Supercritical fluid extraction: A novel method for the resolution of tetramisole

Article abstract of DOI:10.1016/S0957-4166(99)00109-3

A new resolution method, based on the selective distribution of enantiomers between a chiral solid and an achiral supercritical fluid phase, is reported. The chiral solid phase is formed from the optically active dicarboxylic acid derivative, (2R,3R)-O,O'-dibenzoyltartaric acid, and the racemic base (tetramisole). A new method is also described for the enrichment of enantiomeric mixtures which have an enantiomeric ratio other than 1:1. This is based on the partial salt formation of the enantiomeric mixture with an achiral substance, which is then followed by supercritical fluid extraction of the free enantiomer. The extract has an enantiomeric composition which is different from the starting mixture. The method is applied to an enantiomeric mixture of tetramisole with hydrochloric acid.

Full text of DOI:10.1016/S0957-4166(99)00109-3

Pergamon
Tetrahedron: Asymmetry 10 (1999) 1275–1281
Supercritical fluid extraction: a novel method for the resolution of
tetramisole
Sándor Keszei,a Béla Simándi,a,∗ Edit Székely,a Elemér Fogassy,b János Sawinsky a and
Sándor Kemény a
aTechnical University of Budapest, Department of Chemical Engineering, H-1521 Budapest, Hungary
bTechnical University of Budapest, Department of Organic Chemical Technology, H-1521 Budapest, Hungary
Received 23 February 1999; accepted 25 February 1999
Abstract
A new resolution method, based on the selective distribution of enantiomers between a chiral solid and an achiral
supercritical fluid phase, is reported. The chiral solid phase is formed from the optically active dicarboxylic acid
derivative, (2R,3R)-O,O0-dibenzoyltartaricacid, and the racemic base (tetramisole). A new method is also described
for the enrichment of enantiomeric mixtures which have an enantiomeric ratio other than 1:1. This is based on the
partial salt formation of the enantiomeric mixture with an achiral substance, which is then followed by supercritical
fluid extraction of the free enantiomer. The extract has an enantiomeric composition which is different from the
starting mixture. The method is applied to an enantiomeric mixture of tetramisole with hydrochloric acid. © 1999
Elsevier Science Ltd. All rights reserved.
1. Introduction
The production of chiral compounds in an optically active form is continuously growing, due to
demand by both the pharmaceutical and agrochemical industries, as well as researchers.1 The fact
that stereoisomers of certain molecules may have different physiological effects has been known for
some time.2 Many chiral drugs are still administered as racemic mixtures, which usually include the
enantiomers with lower therapeutic activities and possible toxic side-effects. Even if an ‘unwanted’
enantiomer of a racemate is inactive it should be considered as metabolic ballast.3 This is also true
for agrochemicals where the use of enantiomerically pure pesticides could reduce pollution in the
environment.4 Therefore, utilization of enantiomerically pure substances is preferred.
Supercritical fluid chromatography (SFC), is a recently developed method. In this method a chiral
complexing agent, bonded to a stationary phase, enables analytical and preparative scales, and separation
∗
Corresponding author. E-mail: simandi.vmt@chem.bme.hu
0957-4166/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00109-3

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for several important types of racemic compounds5 (e.g. racemic bases6). SFC has several advantages
over liquid chromatography, for example, highly selective enantiomeric separation, rapid mass transfer
in the supercritical mobile phase, and high-speed separation with high resolution.
The high efficiency of SFC suggests that a supercritical fluid extraction (SFE) may also be developed
as a resolution method applicable to a preparative and, perhaps, even an industrial process. In earlier
publications, we have shown that SFE can be applied to the resolution of some acids (e.g. chrysanthemic
and permetric acids).7
In supercritical fluid extraction, the solvent is a fluid at a temperature and pressure above its critical
temperature and pressure. It has been widely applied commercially in the food, pharmaceutical and
cosmetic industries.8 SFE is quite simple and offers an easy extraction technique. Carbon dioxide
is the most widely used SFE solvent because it is inexpensive, nontoxic, nonflammable, and has a
comparatively low critical temperature of 31.3°C and pressure of 7.3 MPa. Changing the extraction
conditions, temperature, and pressure of carbon dioxide, results in extracts of different components as
though several different solvents were being used in the solvent extraction. Furthermore, separation of the
extract from carbon dioxide is achieved by reducing the pressure. As this is done at room temperature, the
extract is cold when recovered, which is very desirable when thermally sensitive substances are extracted.
Compound 6-phenyl-2,3,5,6-tetrahydroimidazo-[2,1-b]thiazole 1 is prescribed as tetramisole (rac-
emate) or as levamisole (S)-(−)-enantiomer. The latter is the biologically active enantiomer known to be
an effective anthelmintic drug with an immunomodulatory activity. On the other hand, systemic toxicities
of (S)-(−)-1 and (R)-(+)-1 are of approximately the same magnitude. It follows that administration of pure
(S)-(−)-1 (levamisole) gives pharmaceutical activity with substantially reduced risks of toxic reaction.9
In the last few decades, separation of the isomers of synthetic tetramisole (RS-1) has come into
prominence. Conventional resolution methods which are based on diastereomeric salt formation and
fractional crystallization using various optically active acids have been described in the literature (see
Table 1). To our knowledge, resolution of racemic bases with SFE has not yet been reported. The present
study was undertaken with the purpose of demonstrating the applicability of this method, and also to
investigate the influence of some parameters on the resolution process of racemic 1.
2. Results and discussion
2.1. Solubility of the components
In the preparatory stage, the loading capacity of the supercritical fluid was obtained by extraction of
pure components under different extraction parameters. The loading concentrations were calculated from
mass of extracts and mass of CO2 passed through the system (see Table 2).
The solubility tests showed that the resolving agent (2R,3R)-O,O0-dibenzoyltartaric acid monohydrate
and the supporting material (Perfilt) are insoluble in supercritical carbon dioxide under the conditions of
the resolution experiments.

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Table 1
Production of levamisole (S)-(−)-1 or its hydrochloric salt by conventional resolution method
Table 2
Loading concentration of 1 in supercritical CO2

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Table 3
Effects of the resolving agent (acid)/racemic mixture (base) molar ratio on the yield, the enantiomeric
excess (ee) and the Fogassy parameter (S) at the resolution of 1 (p=160 bar, T=39°C)
2.2. Partial resolution of tetramisole 1 by SFE
In the new method, the well-known technique for resolving the racemic mixture (in our case 1) with
less than the equivalent of the resolving agent (in our case 2-monohydrate) was used.10 In this procedure,
a complex equilibrium is established which involves both the enantiomers of the free base 1 and two
diastereomeric complexes 1 and 2.
The higher stability of one of the diastereomeric complexes results in the preferential formation of that
product. Consequently, the free base becomes optically active. Thus, the poor solubility of the complexes
in the supercritical CO2 permits the selective extraction of the free base. Also, if the difference in stability
of the complexes is large enough, a remarkable resolution can be achieved in a single extraction step. The
extent of the enantiomeric separation depends on the temperature and pressure, since the dissociation
constant of the diastereomeric complex, an important factor influencing the enantiomeric purity of the
extract, also depends on the state of the solvent.
Mixtures of 1- and 2-monohydrate in different molar ratios were prepared and extracted with super-
critical carbon dioxide at the selected pressure and temperature. The extract contained the enantiomeric
mixture of the free base, whereas the raffinate was the mixture of the diastereomeric complexes. After a
single extraction, the (+)- and (−)-enantiomers were enriched in the extract and the raffinate, respectively.
For comparison, the extractions at different pressures and temperatures, and the resolving capability of
the resolution method, were characterized by the modified Fogassy parameter (S). This parameter is the
product of the chemical yield (yield=mass of the recovered product/mass of the initial racemic acid)
and the enantiomeric purity of the material (S=2×yield×ee/104). The S-value is negative for the (−)-
enantiomer.
2.3. Effects of molar ratio, pressure and temperature
Variations of the resolving agent:racemic mixture (2:1) molar ratio led to establishing the optimum at
the ratio of 0.25. Any further increase of the resolving agent fraction resulted in considerably reduced
yields, although the ee values were higher (see Table 3).
Designed SFE experiments were carried out to map quantitative effects of the pressure and temperature
on extraction at an optimum molar ratio of 2:1. The well-known 32 full factorial design11 was realized and
three repeated experiments were performed in the centre of design (Table 4). In these tests the S-parameter
was used as the dependent variable. The three-dimensional response surface plots for experiments at the
molar ratio of 0.25 are shown in Fig. 1. The results indicate that the best choice for producing both
the (+)-1- and (−)-1-enantiomers is the combination of the highest temperature (45°C) with the highest
pressure (200 bar).

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Table 4
Effects of the pressure (p) and temperature (T) on the enantiomeric excess (ee), yield and Fogassy
parameter (S) at the resolution of 1 at the molar ratio of 0.25
2.4. Improving the enantiomeric purity of partially resolved mixtures
Since the best ee values achieved by the single extraction step at the optimal conditions were only
60% for the (+)-enantiomer, and 54% for the (−)-enantiomer, it was necessary to purify the partially
resolved base. As the racemic 1-hydrochloride is much more stable than the correspondingly resolved
hydrochloride, and is insoluble in CO2, addition of a limited quantity of hydrochloric acid may allow
for the recovery of the enantiomer in excess by SFE. This procedure was used to achieve a satisfactory
resolution of the (+)- and (−)-enantiomers. The results of the purifications obtained from the experiments
involving a single extraction at the optimal pressure and temperature are summarized in Table 5.
3. Conclusion
Supercritical fluid extraction was found to be an efficient separation method for resolution of tetra-
misole. The first extraction of the base-resolving agent mixture resulted in ‘breaking’ the racemic
composition. Enantiomerically pure products were obtained by repeated extraction of the partially
resolved mixtures, after addition of an achiral salt-forming reagent. The choice for carrying out this
separation, with a supercritical fluid replacing toxic industrial solvents, is advantageous for implementing
environmentally benign separation processes.
4. Experimental
4.1. General
Both the racemic mixture of the base (1-hydrochloride) and the resolving agent (2-monohydrate) were
purchased from Sigma–Aldrich Chemie, GmbH. Other analytical-grade reagents were obtained from
Reanal Ltd, Budapest. The samples were prepared by mixing chiral acid with a racemic base (liberated
from its hydrochloric salt) in different molar ratios. Perfilt, a porous supporting material, was impregnated
with the mixtures and placed into the extractor vessel. Carbon dioxide of 99.5% (w/w) purity was
supplied by Messer Griesheim, Hungaria. Optical rotations of extracts and raffinates were measured

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Figure 1. Effects of the pressure and temperature (a) on the Fogassy parameter of the extract (SE); and (b) on the Fogassy
parameter of the residue (SR) obtained by the resolution of tetramisole (1) by SFE (specific CO2 consumption: 600 g g−1)
Table 5
Resolution of the enantiomeric mixture of 1 by SFE using an achiral reagent (hydrochloric acid;
p=200 bar, T=45°C)

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on a Perkin–Elmer 241 polarimeter. Enantiomeric excesses (ees) were determined from optical rotations
based on calibration with enantiomeric mixtures of known purities. Experimental setup and extraction
methods have been extensively described in earlier publications.7
4.2. Resolution of tetramisole 1
Racemic 1 (1.50 g, 7.40 mmol) and (2R,3R)-O,O0-dibenzoyltartaric acid monohydrate (0.69 g, 1.85
mmol), Perfilt (2.00 g) and ethanol (30 mL) were mixed and then evaporated. The residue was extracted
with supercritical carbon dioxide. Extraction was done at 45°C and 200 bar. The quantity of carbon
dioxide used for extraction was 900 g. After the removal of carbon dioxide, (+)-1 (0.60 g, 40%) was
obtained. [α]D20=+64 (c 5, MeOH). The raffinate was suspended in water (40 mL) and cc. NH4OH was
added to pH 9.5. The reaction mixture was stirred for 30 min, and cooled to 10°C. The precipitate was
filtered to give (−)-1 (0.63 g, 42%). [α]D20=−58 (c 5, MeOH).
To a solution of (−)-1 (1.50 g, 7.40 mmol, 54% ee) in ethanol (30 mL) was added 1 M aqueous
HCl (3.0 mL) and Perfilt (2.00 g) and then the solvent was evaporated. The residue was extracted with
supercritical carbon dioxide (900 g, 45°C, 200 bar). After the removal of carbon dioxide, (−)-1 (0.75
g, 50%), [α]D20=−107 (c 5, MeOH) and (+)-1-hydrochloride (0.56 g), [α]D21=+9 (c 0.9, H2O) were
obtained in the extract and raffinate, respectively. Similarly, the mixture of (+)-1 (1.50 g, 7.40 mmol,
60% ee), 1 M aqueous HCl (2.6 mL) and Perfilt (2.00 g) was extracted to give (+)-1 (0.68 g, 45%),
[α]D20=+107 (c 5, MeOH) in the extract and (−)-1-hydrochloride (0.65 g), [α]D21=−6 (c 0.9, H2O) in
the raffinate.
Acknowledgements
This work was supported by the Hungarian National Foundation (OTKA), Grant No. T016880, and
by the Ministry of Education and Culture (MKM, FKFP 0269/1997). We would like to thank David
Zudkevitch, Ph.D., P.E., for helpful discussion.
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