A.; Ramana, K. V.; Ramana, A. V.; Babu, A. H. Tetrahedron: Asymmetry 2003, 14, 2587-2594.

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(a). Gutman, A. L.; Brenner, D.; Boltanski, A. Tetrahedron: Asymmetry 1993, 4, 839-844. (b). Fukusaki, E.; Satoda, S.; Senda, S.; Omata, T. J. Ferment. Bioeng. 1998, 86, 508-509. (c). Rasalkar, M. S.; Potdar, M. K.; Salunkhe, M. M. J. Mol. Catal. B: Enzym 2004, 27, 267-270. (d). Bouzemi, N.; Debbeche, H.; Aribi-Zouioueche, L.; Fiaud, J. Tetrahedron Lett. 2004, 45, 627-630. (e). Zada, A.; Dunkelblum, E. Tetrahedron: Asymmetry 2006, 17, 230-233.

13.

Lipase PS SD and lipase PSIM from Amano Enzyme Inc., Sprin lipo and Sprin epobond from Sprin Technologies, lipase SL from Meito-Sangyo Co. Ltd., IMMABC-T2-150 from ChiralVision and lipase P1 and lipase P2 from Julich Enzyme Products GmbH.

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(a). Gotor-Fernandez, V.; Busto, E.; Gotor, V. Adv. Synth. Catal. 2006, 348, 797-812. (b). Idris, A.; Bukhari, A. Biotechnol. Adv. 2012, 30, 550-563. (c) Truppo, M. D.; Hughes, G. Org. Process Res. Dev. 2011, 15, 1033-1035. (d) Anderson, N. G. Org. Process Res. Dev. 2012, 16, 852-869.

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Skliar, D.; Laporte, D. L.; Chuang, S.; Singh, A.; Cuniere, N.; Goswami, A. AIChE conference Nov 17, 2014, Atlanta. Process Intensification Using a Continuous Packed Bed Reactor for Enzymatic Hydrolysis (publication under preparation).

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Scheme 1: Enzymatic approaches to prepare intermediate S-1

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Scheme 2: Lipase PS 30 catalyzed resolution to prepare intermediate S-1

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Scheme 3: Novozym 435 catalyzed preparation of chiral intermediate R-5

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Figure 1: Novozym 435 catalyzed hydrolysis of RS-4a at different water concentration

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Figure 2: Novozym 435 catalyzed hydrolysis of RS-4a at pilot plant scale

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Figure 3: Novozym 435 catalyzed resolution of RS-4a at 20 g/L, 40 g/L, 60 g/L, 80 g/L and 100 g/L concentrations in 1% water

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Figure 4: X-Ray crystallography of 6

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Table 1: Evolution of enzymatic process to prepare R-5

Description

Campaign 1

Campaign 2

Projected campaign

Reaction

RS-Acetate RS-4a

RS-Acetate RS-4a

RS-Hexanoate RS-4d

conditions

20 g/L

60 g/L

100 g/L

E:S 1:4, 25%

E:S 1:25, 4%

1:200, 0.5%

CH3CN 90%

CH3CN 99%

CH3CN 98%

Water 10%

Water 1%

Water 2%

Temp. 25 °C

Temp. 25 °C

Temp.45 °C

Starting RS-ester

13 Kg (2 batches)

17 Kg (2 batches)

Lab scale batches*

Reaction time

~22 h

~ 24 h

~ 15 h

Solvent swap time

~ 16 h

Not required

Not required

Workup**

Chromatography

Chromatography

Extraction

Yield R-alcohol R-5

4.1 Kg, 36.5%

5.5 Kg, 37.4%

35-40% lab scale

Ee R-alcohol R-5

>99.4%

>99.5%

>99.6 lab scale

Enzyme g/Kg of RSester

250 g

40 g

5g

for water removal

*See table S2 in supporting information **The final step is crystallization of the R-alcohol R-5

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Enzymatic Process for N-Substituted (3S)- and (3R ... - ACS Publications

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