Application Note

Identifying a diastereomeric salt for a challenging
chiral resolution
Advancing a therapeutic for
malaria using the freeslate system

MMV used Unchained Lab’s high-throughput
techniques to deliver successful results inapproximately two weeks to a problem thatthey had been trying to solve for more than one
year using traditional methods.

Researchers at the Medicines for Malaria Venture were pursuing a promising candidate for thetreatment of malaria when they ran into a roadblock. For over a year they attempted to develop
a more direct method for isolating a key chiralintermediate. Until this challenge was overcome,the program was stalled and could not progress. Leveraging the power of Unchained Labs'
freeslate system configured for preformulation,
they overcame this roadblock by identifying andscaling-up a viable chiral resolution method in just
two weeks.

The mission of MMV is to reduce the burden ofmalaria in disease-endemic countries by discovering, developing and facilitating the delivery of new,effective and affordable anti-malarial drugs. They
envision a world in which innovative medicines will
cure and protect the vulnerable and under-served
populations at risk of malaria, and help to ultimately eradicate this terrible disease. In 1999,
there were no compounds in the MMV portfolio.
Ten years later, MMV had a robust portfolio consisting of 42 development programs, including fouranti-malarial compounds in Phase 3 clinical trials
and three compounds in registration—all fundedsolely by MMV. The MMV portfolio has continued
to grow, with new compounds from translational
research moving forward and six drugs approved
for the treatment of malaria.

The Medicines for Malaria
VentureWorldwide, 3.3 billion people are at risk for malaria, which is prevalent in tropical and subtropicalcountries. According to the Centers for Disease
Control, in 2010 an estimated 219 million cases ofmalaria occurred worldwide and 660,000 people
died, 91% of whom lived in Africa. Globally, 86% of
those who died from malaria were children. Malaria is a disease caused by several species of parasites in the genus Plasmodium. These parasites
are carried by infected Anopheles mosquitoes thatfeed on humans. When diagnosed early, malaria
is a preventable and treatable disease; however,
the emergence of parasites that are resistant toanti-malarial drugs has compounded the problem of malaria infection. In 1999, a group from theWorld Health Organization formed the Medicinesfor Malaria Venture (MMV), a not-for-profit public
partnership.

Discovering novel chemotypes
MMV identified several potential anti-malarialchemotypes using high-throughput screening.
One screen, performed at St. Jude Children’sHospital, identified multiple validated series ofhits.1 Of these, a series of SJ557733, such as thecompound in Figure 1, had progressed to the lead
optimization stage. Studies of this series haveidentified the chiral carboxylic acid (+)-2 as a key
intermediate (Figure 2).
1

IDENTIFYING A DIASTEREOMERIC SALT FOR A CHALLENGING CHIRAL RESOLUTION

MMV and Unchained Labs'
collaboration

Initial assessments by MMV to determine theviability of using a chiral resolution method for thekey carboxylic acid enantiomer proved challenging. Figure 3 shows their original route used toprepare the desired chiral carboxylic acid (+)-2 ona relatively large scale (30 g). The resolution wasachieved via two-step synthesis (esterification andsubsequent hydrolysis) and a preparative supercritical fluid chromatography (SFC) chiral separation. This route resulted in good recovery andenantiomeric excess (ee), but the two synthetic
steps and preparative chiral column purification
made the process cumbersome.

To advance development of this novel chemotype,an efficient, facile method to resolve the desiredenantiomeric intermediate from its racemate wasneeded. MMV and Unchained Labs worked together to develop a chiral resolution method to prepare carboxylic acid (+)-2 in excellent yield and ee.
Using the freeslate system, the team developed
the screening strategy, designed the experiments,
performed 144 chiral resolution experiments, andanalyzed and reported the results (Figure 5).

Another promising approach was chiral resolution
via diastereomeric salt formation (Figure 4) withamino alcohols. This approach used chiral trans-1amino-2-indanol to form the diastereomeric salts
and a 1:1 propionitrile (EtCN):methyl tert-butyl ether(MTBE) solvent system to achieve separation basedon solubility differences. However, the recovery waspoor and insufficient for a large-scale separation.

High-throughput chiral resolution
screeningWith the prior chiral resolution results in mind, thefocus was to gain a better understanding of thesolubility of the diastereomeric salts formed, withthe goal of isolating the desired (+)-2 salt as a solid. To do this, a screen using chiral amino alcohols(Table 1), including chiral trans-1-amino-2-indanol,and 12 different solvents and solvent mixtures(Table 2) was undertaken.

CF3 ONNO

On the first day of screening, the diastereomericsalts of racemic-2 were formed by dispensing
32.5 mg (93 µmol) of racemic-2 into a 96-wellmicroplate using the powder dispense capabilityof the system. Next, 500 µL of methanol solution containing 93 µmol (1 equivalent) of chiralamino alcohol were added to the rows of themicroplate as per Table 1. The plate was sealed
and stirred for two hours at 45 °C to encourage
diastereomeric salt formation. At the conclusionof the first day, the methanol was removed byevaporation using a Genevac HT-12 Evaporator
and stored under high vacuum overnight.

NHCNF
Figure 1: SJ557733.

CF3 O

On the second day, 500 µL of the 12 resolution
solvents and solvent mixtures were added to thecolumns of the 96-well microplate as per Table 2.
The microplate was sealed for 20 hours under theconditions shown in Figure 6.

N(S)
(S)

NO

OH

Figure 2: Injection and sampling with
controlled atmosphere sampling port
technology.

2

IDENTIFYING A DIASTEREOMERIC SALT FOR A CHALLENGING CHIRAL RESOLUTION

CF3O

CF3NO

CF31) SFC (30% MeOH in CO2)
Chiral Pak AD-H

NO

OH

N

racemic-2

OOMe

CF3NN

30 g scaleO(R)(R)

OHN(+) -2; ca 100% ee
Desiredracemic-3ON+(S)(S)2) LiOH, MeOH, H20N

OOOH(-) -2; ca 98% ee
ca. 13 g each

Figure 3: Original large-scale route to obtain (+)-2. SFC: supercritical liquid chromatography, MeOH: methanol.

CF3 ON3) Recrystallization using
1:1 EtCN: MTBEOOH

4) AcOH/aqueous solution;then EtOAc to extract

racemic-2
3.1 g, 8.9 mmol

(1S, 2S)-trans-1-amino-2-indanol
1 eq.

ON

2) Cool and stand for 16 hrs;

OH+NH2N

CF31) 1:1 EtCN: MTBE then reflux;

(S)
(S)

NOOH(+) -20.45 g, 1.3 mmol (29%), 98% ee

Figure 4: Original chiral resolution using trans amino indanol. (EtCN: propanenitrile, MTBE: methyl tert-butyl ether, AcOH: aceticacid, EtOAc: ethyl acetate).

1234567Figure 5: The deck elements of the freeslate system configued for preformulation, (1) 5-place balance with integrated camera, (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

RowOH NH22OH
H3C OH NH2NH2
H 3C
NH
NH222
OH
H 3C
H3C OH NH2NH2
H 3C
OH NH2H3C OHOH NH2N
H
OH
HC
H233N
C OH NH2H
OH2NH
2OH
OH
H2N OH
OH OH
H 2N
OH
H 2N
OH
OH
OH OH
H 2N
OH
HOHN
H22N
OH OH
OH
OH
OH
NH2 OHOH
NH2 OHABC

DE

FO2NO2N
O2N
O2N
O2N

G

H

NH2OH NH
Chemical
2OH
OH
NH2structure
NH

O2N
O
O2N
N
2NH2 OH
2NHNH
2NH
OH
NH2 2 OHNH2NH2NH
NHNH
22NHOH
22OH
NH2NH
2NHOH
OH
2OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
NH2OHNH2 OH
OH
OH
OH
OHNH2NH2 OH
NH2NH2 NH OH
OH
2NH2NH
2OH
2NH2 NH
NH2 OH
NH2OH
NH2 OHOH
NH
NH22 OH
OH
OH
OH
OH

ColumnAmino alcohol(R)-(-)-2-amino-1phenylethanol(R)-(-)-2-amino-2propanol(S)-3-amino-1,2propanediol(1R,2R)-(-)-trans-1amino-2-indanol(1R,2S)-(-)-2-amino1,2-diphenylethanol

Solvent / solvent mixture

1

EtCN:MTBE (1:1)

2Acetone

3Acetone:H2O (9:1)

4Acetone:Heptane (1:1)

5

MeOH

6

MeOH:H2O (9:1)

7IPA

8

IPA:H2O (9:1)

9THF:H2O (9:1)

10MeCN11

MeCN:H2O (9:1)

12

EtOAc

Table 2: Solvents and solvent mixtures for first screen, listed
by microplate column. EtCN: propanenitrile, MTBE: methyl
tert-butyl ether, MeOH: methanol, IPA: isopropyl alcohol, THF:tetrahydrofuran, MeCN: acetonitrile and EtOAc: ethyl acetate.

(1R,2R)-(-)-2-amino1-(4-nitrophenyl)1,3-propanediol

Temperature profile(R)-(-)-2phenylglycinol

5040Temp (°C)

OHOH

(S)-(+)-2pyrrolidinemethanol

NHN
H
OH
NOH
N
OH
H
N
H
Table 1: Chiral aminoalcohols
for first screen, listed by microH
OH
Nplate row.
HOH
N
OH
N
H
H

3020No stirring

1000510Time (hour)

1520Figure 6: Temperature and stiring conditions for both screens.

Ideally one of the first day formed diastereomers
would be fully dissolved while the other would remain as a solid. On the third day, using the heated 4-tip liquid dispenser of the freeslate system,
supernatent was transferred to heated filter block.
Post filtration, an HPLC plate was prepared bytransferring 40 µL of filtrate to a new 96-well
microplate, and diluted to an appropriate concentration for analysis. The original wells of the 96-well

microplate were visually inspected for solids, andthe supernatant from all wells with solid were analyzed via chiral HPLC to assess the difference in thesolubility of the diastereomers (Figure 7).
From the first round of screening, only one moderate hit was identified, (1R,2R)-trans-1-amino2-indanol in ethyl acetate (EtOAc), with an overallsolution yield of 38% and 72% ee.

4250200mAU

150

7.422 - MMV2(–)

5.642 - MMV2(+)

IDENTIFYING A DIASTEREOMERIC SALT FOR A CHALLENGING CHIRAL RESOLUTION

100

500012345678910Time (minutes)• Column:• Mobile phase:• Flow rate:• Column temp:• Injection volume:• Detection:• LC duration:• Isocratic mobile phase:

Chiralpak AD-H (4.6x150 mm, 5.0 µm)
Solvent A: Heptane
Solvent B: Reagent alcohol with 0.1% TFA
1.2 mL/min
35 °C
3 µL
210 nm
10 mins +2 min post run
30% Solvent B

Figure 7: Chiral HPLC analysis of racemic-2 standard.

Following up on this moderate hit, a more focused
screen was planned, which included all four isomersof 1-amino-2-indanol (Table 3). In addition, the primary screen displayed high solubility within many
fully dissolved wells. In order to overcome this issue,
the hope was to lower overall solubility by adding
50% Heptane to the affected wells on the secondary screen (Table 4). Previously, MMV conducted aone-off study that indicated 2 eq. of salts might beneeded for complete salt formation. Both 1:1 and 2:1
chiral 1-amino-2-indanol to racemic-2 were testedon the secondary screen as well (Table 5).

The chiral HPLC analysis of the best hit, well A5
is shown in Figure 8. From the HPLC analysis, the(-)-2 salt had a solubility of 27.5 mg/mL and the(+)-2 salt had a solubility of ~0.03 mg/mL. In effect, (-)-2 salt was about 1,000 times more soluble
than the (+)-2 salt, and this dramatic differencein solubility provided excellent resolution of thediastereomers.

Scale-up by MMV and Rutgers
UniversityBased on the parameters from the follow-up
screen, MMV and Rutgers University scaled-up
the experiment to prepare the (+)-2 acid (Table 5).
To do this, they suspended 16.4 g of racemic acid(±)-2 and 14 g of (1S,2S)-trans-1-amino-2-indanol(2 eq.) in 500 mL of 1:1 EtCN:heptane and refluxed
the mixture for 3.5 hours. The solubility of the 2:1(1S,2S)-1-2-indanol:(+)-2 salt was low enoughthat the mixture was a slurry, even under reflux

From the results, three conclusions were clear:• EtCN:heptane (1:1) was identified as the best
solvent mixture• Only trans-1-amino-2-indanols resolved racemic-2• A 2:1 ratio of trans-1-amino-2-indanol:racemic-2
was required to form the diastereomeric salt
5

IDENTIFYING A DIASTEREOMERIC SALT FOR A CHALLENGING CHIRAL RESOLUTION

Summary

conditions. After 3.5 hours, the solution was cooled
to room temperature and filtered. To ensure that allthe (-)-2 salt was removed, the solid was washedwith an additional 200 mL of 1:1 EtCN:heptane.
The scale-up yielded 14.3 g of the 2:1 1-amino2-indanol:(+)-2 salt (94% theoretical yield) witha 99.3% ee. To generate the free (+)-2 acid, the2:1 1-amino-2-indanol:(+)-2 was washed with
20% acetic acid and then extracted with EtOAc.
The EtOAc extracts were combined, dried andconcentrated. This method generated 7 g of the (+)2 acid (85% of theoretical amount) with a 99.3% ee.

Row

ChemicalstructureOHOHOHNH2 OHNH2NH2NH2 OHOH
OH
NH2 OHNH2NH2NH2NH
NH22NH2NH2OHOH
OH
NH2OH
NH2NH2NH2OHOHOHOHABCDThe work described in this paper used Unchained
Labs' high-throughput techniques to deliver successful results in approximately two weeks to aproblem that MMV had been trying to solve forover a year using traditional methods.
During those two weeks, Unchained Labs andMMV developed the screening strategy, designed
the experiments, ran two screens (144 experiments) using the freeslate system, performed thechiral HPLC analysis and analyzed the results. InSolvent ID

Amino alcohol(1S, 2S)-trans-1amino-2-indanol(1R, 2R)-trans-1amino-2-indanol(1S, 2R)-cis-1-amino2-indanol

Solvent / solvent mixture

S1

EtOAc

S2

BuOAc

S3

EtOAc:Heptane (1:1)

S4

EtOH:Heptane (1:1)

S5

EtCN:Heptane (1:1)

S6

DME:Heptane (1:1)

S7

MIBK:Heptane (1:1)

S8

Me-THF:Heptane (1:1)

S9Toluene

Table 4: Solvents and solvent mixtures for follow-up screen.
EtOAc: ethyl acetate, BuOAc: butyl acetate, EtOH: ethanol,
EtCN: propanenitrile, DME: dimethoxyethane, MIBK: methyl
isobutyl ketone and Me-THF: 2-methyl tetrahydrafuran.

(1R, 2S)-cis-1-amino2-indanol

Table 3: Chiral 1-amino-2-indanols for follow-up screen, listed
by microplate row.

Concentrations of 2:1 1-Amino-2-Indanol (+)-2 and (-)-2 salts (mg/mL) in solution
123

456

78

9

10

1-Amino-2-Indanol:2 Ratio:2:1

1112

1:1

EtOAc

t-BuOAc

EtOAc:
Heptane
(1:1)

EtOH:
Heptane
(1:1)

EtCN:
Heptane(1:1)

DME:
Heptane(1:1)

MIBK:Heptane(1:1)

Me-THF:
Heptane(1:1)

Toluene

EtOAc

EtOH:
Heptane(1:1)

TolueneA(1S, 2S)trans0.7 (+)
14.6 (–)

0.0 (+)
2.9 (–)

0.0 (+)
0.1 (–)

3.4 (+)
25.2 (–)

0.03 (+)
27.5 (–)

0.0 (+)
3.4 (–)

0.0 (+)
0.3 (–)

1.2 (+)
1.8 (–)

0.0 (+)
0.0 (–)

8.4 (+)
20.9 (–)

26.2 (+)
25.7 (–)

0.0 (+)
0.0 (–)

B(1R, 2R)trans24.9 (+)
0.0 (–)

3.2 (+)
0.0 (–)

1.4 (+)
0.0 (–)

25.7 (+)
25.4 (–)

30.9 (+)
0.00 (–)

5.3 (+)ND (–)

0.6 (+)
0.0 (–)

0.9 (+)ND (–)

0.1 (+)
0.0 (–)

27.1 (+)
26.3 (–)

29.8 (+)
29.4 (–)

1.1 (+)
1.0 (–)

C(1S, 2R)cis

4.4 (+)
4.1 (–)

2.4 (+)
2.2 (–)

38.8 (+)
38.3 (–)

6.1 (+)
5.9 (–)

0.1 (+)
0.0 (–)

1.1 (+)
1.0 (–)

1.1 (+)
1.0 (–)

2.5 (+)
2.4 (–)

D(1R, 2S)cis

12.2 (+)
11.7 (–)

2.5 (+)
2.2 (–)

43.3 (+)
42.7 (–)

3.0 (+)
3.0 (–)

0.9 (+)
0.8 (–)

2.6 (+)
2.0 (–)

7.9 (+)
7.3 (–)

ND (+)ND (–)

Table 5: Results of follow-up screen. EtOAc: ethyl acetate, BuOAc: butyl acetate, EtOH: ethanol, EtCN: propanenitrile, DME: dimethoxyethane, MIBK: methyl isobutyl ketone and Me-THF: 2-methyl tetrahydrafuran.

6

IDENTIFYING A DIASTEREOMERIC SALT FOR A CHALLENGING CHIRAL RESOLUTION

50

7.422 - MMV2(–)

405.648 - MMV2(+)

mAU

3020100012345678910Time (minutes)Figure 8: Chiral HPLC analysis of well A5 from the follow-up screen.

total, 11 chiral amino alcohols, 20 solvents andsolvent mixtures and two equivalent-levels ofcounterions were screened. This study identified
a chiral resolution condition to isolate a key (+)2 intermediate with both excellent yield and ee.
This condition discovered via high-throughput
screening was also amenable to scale-up, whichcarboxylic acid (+)-2 with 85% yield and 99.3% ee.
Until the challenge of purifying this key intermediate was overcome, MMV’s development program

was unable to progress. As a result of this work,
MMV has begun using this method to prepare thedesired (+)-2 carboxylic acid, which enabled theadvancement of their promising anti-malaria drugcandidate, SJ557733.

References
1 Guiguemde, W. A. et al. Chemical genetics ofPlasmodium falciparum. Nature. 2010; 465;
311–315.

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