APPLICATION NOTE Identifying a diastereomeric salt for a challenging chiral resolution Advancing a therapeutic for MMV used Unchained Lab’s high-throughput malaria using the Big Kahuna techniques to deliver successful results in system approximately two weeks to a problem that Researchers at the Medicines for Malaria Ven- they had been trying to solve for more than one year using traditional methods. ture were pursuing a promising candidate for the treatment of malaria when they ran into a road- block. For over a year they attempted to develop a more direct method for isolating a key chiral The mission of MMV is to reduce the burden of intermediate. Until this challenge was overcome, malaria in disease-endemic countries by dis- the program was stalled and could not progress. covering, developing and facilitating the delivery Leveraging the power of Unchained Labs' Big Kahuna of new, effective and affordable anti-malarial system configured for preformulation, they over- drugs. They envision a world in which innovative came this roadblock by identifying and scaling-up medicines will cure and protect the vulnerable a viable chiral resolution method in just two and under-served populations at risk of malar- weeks. ia, and help to ultimately eradicate this terrible disease. In 1999, there were no compounds in the MMV portfolio. Ten years later, MMV had a The Medicines for Malaria robust portfolio consisting of 42 development Venture programs, including four anti-malarial com- Worldwide, 3.3 billion people are at risk for pounds in Phase 3 clinical trials and three com- malaria, which is prevalent in tropical and pounds in registration—all funded solely by MMV. subtropical countries. According to the Centers The MMV portfolio has continued to grow, with for Disease Control, in 2010 an estimated 219 new compounds from translational research million cases of malaria occurred worldwide and moving forward and six drugs approved for the 660,000 people died, 91% of whom lived in Afri- treatment of malaria. ca. Globally, 86% of those who died from malaria were children. Malaria is a disease caused by Discovering novel chemotypes several species of parasites in the genus Plas- MMV identified several potential anti-malarial modium. These parasites are carried by infected chemotypes using high-throughput screening. Anopheles mosquitoes that feed on humans. One screen, performed at St. Jude Children’s When diagnosed early, malaria is a preventable Hospital, identified multiple validated series of and treatable disease; however, the emergence hits.1 Of these, a series of SJ557733, such as the of parasites that are resistant to anti-malarial compound in Figure 1, had progressed to the lead drugs has compounded the problem of malar- optimization stage. Studies of this series have ia infection. In 1999, a group from the World identified the chiral carboxylic acid (+)-2 as a key Health Organization formed the Medicines for intermediate (Figure 2). Malaria Venture (MMV), a not-for-profit public partnership. Initial assessments by MMV to determine the viability of using a chiral resolution method for the key carboxylic acid enantiomer proved chal- 1 IDENTIFYING A DIASTEREOMERIC SALT FOR A CHALLENGING CHIRAL RESOLUTION lenging. Figure 3 shows their original route used MMV and Unchained Labs' to prepare the desired chiral carboxylic acid (+)-2 collaboration on a relatively large scale (30 g). The resolution was achieved via two-step synthesis (esterifica- To advance development of this novel chemotype, tion and subsequent hydrolysis) and a preparative an efficient, facile method to resolve the desired supercritical fluid chromatography (SFC) chiral enantiomeric intermediate from its racemate separation. This route resulted in good recovery was needed. MMV and Unchained Labs worked and enantiomeric excess (ee), but the two synthet- together to develop a chiral resolution method ic steps and preparative chiral column purification to prepare carboxylic acid (+)-2 in excellent yield made the process cumbersome. and ee. Using the Big Kahuna system, the team developed the screening strategy, designed the Another promising approach was chiral resolution experiments, performed 144 chiral resolution ex- via diastereomeric salt formation (Figure 4) with periments, and analyzed and reported the results amino alcohols. This approach used chiral trans-1- (Figure 5). amino-2-indanol to form the diastereomeric salts and a 1:1 propionitrile (EtCN):methyl tert-butyl ether (MTBE) solvent system to achieve separation based High-throughput chiral resolution on solubility differences. However, the recovery was screening poor and insufficient for a large-scale separation. With the prior chiral resolution results in mind, the focus was to gain a better understanding of the solubility of the diastereomeric salts formed, with the goal of isolating the desired (+)-2 salt as a sol- id. To do this, a screen using chiral amino alcohols CF3 O (Table 1), including chiral trans-1-amino-2-indanol, and 12 different solvents and solvent mixtures N (Table 2) was undertaken. On the first day of screening, the diastereomeric salts of racemic-2 were formed by dispensing 32.5 mg (93 µmol) of racemic-2 into a 96-well N O NH microplate using the powder dispense capability of the system. Next, 500 µL of methanol solu- tion containing 93 µmol (1 equivalent) of chiral amino alcohol were added to the rows of the CN microplate as per Table 1. The plate was sealed F and stirred for two hours at 45 °C to encourage Figure 1: SJ557733. diastereomeric salt formation. At the conclusion of the first day, the methanol was removed by evaporation using a Genevac HT-12 Evaporator CF3 O and stored under high vacuum overnight. N On the second day, 500 µL of the 12 resolution (S) solvents and solvent mixtures were added to the (S) columns of the 96-well microplate as per Table 2. The microplate was sealed for 20 hours under the conditions shown in Figure 6. N O OH Figure 2: Injection and sampling with controlled atmosphere sampling port technology. 2 IDENTIFYING A DIASTEREOMERIC SALT FOR A CHALLENGING CHIRAL RESOLUTION CF3 O CF3 O CF3 O CF3 O 1) SFC (30% MeOH in CO ) N N 2 N N Chiral Pak AD-H (S) (R) (S) + (R) 2) LiOH, MeOH, H20 N O OH N O OMe 30 g scale N O OH N O OH racemic-2 racemic-3 (+) -2; ca 100% ee (-) -2; ca 98% ee Desired ca. 13 g each Figure 3: Original large-scale route to obtain (+)-2. SFC: supercritical liquid chromatography, MeOH: methanol. CF O CF3 O 3 1) 1:1 EtCN: MTBE then reflux; N 2) Cool and stand for 16 hrs; N OH (S) + (S) 3) Recrystallization using NH2 1:1 EtCN: MTBE N O OH 4) AcOH/aqueous solution; N O OH then EtOAc to extract racemic-2 (1S, 2S)-trans-1-amino-2-indanol (+) -2 3.1 g, 8.9 mmol 1 eq. 0.45 g, 1.3 mmol (29%), 98% ee Figure 4: Original chiral resolution using trans amino indanol. (EtCN: propanenitrile, MTBE: methyl tert-butyl ether, AcOH: acetic acid, EtOAc: ethyl acetate). 1 2 3 4 5 6 7 Figure 5: The deck elements of the Big Kahuna system configued for preformulation, (1) 5-place balance with integrated cam- era, (2) tip rack holder, (3) vortexing station, (4) combination rack, (5) two heating/cooling/stirring stations — 1 cooled position, (6) capping/decapping station, and (7) wash station. The arm elements (not shown) include a vial/plate gripper, 1-tip liquid dispenser, heated 4-tip liquid dispenser and flexible tool changer. 3 IDENTIFYING A DIASTEREOMERIC SALT FOR A CHALLENGING CHIRAL RESOLUTION OH OH NH OH 2 ChemicalOH NH 2 Column Solvent / solvent mixture OH Row structureNH2 Amino alcohol OH NH2 NH2 1 EtCN:MTBE (1:1) OH H C NH2 3 NH 2 Acetone H C NH2 3 NH 2 (R)-(-)-2-amino-1- A OH 22 H3C phenylethanol 3 Acetone:H2O (9:1) H3C OH NH2 H3C NH2 OH NH2 H3C OH 4 Acetone:Heptane (1:1) OH NH2 HH23NC OH H3C NH2 (R)-(-)-2-amino-2- H2N OH NHOH2 5 MeOH B OH H N OH OH propanol H2N OHOH OH H2N OH 6 MeOH:H2O (9:1) 2 OH OH H2N OH (S)-3-amino-1,2- C OH 7 IPA H2N OH H2N OH OH propanediol OH OH 8 IPA:H2O (9:1) OH NH2OH NH2OH (1R,2R)-(-)-trans-1- 9 THF:H2O (9:1) D NH OH NH2 amino-2-indanol NHNH2 2 10 MeCN 2OH NH2 NH2 NH2 11 MeCN:H2O (9:1) NHNH2 2 NHOH2 2 (1R,2S)-(-)-2-amino- 12 EtOAc E NHOH2 1,2-diphenylethanol NHOH2 NHOH2 OH OH Table 2: Solvents and solvent mixtures for first screen, listed OH OH OH by microplate column. EtCN: propanenitrile, MTBE: methyl OH OH OH (1R,2R)-(-)-2-amino- tert-butyl ether, MeOH: methanol, IPA: isopropyl alcohol, THF: NH2 F O2N OH OH 1-(4-nitrophenyl)- tetrahydrofuran, MeCN: acetonitrile and EtOAc: ethyl acetate. O N NH2 OH 2 OH OH 1,3-propanediol O N OHNH2 2 NH2 OH O2N NH2 O2N OH NH2 OH NH2 O2N NH 2 Temperature profile NHOH2 O2N NH NH2 (R)-(-)-2- O2N 2 50 G NH2 OH NH2 phenylglycinol OH NH OH 40 2 OH NH2 NH2 OH 30 OH (S)-(+)-2- H OHOH 20 N pyrrolidinemethanol NH OH (°C) Temp No stirring H 10 N OH N OH NH OH 0 Table 1: Chiral aminoH alcohols for first screen, listed by micro- H OH 0 5 10 15 20 plate row. N H OH Time (hour) N OH H H Figure 6: Temperature and stiring conditions for both screens. Ideally one of the first day formed diastereomers microplate were visually inspected for solids, and would be fully dissolved while the other would re- the supernatant from all wells with solid were ana- main as a solid.