Communication pubs.acs.org/OPRD
Development of a Two-Step, Enantioselective Synthesis of an Amino Alcohol Drug Candidate Michael A. Schmidt,* Emily A. Reiff, Xinhua Qian, Chao Hang, Vu Chi Truc, Kenneth J. Natalie, Chenchi Wang, Jacob Albrecht, Andrew G. Lee, Ehrlic T. Lo, Zhiwei Guo, Animesh Goswami, Steven Goldberg, Jaan Pesti, and Lucius T. Rossano* Chemical Development, Bristol-Myers Squibb, One Squibb Drive, New Brunswick, New Jersey 08903, United States of the material is lost as the undesired enantiomer and must be purged in a separate resolution step, with no opportunity to recycle. Ultimately, the issue of low material throughput (∼25% overall yield) and the number of required operations for such a short sequence were strong factors in the decision to develop alternative routes to 1. Amino Acid through an Enzymatic Aldol Reaction. A myriad of different catalytic, asymmetric routes to 1 were considered such as a dynamic, kinetic reduction of a beta−keto amide, an asymmetric Henry reaction, an asymmetric Darzens reaction, an enantioselective [3 + 2] cyclization akin to Scheme 1 and an enzymatic aldol reaction.2 Early work showed significant promise for the enzymatic aldol process, delivering both stereogenic centers in a single operation from commercially available materials (Scheme 2). Specifically, a Dthreonine aldolase catalyzed aldol reaction between 4-pyridine carboxaldehyde (4) and glycine (8) afforded the desired hydroxy amino acid 9 with good yield and excellent chiral purity. The enzyme was produced in recombinant E. coli cells and partially clarified cell extracts were used in the reaction. The optimal pH for the reaction, pH = 8.0, was also close to the isoelectric point of 9, and the desired hydroxy amino acid diastereomer precipitated from the reaction mixture allowing for a simple isolation by filtration upon reaction completion. This greatly simplified an otherwise tedious separation from the cofactor and proteinaceous components of the enzymatic reaction mixture. After extensive optimization, a 72% yield of 9 was realized with 99.1% de and 99.7% ee (Scheme 2).3 Unfortunately, the pyrrolidine amide of glycine was not a suitable substrate for the aldolase enzyme, therefore the pyrrolidine ring had to be appended through an additional amide bond formation step. Development Synthesis 1: Synthesis of 1 via Boc Carbamate. Conversion of the amino acid 9 to 1 was initially demonstrated using standard peptide coupling chemistry. This began with the protection of the amino group with Boc2O to avoid side reactions such as retro-aldol decomposition and dimerization. As an additional benefit, protection also significantly increased the solubility of the amino acid derivative in organic media. Thus, Boc protection of 9 afforded the carbamate 10, which was converted to the pyrrolidine amide 11 with EDC in the presence of N-hydroxysuccinimide. The Boc group of 11 was subsequently cleaved with methanolic HCl, the resulting HCl salt neutralized, and the free base was crystallized
ABSTRACT: The process development and large scale synthesis for the β-hydroxyamino amide 1 is described. The route evolved from a multistep sequence utilizing a classical resolution to a two-step enantioselective process involving an enzyme-catalyzed aldol reaction and a direct amidation of a carboxylic acid. By utilizing a siliconmediated direct amidation strategy, the route was devoid of protecting and deprotecting steps while retaining the stereochemical integrity of a highly sensitive β-hydroxyamino acid. The two-step strategy employed herein significantly improved the yield, process greenness, cycle time, and estimated cost in the production of 1.
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INTRODUCTION The β-hydroxyamino amide L-tartrate salt 11 is a drug candidate with a high projected human dose. As a result, a synthesis that would reliably produce metric ton quantities of 1 is needed. This report details the identification and development of a twostep synthesis which enabled the realization of this goal.
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RESULTS AND DISCUSSION
Previous Synthesis. The initial synthesis (Scheme 1)1c that was developed used a [3 + 2] cyclization between isocyanide 3 and aldehyde 4 to construct an oxazoline 5 diastereoselectively, albeit racemically. After hydrolysis of the oxazoline ring in 5, the amino alcohol was isolated as a bishydrochloride salt. The enantiomers were then separated through a classical resolution with di-p-toluoyl-L-tartaric acid (LDTTA). The desired enantiomer was liberated from the resolving acid with KOH, and the crude free base stream was passed though a resin, then crystallized as the L-tartrate salt to afford 1 in an average overall yield of 25%. The anticipated high clinical dose for compound 1 required metric tons to support late stage clinical trials. While this synthesis performed well, a number of inherent inefficiencies of the process were not suitable for larger scale campaigns. The first reagent in the synthesis, ethyl isocyanoacetate (2) is toxic, unstable, expensive, and not readily available. The subsequent cyclization with 2 affords a racemic oxazoline 5; therefore, 50% © 2015 American Chemical Society
Received: June 12, 2015 Published: July 2, 2015 1317
DOI: 10.1021/acs.oprd.5b00192 Org. Process Res. Dev. 2015, 19, 1317−1322
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Communication
Scheme 1. Initial Synthesis of 1
venting capacity. The EDC-mediated amidation of 10 with pyrrolidine was scaled without issue; the urea and hydroxysuccinimide byproducts were conveniently washed away with an aqueous pH 7 buffer. A solvent swap into acetonitrile crystallized the pyrrolidinoamide 11 in 88% yield, >99.9% purity, and 98.8% potency. The final Boc deprotection was carried out using TMSCl in methanol to generate HCl in situ. Upon reaction completion, the acids were quenched with methanolic KOH, and the insoluble portion of KCl was removed by filtration. The soluble portion of KCl was removed by treating the filtrate with 1.3 equiv of L-tartaric acid, which precipitated potassium bitartrate immediately and was removed by filtration. This step was conducted as residual KCl impacted the downstream crystallization of 1. After filtration, 1 was crystallized by addition of ethanol as the antisolvent at 35 °C to afford the product in 83% yield, >99.9% purity, and 99.7% potency. This sequence (Scheme 3) achieved the goals of eliminating the use of ethyl isocyanoacetate (2) and avoiding a chiral resolution. However, there was still multiple unit operations to this process, therefore further improvements were sought to increase material throughput. Our goal was to eliminate the Boc protection and deprotection steps.4 Additionally, it was desirable to avoid handling 1 in the free base form because, like 9, facile retro-aldol decomposition occurred in solution. In considering alternative strategies that allowed for protectinggroup free approaches, we sought to exploit the higher nucleophilicity of pyrrolidine compared to the amine of 9. The Fischer esterification of the amino acid 9 to a methyl ester was studied, followed by the direct amidation of the ester with pyrrolidine. Development Synthesis 2: Direct Amidation of a Methyl Ester. The foray into a protecting-group free route began with a Fischer esterification of 9, initially performed by heating in MeOH at 55 °C with anhydrous HCl. Under these
Scheme 2. Enzymatic Aldol Reaction
as before with L-tartaric acid to afford the desired compound 1 (Scheme 3). Scheme 3. Three-Step Conversion of 9 to 1
This sequence was successfully demonstrated at kilogram scale. The Boc protection of 9 required careful monitoring of the reaction pH to allow for the controlled release of carbon dioxide from the breakdown of the Boc anhydride. Maintaining the pH between 8 and 10 led to a metered rate of gas generation while also minimizing Boc anhydride hydrolysis. This control was used to safely run the process on scale, as uncontrolled gas evolution may have exceeded the equipment’s Scheme 4. Synthesis of 1 via Methyl Ester 12
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DOI: 10.1021/acs.oprd.5b00192 Org. Process Res. Dev. 2015, 19, 1317−1322
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conditions, the heterogeneous reaction would stall at ∼65% conversion and usually required additional charges of acid due to the loss of HCl vapors. To increase the rate of esterification, the solubility of 9 was increased by using aqueous concentrated HCl and a phase transfer catalyst, optimally tetrabutylammonium triflate or sodium triflate (Scheme 4). While the rate of the esterification did increase, the reaction only achieved ∼85% conversion due to the presence of excess water. The inclusion of dehydrating agents such as orthoformates had minimal impact. Other acids such as sulfuric acid and methanesulfonic acid improved conversion to >90%, but complicated the isolation of the ester due to poor crystallinity of the corresponding salts and would possibly introduce genotoxic sulfate esters into the penultimate step of the process. The methyl ester dihydrochloride 12 could be easily isolated in 85− 92% purity (remainder is 9) by cooling the reaction mixture and filtering. Further purification of the salt to >95% could be achieved through a reslurry in methanol containing 1−5% water; however, this was not necessary as the remaining 9 could be purged in the next amidation step. In an initial experiment, ester 12 was successfully converted to the pyrrolidine amide by simple incubation in neat pyrrolidine. However, significant decomposition via retroaldol and other unidentified pathways was observed. In addition, a variable amount (∼5−10%) of C2 epimerization was observed. After screening a variety of conditions, the choice of solvent had the most profound effect on the impurity profile. A screen of a variety of solvents revealed that acetone and methyl isobutylketone (MIBK) afforded the desired product with minimal decomposition or epimerization. Optimally, the methyl ester 12 was amidated by treatment with 5 equiv of pyrrolidine in MIBK for 24−48 h at room temperature. By monitoring the reaction through NMR and mass spectrometry, the reason for the enhanced chemical stability and stereochemical retention was discovered. A MIBK oxazolidine 13 (55:45 ratio of isomers at the ketal center, Figure 1) formed
with aqueous NaCl was necessary; however, this resulted in an additional ∼4% loss of 1. Addition of ethanolic L-tartaric acid affected the crystallization of 1 in 53% yield and 95−98% purity. After recrystallization from an ethanol, methanol, and water mixture, 1 was obtained in 99.9% purity. This approach represented a possibility to prepare 1 with no formal protecting group steps by using the solvent to stabilize the molecule in situ as an oxazolidine. However, the difficulties encountered in achieving a full conversion in methyl ester preparation as well as the high loss of the product during isolation resulted in only a moderate overall yield (∼47%). We considered leveraging this result in a new way by exploring an approach where an easily removable silicon group could be used to stabilize and possibly rigidify the hydroxyethylamine motif, while simultaneously activating the carboxyl group for amidation. We anticipated the salt forming step could remove the silicon group, thus potentially obviating an aqueous work up entirely. Development Synthesis #3: Direct Amidation of an Amino Acid. The direct amidation of carboxylic acids with amines can be affected under a variety of conditions;5 however, our attention was drawn to the use of silicon reagents via activation of carboxylates as silylesters as they are safer than the alternatives5 and readily available.6 Remarkably, reacting 9 with 4 equiv of Me2SiCl26a,b and 24 equiv of pyrrolidine afforded 15 in 96% purity and with 0.7% of the diastereomer (Scheme 5). Scheme 5. Dichlorodimethylsilane-Mediated Amidation
Interestingly, this diastereomer was the C3 epimer, not the C2 epimer.7 Potentially, Me2SiCl2 may stabilize 9 by formation of a chelate with the amino acid, while activating the carboxylic acid for coupling. In support of this, we found that performing the reaction with hexamethyldisilazane,8 which cannot form a chelate, led to 15 with significant (∼20%) epimerization. The C3 diastereomer (0.7%) completely purged in the downstream crystallization and was of no concern. In this new process, pyrrolidine functions as both solvent and reagent which effectively dissolves the amino acid 9 which is otherwise insoluble in most organic solvents. Even so, under these conditions, the reaction was heterogeneous due to the formation of pyrrolidine hydrochloride. The presence of these solids was a concern since mass transfer in the reaction was impacted; scale-ups were difficult to model, and rate calculations were not possible. To obviate the presence of the insoluble salts during the reaction, dimethyldipyrrolidinosilane (16) was synthesized which would preclude the need for a HCl acceptor (Scheme 6).9 This strategy allowed us to remove the
Figure 1. Oxazolidine intermediates (55:45 ratio of isomers at the ketal carbon).
instantaneously when 12 was mixed with pyrrolidine and MIBK. Further reaction monitoring showed that 13 then reacts with pyrrolidine to form 14. The formation of the oxazolidines 13 and 14 rationalized the enhanced reaction profile when MIBK was used as solvent. By forming 13 and 14, the chemical stability toward base induced retro-aldol decomposition was significantly increased. In addition, the stereochemical arrangement of 9 leads to the formation of thermodynamically favored trans-oxazolidines 13 and 14; therefore, the compounds resist epimerization. The majority of the excess pyrrolidine, pyrrolidine hydrochloride, and 9 was removed by a postreaction phase split and a 6 N HCl wash. One challenge to this process was the high water solubility of 1; in spite of saturating the HCl wash solution with NaCl, the loss of 1 was still ∼10%. In order to remove the remaining impurities, which hamper the isolation of 1, a subsequent wash
Scheme 6. Synthesis of 16
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by reverse phase HPLC using a Shimadzu Prominence LC equipped with a Waters Xbridge C8 (3.5 μm, 4.6 × 150 mm) column at a 1.0 mL/min flow rate and 210 nm detector wavelength. The mobile phases consisted of A) 0.05% ammonium hydroxide in 95:5 water−methanol and B) 0.05% ammonium hydroxide in 5:95 water: methanol. A gradient of t = 0 min, 0% B, t = 10 min, 13% B, t = 15 min, 100% B, t = 20 min, 100% B was used. Approximate retention times are (9) 1.3 min and (1) 12.2 min. Enantiomeric and diastereomeric ratios were obtained by HPLC using a Chiralpak AD-H (5.0 μm, 4.6 × 250 mm) column at a 1.0 mL/min flow rate and 257 nm detector wavelength. The mobile phases consisted of (A) 0.2% triethylamine in 90:10 acetonitrile: methanol and (B) 0.2% triethylamine in 70:30 acetonitrile−methanol. A gradient of t = 0, 0% B, t = 11 min, 0% B, t = 11.5 min, 100% B, t = 20 min, 100% B, t = 20.5 min, 0% B, t = 35 min, 0% B was used. Approximate retention times are (2R, 3R) 7.9 min, (2S, 3S) 9.0 min, (2R, 3S, 1) 11.0 min, (2S, 3R) 19.9 min. (2R,3S)-2-Amino-3-hydroxy-3-(pyridin-4-yl)propanoic Acid Dihydrate (9). To a reactor was added a 100 mM pH-8 potassium phosphate buffer solution (35.0 L,), glycine (4.15 kg, 55.30 mol, 1.90 equiv), pyridoxal-5-phosphate (3.65 g, 13.76 mmol, 0.00047 equiv), and manganese(II) chloride tetrahydrate (1.42 g, 8.49 mmol, 0.00029 equiv). The mixture was stirred until dissolved. 4-Pyridine carboxaldehyde (4, 2.80 L, 29.27 mol, 1.00 equiv) was added followed by approximately 175 u/mL D-threonine aldolase,3 and the solution was diluted with DI water to bring the 4 concentration to 4 vol %. The amount will vary based on the titer of the enzyme solution. The reaction mixture was heated to 25−30 °C and held for 5−6 h. The product 9 precipitates from the reaction mixture and is filtered directly onto a 20 μm polypropylene cloth and dried at 40 °C for 24 h. Isolated yields are generally in the 70% range. To dry 9 below KF of 1 wt %, 9 was dried in a tray dryer for 24−48 h at 45−50 °C with intermittent agitation. 1H NMR (400 MHz, D2O, 20 °C): δ 8.53 (d, J = 6.3 Hz, 2H), 7.50 (d, J = 6.3 Hz, 2H), 5.32 (d, J = 4.1 Hz, 1H), 3.93 (d, J = 4.1 Hz, 1H). 13C NMR (100 MHz, D2O, 20 °C): δ 150.0, 149.1, 121.6, 70.1, 60.2. IR (neat, cm−1): 3408 (s), 3208 (br s), 1647 (s), 1519 (s), 1408 (s). HRMS (ESI) m/z: Calcd for: C8H11N2O3 [M + H]+: 183.07642. Found: 183.07678. Elemental analysis: Calcd for: C8H14N2O5: C, 44.03; H, 6.46; N, 12.83. Found: C, 44.59; H, 6.30; N, 12.97. [α]D20 (c = 0.386, H2O): +33.98°. M.p.: 212 °C (decomp). (2R,3S)-2-((tert-Butoxycarbonyl)amino)-3-hydroxy-3(pyridin-4-yl)propanoic Acid (10). The amino acid 9 (3.80 kg, 17.42 mol, 1.00 equiv) was dissolved in water (19.0 kg), and the solution is basified to pH ∼ 11 with an aqueous solution of 5 N NaOH (4.45 kg). A solution of di-tert-butyl dicarbonate (6.08 kg, 27.86 mol, 1.60 equiv) in acetonitrile (6.80 L) was slowly charged while simultaneously controlling the pH of the reaction mixture between 8.3 and 9.0 with 5 N NaOH. After the reaction is complete, the slurry is slowly acidified with 6 N aqueous HCl (16.47 kg) to decompose the bicarbonate and crystallize 10. The mixture is filtered and washed with water to a conductivity 99.9% e.e. For the first time, the salt formation was fully integrated into the overall process. This process has been successfully demonstrated up to 450 g scale (Scheme 7). Scheme 7. Direct Amidation of 9 to 1 on 450 g Scale
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CONCLUSION Through the use of a D-threonine aldolase catalyzed aldol reaction followed by a silicon-mediated amidation, a short synthesis of 1 has been developed. By utilizing this chemoenzymatic route, 1 could be synthesized in two linear steps in 60% overall yield from aldehyde 4 and glycine 8, representing a greater than 2-fold improvement in yield versus the original classical resolution route. This route also had a much higher process greenness10 score due to the implementation an enantioselective step to address the stereochemistry and the elimination of waste from the reduced step count. This resulted in higher material throughput leading to a significant improvement in the overall efficiency in the synthesis of 1.
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EXPERIMENTAL SECTION All reagents were used as received. The D-threonine aldolase enzyme solution was prepared as described.3 Proton and carbon nuclear magnetic resonance shifts are reported in ppm. The reactions involving synthesis of 1 from 9 were monitored 1320
DOI: 10.1021/acs.oprd.5b00192 Org. Process Res. Dev. 2015, 19, 1317−1322
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d, J = 7.9 Hz, 1H), 1.23 (s, 9H), 1.11* (s, 9H). 13C NMR (125.8 MHz, DMSO-d6, 20 °C): δ 171.6, 155.3, 150.9, 149.0, 121.4, 78.3, 71.4, 59.0, 28.0. IR (neat, cm−1): 3308 (br s), 1707 (s), 1661 (m), 1636 (m), 1541 (m). HRMS (ESI) m/z: Calcd for: C13H19N2O5 [M + H]+: 283.12958 Found: 283.12885. Elemental analysis: Calcd for: C13H18N2O5: C, 55.31; H, 6.42; N, 9.92. Found: C, 55.63; H, 6.53; N, 9.91. [α]D20 (c = 0.965, DMSO): +0.92°. M.p.: 218 °C (decomp). tert-Butyl-((1S,2R)-1-hydroxy-3-oxo-1-(pyridin-4-yl)-3(pyrrolidin-1-yl)propan-2-yl)carbamate (11). Acid 10 (3.20 kg, 11.34 mol, 1.00 equiv) and N-hydroxysuccinimide (0.20 kg, 1.70 mol, 0.15 equiv) were dissolved in dichloromethane (25.6 L). Pyrrolidine (1.01 kg, 14.17 mol, 1.25 equiv) was added, followed by EDC (2.72 kg, 14.17 mol, 1.25 equiv). The reaction was stirred for 3 h at 20 °C before being quenched by the addition of a 1 M potassium phosphate buffer (pH = 7, 32.0 L). The layers were separated, and the organic layer was washed with water (2 × 16 L). The dichloromethane was removed and replaced with acetonitrile in a constant volume distillation (48.0 L), causing the product to crystallize. The slurry was cooled to 5 °C and held for 1 h. The product was collected on a polypropylene filter and washed with acetonitrile (6.5 L), then dried at 40 °C at 25 Torr for 24 h to afford 11 as a white crystalline solid (3.4 kg, 88%). 1H NMR (400 MHz, CDCl3, 20 °C): δ 8.55 (d, J = 6.0 Hz, 2H), 7.35 (d, J = 6.0 Hz, 2H), 5.61 (d, J = 9.5 Hz, 1H), 5.16 (s, 1H), 5.06 (s, 1H), 4.62 (d, J = 9.5 Hz, 1H), 3.25−3.60 (m, 4H), 1.70−2.00 (m, 4H), 1.29 (s, 9H). 13C NMR (100 MHz, CDCl3, 20 °C): δ 169.4, 155.6, 149.5, 148.6, 121.3, 80.2, 72.8, 56.1, 46.7, 46.0, 28.0, 25.8, 24.0. IR (neat, cm−1): 3369 (s), 1706 (s), 1624 (s), 1529 (s), 1168 (m). HRMS (ESI) m/z: Calc’s for: C17H26N3O4 [M + H]+: 336.19178 Found: 336.19245. Elemental Analysis: Calc’d for: C17H25N3O4: C, 60.87; H, 7.51; N, 12.52. Found: C, 60.67; H, 7.76; N, 12.47. [α]D20 (c = 0.339, CH2Cl2): +45.54°. M.p.: 187−189 °C. (2R,3S)-2-Amino-3-hydroxy-3-(pyridin-4-yl)-1-(pyrrolidin-1-yl)propan-1-one L-Tartrate (1). To a reactor was charged methanol (5.0 L) and was cooled to 10 °C. Chlorotrimethylsilane (680.00 mL, 5.35 mol, 3.50 equiv) was added, and the mixture was stirred for 30 min at 20 °C. Amide 11 (500.00 g, 1.49 mol, 1.00 equiv) was added, and the solution was heated to 40 °C and held for 3 h. The batch was cooled to 20 °C, and a 2.58 M solution of potassium hydroxide in methanol was added to reach a solution pH of 7.5 (1.95 L). The potassium chloride that precipitates during the KOH charge is filtered off using a Buchner funnel; then the batch is distilled under vacuum to 1.50 L. Additional KCl is filtered off as above to obtain a concentrated solution of 1 as the freebase 15 (332.06 g, 1.41 mol, 94.6%). A solution of L-tartaric acid (275.4 g, 1.83 mol, 1.30 equiv. wrt 15)in water (333 mL) is added to the freebase solution, resulting in a small precipitate of potassium bitartrate that was filtered off. The filtrate is heated to 40 °C, and ethanol (823.0 mL) is added. The solution was seeded (1 wt %); then ethanol (4.43 L) was added over 4 h. The slurry is cooled to 18 °C over 3 h and held overnight. The slurry is filtered and the cake washed with ethanol (2 × 133 L), then dried at 50 °C and 20 Torr for 24 h to afford 1 as a white crystalline solid (477.2 g, 83%, 99.9% HPLC purity, 99.7% potency). For characterization data see below. (2R,3S)-Methyl 2-amino-3-hydroxy-3-(pyridin-4-yl)propanoate Dihydrogen Chloride (12). To a slurry of 9 (40.0 g, 183.33 mmol, 1.00 equiv) in methanol (560 mL) was added concentrated aqueous HCl (98.54 g, 916.65 mmol, 5.00
equiv) and tetrabutylammonium triflate (9.33 g, 23.83 mmol, 0.13 equiv). The suspension was heated to 65 °C for 66 h. The slurry was cooled to room temperature and filtered to afford 12 as a solid (41.9 g, 85% as a 8:2 ratio of 12 to 9). 1H NMR (400 MHz, D2O, 20 °C): δ 8.86 (d, J = 6.9 Hz, 2H), 8.25 (d, J = 6.9 Hz, 2H), 5.29 (d, J = 3.5 Hz, 1H), 4.69 (d, J = 3.5 Hz, 1H), 3.89 (s, 3H). 13C NMR (100 MHz, D2O, 20 °C): δ 167.7, 159.3, 141.8, 125.2, 68.9, 57.8, 54.3. IR (neat, cm−1): 3149 (m), 1750 (s), 1597 (m), 1506 (s), 1234 (m). HRMS (ESI) m/z: Calcd for: C9H13N2O3 [M + H]+: 197.09207. Found: 197.09232. Elemental analysis: Calcd for: C9H14Cl2N2O3: C, 40.16; H, 5.24; N, 10.41; Cl, 26.34. Found: C, 40.16; H, 5.06; N, 10.25; Cl, 26.05. [α]D20 (c = 0.385, H2O): +30.15°. M.p.: 200 °C (decomp). (2R,3S)-2-Amino-3-hydroxy-3-(pyridin-4-yl)-1-(pyrrolidin-1-yl)propan-1-one L-tartrate (1). To a suspension of 12 (10.0 g, 37.16 mmol, 1.00 equiv) in methyl isobutyl ketone (55.0 mL) was added pyrrolidine (13.48 g, 189.52 mmol, 5.10 equiv). The resulting hazy solution was agitated for 48 h at 20 °C which gradually separated into two layers. The conversion was typically >95% according to HPLC monitoring. The bottom layer was removed and discarded, and the remaining layer was diluted with MIBK (10.0 mL). The pH of the mixture was adjusted to 7−8 with concentrated aqueous HCl (5.20 mL). The denser aqueous layer was removed and discarded. The organic layer was washed with brine (10.0 mL) then diluted with ethanol to a total volume of ∼140 mL. This solution was added slowly (
