Lipase Catalysed Kinetic Resolution of Stiripentol

Kinetic resolution of rac-Stiripentol, catalysed by lipase A from Candida antarctica by esterification with vinyl butanoate was performed with an E-value of 24. This allowed isolation of (3S)-Stiripentol with an ee of 86 % and the corresponding (3R)-butanoate with an ee of 87 %. Enzymatic hydrolysis of the ester product gave (3R)-Stiripentol with 94 % ee.


Introduction
dioxol-5-yl)-4,4-dimethylpent-1-en-3-ol (rac-1) is a novel antiepileptic drug structurally unrelated to any other antiepileptic drugs.It has been suggested that Stiripentol induced increase in gamma amino butanoic acid (GABA) concentration through at least two independent neurochemical mechanisms (Trojnar et al., 2005).Recently, it was reported that Stiripentol acts directly on the GABA A receptor as a positive allosteric modulator (Fischer et al., 2009).In addition, its anticonvulsant potency had been proven in different types of animal seizures, as well as in clinical trials (Chiron, 2005).Stiripentol is a secondary alcohol containing one stereogenic center.So far, it is marketed as a racemic mixture, albeit, there are marked differences in pharmacokinetics and antiepileptic potency between the enantiomers (Trojnar et al., 2005).
It is well known that enantiomers of a racemic drug may have different pharmacokinetic and pharmacodynamic effects.The body will interact with and metabolise each enantiomer differently to produce different pharmacological activities.Thus, one isomer (eutomer) may produce the desired therapeutic activities, while the other (distomer) may be inactive or, in worst cases, produce unwanted negative effects (Sheldon, 1993).US Food and Drug Administration (FDA) considers one enantiomer (distomer) as an impurity of the other enantiomer (eutomer).This requires independent investigations of both enantiomers of chiral drugs.Therefore, development of new analytical and preparative techniques to obtain pure enantiomers has become of particular importance for pharmaceutical industry (Collins et al., 1992;Collins et al., 1997).
Resolution is one of the major strategies for providing enantiopure chiral building blocks (CBB's) for drug synthesis.Enzyme catalysed kinetic resolution is a common method in order to obtain CBB's.Enzymes are remarkable catalysts, capable of accepting a wide range of substrates, at the same time exhibiting chemo-, regio-, and enantio-selectivity (Fessner et al., 2009;Bommarius et al., 2004).Moreover, the availability and low price of most classes of enzymes have significantly made them more economically attractive catalysts for the production of biologically active compounds.Hydrolytic enzymes, in particular lipases, are most commonly used as biocatalysts for enzymatic resolution.Most lipases accept a broad range of non-natural substrates and are thus very versatile for applications in organic synthesis.They do not require cofactors and are commercially available in free and immobilised forms.In many cases, they exhibit good to excellent stereoselectivity (Bornscheuer et al., 2005).

General
All solvents used were analytical grade (p.a.) and purchased from Sigma-Aldrich (Steinheim, Germany).Immobilized Candida antarctica lipase B (Novozym 435, activity 10 000 PLU/g, lot no.LC 200205) was bought from Novozymes (Bagsvaerd, Denmark).Lipase A from Candida antarctica immobilized on Immobead 150, (activity 500 U/g, lot no.1388471) was bought from Sigma-Aldrich. 1 H-NMR spectra were carried out on Jeol 500 and Bruker 400 MHz spectrophotometers using TMS as an internal standard.Chemical shift values are recorded in ppm δ scale.Optical rotations ([] D ) were determined at 20 ℃ using a Perkin-Elmer 341 instrument, concentrations are given in g/100 mL.All melting points are uncorrected and measured by an Electrothermal Capillary melting point apparatus.A IKA KS 4000 shaker incubator was used for the enzyme reactions.Enantiomeric ratios, E, were calculated based on ping-pong bi-bi kinetics using the computer program E & K Calculator 2.1b0 PCC (Anthonsen et al., 1996) based on the calculations of Chen and Rakels.(Chen et al., 1982;Rakels et al., 1994).

Small-scale lipase catalysed reactions 2.4.1 Transesterification of stiripentol
Racemic Stiripentol (rac-1) (10 mg, 0.042 mmol), acyl donor (0.12 mmol, 3 equiv.)and 2.0 mL n-hexane were added and stirred in a 4 mL reaction vial.Lipase A from Candida antarctica (CALA) (200 mg) was added and the reaction mixture was thermostated at 35 .Samples (10 µL) were withdrawn, filtered, diluted to 1 mL and ℃ injected on HPLC column at several time intervals.Several other lipases were used, however only CALA showed enantioselectivity.Of the acyl donors used, (ethylmethoxy acetate, isopropenyl acetate, vinyl benzoate, vinyl propanoate and vinyl butanoae) vinyl butanoate was chosen as the best.

Transesterification of stiripentol wih controlled water activity
Racemic Stirpentol (rac-1) (10 mg, 0.042 mmol), vinyl butanoate (0.12 mmol, 3 equiv.)and 2.0 mL n-hexane were added and stirred in a 4 mL reaction vial.The salt hydrates Na 2 HPO 4 x 2 hydrate and Na 2 HPO 4 x 0 hydrate (0.17 g of each) were added to give the water activity (a w ) value 0.18 (at 35 ).Lipase A from ℃ Candida antarctica (200 mg) was added and the reaction mixture was thermostated at 35 .Samples (10 µL) were ℃ withdrawn, filtered, diluted to 1 mL and injected on HPLC column at several time intervals.

Results and Discussion
Stiripentol rac-1 was prepared in good yields from piperonal and pinacolone followed by reduction using sodium borohydride (Scheme 1).The racemic alcohol 1 was resolved using vinyl butanoate in hexane and catalysed by Lipase A from Candida antarctica (CALA) (Scheme 2).Several lipases were tested for the reaction, but as expected, CALA was the best suited catalyst (Kirk et al., 2002).CALA is in the collection of lipases which exhibit strong restriction on the acid part having a narrow tunnel to accommodate the acyl group, but a wider alcohol binding site (Naik et al., 2010).We have previously demonstrated that substrates with bulky groups around the secondary stereogenic center needs CALA for the desired reaction to take place.(Riise Moen et al., 2007;Fuglseth et al., 2006;Tjosås et al., 2008) The alternative, lipase B, has been shown by Uppenberg et al. (1995) to have a stereospecificity pocket in the active site of limited size.Jacobsen et al. (2000) showed that the maximum size of the small group was three carbons counted from the stereocenter.This pocket accommodates the smaller of the two groups at stereogenic center.For transesterification, five different acyl donors have been used for the CALA catalysed transesterification of racemic Stiripentol (rac-1) in n-hexane.Vinyl butanoate gave selective reactions with high rate for ester formation and with an E-value of 24.(Figure 1) In order to increase the E-value, the water activity was adjusted to a w 0.18, however, this did not increase the E-value.The reaction time was also slowed down by addition of the salt hydrate pairs.Hydrolysis in phosphate buffer and in buffer and co-solvent (50 % THF or 30 % acetone) of the butanoate (rac-2), acetate (rac-3) and propanoate (rac-4) esters catalysed by CALB did not show any conversion after seven days.With CALA as the catalyst the reaction was slow and non-selective.We have previously observed that acetone as a co-solvent in hydrolysis of straight-chained secondary alcohols catalysed by CALB increased the E-value significantly compared to hydrolysis in buffer alone.This co-solvent effect was explained as an enantiospecific inhibition by the liberated alcohol due to increased solubility of the product (Lundhaug et al., 1998).Since no selectivity was observed in similar hydrolysis reactions of Stiripentol esters by CALA even after use of co-solvents, restrictions due to solubility problems of organic substrate and products should be excluded.Further investigations are under way.
The secondary alcohol produced by CALA catalysed hydrolysis of the produced butanoate had optical rotation [a] D +23.9 (c 10 CHCl 3 ) for 94 % ee.Hence the absolute configurations of the faster reacting enantiomer was established as being (R) since it has been reported that (R)-Stiripentol has specific optical rotation [a] D +24.9 (c 2.61 MeOH) (Zhang et al., 1994).The slower reacting alcohol showed [a] D -18.1 (c 10 CHCl 3 for 86 % ee.The (R)-butanoate eluted as the second isomer in chiral HPLC as compared to the (R)-alcohol, which was the first eluted enantiomer (Figure 2).Also, it showed levorotatory effect [a] D -68.0 for 92 % ee, whereas the (R)-alcohol has a dextrorotatory effect.
We have optimised the simultaneous chiral HPLC separation of Stiripentol (rac-1) and its acetate (rac-3), propanoate (rac-4) and butanoate (rac-2) in one run with baseline separation.n-Hexane, 2-propanol (2-PrOH) and/or MTBE have been used as a mobile phase component at different ratios on Chiralcel OD-H column.Examples of separation of rac-1, rac-2 and rac-3 are shown in Figure 2.