Molecular imprinted polymers for the chiral separation of pharmaceutically relevant heterocycles: First results of the separation of the anti-Alzheimer drug Galanthamine

Molecular imprinted polymers for the chiral separation of pharmaceutically relevant heterocycles: First results of the separation of the anti-Alzheimer drug Galanthamine.

Jordis, Ulrich*); Rizzi, Andreas**); Grohmann, Franz*);

*) Institute of Organic Chemistry, Vienna University of Technology, Getreidemarkt 9, A 1060 Vienna, Austria;
**) Institute of Analytical Chemistry, University Vienna, Währingerstr. 8, A 1090 Vienna;

Abstract

Polymerization of methacrylic acid and trimethylolpropanetrimethacrylate in the presence of (-)-galanthamine as imprinting template resulted in a polymer that, after extractive removal of the template, was used for batch- and preliminary HPLC-separation of (+-)-Galanthamine.

Introduction

The concept of imprinting of synthetic polymers using molecular templates has been reviewed [1] and patents for applications have been published [2].

The aim of our work was to investigate the possibility of preparing custom-designed polymers for the chiral separation of a target compound. While many chiral solid phases exist already for the separation of enantiomers by chiral HPLC, there remains enough trial and error in finding a suitable chiral stationary phase, and typically for new separations a number of (expensive) phases must be evaluated. We wanted to circumvent this problem by rationally designing solid phases for the resolution for a defined separation problem. Such an approach would be justified, if the separation problem was to be repeated on a routine basis, as might be the case e.g. for the separation of biologically active compounds or for the routine batch analysis in a production scenario. In addition it is conceivable to use such imprinted polymers in a "polishing step" for the removal of undesired enantiomeric impurities.

The recognition of the template within the polymer matrix can be achieved via non-covalent bonds comprising electrostatic-, hydrophobic-, charge-transfer- (e.g. hydrogen bonds) or coordinated metal-bonds. For a chiral recognition a minimum of three binding sites for each molecule must exist:

Fig. 1: Chiral recognition

Galanthamine was chosen for the current investigations because it is currently under being developed as a anti-Alzheimer drug [5], synthesized industrially on a large scale [6] and marketed in > 99.7% enantiomeric purity.

Results and Discussion

It was demonstrated, that a polyacrylate imprinted with (-)-galanthamine preferentially binds (-)-galanthamine from a solution of rac.-galanthamine.


(-)-Galanthamine	(+)-Galanthamine

The best result obtained so far were enantioselectivity coefficients defined as mass ratios of polymer-bound anantiomers to non-bound enantiomers [(m_(-)^pol / m_(-)^sol)/m₍₊₎^pol/m₍₊₎^sol)] between 1.3 and 2.0 depending on the preparation procedure of the polymers. With the phase ratio chosen such a selectivity corresponds to a 2:1 enrichment of the non-imprinted enantiomer in the supernatant as analyzed by chiral capillary electrophoresis (CE). A typical electropherogram is shown in fig.2.

Fig. 2: Chiral CE of (-)- and (+)-galanthamine [4]

Thus the concept of the preparation of a custum designed polymer for solution of a defined chiral separation problem could be verified for this important example of Amaryllidaceae alkaloids. For the development of a material suited for chiral HPLC separations (imprinted polymers with enantioselectivity coefficients significantly beyond 2) more experiments are needed.

First attempts to determine the retention volume for (-)-galanthamine were unsuccessful so far due to problems with the packing of the column and dependence of polymer swelling on mobile phase composition is still under investigation.

Experimental

Molecular imprinted polyacrylate

A solution of (-)-galanthamine (129 mg, 0.45 mmol), methyl acrylate (370 mg, 4.4 mmol), trimethylolpropane trimethacrylate (4.6g, 13.6 mmol) 2,2'-azobis(isobuyronitrile) (30 mg) in chloroform (6 ml) was degassed for 15 min using argon. The solution was polymerized for 18 hrs. at 0-20° using a UV lamp resulting in a yellowish, clear, brittle powder. The polymer was filtered, washed, dried and ground, first manually using a porcelane mortar and piston, than in a mechanically driven agate mortar for15 min at 100 rpm. The powder was wet-sieved by brushing a suspension of the polymer in 800 ml EtOH/water 1:1) over a 45mm sieve. The residue from this sieving procedure was used for batch-binding experiments (see below).

Batch-binding of rac.-Galanthamine

For these experiments the >45mm particles were used and any remainig template was removed by Soxleth-extraction (24 hrs., 15% AcOH in chloroform). The molecular imprinted polymer was dried in vacuo. 30 mg of this material was sonicated after addition of 1 ml of rac.-galanthamine (1.6 mmol/ml) for 5, 10, 30, 60 and 180 min and the supernatant analyzed by chiral CE for the (-)- and (+)-galanthamine ratio.

First attemps for the HPLC resolution of rac.-galanthamine using a molecular imprinted polymer

To prepare material suited for HPLC-columns, the > 45mm from residue was dried under an IR-lamp and the grinding and sieving procedure was repeated three times. The combinded fines thus obtained using the 45mm sieve were wet-sieved using a 25mm sieve. The 25mm-45mm particles were further purified by sedimentation of a sonicated suspension in acetonitrile using a glass column (20 x 560 mm) [3]. The material thus obtained contained very few particles in the 3mm range as viewed by microscope. 980 mg of this material were used to fill a 4mm HPLC column and any remaining template was removed using MeOH/AcOH 9:1 (250 ml).