Effect of β -cyclodextrin on the extraction of isoxazolines from aqueous ethanol into chloroform

Regioisomeric pairs of isoxazolines were prepared through reaction of nitrile oxides with ethyl trans-cinnamate. The effect of β -cyclodextrin on the solubility of these compounds in aqueous ethanol and their extraction into chloroform has been examined. Somewhat anomalously, the compound which is least soluble in the aqueous ethanol is least efficiently extracted into the chloroform, presumably because it is most extensively complexed by the cyclodextrin. In one case, extraction of a 1:1 mixture of a regioisomeric pair of isoxazolines resulted in the separation of ca. 50% of each of the compounds in ca. 90% purity, from a single partitioning.


Introduction
In a series of articles published in the early 1990s, Rama Rao et al. 1 reported that baker's yeast catalysed 1,3-dipolar cycloaddition reactions of nitrile oxides with cinnamates, vinylpyridines, acrylates and vinylcarbazoles, and that β-cyclodextrin affected the regioselectivity and stereoselectivity of some of these processes.Our preliminary investigations in this area 2 showed that neither yeast nor β-cyclodextrin significantly affected the cycloadditions, but β-cyclodextrin did have some effect on the isolation of the cycloadducts from the product mixtures.The effects could be duplicated in simple extraction procedures.Accordingly, when a 1:1 mixture of the isoxazolines 6a and 7a, or 6b and 7b, in ethanol-water (1:4, v/v) containing β-cyclodextrin (0.05 mol dm -3 ) was extracted with organic solvents, samples enriched in either 6a or 6b were obtained.No enrichment was observed in the absence of a cyclodextrin.To examine these effects in more detail, we have now included the isoxazolines 6c,d and 7c,d in our studies.These were chosen because compounds containing the tert-butylphenyl moiety are known to form very stable cyclodextrin inclusion complexes, while a tert-butyl group associates with β-cyclodextrin only weakly. 3,4We have studied the effect of β-cyclodextrin on the extraction of the isoxazolines 6a-d and 7a-d from aqueous ethanol into chloroform, and correlated the trends seen in these experiments with the solubility of the isoxazolines 6a-d and 7a-d in aqueous ethanol, in the presence and absence of the cyclodextrin.

Results and Discussion
The isoxazolines 6a-d and 7a-d were prepared as shown in Scheme 1. 5 The aldehydes 1a-d were treated with hydroxylamine to give the corresponding oximes 2a-d, which reacted with Nchlorosuccinimide to give the hydroximinoyl chlorides 3a-d, respectively.The nitrile oxides 4ad were prepared in situ from the corresponding hydroximinoyl chlorides 3a-d, through treatment with triethylamine, and they reacted with ethyl trans-cinnamate 5 to give the cycloadducts 6a-d and 7a-d, respectively, which were separated using flash column chromatography.

Scheme 1
The regioisomers 6a-d and 7a-d were distinguished on the basis of their 1 H NMR spectra.As reported previously for similar compounds, 6 the resonances for the C-4 and C-5 protons of the 5-ethoxycarbonyl-4-phenylisoxazolines 6a-d are closer in chemical shift (∆δ < 0.5 ppm) and show smaller coupling constants (J < 5.5 Hz) than those observed for the corresponding 4ethoxycarbonyl-5-phenylisoxazolines 7a-d (∆δ > 1.5 ppm, J > 6.0 Hz).The structure of the isoxazoline 7a was confirmed through X-ray crystallographic analysis. 7Polar effects generally dominate the regioselectivity in reactions of nitrile oxides with 1,2-disubstituted dipolarophiles 5 and, as expected on this basis, the 4-ethoxycarbonyl-5-phenylisoxazolines 7a, 7c and 7d formed in preference to their regioisomers 6a, 6c and 6d, through selective reaction of the electron-rich oxygen of the nitrile oxides 4a, 4c and 4d at the electron-deficient carbon of the dipolarophile 5.By contrast, the isoxazolines 6b and 7b formed in approximately equal quantities, presumably as a result of the steric bulk of the mesityl group of the nitrile oxide 4b, increasing the tendency of the oxygen of the dipole 4b to add to the least hindered carbon of the dipolarophile 5. 5 Solutions of the regioisomeric pairs of the isoxazolines 6a-d and 7a-d in ethanol/water (1:4, v/v) containing β-cyclodextrin (0.05 mol dm -3 ) were extracted with chloroform, and the results are shown in Table 1.The isoxazolines 6d and 7d were extracted effectively and without selectivity.By comparison, with the isoxazolines 6a,b and 7a,b, a modest selectivity for phase transfer of 6a over 7a, and of 6b over 7b was observed, while the experiments with 6c and 7c showed a more substantial selectivity and the phase transfer of 7c was significantly retarded.In practical terms it was possible to obtain approximately 50% of each of the isoxazolines 6c and 7c in approximately 90% purity, from a single partitioning of a 1:1 mixture.To explore the origin of these effects, the solubility of the isoxazolines 6a-d and 7a-d in ethanol/water (1:4, v/v) was measured, in the presence and absence of β-cyclodextrin (0.05 mol dm -3 ), and the results are shown in Table 2.At this concentration β-cyclodextrin either increased or had no effect on the solubility of each of the isoxazolines 6a, 6c, 6d and 7a-d in the aqueous ethanol, while lower concentrations of β-cyclodextrin resulted in similar but proportionately reduced effects.When β-cyclodextrin was added to a solution of the isoxazoline 6b, the concentration of the isoxazoline 6b decreased, presumably due to precipitation of the βcyclodextrin-isoxazoline 6b complex.Consequently it was not practical to quantify the effect of β-cyclodextrin on the solubility of the isoxazoline 6b.Table 2. Solubility of the isoxazolines 6a-d and 7a-d in ethanol/water (1:4, v/v) at 30 °C, in the presence and absence of β-cyclodextrin (0.05 mol dm -3 )

Isoxazoline
Solubility (mol dm 2.8 × 10 -6 5.2 × 10 -5 6c 4.9 × 10 -6 1.0 × 10 -4 7c 9.4 × 10 -9 1.1 × 10 -5 6d 2.5 × 10 -4 1.7 × 10 -3 7d 6.7 × 10 -4 4.1 × 10 -3 *Not determined -see text for explanation The results shown in Tables 1 and 2 are anomalous in that the isoxazoline 7c which is the least soluble in the aqueous ethanol is least effectively extracted into chloroform, while compounds 6d and 7d which are the most soluble in the aqueous ethanol are among those which most readily transfer.Presumably the cause of this anomaly is the cyclodextrin.The effect of the cyclodextrin on the solubility of the isoxazolines 6a, 6c, 6d and 7a-d indicates the extent to which they are complexed by the cyclodextrin.The data in Table 2 show that of the isoxazolines 6c and 7c, the latter is much less soluble in the aqueous ethanol and much more extensively complexed by the cyclodextrin.Consequently, at any time there will be less present uncomplexed in solution and available for extraction into chloroform.It appears that the complexed isoxazolines are not extracted and that dissociation of the isoxazolines from the cyclodextrin complexes is sufficiently slow to retard the extraction process.The selectivity of phase transfer is less with the dichlorophenylisoxazolines 6a and 7a, and negligible with the tertbutylisoxazolines 6d and 7d, decreasing as the solubility in the aqueous ethanol increases and the tendency to be complexed by the cyclodextrin decreases.The partitioning of the isoxazolines 6b and 7b is a particular case, where it seems likely that chloroform dissolves the isoxazoline 6b from the precipitated cyclodextrin-isoxazoline 6b complex.

Experimental Section
General Procedures.Melting points were determined on a Kofler hot-stage apparatus under a Reichert microscope and are uncorrected. 1H NMR spectra were recorded on either a Bruker ACP-300, a Varian Gemini 300 or a Varian Mercury 300 spectrometer, as dilute solutions in CDCl 3 .Electron impact mass spectra were recorded on either a VG Micromass 7070F or an AEI MS-30 spectrometer, operating at an ionisation potential of 70 eV.Elemental analyses were performed by either the Microanalytical Laboratory, Research School of Chemistry, Australian National University, or Chemical and Microanalytical Services Pty.Ltd., Melbourne, Australia.Chromatography was performed using Merck-Keiselgel 60 (230-400 mesh ASTM).The aldehydes 1a-d and ethyl trans-cinnamate 5 were purchased from Aldrich Chemical Co.

General procedure for preparation of the oximes 2a-d from the corresponding aldehydes 1a-d
Hydroxylamine hydrochloride (0.45 g, 6.5 mmol) was added to a solution of the aldehyde (5.7 mmol) in water (1.5 mL), ethanol (2 mL) and ice (2.5 mL).Aqueous sodium hydroxide (50%, 1.43 mL) was then added and the mixture was stirred for 1.5 h, then it was washed with ether (1 × 10 mL), acidified (concd HCl) and extracted with ether (2 × 10 mL).The combined extracts were dried (MgSO 4 ) and concentrated under reduced pressure.The resulting solid was recrystallised from aqueous ethanol.General procedure for preparation of the hydroximinoyl chlorides 3a-d from the corresponding oximes 2a-d N-Chlorosuccinimide (3.51 g, 26.3 mmol) was added to a stirred solution of the oxime (26.3 mmol) in N,N-dimethylformamide (22 mL).The reaction was initially slightly endothermic but soon became exothermic and was kept at 25-35 °C in an ice bath.When the exothermic reaction ceased the solution was poured into ice/water (100 mL) and the mixture was extracted with ether (2 × 50 mL).The combined extracts were washed with water (3 × 50 mL), dried (MgSO 4 ) and concentrated under reduced pressure.The resulting solid was recrystallised from ether/n-hexane.

General procedure for preparation of the isoxazolines 6a-d and 7a-d from the corresponding hydroximinoyl chlorides 3a-d via the nitrile oxides 4a-d
A solution of triethylamine (3.2 ml, 21.5 mmol) and ethyl trans-cinnamate 5 (4.77 g, 27.1 mmol) in dry THF (60 mL) was added dropwise with stirring to the hydroximinoyl chloride (16.3 mmol) in dry THF (30 mL).The mixture was stirred at room T for 1 h, heated to reflux for a further 3 h, then cooled and concentrated under reduced pressure.The residue was taken up in chloroform, and the solution was washed with water (3 × 50 mL), dried (MgSO 4 ) and concentrated under reduced pressure.Analysis of the residue using 1 H NMR spectroscopy was carried out to determine the ratio of the two isoxazolines present in the product mixture.They were separated by flash chromatography on silica, eluting with ethyl acetate/n-hexane (1:5) and each was then recrystallised from ethyl acetate/n-hexane.

General extraction procedure
A solution of an approximately equimolar mixture of a regioisomeric pair of the isoxazolines 6ad (0.5 mmol) and 7a-d (0.5 mmol) in ethanol/water (1:4, v/v, 100 mL) containing β-cyclodextrin (5 mmol) was shaken vigorously with chloroform (100 mL) for 5 s, then the organic layer was separated, dried (MgSO 4 ) and concentrated under reduced pressure.The residue was weighed and analysed using 1 H NMR spectroscopy.The results are shown in Table 1.

Determination of the solubility of the isoxazolines 6a-d and 7a-d
A solution of an approximately equimolar mixture of a regioisomeric pair of the isoxazolines 6ad (0.5 mmol) and 7a-d (0.5 mmol) in ethanol (12.5 mL) was added dropwise and with vigorous stirring to a mixture of ethanol (12.5 mL) and water (75 mL), equilibrated at 30 °C.The resultant suspension was left to stand at 30 °C for 24 h, then it was filtered twice through 0.2 µm syringe filters (Sartorius Ministart ® ) and once through a 0.02 µm syringe filter (Whatman Anotop 25 Plus ® ).The filtrate was extracted with chloroform (3 × 50 mL) and the combined extracts were dried (MgSO 4 ) and concentrated under reduced pressure.The residue was dissolved in acetonitrile (50 µL) and analysed by HPLC [Waters Symmetry ® C18 3.5 µm (75 mm × 4.6 mm) column, eluting with acetonitrile/water (7:3, v/v)], using one of the other isoxazolines 6a-d and 7a-d as a standard.A similar procedure was used to determine solubility in the presence of βcyclodextrin (5 mmol), which was added to the aqueous ethanol before adding the solution of the

Table 1 .
Percentage recovery of the isoxazolines