IES980221A2 - Separation of enantiomers - Google Patents

Abstract

A method of separating enantiomers of a compound from a mixture of said using one enantiomer of a di-naphthylprolinol calix[4] arene derivative of general Formula I. wherein R is selected from H, C|-C|Oalkyl, Cj- C|OaIkenyI, C|-C|0alkoxy and groups which enhance solubilisation of the calix[4]arene derivative and/or enhance immobilisation on a substrate surface, and the asterisk represents a chiral centre. More particularly, R may be H or a sterically bulky group such as t-butyl, or a group capable of forming a bond to a silica substrate for example a glass substrate, or may be an ionic group such as SO.iH which increases the solubility of the calixarene in a polar solvent such as water, or is Δη ethylenically unsaturated group.

Description

Separation of enantiomers _ S 9 8 0 2 2 1 APPLICATION mil mi MiiiWWW ifiVShBon relates to a method of separation of enantiomers of a compound from a mixture of such enantiomers particularly for use in pharmaceutical, biomedical and 5 general drug analysis. The invention also relates to novel calixarene derivatives for use in the method.

Background to the Invention In general, chiral separation methods utilise a mobile phase into which the sample is introduced (e g. by injection), and a column/capillary with which the components of the sample interact to varying degrees, leading to differing rates of progression of these components through the column/capillary. There are several methods by which the separation may be achieved: 1. Addition of an active agent (or derivatising agent or chiral selector) to the mobile phase and the sample. The active agent has the ability to distinguish enantiomers of the same compound from each other by interaction with only one of the enantiomers. This generally means that the active agent itself must possess chirality and it must be soluble in the mobile phase. The enantiomeric components of the sample may form derivatives which are stereoisomers of each other to differing degrees (e.g. in the ideal case one enantiomer reacts with the active agent while the other does not), and the derivatives formed progress at different rates through the column/capillary, leading to separation. The different rates of progression may result from physical and/or chemical interactions between the active agent and one enantiomer of the sample. For instance the physical interaction may result in the steric hindrance of polar groups on an enantiomer of the sample thus affecting its progression through the column/capillary by interfering with the interaction between the enantiomer and the column/capillary. 2. Immobilisation of one enantiomer of the active agent in the column/capillary (e.g. on beads in a packed high performance liquid chromatography (HPLC) column or on the walls of a capillary electrophoresis (CE) capillary) Again the active agent is normally itself chiral Differing degrees of affinity of the enantiomers of a racemic sample for the immobilised active aeent leads to difieiiim rates of __ _ Ul··-Ll- IL-T~ '1 OPEN TO PUBIX INSPECTION UNDER SECTION 26 AND RULE 23 JML • β«βοο·β·«»·βΟ&Λ·Μ·*·· IE 980221 progress for each enantiomer through the column/capillary thus effecting separation of the enantiomers.

Capillary electrophoresis (CE) is a powerful separation technique for many compounds. An approach often adopted in chiral separations in CE is the addition of a chiral reagent to the mobile phase (run buffer). The separation of enantiomers of a chiral compound is based on differing degrees of interaction with the chiral reagent. This has been demonstrated previously with cyclodextrins (see S.J. Fanali, J. Chromalogr., 1989, 474, and S. Terabe, Trends Anal. Chem., 1989, 8, 129). and crown ethers ( see R. Kuhn el al, Anal, chem., 1992, 64, 2815 and R. Vespalec el al, Electrophoresis, 1994,15,755).

An alternative to the addition of chiral additives to the run buffer is to immobilise the chiral selector (active agent) on the CE column as reported by Armstrong et al., Anal. Chem , 1993, 65, 1114.

Chiral acylcalix[4]arene amino acid derivatives have been used as mobile phase additives in CE in the enantioseparation of three binaphthylderivatives ( see Sanchez et al, Tett. Lett., 1996, 37, 5841 and Sanchez et al, Anal. Chem., 1997, 69, 3239).

The latter paper does not describe the use of the acylcalix[4]arene in CE where the acylcalix[4]arene has been immobilised. This paper refers to capillary zone electrophoresis (CZE). For the purposes of CZE it is stated that chiral separations in CZE require the addition of chiral selectors as mobile phase additives. Normally capillary separations are used to separate relatively small amounts of the enantiomers. Columns are used for larger scale separations.

The use of the molecule ((S)-Di-Naphthylprolinol Calix[4]arene) in an optical sensor to distinguish the enantiomers (chiral recognition) of phenylethylamine (PEA) and norephedrine (NOR - see Table 2) on the basis of fluorescence quenching has been described (see Grady el al, Anal. Chem., 1996, 68, 3775-3782). The authors of this paper, which include the present inventors, were able to measure the enantiomeric composition of phenylethylamine and norephedrine to within an error of 4.1% and 2.6% respectively, based on fluorescence measurements. There is speculation in this paper that it is possible that the receptor may prove to be a useful component in CE or I1PLC chiral separations However this could not be predicted as the fluorescence IE 980221 quenching results cannot be directly extrapolated to other techniques. Chiral recognition and chiral separation are two very highly sensitive processes which are influenced by a large number of factors which include factors such as polarity, pH, hydrogen bonding properties etc.. The separation and recognition processes are distinct and the degree of influence one of these factors has on either the separation or recognition processes cannot be extrapolated to the other.

While the techniques of chiral or enantiomeric separation are well known it is not always possible to obtain actual separation of enantiomers with an agent which has * demonstrated a form of chiral recognition (such as in fluorescence quenching experiments) under different experimental conditions. This is due, in part, to the fact that, in techniques where immobilisation of the active agent is desirable, chiral separation ability such as that referred to above can be lost. In any case, it can prove very difficult if not impossible to immobilise any active agent on a desired substrate such as a CE capillary or on (glass beads of) a separation column. A satisfactory method of immobilisation can often prove elusive and a large amount of time and experimentation can be expended without any immobilisation technique being realised. Achieving immobilisation is not the end of the process as the chiral separation ability of the active agent must not be lost during immobilisation, or on immobilisation. An immobilisation method has possible effects on the active agent which cannot be foreseen and can only be investigated after an immobilisation method has been used. The immobilisation method should not change the chemical structure of the active agent as this may have the undesired consequence of loss of chiral recognition ability of the active agent. Immobilisation may and has been shown to cause steric readjustments to take place in an active agent being immobilised. Loss of a specific steric structure in the active agent can and usually does result in loss of the ability to demonstrate chiral separation. Chiral separation abilities of an immobilised active agent cannot therefore be extrapolated directly from experimental evidence of chiral recognition.

From a commercial perspective, separation of enantiomers to a degree less than about 98% or even 99% would not be useful due to the necessity to have as close as possible to 100% separation of enantiomers, where only one enantiomer has been shown to demonstrate a therapeutic effect. This is most important where another enantiomer IE 980221 displays undesirable side effects. Once immobilisation of an active agent has been achieved without loss of its chiral separation abilities, it must also demonstrate separation abilities of an order which allow it to be used in commercial processes, in order for the active agent to be commercially useful.

It is also desirable to provide an active agent which can be used in a mobile phase separation as described above. The activity of an active agent in a mobile phase cannot be predicted from chiral recognition experiments as the active agent must be soluble in the mobile phase. Secondly the separation ability which must be * demonstrated, in order to allow the active agent to be used commercially must be very high.

Summary of the Invention This invention relates to a method of separating enantiomers of a compound from a mixture of said enantiomers characterised by using one enantiomer of a dinaphthylprolinol calix[4]arene derivative of general Formula I. wherein R. is selected from H,Ci-Clualkyl, Ci-Cioalkenyl, Ci-Cioalkoxy and groups 20 which enhance solubilisation of the calix[4]arene derivative and/or enhance IE 980221 immobilisation on a substrate surface, and the asterisk represents a chiral centre.

In a preferred embodiment of the invention R is H or a sterically bulky group such as t-butyl, or a group capable of forming a bond to a silica substrate for example a glass substrate , or is an ionic group such as SO3H which increases the solubility of the calixarene in a polar solvent such as water, or is an ethylenically unsaturated group.

Suitably the group R is a group containing an alkoxy silyl moiety of the general formula IV -Si-O—R7 wherein R7 is C1-C6 alkyl, more particularly C1-C3 alkyl, for example a group of 10 Formula V —(CH2)—S— (CHjV -Si-O—R7 Rs wherein : n and m are integers from 1 to 4, R4 and Rj are independently OR7 or R7 where R7 is as defined above. The alkoxy 15 silyl moiety may be used to bond the calixarene to a substrate such as glass, in particular the glass beads of a chromatography column.

The invention also relates to a method as described above wherein the enantiomers of the compound of formula I are those wherein the group R is the group -(CH2)3S(CH2)3Si(OCH2CH3)3 in particular where the compound is compound (7) below. ο IE 980221 or the group -(CH2)3SCH2Si (CH3)2OCH2CH3, in particular where the compound is compound (5) below; IE 980221 According to a preferred embodiment of the invention the enantiomers to be separated are those of a compound having the general formula II wherein: Ri is a group capable of forming hydrogen bonds, such as NH2 or OH; R2 is a group which does not interfere with the hydrogen bonding of Ri; R3 is H or CH3; and the asterisk indicates a chiral centre.

In Formula IIR2 may be H, OH, Cl, F, Br, Ci-C4alkyl, Ci-C4alkenyl, Ci-C4alkoxy, or Ci-C4alkyl substituted with one or more of H, OH, Cl, F, Br or N(Rg)(R9) wherein Rg and R9 are independently selected from H, Ci-C4alkyl, Ci-C4alkenyl, or Ci-C4alkyl substituted with H, OH, Cl, F, Br, or C1-C4 alkenyl substituted with one or more of H, OH, Cl, F, Br or N(Rg)(R9) wherein Rg and R9 are independently selected from H, Ci-C4alkyl, Ci-C4alkenyl, or Ci-C4alkyl substituted with H, OH, CI, F, Br, or C1-C4 alkoxy substituted with one or more of H, OH, Cl, F, Br or N(Rg)(R9) wherein Rg and R9 are independently selected from H, Ci-C4alkyl, Ci-C4alkenyl, or Ci-C4alkyl substituted with H, OH, Cl, F, Br. When the compound of formula II is phenylethylamine, phenylglycinol, norephedrine, or ephedrine particularly good separation may be achieved.

Suitably R, in Formula I is Ci-Cj alkyl, C1-C5 alkenyl or C1-C5 alkoxy.

In one embodiment of the invention the group R in Formula I is t-butyl.

In a more preferred embodiment of the invention the enantiomer of a ο IE 980221 The invention also relates to a substrate having immobilised thereon a dinaphthylprolinol calix[4]arene of general formula I as defined above. The invention also relates to novel compounds of general formula I wherein the group R is the group of Formula V, as defined above, particularly the group -(CH2)3S(CH2)3Si(OCH2CH3)3, and in particular the compound of formula (7) shown above, or the group -(CH2)3SCH2Si(CH3)2OCH2CH3, in particular the compound of formula (5) shown above. These novel compounds have particular utility as chiral separation agents adapted for immobilisation on a silica substrate, especially glass The use of the methods of the present invention could not have been predicted to achieve such effective chiral separation ability to a degree and with an efficiency which is unexpected and not predictable on the basis of prior knowledge. As this separation ability has now been demonstrated with phenylglycinol, it is reasonable to conclude, on the basis of fluorescence quenching experiments that this separation behaviour will occur with a range of chiral compounds containing structural features similar to that of phenylglycinol Brief Description of the Drawings.

Figure 1(a) is an electropherogram showing two well separated peaks for the IE 980221 enantiomers of phenylglycinol.

Figure 1(b) is an electropherogram which shows a single peak for the (S) enantiomer of phenylglycinol.

Figure 2(a) shows the linear response range of fluorescence intensity to concentration 5 of calixarene in methanol Figure 2(b) illustrates the Stern-Volmer plots for the quenching of the fluorescence of the calixarene upon addition of 0%, 50% and 100% S-2-phenylglycinol.

Figure 3(a) is an image of an uncoated capillary tube taken by electron microscope Figure 3(b) is an image of a capillary having immobilised on its surface (S)-t-Butyl10 Naphthylprolinol Calix[4]arene taken by electron microscope.

Detailed Description of the Invention The invention will be described in more detail in particular with reference to compound III below. The invention is described particularly with reference to electrophoretic and chromotographic techniques but is not limited to such techniques. ιυ IE 980221 Synthesis of (S)-t-Butyl-DiNaphthylprolinol Calixfdjarene Compound III was synthesised as follows: 2.48g (2.5 mmol) of the corresponding calix[4]arene tetraethyl ester was prepared as described previously (by Arnaud-Neu, et al. J Am. Chem. Soc., 1989, 111, 8681) and hydrolysed to its carboxylic acid potassium salt by refluxing with 2.5g KOH in 50 mL of ethanol followed by filtration and acidification with 37% aqueous HC1 to give 2.1g (95%) of product. This was subsequently converted to the acid chloride by a 2 hour reflux in 10 mL of thionyl chloride followed by removal of volatiles (the last traces under reduced pressure) to give a quantitative yield. To 0.23 8g (0.25 mmol) of the acid chloride in 5 mL of dry THF was added 0.353g (I mmol) of S-Di-2-naphthyIprolinoI and 0.079g (1 mmol) of dry pyridine with stirring at room temperature for 24 hours. 0.528 g (95%) of a pale brown product was isolated which was purified by chromatography on basic alumina using chloroform to give 211 mg (40%) yield of the compound as a buff coloured solid: mp 198-200”C, IR (KBr) y(C-O) 1634 cm'1; *H NMR (400 MHz, CDCh), δ 1.24 [s, 3611, C(C/Y.,b] \ δ 2.0-2 40 [m, 16H C/72C//;], δ 2.90 [m, 4H, NC/AJ, δ 3 25 IE 980221 [m, 4H, NCtf2J; δ 3.50 [d, 2H, NC//]; δ 4.06 [d, 4H, //BArCH2Ar], δ 4.40 [d, 2H, NC//]; δ 4.70 [m, 4H, NCHCO//]; δ 5.16 [d, 4H, //AArCH2Ar]; δ 5.30 [m, 8H, OC//2]; δ 6.76-8.11 [m, 64H, ArH], δ 7.27 [s, CHCb]. Anal. Calcd. for (C38H37O3N)4.CHC13: C, 78.45; H, 6.41; N, 2.39. -Found: C, 78.04; H, 6.59; N, 2.40. [a]n '304 22° (c = 25 mg in 2 mL Chloroform).

Sources of materials .

Both enantiomers of phenylglycinol, R-(-)-2-phenylgIycinol and S-(+)-2phenylglycinol were obtained from the Aldrich Chemical Company. Other chiral amines (see Table 2) were obtained from Aldrich or Sigma, and were of the highest purity available, Methanol (HPLC grade solvent) was obtained from Labscan, Tris buffer reagent from Fluka Biochemika, and concentrated hydrochloric acid from Riedel de Haen. A fused silica capillary was obtained from Composite Metal Services Ltd., (50 pm Type TSPO5O375), cut to the desired length, and inserted in the capillary cartridge unit of the CE system.

Capillary Electrophoresis System A Beckman P/ACE capillary electrophoresis system with system gold software (Version 810) was employed for all separations. A separation voltage of 20 kV with a rise time of 12 seconds was used. A UV Photodiode array spectrophotometer was used to detect the analytes'scanning from 200 - 350 nm, with peak detection at 200 nm. A capillary temperature of 20°C was employed for all separations. Pressure injection was performed at low pressure (0.5 psi) over a time period of 5 seconds.

A fused silica capillary, dimensions 60 cm x 50 pm, 53 cm to the detector window, was employed The capillary was housed in the cartridge unit equipped with a detector aperture window of 100 x 800 pm. The capillary was conditioned by high pressure rinsing with water for 5 minutes, 0.1 M NaOH for 15 minutes, water again for five minutes, and finally 100% methanol for 10 minutes The coating solution was 11 mg of cali.x[4]arene derivative (Compound 111) in 25 ml Tris non-aqueous IE 980221 methanolic buffer. The Tris non-aqueous methanolic buffer was prepared as follows: 0.303 g of Tris was added to 75 ml of methanol and dissolved by stirring The pH was then adjusted to give an apparent pH of 7.4 using small amounts of concentrated HCl.

The total volume was then made up to 100 ml, and the pH rechecked using a pH meter.

The coating solution was pressure rinsed through the capillary under low pressure (0.5 psi) for approximately 2 hours. Following this, the coating solution was displaced by pressure rinsing with nitrogen at low pressure for 20 minutes, followed by high pressure nitrogen rinsing for 20 minutes. Pressure rinsing with nitrogen was accomplished by placing an empty vial in the rinse buffer compartment. Finally the capillary was high pressure rinsed with water for 30 minutes and blown dry with nitrogen for 5 minutes. This left a coating of the calix[4]arene on the walls of the capillary (confirmed by the electron microscope pictures in Figures 3(a) and 3(b) which show respectively the uncoated and the coated capillary wall).

Example 1 The capillary electrophoresis system described above was used. An applied voltage of 25 kV was used giving a current of 2.1 μΑ. Detection was by photodiode array. Scan controls were set from 200 - 350 nm, with peak detection at 200 nm and a band width of 4 nm. Data collection rate was 2 Hz. The total run time employed was 25 minutes The coated capillary was conditioned as follows. A high pressure rinse with 0.1 M NaOH was performed for 20 minutes, followed by water for 10 minutes. The water was displaced by high pressure rinsing with methanol for 3 minutes, and run buffer for 10 minutes. 2.5 mM Tris in methanol was employed as the run buffer, at a pH of 9.3.

Sample preparatiorr A racemic mixture of phenylglycinol was prepared by adding 0.1 mg/ml of each enantiomer to methanol. Samples were introduced onto the capillary by 5 second, low pressure (0.5 psi) injection.

While racemic mixtures have been used in the examples of the inventions, it will be IE 980221 appreciated by those skilled in the art that unequal mixtures of enantiomers (i.e. non-racemic mixtures) may also be separated by the method of the invention.

Results Capillary Electrophoresis results An injection of the racemic mixture of phenylglycinol resulted in the , electropherogram shown in Figure 1(a). Two well separated peaks are obtained for the enantiomers of phenylglycinol at 12.8 minutes and 14.9 minutes, respectively. An injection of the S-(+)-2-phenylglycinol enantiomers (0.1 mg/ml methanol) confirms the first eluting peak to be the S-enantiomer ( see Figure 1(b)).

Procedure for fluorescent measurements All experiments were performed using a Perkin Elmer Luminescence Spectrometer LS 50B interfaced with an Elonex PC-466 that employs fluorescence data manager software. Post-run data processing was performed using Microsoft Excel v. 5.0 after importing the spectra as ascii files.

A 50 pmol dm'3 stock solution of the calixarene (Compound III) was prepared by dissolving 11.7 mg in 100 mL of methanol. A 25 mmol dm'3 solution of the phenylglycinol was prepared by dissolving the required combination of the two enantiomers, totalling 0.3430 g in 10 mL of methanol. A 1 mL volume of this solution was diluted to 10 mL with methanol to a working stock solution. Test solutions were prepared by placing a 0.1 mL aliquot of the calixarene stock solution in a 10 mL volumetric flask, adding 0.5, 1, or 1.5 mL of phenylglycinol stock solution and making up to volume with methanol.

The fluorescence intensity of the solutions was measured at an excitation wavelength of 230 nm. The fluorescent intensity readings were compared to a solution containing 0.5 μιηοΐ dm'3 calixarene and no phenylglycinol This procedure was repeated for other amines invesliuated.

IE 980221 Fluorescence results The linear response range of fluorescence intensity to concentration of the calixarene in methanol is shown in Figure 2(a). It is important to use a concentration of the calixarene within the linear range in order to ensure that no self-quenching occurs and therefore no alternative self-quenching mechanisms are present. A concentration of 0.5 pmol dm'3 was chosen for subsequent experiments to examine the effect of Phenylglycinol, and hence any quenching observed can be related to the effect of the target species on the ligand. Figure 2(b) illustrates the Stern-Volmer plots for the quenching of the fluorescence of the calixarene upon addition of 0%, 50% and 100% S-2-phenylglycinol at a concentration range of 0-3.75 mmol/dm3. The values for Ksv are summarised in Table 1. The Ksv ratio [ 100%(R)/l00% (S)] is 2.463 which is significantly larger than the ratio reported for phenylethylamine (1.856) with the same calixarene (see Grady et al, Anal. Chem., 1996, 68, 3775-3782). which demonstrates the increased enantiomeric discrimination for phenylglycinol compared to phenylethylamine. The size of this ratio suggests a particularly high degree of enantiomeric discrimination is occurring with the enantiomers of phenylglycinol.

These fluorescence results are in agreement with the capillary electrophoresis study, as the Stern-Volmer plots confirm that the R-enantiomer of phenylglycinol interacts more efficiently with the tetra-(S)-di-2-naphthyIprolinol calix[4]arene, to give a higher Ksv value than the S-enantiomer. This increased interaction of the Renantiomer with the calix[4]arene, is the reason for its slower progress through the calixarene coated capillary (14.94 minutes as opposed to 12.4 for the S-enantiomer, see Figures 1(a) and 1(b)).

Results with other amines A number of related chiral amines (see Table 2) were screened using the fluorescence method The results are summarised in Table 2 Chiral discrimination was observed in every case except for phenylalaninol. The structural difference between this molecule and the other amines lies in the presence of the additional methylene spacer between the aryl group and the chiral centre From this, it can be predicted that «J IE 980221 efficient separations will also be obtained for enantiomers with the basic structural features of compounds having the general formula II.

II wherein: Tp Ri is a group capable of forming hydrogen bonds; such as NH2 or OH, R2 is a group which does not interfere with the hydrogen bonding of Ri; R3 is H or CH3; and the asterisk indicates a chiral centre.

While the present inventors do not wish to be limited to any particular theory it is believed that the mechanism for chiral recognition and hence the basis of chiral separation lies in the combination of the following unique structural features in the calixarene: 1. Chiral centre attached adjacent (desirably directly attached) to an aryl group (provided by the naphthyl groups); 2. Hydrogen bonding sites attached adjacent to this chiral centre; 3. A high degree of spatial crowding arising from the restricted space below the calixarene macrocycle.

Previous results (See Grady et al , Anal. Chem., 1996, 68, 3775-3782) have demonstrated that feature (3) above is important, as there is no evidence of chiral recognition of phenylethylamine (PEA) obtained from fluorescence quenching experiments performed with the free (S)-Di-Naphthylprolinol (i.e. not attached to the calix[4]arene backbone).

It is postulated that the recognition process involves a preferred orientation of the guest molecule such that it can fit through the sterically crowded space below the IE 980221 calixarene and hydrogen bond with the calixarene hydrogen bonding sites This preferred orientation is believed to be the basis of the very efficient chiral separation observed with this molecule. Furthermore, the guest species must exhibit complementarity in the spatial arrangement of its groups in order to interact efficiently with the calixarene host.

Table 2 shows the results of fluorescence based screening tests for evidence of chiral recognition for a number of chiral amines. Good discrimination was observed for enantiomers of phenylethylamine (PEA), norephedrine (NOR), ephedrine (EPH) and w phenylglycinol (PGL), but not for phenylalaninol (PAL). The reason for this may lie in the separation of the chiral centre in PAL from the aryl group by a methylene spacer, which results in a spatial arrangement of the aryl group, chiral centre and hydrogen bonding groups which cannot provide efficient interaction with the recognition sites on the (S)-Di-Naphthylprolinol Calix[4]arene. In each of the other cases, the chiral centre with associated hydrogen bonding site(s) is attached directly to the aryl group, thus providing the required complementarity for efficient interaction with the (S)-Di-Naphthylprolinol Calix[4]arene.

ID Similar results are predictable if other immobilisation strategies are used (e.g. covalent bonding to silica surfaces of the (S)-Di-Naphthylprolinol Calix[4]arene via alkoxysilyl derivatisation of the t-butyl groups on the calixarene upper rim (See S.J. Fanali, J. Chromatogr., 1989, 474) 2D Chiral separations can be predicted for this calixarene and similar derivatives using chromatography, either by addition of the calixarene to the mobile phase, formation of stereoisomers of the guest species (derivatisation) or by immobilisation of the calixarene in the column.

Enantiomeric separation with good results comparable to those described above could be achieved using other other inimobilisaton techniques such as bonding the calixarene to a substrate using alkoxysilyl derivatisation of the calixarene to allow the calixarene to bond to silica atoms on a substrate such as glass beads in a chromatography column An ethylenically unsaturated group could also be placed on IE 980221 the calixarene as a convenient method of immobilising the calixarene to a polymeric substrate. Solubility of the calixarene in a mobile phase which is polar, in particular an aqueous mobile phase, can be increased by adding ionic groups such as -SO3H, for example adding a group such as -SO3H as the R group in the calix[4]arene derivative of.the general formula I mentioned above. Solubility in the mobile phase is important where separation is not carried out by immobilisation.

The synthesis of the present invention invention and the separation results obtained have been based on the (S) enantiomer of a compound of formula I (in particular j compound III) It will be appreciated that the (R) enantiomer of that compound 10 would demonstrate a reverse affinity to that demonstrated by the (S) enantiomer while achieving the same degree of separation, for example the (S) enantiomerof phenylglycinol would interact more strongly with the (R) enantiomer of the dinaphthylprolinol calix[4]arene derivative.

A Ο IE 980221 Table I: Stern-Volmer slopes and correlation coefficients for curves in Figure 2(b) (n=4) Composition Ksv .i 0%S,100%R 50%S, 50%R 100%S,0%R 0.054 (0.9911) 0.025 (0.9930) 0.133 (0.9991) Table 2: Results of fluorescence screening tests for evidence of chiral recognition IE 980221 A compound according to the present invention which is suitable for immobilisation is compound (7) whose synthesis is described below in detail through a series of starting material s/intermediates (1)-(6): Preparation of compound (1) ’ To 5 lg (0.0087 mole) p-allylcalix-4-arene prepared following the procedure of C D. Gutsche and J.A. Levine in J.Am. Chem. Soc. 104 p 2652 1982 in 100 ml dry THF (tetrahydrofuran) was added 0.84 g (0.0348 mole) sodium hydroxide and entire was allowed to stir at room temperature under nitrogen atmosphere for 30 minutes following which 5.8g (0.0348 mole) ethyl bromoacetate was added and the entire was refluxed with stirring under nitrogen for 18 hours. After this period of time the reaction mixture was filtered while hot through a layer of celite. The volatiles were removed under reduced pressure and to the heavy brown oily residue was added 100 ml 5% HC1 and 10 ml dichloromethane and the mixture was shaken together. The lower organic layer was then drawn off and dried over dry magnesium sulphate. All volatiles were then removed under reduced pressure to give 8.1 g pale brown oil product. Chromatography on alumina utilising ethyl acetate:dichloromethane 1:1 v/v gave 4.9 g 60% yield of (1) as a pale brown oil.

IE 980221 IR spectroscopy results: v = nSOfsjCm'1 C = 0 Elemental Analysis Calculated for C% Hm On’l^CHiCL C = 69.84, H = 6.74, Found C = 70.05, H = 6.35.

Preparation of compound (2) 2.23 g (0.0025 mol) of (1) was refluxed with 2.3 g (0.042 mole) KOH in 50 ml absolute ethanol for 2 hours following which upon cooling all volatiles were removed under reduced pressure and to the solid residue was added 30 ml ice cold 10% aqueous HCI. The off-white precipitate which formed was then collected and washed with minimum amount ice cold water and air dried overnight to give 2.0 g (98% yield) of (2).

Preparation of compound (3) To 0.412 g (0.0005 mol) of (2) prepared above was added 2g oxalyl chloride and 10 ml dichloromethane and the entire was allowed to stir at room temperature overnight with calcium chloride drying tube attached. After this time all volatiles were IE 980221 removed to give (4) which was not purified due to its moisture sensitivity. 5 ml dry THF was then added to the acid chloride and with storing at room temperature was added 0.706 g (0.002 mole) S-di-2-naphthyl prolinol (Oxford Asymmetry) in 1 ml dry THF and 0.16 g (0.002 mole) dry pyridine. The reaction mixture was allowed to sit at room temperature for 24 hours following which all volatiles were removed under reduced pressed and water added to the buff-coloured residual solid which was filtered off and dried overnight at room temperature to give 1.2 g crude product. This was chromatographed on basic alumina using chloroform as eluent to give 0.421 g (38%) of (3) as an off-white solid. (4) Elemental analysis calculated for Ci48 Hm Oi2 N4 3CHCI3 C = 72.50, H = 5.41, N = 2.23, Found C = 71.64, II = 6.40, N = 2.96%. IR. Analysis v(C = 0) 1634 (S) cm’1 Preparation of compound (5) 0.42 lg (0.00017 mol) of (3) was added to 0.102g (0.00068 mol) of (6) (Fluorochem) Li IE 980221 and 40 mg azoisobutyronitrile free- radical initiator and 1.5 g benzene and the entire were refluxed with stirring under nitrogen for 2 hours. Following this period of time a further 40 mg azoisobutyronitrile was added following by a further 2 hour reflux period under nitrogen to give the product (5) which was stored under nitrogen as a benzene solution Preparation of compound (7) To 0.319g (0.00014 mol) of (3) was added 0.134 g (0.00056 mol) of (HS(CH2)jSi(OCH2CHj)3 (Fluorochem), 40 mg azoisobutyronitrile and 1.5 g 1,2dichloroethane and the entire was refluxed with stirring under nitrogen for 2 hours.

Following this period of time a further 40 mg azoisobutyronitrile was added followed IE 980221 by a further 2 hour reflux period under nitrogen to give (7), which was stored under nitrogen as a 1,2-dichloroethane solution.

Other compounds of Formula 1 wherein R is of Formula IV may be prepared by analogous methods.

Claims (12)

Claims

1. A method of separating enantiomers of a compound from a mixture of said enantiomers characterised by using one enantiomer of a di-naphthylprolinol 5 calix[4]arene derivative of general Formula Γ. wherein: R is selected from H,Ci-Cioalkyl, Ci-Cioalkenyl, Ci-Cioalkoxy and groups 10 which enhance solubilisation of the calix[4]arene derivative and/or enhance immobilisation on a substrate surface, and the asterisk represents a chiral centre.

2. A method according to claim 1 wherein R is H or a sterically bulky group such as t-butyl, or a group capable of forming a 15 bond to a silica atom on a substrate for example a glass substrate , or is an ionic group such as HSO 3 which increases the solubility of the calixarene in a polar solvent such as water, or is an ethylenically unsaturated group

3. A method according to claim 1 or claim 2 wherein the group R is a group containing an alkoxy silyl moiety of the general formula IV ω IE 980221 li—O—R 7 wherein R 7 is Ct-Ce alkyl, for example a group of Formula V Rj —(C n 2 )—S —(CII J—1 i-0 -R 7 Rs wherein : n and m are integers from 1 to 4, R» and Rj are independently OR7 or R 7 where R 7 is as defined above.

4. A method according to claim 3 wherein the group R of Formula I is the group 10 -(CH 2 )3S(CH 2 ) 3 Si(OCH 2 CH3)3

5. A method according to claim 4 wherein the compound of general formula I is compound (7) below: AU IE 980221

6. A method according to any of the preceding claims wherein the enantiomers to be separated are those of a compound having the general formula II 5 wherein: Ri is a group capable of forming hydrogen bonds; such as NH 2 or OH; R 2 is a group which does not interfere with the hydrogen bonding of Ri; Rj is H or CH 3 ; and the asterisk indicates a chiral centre.

7. A method according to claim 6 wherein R 2 is H, OH, Cl, F, Br, C|-C4alkyl, Cr IE 980221 C4alkenyl, Ci-C4alkoxy, or C1-C4 alkyl substituted with one or more of H, OH, Cl, F, Br or N(Rg)(R9) wherein Rs and R9 are independently selected from H, Cj-C4alkyl, Ci-C4alkenyl, or Ci-C4alkyl substituted with H, OH, Cl, F, Br, or > 5 Ci-C4alkenyl substituted with one or more of H, OH, Cl, F, Br or N(Rg)(R9) wherein Rg and R9 are independently selected from H, Ci-C4alkyl, Ci-C4alkenyl, or Ci-C 4 alkyl substituted with H, OH, Cl, F, Br,or » Ci-C4alkoxy substituted with one or more of H, OH, Cl, F, Br or N(Rg)(Rg) wherein Rg and R9 are independently selected from H, Ci-C4alkyl, Ci-C4alkenyl, or Ci-C 4 alkyl 10 substituted with H, OH, Cl, F, Br.

8. A method according to any preceding claim wherein R is t-butyl.

9. A method according to claim 8 wherein the enantiomer of a dinaphthylprolinol calix [4]arene derivative is compound III below:

10. A method according to any of claims 6 to 9 wherein the compound of formula II is phenylethylamine, phenylglycinol, norephedrine, or ephedrine. IE 980221 ll. A substrate having immobilised thereon a dinaphthylprolinol calix[4]arene of general formula I as defined in claim 1. 5 12. A compound of formula I as defined in claim 1 wherein, R is of the formula in which m and n are integers from 1 to 4, and R and Rj are independently OR7 or R7, where R7 is C1-C6 alkyl. 10 13. A compound of formula I, as defined in claim 1, wherein the group R is the group -(CH 2 )3S(CH 2 ) 3 Si(OCH2CH3) 3 , or (CH 2 ) 3 SCH 2 Si(CH3) 2 OCH 2 CH 3 .

11. 14. A compound of formula (7): OCH 2 CH 3 S(H 2 C) 3 -Si-OCH 2 CH 3 S' 2-naphth yl — 2-naphthyl OH (7)

12. 15. A compound of Formula (5): IE 980221 Tomkins & Co. IE 980221