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Title:
SEPARATING ENANTIOMERS BY MOLECULAR IMPRINTING
Document Type and Number:
WIPO Patent Application WO/1998/007671
Kind Code:
A1
Abstract:
The present invention is directed to the separation of optically active compounds into their respective enantiomers. The separation uses imprinted polymers having a desired structure and molecular memory for the corresponding print molecules. The invention is particularly concerned with the separation of ibuprofen, naproxen, and ketoprofen. Associated processes and materials such as certain polymers are also within the scope of the present invention.

Inventors:
MOSBACH KLAUS
MARTIN MARK T
MASSEY RICHARD J
Application Number:
PCT/US1996/013746
Publication Date:
February 26, 1998
Filing Date:
August 22, 1996
Export Citation:
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Assignee:
IGEN INC (US)
International Classes:
C07B57/00; C07C51/47; (IPC1-7): C07B55/00; C07B57/00; C08K5/15; C08K5/48
Domestic Patent References:
WO1993009075A11993-05-13
Other References:
JOURNAL OF CHROMATOGRAPHY A, 1994, Vol. 664, KEMPE M. et al., "Direct Resolution of Naproxen on a Non-Covalently Molecularly Imprinted Chiral Stationary Phase", pages 276-279.
TRENDS IN BIOTECHNOLOGY, April 1989, Vol. 7, EKBERG B. et al., "Molecular Imprinting: a Technique for Producing Specific Separation Materials", pages 92-96.
Attorney, Agent or Firm:
Igoe, Patrick J. (Inc. 16020 Industrial Drvi, Gaithersburg MD, US)
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Claims:
CLAIMS
1. A process for separating enantiomers of a particular optically active compound, comprising: (a) polymerizing a selected enantiomer of the compound as a print molecule in molecular imprinting polymerization reactions so that the resulting imprint polymer contains has memory recognition corresponding to the selected enantiomer, (b) contacting the resulting polymer from step (a) with a solution containing differing enantiomers of the compound and thereby allowing the selected enantiomer to bind to the imprint polymer, (c) separating the selected enantiomer from the differing enantiomers based on the binding of the selected enantiomer A process as claimed in claim 1 , wherein the active compound is either ibuprofen, naproxen, or ketoprofen The process of claim 2, wherein the selected enantiomer is either the R or S enantiomer of ibuprofen The process of claim 3, wherein the selected enantiomer is the R enantiomer of ibuprofen The process of claim 3, wherein the selected enantiomer is the S enantiomer of ibuprofen The process of claim 2, wherein the selected enantiomer is either the R or S enantiomer of naproxen The process of claim 6, wherein the selected enantiomer is the R enantiomer of naproxen The process of claim 6, wherein the selected enantiomer is the S enantiomer of naproxen The process of claim 2, wherein the selected enantiomer is either the R or S enantiomer of ketoprofen 10 The process of claim 9, wherein the selected enantiomer is the R enantiomer of ketoprofen 1 The process of claim 9, wherein the selected enantiomer is the S enantiomer of ketoprofen 12 The process of claim 1 , wherein the monomers in the imprinted polymer are crosslinked 13 The process of claims 1 or 13, wherein the separating is performed by using the imprinted polymer as chiral stationary phase packing matenal in a chromatography separation apparatus 14 The process of claim 13, wherein the imprinted polymer is mechanically grounded prior to being used as the packing material 15 The process of claim 14, wherein the imprinted polymer, after being mechanically grounded, is additionally processed by being wetsieved prior to being used as the packing material in the chromatography separation apparatus 16 An imprint polymer capable of separating enantiomers of an optically active compound, comprising (a) a plurality of monomeπc units chemicallv bonded to each othei , (b) wherein the monomeπc units are stet eochemically configured so that imprint recognition sites aie encompassed within and defined by the polymer structure, and (c) wherein the recognition sites retain a molecular memory of the selected enantiomer that was used as a print molecule in the molecular imprinting polymerization reactions 17 The imprint polymer as claimed in claim 1 , wherein the monomeπc units are crosslinked 18 The imprint polymer of claim 17 wherein the cross linkages are through ethyiene glycol dimethacrylate and/oi 1 ,4bιs acryloylpiperazine linkages The imprint polymer as claimed in claims 16 or 17 wherein the optically active compound is selected from the group consisting of ibuprofen, naproxen, and ketoprofen The imprint polymer of claim 19, wherein the monomenc units are based on either monomers containing a carboxylic acid group and/or on cationic monomers The imprint polymer of claim 20, wherein the monomers are chosen from any suitable combination of acrylic acid, methacrylic acid, itaconic acid, 1 vinyl imidazol, and/or 4vιnyl pyπdine The process as claimed in claim 2, wherein the separating step isolates the R and S enantiomers of the active compound while simultaneously separating into two different groups mixtures of the R and S enantiomers of the remaining two optically active compounds The imprint polymer of claim 19, wherein the polymer is capable of performing the simultaneous separations of claim 22 An imprint polymer capable of simultaneously separating enantiomers of differing optically active parent compounds comprising (a) monomenc units encompassing and defining molecular memory recognition sites within the imprint polymer, and (b) wherein the molecular memory recognition sites can differentiate among the differing enantiomers of structurally related differing parent compounds A process for separating enantiomers of optically active compounds, comprising (a) contacting an imprint polymer with a mixture of optically active compounds, and (b) separating various enantiomers based on varying binding affinities of the enantiomers to the imprint polymer.
Description:
SEPARATING ENANTIOMERS BY MOLECULAR IMPRINTING

The present invention is directed to the separation of optically active compounds into their respective enantiomers The invention is particularly concerned with the separation of 4-ιsobutyl-α-methylphenylacetιc acid

(ibuprofen) into its enantiomers in addition to separations for two structurally related compounds (naproxen and ketoprofen) Associated processes and materials such as certain polymers are also within the scope of the present invention In describing the invention, certain documents ai e discussed These documents are indicated by number (e g "document 1 ") throughout the remainder of the specification These documents are identified immediately prior to the claims and each is incorporated by reference in its entirety Background of the Invention A compound is optically active if it contains at least one central atom to which four different atoms or gioups of atoms (collectiveK ' substituents") are chemically bonded This central atom is also known by the following interchangeable terms (i) a chiral atom, (n) a chiial center, (in) an asymmetric atom, and (iv) an asymmetπc center Frequently the chπal atom is carbon and is denoted as C* Possible ways to represent the existence of multiple chiial atoms within the same molecule include (i) C l *, C2"\ C> * , or (n) C I *, C2*, C3*, By definition, an optically active compound (i) contains at least one chiral atom, and (n) exists in at least two different enantiomeπc forms (alternatively, "enantiomers") These two enantiomers are a d-form and a 1-lorm 1 he d-enantiomer is alternativeh termed the dextro-enantiomer while the 1-enantιomer is alternativeK termed the levo-enantiomer By definition (i) d-enantiomers rotate to the right a plane of polarized light passing thiough a solution containing that d-enantiomer relative to the original orientation of the incoming

light, (π) 1-enantιomers rotate to the left this plane of polarized light, and (in) a racemic mixture (racemate) of the corresponding d- and I- enantiomers does not rotate this plane of polarized light to either the right or the left

The rotation of the plane of polarized light effected by any particular optically active compound can be defined as [α] = rv/nl wherein

• (1) [α] is the specific rotation,

• (n) r is the observed rotation in degrees,

• (in) v is the volume in cubic centimeters of the solution

• (iv) n is the amount in grams of the compound in the solution and • (v) I is the length of the measurement tube in decimeters

Thus for any particular optically active compound the optical rotation of the d-enantiomer differs from the corresponding rotation of the I- enantiomer Optical rotation values for πghtward rotations die assigned positive (-+ ) values for the experimentally observed rotation in degrees Likewise, these values aie negative (-) for the experimentally measured leftward rotations

The three-dimensional configuration of any given optically active compound is represented with the notation R or exclusively, S on a per chiral atom basis Thus, for example, an optically active compound containing five chiral carbons (hereinafter, "OA5/4 ") could be represented as [ l * C2*, C3*, C4*, C5*](OA5/R,S) = [ Rl , R2, R3, R4, S5](OAS/4, l ) if the first four chiral carbons have the "R" stereochemical configuration while the fifth chiral carbon has the "S" stereochemical configuration Note that this stereochemical information (ι e , [C1 * C2*, C3 *, C4*, C5*](OA5/R,S) - [R l R2 Rl, R4, S5](OA5/4, l )) defines the three-dimensional structure of O AS/4 1 However, this information does not a priori determine whether the optical rotation of O ASM, 1 will be dextro or, exclusively, levo Thus, if OA5/4, 1 was experimentally determined to have le\ o optical actively, then OA5/4, 1 would be a 1-enantιomer of the

parent compound and the optical and stereochemical features of OA5/4, 1 could be represented by the following [dl/Cl *, C2*, C3*, C4*, C5*](OA5/R,S) = [1/ Rl , R2, R3, R4, S5](OA5/4, 1 ) Applicants use this expression to indicate that the compound to the right of the equals sign (=) is a specifically defined enantiomer having the indicated optical activity and absolute stereochemical configuration of the parent compound to the left of the equals sign The expression OA5/4, 1 is an abbreviated name for this specific enantiomer

As explained above, the notations R and S represent, for any given chiral atom, the three-dimensional configuration of the four different substituents chemically bonded thereto Once such a spatial configuration is known the notation R or, exclusively, S is then assigned to that chiral atom These assignment procedures are well-known in the art and, in brief, involve relative ordering of the four substituents along with comparisons to established rankings of types of substituents (e g , amino, alkyl, alcohol, aromatic, ) Returning to the prior discussion of the chiral compound OA5/4, 1 wherein [dl/C l *, C2*, C3*, C4*, C5*](OA5/R,S) = [1/ R l . R2, R3, R4, S5](OAS/4, 1 ), we assume for purposes of explanation that a d-enantiomer of the compound can be represented by the following optical and stereochemical features [dl/C l *, C2*, C3*, C4*, C5*](OA5/R,S) - [άl Rl , R2, R3, S4, S5J(OAS/3,2) This d-enantiomer of the parent compound OA5/R,S has the first three chiral carbon atoms in the R configuration and the last two chiral carbon atoms in the S configuration Because of the difference between the spatial configuration at the fourth chiral carbon (I e , at C4*), the compounds OA5/4, 1 and OA5/3,2 are nonsupeπmposable mirror images of each other and are different enantiomers

And finally, as the number of chiral atoms in any optically active compound increases, so does the number of theoretically possible different enantiomers due to possible combinations of R and S for each chiral atom Thus, although there aie only two different enantiomers (l e , R and S) for a

one-chiral atom compound, there are eight different enantiomers for a three chiral atom compound (1 e , RRR. RRS, RSR, SRR, RSS. SRS. SSR, SSS)

Having explained the meaning and context of optical activity, chirality/asvmmetry, enantiomenc forms, dextro- and levo- notations, and R or S designations the question remains as to what is the significance of these d/l and R S distinctions as applied to actual compounds The answer is that the activity (defined below) of any optically active compound depends greatly upon the actual enantiomenc form of that compound In other words, the activity is dependent upon the combined R and/or S configurations for each of the chiral atoms of that compound

Activ ity may be defined as any measurable property of the optically active compound which depends upon and/or varies with the enantiomer Possible activities include

• (i) pharmacological, • (n) toxicological,

• (in) catalytic, and

• (iv) reactive

For example, returning to the compound O S/4 1 the activity associated with this specific enantiomenc foi m might be the combination of increased pharmacological, catalytic, and reactive activities along with decreased toxicological activities The compound O S/3 2 however, might have the following activity profile increased toxicological activities in combination with decreased pharmacological, catalytic and reactive activities The separation of optically active compounds into their respective enantiomers is frequently a critical step in obtaining a desired enantiomer having a sought-for activity relative to the other enantiomenc forms of that compound Separation techniques may include (Document 1 p i , lines 1 1 -22)

• (i) asymmetric synthesis reaction mechanisms

• (n) biocatalysts,

• (in) fractional crystallizations, and

• (iv) indirect or direct chromatographic separation of the enantiomers

In general, these separation techniques do not enjoy the advantages afforded by the present invention nor do they anticipate/suggest the present invention As explained more fully below, the present invention represents a patentable advance in the field of enantiomenc separation technology Summary of the Invention

This invention is a method for the separation of the enantiomers of 4- lsobutyl- -methylphenylacetic acid (genencally known as ibuprofen) The method is based on the preparation, employing the molecular imprinting technique, of a polymer material exhibiting selective adsorptive properties for one of the enantiomers (Document 1 , from p 1 , line 23 to p 9, line 29, Documents 2- 13) This polymer material can thereafter be used by a liquid chromatographical approach for the separation of the enantiomers of 4- isobutyl-α-methylphenylacetic acid Only the (d,S) enantiomer (sometimes referred to as "the (+,S) enantiomer") of 4-ιsobutyl-α-methvlphenylacetιc acid is pharmaceutically active (in vitro) However, in vivo, smaller amounts of the (1,R) enantiomer can be metabolized into the (d.S) enantiomei to thus provide some pharmaceutical effects The present invention can also be used to separate the enantiomers of two additional compounds that are structurally i elated to of 4-ιsobutyl- - methylphenylacetic acid Specifically, each of naproxen and ketoprofen exists in exactly two enantiomenc forms that can be separated according to the techniques of the present invention Brief Description of the Drawings

• Figure Ha) depicts the chemical formula of 4-ιsobutyl-σ-methylphenylacetιc acid (hereinafter, compound "1 C * ") without depicting either the R or S configuration for the chiral carbon atom

• Figure Kb) depicts the stereochemical formula of the (d,R) enantiomer

(hereinafter, compound "l C*(d,R)") of compound 1C*

• Figure 1 (c) depicts the stereochemical formula of the (1,S) enantiomer (hereinafter, compound "1 C*(1,S)") of compound 1 C*

• Figure 2(a) depicts the chemical formula of naproxen without depicting either the R or S configuration for the single chiral carbon atom

• Figure 2(b) depicts the chemical formula of ketoprofen without depicting either the R or S configuration for the single chiral carbon atom

• Figure 3 depicts the generalized reaction scheme for the preparation of a polymer useful in carrying out the enantiomenc separation of the present invention in terms of thiee different monomers having different shapes and a single print molecule

• Figure 4(a) depicts the resolution of (R,S)-naproxen on the naproxen- lmpπnted polymer as described in the examples

• Figure 4(b) depicts the chromatographical resolution of 2 μg (R,S)- naproxen on the naproxen-impπnted polymei

• Figure 4(c) depicts the separation of a mixture of 4-ιsobutyI-9- methylphenylacetic acid (2 μg), ketoprofen (0 2 μg) and naproxen (2 μg) on the naproxen-impnnted polymer Only naproxen was resolved into its enantiomeis Detailed Description of the Invention

4-ιsobutyl- -methylphenylacetιc acid (alternatively referred to as p- lsobutylhydratropoic acid) [see Figure l (a),(b)(c)] with the generic name ibuprofen is an active ingredient in several drugs (Ipren by ACO. Brufen by Boots/ Astra, Nurofen by Ci ookes/Astra and many more) showing anti lnflammatoπc, analgethic and antipyretic effects As can be seen in Figure 1 (a),(b),(c) there is a single asymmetric carbon atom in 4-ιsobutyl-9- methylphenvlacetic acid which explains why this compound has two optical isomers, R and S As for many other pharmaceuticals that have two optical isomers, only one of these is phaimacologically active The drug 4-ιsobutyl-9-

methylphenylacetic acid is administered as a racemate but only the S- enantiomer is active It is therefore of a great inteiest to be able to administei the drug in an optically clean form (1 e , a specific enantiomer) The present invention is a method for the separation of the enantiomers of 4-ιsobutyl-α- methylphenvlacetic acid (ibuprofen) as well as other structurally similar compounds such as naproxen and ketoprofen The method is based on the use of a polymer material, exhibiting selective adsorptive properties for one of the enantiomers of the optically active compound being considered in a liquid chromatographical approach for the separation of the enantiomers According to the molecular imprinting technique (Documents 1 -1 1 , 13), it is possible to prepare polymers exhibiting a molecular memory for a chosen substance This memory function can be used for the purification of this substance, which can be present in a mixture of various substances, as it opposed to the other substances, binds to the polymer The polymer is prepared by having the substance of interest (the print molecule) mixed with carefully chosen functional monomers, with the ability to complementary bind to the print molecule, (step a in Figure 3) and that are thereafter allowed to polymerize in the presence of a cross-linker (step b), after which the print molecule is extracted from the so formed polymer (step c) Due to the fact that the print molecule was present as the polymer was formed (step b) the polymei contains a memory function on the moleculai level for the print molecule When the polymer is allowed to interact with various substances (step d) it will bind most strongly to the original substance (print molecule) The prepared polymer is mechanically and thermally stable and can be reused for the purification of the substance

In the presented invention one enantiomer of either ibuprofen or naproxen or ketoprofen is used as the print molecule In this particular case functionahzed monomers, a carboxylic acid group containing monomer (such as acrylic acid, methacrylic acid or itaconic acid) or potentially cationic

monomers (such as 1 -vinyl lmidazol or 4-vιnyl pyndine), can be used (step a in Figure 3) for the complementary interactions with the punt molecule The polymerization can be done in aqueous phase, organic phase or a mixture thereof For instance, ethylene glycol dimethacrylate or 1 , 4- bisacryloylpiperazine can be used as cross-linkers (step b) The polymerization can be initiated thermally or photochemically The print molecule u e , the selected enantiomer for a particular optically active compound) can be separated from the so formed polymer by repetitive extraction The polymer can thereafter primarily be used for liquid chromatographic separations of racemic mixtures of various combinations of ibupiofen naproxen and ketoprofen

After obtaining the desired polymer having the sought-for moleculai recognition of the selected enantiomer (alternatively, the imprint polymer"), the imprint polymer is processed to prepare it for use in separation processes Mechanically grinding and wet-sieving are typically used to process the imprint polymer prior to its use as a chiral stationary phase packing material in chromatography separation apparatus

When used as a CSP packing material in the separation processes of the present invention, the binding between the selected enantiomer and the recognition sites within the imprint polymer result in distinct adsorption properties of that selected enantiomer This results in different adsorption and/or elution rates of the selected enantiomer in comparison to either different enantiomers of the same parent compound or enantiomers of a different parent compound In another aspect of the present invention an imprinted polymer containing molecular memory recognition sites that are specific for a selected enantiomer of an optically active parent compound may be used to simultaneously perform the following processes • (i) separating the selected enantiomer from differing enantiomers of the

same parent compound,

• (n) separating the selected enantiomer from differing enantiomers of differing parent compounds (e g , first parent compound, second parent compound, ), and • (in) separating as a group a first mixture of enantiomers of a different first parent compound from a second mixture of enantiomers of a different second parent compound

For an imprinted polymer to perform these separation processes simultaneously, the selected parent compound (from which the print molecule is isolated) and the other different parent compounds should share certain stereochemical features In particular, each of the parent compounds should have the same number and type of chiral atoms In other words, each parent compound should have, for example, (I) exactly one chiral carbon atom, or (n) exactly two chiral carbon atoms and one chiral sulfur atom In addition, there must be sufficient similarity among the substituents ,as a group at each of the chiral atoms on each of the parent compounds so that the relative adsorption/elution rates among the enantiomei permit these simultaneous separations

The three compounds ibuprofen, naproxen and ketoprofen satisfy the above criteria for such simultaneous separations using an imprinted polymer as a chiral stationary phase (CSP) packing material in a chi omatography separation apparatus As illustrated in Figures 1 (a), 2(a), and 2(b), each compound has one chiral carbon atom Additionally, three out of the four substituents for the chiral carbon atom are identical, namely • (i) a methyl group (i e , -CH3),

• (n) a carboxylic acid group (I e , -COOH), and

• (in) a hvdrogen atom (i e , -H)

The fourth substituent, although not identical is similar in that for each of the three compounds the moiety that is directly bonded to the chiral carbon

atom is a benzene ring The differences among this fourth substituent arise from the differing replacements at the meta- and para- positions of this common benzene ring

Figure 4(a),(b),(c) illustrate the simultaneous separations among naproxen, ibuprofen, and ketoprofen Figure 4(c) details the chromatographic separation obtained with an imprinted polymer that used an enantiomer of naproxen as the print molecule The separation between the R-enantiomer of naproxen (denoted "(R)- l ") and the S-enantiomer of naproxen (denoted "(S)- 1") is evident Likewise, the group separations of mixed enantiomers of ibuprofen (denoted "R,S)-2") and ketoprofen (denoted "(R,S)-3") are seen Although the above-description of the invention provides an enabling disclosure to the skilled artisan, applicants additionally piovide the following specific examples of the embodiments of this invention These examples ate provided for the convenience of the reader and are in no way intended to be limiting with respect to the interpretation of the appended claims

Example 1 NAPROXEN Chemicals

(R)-Naproxen was a gift from Syntex Nordica (Sodertalie, Sweden) (S)-Naproxen, 4-ιsobutyl-9-methylphenylacetιc acid and ketoprofen were from Sigma (St Louis, MO, USA) 4-Vιnylpyrιdιne, ethyiene glycol dimethacrylate and 2,2 , -azobιs(2-methylpropιonιtnle) were purchased from Merck-Schuchaidt (Germany) All organic solvents were of analytical or HPLC grades Equipment

The bulk polymers were ground in a Retsch end runner mill Model RM O (Haan, Germany) A 25-μm Retsch sieve was used for particle sizing The HPLC analyses were performed using a Kontron HPLC system comprising a pump 420, a gradient former 425 and a variable-wavelength detector 432 The column was packed using an air-driven fluid pump from Haskel Engineering (Burbank, CA, USA)

Preparation of the polymer

A 0 46-g amount of (S)-naproxen (2 mmol), 1 26 g 4-vinylpyridine (12 mmol), 1 1 89 g ethyiene glycol dimethacrylate (60 mmol) and 0 1 15 g 2,2'- azobιs(2-methylpropionitrile) (0 7 mmol) were dissolved in tetrahydrofuran (THF) (18 ml) The mixture was sonicated and deoxygenated with UV light (366 nm) at 4 C for 48 h The bulk polymer was ground in a mechanical mortar and wet-sieved by hand with water and ethanol through a 25-μm sieve The particles which passed the sieve were collected, dried on a sintered glass funnel and allowed to sediment (5 x 20 min) in acetonitrile (300 ml) The particles that did not sediment were discharged High-performance liquid chromatography

The sieved and scdimented polymer particles were packed at 300 bar into a stainless-steel HPLC column (200 x 4 6 mm) using acetonitrile as solvent After packing, the column was eluted with THF-acetic acid (7 3, v/v) at 1 ml/ in until a stable baseline was achieved The eluent used for the separation studies was THF-heptane-acetic acid (250 250 1 , v/v/v) The flow- rate was 0 1 ml/min, the elution was monitored at 260 nm and the separation was performed at ambient temperature

The separation factor ( ) was determined using the relationship α - k's/k'R, where k's is the capacity factor of the S enantiomer and k'R, is the capacity factor of the R enantiomer The capacity factors were determined according to k'S = (ts - to)/ to, where ts is the retention time of the S enantiomer and to is the retention time of the void, which was determined by injection of toluene The resolution factor was determined according to Meyer (Document 9)

Results and discussion

The aim of this study was to use molecular imprinting for the preparation of a synthetic polymer selective for naproxen The clinically useful (S)-naproxen was used as template molecule 4-Vinylpyridine was chosen as

the functional monomer, on the basis that it has previously been shown to be efficient in the preparation of molecularly imprinted polymers selective for N- protected amino acids (Document 13) It is assumed that 4-vinylpyridine interacts with the carboxy group in naproxen by ionic interactions (R,S)- Naproxen was well resolved on this CSP, as can be seen in Figure 4(a) and Figure 4(b) The flow-rate was 0 1 ml/min Higher flow-rates resulted in less resolved peaks A separation factor of 1 65 and a resolution factor of 0 83 were obtained when 2 μg of the racemate was loaded on the column (Figure 4(b)) When the loaded amount was increased to 20 μg. the separation factor was 1 26 and the resolution factor was 0 69 These values compare well with those previously reported for direct resolution of naproxen on conventional chiral stationary phase media ( = 1 71 )

The imprinting procedure gives rise to specific recognition sites in the polymer It was of interest to investigate if this CSP, designed specifically for naproxen, was able to resolve other 2-APA-NSAIDs 1 he methyl and the carboxy groups attached to the chiral carbon are common to all 2-APA- NSAIDs, though the aryl-substituents vary The sti uctui es of ibuprofen at Figure 1 (a) and of naproxen and ketoprofen at Figure 2( a),(b) illustrate the precise differences among these three optically active compounds The literature reports that " a three-point" interaction is necessary foi stereochemical specificity (Document 12) Therefore it is required that not only the carboxy group attached to the chii al carbon intei cts with the imprinted polymer, as discussed above, but also the methyl and aryl substituents The role of the aryl substituent in the recognition mechanism was elucidated by investigation of whether the polvmer was able to resolve related 2-APA-NSAIDs It was shown that neither the racemate of 4-ιsobutyl-α- methylphenvlacetic acid nor ketoprofen were resolved on this CSP [Figure 4(a),(b),(c)] Accordingly, the shape, size and nature of the aryl substituent is important for the recognition under the conditions chosen

Example 2 IBUPROFEN and KETOPROFEN Based on the structural similarity of ibuprofen and ketoprofen to naproxen and the detailed theoretical basis developed above, a skilled artisan would be able to accomplish the following individual tasks that would in total allow that artisan to practice the present invention specifically for each of these other two optically active compounds

• (i) determine appropuate monomers and/or cross-linkers for the selected enantiomer,

• (n) conduct the imprint polymerization reactions to obtain the linpiint polymer having the desired molecular recognition for the selected enantiomer

• (in) treat the imprint polymer to prepare it for use as a chiral stationary phase packing material for use in chromatography separation apparatus, and

• (iv) perform the desired CSP separations using the imprinted polymer on desired sample solutions containing mixtures of enantiomers

Although the present invention has been described in detail in the above specification including particular references to specific embodiments and/or examples, a skilled artisan will clearly envision many alternatives and variations in light of the disclosure herein Accordingly, the present invention is intended to cover all possible embodiments that fall within the spirit and scope of the appended claims Applicants thus desne the full extent of the patent protection to which the invention is entitled

Identification of Documents

1 Intl App No PCT/SE92/007 1 Intl Filing Date October 30, 1992

Intl Pub Date May 13, 1993 (as WO 93/09075)

2 Intl App No PCT/SE93/01 107

Intl Filing Date December 27, 1993

Intl Pub Date July 7, 1994 as (WO 94/14835) Intl App No PCT/SE93/00960 Intl Filing Date November 1 1 , 1993 Intl Pub Date May 26, 1994 (as WO 94/1 1403) Intl App No PCT/SE92/00610 Intl Filing Date September 4, 1992

Intl Pub Date March 18, 1993 (as WO 93/05068) U S Patent No 5, 1 10,833 to Klaus MOSBACH issued May 5, 1992 Ekberg et al /'Molecular imprinting a technique for producing specific separation materials", Trends in Biotechnology vol 7, pp 92-96 ( 1989) Kempe et al , "Direct resolution of naproxen on a non-covalently molecularly imprinted chiral stationary phase". Journal of Chromatography A, vol 664, pp 276-279 ( 1994) Kempe et al , "Review Molecular imprinting used for chiral separations", Journal of Chromatography A, vol 694, pp 313 ( 1995) V R Meyer, Chromatographia, vol 24, p 639 ( 1987) Andersson et al , Chapter 24, pp 383-394 "Bioseparation and Catalysis in Molecularly Imprinted Polymers", in Molecular Interactions in

Bioseparations, That T Ngo, ed , Plenum Pi ess, NY, NY ( 1993) Pharmaceutical specialties in Sweden, Lakemedelsinformation AB, 1992 C E Dalgliesh, J Chem Soc , vol 137, p 3940 ( 1952) Kempe et al , J Mol Recogn , vol 6, p 25 ( 1 93)