METHOD FOR ISOLATING (R)-TOFISOPAM

Field of the Invention

The present invention relates to the preparative-scale isolation of (R)- tofisopam via chromatographic resolution of a mixture of tofisopam enantiorners employing a chiral separation medium.

Background of the Invention

Tofisopam- Physical Properties/Chemistry Tofisopam (structure shown below, with the atom numbering system indicated) demonstrates potent CNS modulating activity. Tofisopam is l-(3,4- dimethoxyphenyl)-4-methyl-5-ethyl-7,8-dimethoxy-5H-2,3-benzo diazepine.

Tofisopam exists as a racemic mixture of (R)- and (S)- enantiomers. This is due to the asymmetric carbon (indicated by *) at the 5-position of the

benzodiazepine ring. An asymmetric carbon is a carbon atom with four different groups attached.

The molecular structure and conformational properties of tofisopam have been determined by NMR, CD and X-ray crystallography. See, Visy et ah, Chirality 1:271-275 (1989); the entire disclosure of which is incorporated herein by reference. In addition to existing as (R)- and (S)-enantiomers, each enantiomer of tofisopam exists in two stable conformations that may be assumed by the benzodiazepine ring as generally depicted below.

The sign of the optical rotation is reversed upon inversion of the diazepine ring from one conformer to the other. The major conformers, (R)-(+) and (S)-(-) have the 5-ethyl group in a quasi-equatorial position, while in the minor conformers, (i?)-(-) and (5)-(+), the 5-ethyl group is positioned quasi- axially. Thus, racemic tofisopam can exist- as four molecular species, i.e., two enantiomers, each of which exists in two conformations. In crystal form, tofisopam exists only as the major conformations, with dextrorotatory tofisopam being of the (R) absolute configuration. See, Toth et al., J. Heterocyclic Chetn., 20:709-713 (1983); Bioorganic Heterocycles, Van der Plas, H.C., Otvδs, L, Simongi, M., eds. Budapest Amsterdam: Akademia; Kiado-Elsevier, 229:233 (1984); the entire disclosures of which are incorporated herein by reference.

The (R)- and (S)-enantiomers of tofisopam have been shown to possess different biological activity profiles. In particular, the use of the (/-)-enantiomer of tofisopam, substantially free of the 0-O-enantiomer of tofisopam, has been shown to be useful in the treatment of anxiety, resulting in diminished adverse effects and accordingly an improved therapeutic index as compared to

administration of racemic tofisopam. See, US Patent No. 6,080,736, the entire disclosure of which is incorporated herein by reference. Tofisopam - Synthetic Preparation of the Racemate

Racemic tofisopam is prepared by reacting 3,4,3',4'-tetramethoxy-6-(α- aceto-propyl)benzophenone with hydrazine hydrate to form the corresponding hydrazone, 3,4,3',4'-tetramethoxy-6-(l-ethyl-2-hydrazono-propyl) benzophenone. The hydrazone is then cyclized in the presence of methanol (MeOH) and gaseous hydrogen chloride to yield racemic tofisopam. See, US Patent Nos. 3,736,315 and 6,080,736, the entire disclosure of which are incorporated herein by reference. The synthetic route to racemic tofisopam is depicted below :

The main impurities in the product resulting from the above reaction are the starting 3,4,3',4'-tetramethoxy-6-(α-aceto-propyl)benzophenone and the hydrazone intermediate.

The resolution of tofisopam by chiral chromatography using macrocyclic glycopeptide as a stationary phase on a Chirobiotic V™ column (ASTEAC, Whippany, NJ) and using 10% MeOH in tert-butylmethyl ether as the mobile phase, is disclosed in US Patent 6,080,736. In this method, the (R)-(+) enantiomer was the first compound to elute from the column, (R)-(-)-tofisopam, (S)-(-/+) tofisopam, and residual (i-)-(+)-tofϊsopam co-eluted and were collected in a subsequent fraction. Fitos et at. (J. Chromatogr., 709 265 (1995)), discloses another method for resolving racemic tofisopam by chiral chromatography using a chiral oci-acid glycoprotein as a stationary phase on a CHIRAL-AGP™ column (ChromTech, Cheshire, UK), and 10% ACN in a pH 7.0 phosphate buffer as the mobile phase. Zsila et al., disclose another resolution of tofisopam using Chiralcel ^® OJ ^® (Daicel) as a stationary phase and

n-hexane, 2-propanol and MeOH (72:1.5:3) as a mobile phase, in a method taking more than 40 minutes to elute the enantiomers. The methods of Fitos et ah and Zsila et a are analytical methods to analyze samples where nanogram quantities of tofisopam are applied to a column. These methods are not suitable for isolation of large quantities of materials, such as for production purposes.

None of the disclosed methods has been optimized for production of large quantities of (i?)-tofisopam of a chemical and enantiomeric purity suitable for preparation of a drug formulation. What is needed is an industrially applicable procedure for isolation of (i?)-tofisopam, substantially free of the (S)- enantiomer of tofisopam, which:

(a) provides high chemical purity;

(b) provides high enantiomeric purity;

(c) provides high yield of the (/?)-tofϊsopam; and

(d) provides the above features in an economically feasible process.

Summary of the Invention

A method is provided for separating (R)-tofϊsopam, substantially free of the (S)-enantiorner of tofisopam, from a mixture of tofisopam enantiomers comprising introducing a feedstock solution of a mixture of tofisopam enantiomers into a non-simulated moving bed system comprising a chiral separation medium and isolating (R)-tofisopam in an eluent from said chiral separation medium.

In one embodiment of the invention, the method comprises the steps of: a) forming a feedstock solution of a mixture of tofisopam enantiomers; b) introducing said feedstock solution of racemic tofisopam into a non- simulated moving bed system maintained at a substantially constant temperature, said system comprising a plurality of chromatographic columns, each column containing an adsorbent, wherein said columns are arranged in series and in a loop, said loop comprising a feedstock inlet point, a raffinate outlet point, a solvent inlet point, and an extract outlet point, the portion of the loop between an inlet point and an outlet point defining a chromatographic zone;

c) introducing a solvent into said system at said solvent inlet point; d) shifting the location of said inlet points and said outlet points, wherein shifting said inlet points occurs at a substantially different time than shifting of said outlet points; e) removing (R)-(-)-tofisopam, (S)-(+)-tofisopam, and (S)-(-)-tofisopam from said loop at said raffϊnate draw-off point; and f) collecting (R)-tofisopam from said extract outlet point. In one embodiment, the adsorbent comprises a chiral stationary phase comprised of at least one sugar derivative. In another embodiment, the adsorbent comprises a chiral stationary phase selected from the group consisting of cellulose tris 4-methylbenzoate; cellulose tricinnamate; amylose tris [(S)-α-methyl benzylcarbamate]; amylose tris-(3,5-dimethylphenyl) carbamate; and an amylose derivative of formula I:

wherein each R ¹ is a radical of the formula II:

wherein R ² and R ³ are independently selected from the group consisting of chloro and — CH3; n is an integer from about 2 to about 250, preferably from about 10 to about 150; more preferably from about 10 to about 100; and said amylose derivative is coated on, or covalently bonded to, a porous inorganic carrier or a porous organic carrier.

In a one embodiment the adsorbent comprises a chiral stationary phase of amylose tris-(3,5-dimethylphenyl) carbamate.

In a preferred embodiment, the adsorbent comprises an amylose derivative of formula Ia:

wherein n is an integer from about 10 to about 100.

In one embodiment of the present invention, the solvent is selected from the group consisting of Cl-ClO alkanes, C1-C6 alcohols, acetates of C1-C6 alcohols, propionates of C1-C6 alcohols, Cl-ClO ketones, Cl-ClO ethers, halogenated Cl-ClO hydrocarbons, trifluoroacetic acid, dimethylformamide, dimethylacetamide, acetonitrile and combinations thereof.

In a preferred embodiment the solvent is selected from the group consisting of hexane, heptane, methanol, ethanol, propanol, isopropanol, butanol, methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, methyl propionate, ethyl propionate, propyl propionate, isopropyl propionate, acetone, butanone, isopropyl methylketone, diethyl ether, diisopropyl ether, tertbutylmethyl ether, tetrahydrofύran, dioxane, methylene chloride, chloroform, chlorobenzene, trifluoroacetic acid, dimethylformamide, dimethylacetamide, acetonitrile and combinations therein. In a more preferred embodiment, the solvent is a mixture of acetonitrile and methanol. In the most- preferred embodiment the solvent is acetonitrile.

In another embodiment of the present invention, the solvent is selected from the group consisting of acetonitrile, methanol, and mixtures thereof, and the chiral stationary phase is selected from the group consisting of cellulose tris 4-methylbenzoate; cellulose tricinnamate; amylose tris [(S)-α-methyl benzylcarbamate]; amylose tris (3,5-dimethylphenyI) carbamate; and an amylose derivative of formula I ^v as described above.

In one embodiment the solvent is acetonitrile and the chiral stationary phase is amylose tris (3,5-dimethylphenyl) carbamate.

In a preferred embodiment, the solvent is acetonitrile and the chiral stationary phase is an amylose derivative of formula Ia as described above. In certain embodiments of the present invention the substantially constant temperature is from about 0 ⁰ C to about 60 ⁰ C, more preferably from about 25 ⁰ C to about 45 ⁰ C, and most preferably about 40 ⁰ C.

In some embodiments, the mixture of tofϊsopam enantiomers to be resolved may be a racemic mixture. In other embodiments, the mixture of tofisopam enantiomers may be other than a racemic mixture.

In some embodiments, the solvent system is isocratic throughout the chromatographic separation. In other embodiments, the solvent system may comprise a gradient from a first solvent composition to a second composition.

In some embodiments of the invention, the chiral separation medium is contained in a chromatography column.

In some embodiments of the invention, the chiral separation medium is contained in a plurality of chromatography columns.

In one sub-embodiment thereof, the (λ)-enantiomer of tofisopam, substantially free of the (S)-enantiomer of tofϊsopam, is isolated in a yield of at least about 65% by weight, preferably at least about 70% by weight, more preferably at least about 75% by weight, even more preferably at least about

85% by weight.

In another sub-embodiment thereof, the (iϊ)-enantiomer of tofisopam, substantially free of the (S)-enantiomer of tofisopam, is isolated in an enantiomeric purity of greater than 98% enantiomeric excess.

Suitable organic carriers for supporting the amylose derivative of formula I or formula Ia include, for example polystyrene, polyacrylamide and polyacrylate. Suitable porous inorganic carriers for supporting the amylose derivative of formula I include, for example, silica, alumina, magnesia, glass, kaolin, titanium oxide and silicates. The amylose derivative of formula I or formula Ia may be coated on, or covalently bonded to, the carrier. The carrier for supporting the amylose derivative of formula I or formula Ia comprises a

particle size of from about 10 to about 100 microns, preferably from about 10 to about 50 microns, more preferably from about 10 to about 30 microns, most preferably about 20 microns.

In another embodiment of the invention, there is provided an (R)- enantiomer of tofisopam substantially free of (>S)-enantiomer of tofisopam, said (i?)-enantiomer having an enantiomeric excess of greater than 95%.

In another embodiment of the invention, there is provided an (R)- enantiomer of tofisopam substantially free of (5)-enantiomer of tofisopam, said (ii)-enantiomer having an enantiomeric excess of greater than 98%. In another embodiment of the invention, there is provided an (R)- enantiomer of tofisopam substantially free of (^-enantiomer of tofisopam, said (i?)-enantiomer having an enantiomeric excess of greater than 99%.

In another embodiment, the invention provides an improved process for the isolation of the (R)-enantioner of tofisopam with high enantiomeric purity and high overall yields.

The present invention provides processes for obtaining the (R)-tofisopam by forming a solution of tofisopam enantiomers and separating the enantiomers of tofisopam by non-simulated moving bed chromatography. A solution of topisopam enantiomers is passed through at least two columns linked in a loop, where the columns contain a solid chiral stationary phase.

Brief Description of the Figures

Fig. 1 shows a diagram of a reconstructed chromatographic resolution of racemic tofisopam using a chiral separation medium comprising an amylose derivative of formula Ia and a solvent system comprising 100% ACN, illustrating the elution order of the enantiomers of tofisopam.

Fig. 2 shows a diagram of a chromatographic resolution of an overload injection of racemic tofisopam using a chiral separation medium comprising an amylose derivative of formula Ia and a solvent system comprising 100% ACN. Fig. 3 shows a diagram of a chromatographic resolution of a dilute injection of racemic tofisopam using a chiral separation medium comprising an

amylose derivative of formula Ia and a solvent system comprising 85% ACN/15% MeOH (VAO-

Fig. 4 shows a diagram of a chromatographic resolution of an overload injection of racemic tofisopam using a chiral separation medium comprising an amylose derivative of formula Ia and a solvent system comprising 85% ACN/15% MeOH (VAT).

Fig. 5 shows a diagram, of a chromatographic resolution of an overload injection of racemic tofisopam using a chiral separation medium comprising an amylose derivative of formula Ia and a solvent system comprising 81% ACN/19% MeOH (VAO (V/V).

Detailed Description of the Invention

A selection of chiral separation media were experimentally assessed for use in the resolution of tofisopam. It was found that most of the experimental separations were unsatisfactory because the chiral separation media were either incapable of providing satisfactory resolution of tofisopam enantiomers, or demonstrated long retention times when optimized to resolve the tofisopam enantiomers.

Retention time for the (2?)-enantiomer of tofisopam is a significant factor in resolution of tofisopam enantiomers. This is because the (/?)-enantiomer in solution demonstrates an equilibrium between the (/?)-(+) and (i?)-(-) conformers. The equilibrium composition of (λ)-tofϊsopam in solution is about 85% of the (λ)-(+) conformer and about 15% of the (R)-(-) conformer. The equilibration continues during chromatographic separation. As the tofisopam enantiomers and conformers resolve from the mixture on the separation medium, they form areas of purified single isomers, referred to as "bands" that move with the flow of the solvent system. The major (λ)-enantiomer conformer, (iJ)-(+), is retained most by the separation medium, and the (i?)-(-), conformer is retained least. Throughout the separation, the major (R)-(+)- conformer equilibrates at a finite rate to the minor (iϊ)-(-) conformer. This results in broadening of the bands and may serve to significantly lower yields of (/?)-tofisopam isolated via chromatographic resolutions. Thus, separation

conditions that achieve resolution of (jR)-tofisopam in a shorter time interval are advantageous because the equilibration of the conformers of (λ)-tofϊsopam as a function of time is minimized, and the yield of the major conformer of (R)- tofϊsopam is increased. The present invention provides a chiral chromatographic method for isolating (i?)-tofisopam, substantially free of the (5)-enantiomer of tofisopam, via chromatographic resolution of a mixture of tofisopam enantiomers. The method of the present invention is applicable to small scale samples for analytical applications and is particularly useful in the preparation of large quantities of (λ)-tofisopam. The method is rapid, high yielding, and provides (22)-tofisopam of high chemical and high enantiomeric purity. The method may be used for resolving racemic mixtures of tofisopam enantiomers and tofisopam mixtures other than racemic mixtures, for example partially resolved tofisopam. The tofisopam feed for the method may contain additional contaminants other than the (R)- and (S)- enantiomers and their respective conformers. Such additional contaminants may include, for example, synthetic intermediates in the tofisopam synthesis.

The expression "separation medium" means the material on which a mixture of components to be separated is differentially adsorbed during a chromatographic separation. This material is often referred to as a "stationary phase" when chromatographic separation is performed in a chromatography column.

The expression "chiral separation medium" means that the separation medium comprises a chiral functionality such that the separation medium may interact differently with the two enantiomers of an optically active compound.

The expression "solvent system" means the solvent, or mixture of solvents, which elutes a mixture of components to be separated from a separation medium on which the mixture of components is differentially adsorbed. In conventional column chromatography, this material is referred to as a "mobile phase."

The terms "adsorb" and "adsorption" refer to an interaction between the separation medium and a component of a mixture to be separated by

chromatography on the separation medium. The forces involved are relatively small, on the order of van der Waals forces.

The term "isocratic" means that the composition of the solvent system remains constant throughout the chromatographic method. The term "gradient," when used as a parameter in chromatographic separation, means that the composition of the solvent system is varied according over at least one time interval during the chromatographic method. The gradient may be linear or may be stepped.

The expression "adsorption constant" and the symbol K describe the degree to which a compound is retained by a separation medium in a chromatographic separation. The adsorption constant for a compound "a" in a dilute single-component system is equal to the ratio of the concentration of [a] in the mobile phase (solvent system) C _a (g/1) to the concentration of [a] in the stationary phase (separation medium) . In the case of a multi- component system, the different components compete with each other for the finite number of adsorption sites on the separation medium. Thus, the concentration of a given species [a] depends not only on its mobile phase concentration, but also on the mobile phase concentration ' of all other components in the system. For a mixture of two components (A and B) at low concentration, the retention times I _R (A) and tR(B) are related to the adsorption constants according to:

wherein: the term S _E is the "external porosity," defined as VuJVcou where V _ML is the volume of moving (non-stagnant) mobile phase and F _COL is the volume of the chromatography column; and the term to is the "zero retention time" defined by the expression:

wherein Q is the flow rate the mobile phase. Thus for a 250 mm x 4.6 mm chromatography column, operated at a flow rate of 1 mL/min, with an external porosity of 0.4, the calculated zero retention time is:

The term "selectivity" between two components on a separation medium is a measure of the degree to which two components are resolved in a chromatographic separation. Selectivity is symbolized as "α" and is defined as the ratio of the respective retention factors of the two components:

The expression "optically active" refers to a property whereby a material rotates the plane of plane-polarized light. A compound that is optically active is nonsuperimposable on its mirror image. The property of nonsuperimposablity of an object on its mirror image is called chirality. The property of "chirality" in a molecule may arise from any structural feature that makes the molecule nonsuperimposable on its mirror image. The most common structural feature producing chirality is an asymmetric carbon atom, i.e., a carbon atom having four nonequivalent groups attached thereto.

The term "enantiomer" refers to each of the two nonsuperimposable isomers of a pure compound that is optically active. Single enantiomers are designated according to the Cahn-Ingold-Prelog system, a set of priority rules that rank the four groups attached to an asymmetric carbon. See March, Advanced Organic Chemistry, 4 ^th Ed., (1992), p. 109. Once the priority ranking of the four groups is determined, the molecule is oriented so that the lowest ranking group is pointed away from the viewer. Then, if the descending rank order of the other groups proceeds clockwise, the molecule is designated (R) and if the descending rank of the other groups proceeds counterclockwise, the molecule is designated (S). In the example below, the Cahn-Ingold-Prelog

ranking sequence is A > B > C > D. The lowest ranking atom, D is oriented away from the viewer.

The term "racemate" or the phrase "racemic mixture" refers to a 50-50 mixture of two enantiomers such that the mixture does not rotate plane- polarized light.

The expression "enantiomeric excess," generally reported as a percentage, is a means of expressing the degree of enantiomeric purity of a non- racemic mixture, i.e., a resolved or partially resolved enantiomer. The percent enantiomeric excess (% e.e.) is defined as:

The expression "(/?)-tofisopam, substantially free of the (<S)-enantiomer of tofisopam," means a composition that comprises at least about 80% e.e. of the (ϋ)-enantiomer of tofisopam. Preferably, such a composition comprises at least about 90% e.e. of the (i?)-enantiomer of tofisopam. More preferably, such a composition comprises at least about 95% e.e. of the (/2)-enantiomer of tofisopam. Most preferably, such a composition comprises greater than 98% e.e. of the (i?)-enantiomer of tofisopam.

The term "linker," when used to describe a chiral separation medium, refers to the functional group that serves as a tether to link the chiral functionality to a carrier such as silica.

As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

The Chiral Separation Media

The method of the invention employs a chiral separation medium which comprises a chiral stationary phase derivatized with at least one sugar derivative

coated on, or covalently bonded to, a porous inorganic carrier or a porous organic carrier. In a preferred embodiment, the sugar derivative is a derivatized polysaccharide that is selected from the group consisting of amylosic, cellulosic, chitosan, xylan, curdlan, dextran, and inulan polysaccharides, more preferably amylosic and cellulosic polysaccharides. In a preferred embodiment, the sugar derivative is an ester or carbamate of a polysaccharide. In a more preferred embodiment, the chiral stationary phase is an amylose derivative of formula I, coated on, or covalently bonded to, a porous inorganic carrier or a porous organic carrier. Non-limiting examples of chiral stationary phases include cellulose phenyl carbamate derivatives, such as cellulose tris (3,5-dimethylphenyl) carbamate (available from Daicel Chemical Industries, Ltd. as "Chiralcel OD"); cellulose tribenzoate derivatives, such as cellulose tris 4-methylbenzoate (available from Daicel Chemical Industries, Ltd. as "Chiralcel OJ"); cellulose tricinnamate (available from Daicel Chemical Industries, Ltd. as "Chiralcel OK"); amylose phenyl and benzyl carbamate derivatives, such as amylose tris [(S)-α-methyl benzylcarbamate] (available from Daicel Chemical Industries, Ltd. as "Chiralpak AS"), amylose tris-(3,5-dimethylphenyl) carbamate (available from Daicel Chemical Industries, Ltd. as "Chiralpak AD", or as "Chiralpak IA", amylose 3,4-substituted phenyl carbamate, amylose 4- substituted phenyl-carbamate; and amylose tricinnamate.

Chiral separation media comprising amylose derivatives are disclosed in US Patent No. 5,202,433, the entire disclosure of which is incorporated herein by reference. Several examples of chiral separation media comprising an amylose derivative of formula I, are disclosed by Chankvetadze et ah, J. Chromatography A, 694 (1995), pp 101-109, the entire disclosure of which is incorporated herein by reference.

Synthesis of phenyl carbamate derivatives of amylose according to formula I may be carried out by reacting amylose with an amount of a phenyl isocyanate substantially in excess of the stoichiometric amount required to completely react the free hydroxy groups of amylose, in the presence of an acid scavenger such as, for example pyridine. See, Chankvetadze et ah, Id at page

102. The derivatized amylose may be coated onto a porous organic or inorganic support, by methods known in the art. See, Okamoto et ah, J. Chromatography, 363 (1986), pg. 106, the entire disclosure of which is incorporated herein by reference. Derivatized polysaccharides may be bonded to a carrier, as described by Okamoto, et al. (US 5,679,572), the entire disclosure of which is incorporated herein by reference.

The amylose derivative, coated on, or bonded to, a porous support may be packed in a chromatography column using dry packing or slurry packing methods known in the art. In one embodiment, the polysaccharide derivative is coated on, or bonded to, silica gel, zirconium, alumina, ceramics and other silicas, preferably silica gel. The average particle diameter of the packing material is between 1 to 300 μm, preferably 2 to 100 μm, more preferably 5 to 75 μm and most preferably 10 to 30 μm. Other diameters are possible. The average particle diameter of the packing material may be selected to adjust the pressure drop in the continuous chromatographic process and the efficiency of the packing material.

The chiral separation medium may be packed in a chromatography column, and the solvent system passed through the column.

The present invention utilize a non-simulated moving bed chromatographic method, such as the "VariCol™ process," to separate (R)-(+)- tofisopam from a mixture of tofisopam enantiomers. The process employs a plurality of fixed-bed columns packed with a solid stationary phase connected in a loop. Within the loop, a plurality of inlet points and outlet points are located between the columns. The mixture of tofisopam enantiomers is introduced into the loop through a feed inlet point, while the solvent (mobile phase) is introduced into the loop through a solvent inlet point. A solution comprising co- eluting (S)-tofisopam and (R)-(-)-tofisopam is removed from the loop through a raffinate outlet point, while (R)-(+)-tofisopam is removed from the loop at an extract outlet point. The locations of the inlet and outlet points within the loop are shifted at substantially fixed time intervals in the fluid flow direction, where the shifts of the locations of inlet points occur at a different time than the shifts of location of

outlet points. In shifting the location of the feed inlet point for introducing a solution of a mixture of tofϊsopam enantiomers into the loop, the introduction of the solution the mixture of tofisopam enantiomers at the original inlet point is halted and introduction of the solution of the mixture of tofisopam enantiomers commences at the next feed inlet point in the loop. The location of the solvent inlet point is shifted by halting the introduction of solvent at the original solvent inlet point in the loop and introducing the solvent at the next solvent inlet point in the loop. The location of the raffinate outlet point is shifted by halting removal of raffinate at the original raffinate outlet point in the loop and removing the raffinate at the next raffinate outlet point in the loop. The location of the extract outlet point is shifted by halting removal of solution at the original extract outlet point in the loop and removing the solution at the next extract outlet point in the loop. Methods of directing the flow of fluid from one point to another point within the loop are well know to one of ordinary skill in the art. For example, a valving system can be used to shift flows from an original point in the loop to a new point in the loop. Shifting the location of the inlet and outlet points creates zones within the loop where each zone contains different distributions of tofisopam isomers. Zone lengths are not constant over time and the number of columns per zone is not constant during a time period. Depending upon the configuration of the columns and the locations of inlet and outlet points, a plurality of zones may be established.

The following is a representative configuration for a four-zone system for separating tofisopam enantiomer.

Zone 1, located between the solvent inlet point and the extract outlet point, contains essentially (R)-(+)-tofisopam;

Zone 2, located between the extract outlet point and the feed inlet point, contains a mixture of the four isomers of tofisopam, enriched in (R)-(+)- tofisopam;

Zone 3, located between the feed inlet point and the raffinate outlet point, contains the a mixture of the four isomers of tofisopam, enriched in (R)-(-)-tofisopam, (S)-(+)-tofisopam, and (S)-(-)-tofisopam; and

Zone 4, located between the collection point of the raffinate and the injection point of the eluent, contains essentially (R)-(-)-tofisopam, (S)- (+)-tofisopam, and (S)-(-)-tofisopam.

Non-simulated moving bed separation processes and apparatus for use in such processes are more fully described in U.S. Pat. Nos. 6,136,198, 6,375,839; 6,413,419; and 6,712,973, the entire disclosure of which are incorporated herein by reference.

The Solvent System

When the separation medium is a sugar derivative, or an amylose derivative of formula I, coated on, or covalently bonded to, a porous inorganic carrier or a porous organic carrier, the solvent system preferably comprises a mixture of ACN and an alkyl alcohol. The alkyl alcohol is preferably a (Ci-C ₄ ) alcohol. The alcohol is most preferably MeOH. The solvent system preferably comprises from about 5% (VAO to about 40% (VAO MeOH in ACN, more preferably from about 10% (VAO to about 35% (VAO MeOH in ACN, even more preferably from about 15% (VAO to about 25% (VAO MeOH in ACN, most preferably about 19% (VAO MeOH in ACN. Alternatively, the solvent system is ACN alone.

The addition of an alkyl alcohol to the ACN solvent system is observed to have a significant effect on the resolution of tofisopam. All four isomers of tofisopam are resolved. However, though selectivity is improved between the enantiomers of tofisopam and their respective conformers, the solubility of tofisopam decreases with higher concentrations of alkyl alcohol in the solvent system. Thus the concentration of an alkyl alcohol in the solvent system is preferably no more than about 40% by volume.

Where (R)-(+)-tofϊsopam is isolated, the solvent system of acetonitrile is used to co-elute (R)-(-)-tofisopam, (S)-(+)-tofisopam, and (S)-(-)-tofisopam before (R)-(+)-tofisopam is eluted. Depending upon the columns and conditions used, other solvents or solvent mixtures may produce similar results. Solubility studies have determined that the solubility of racemic tofisopam in a mixture of 19% (WV) MeOH in ACN is about 135.5 g/L. Thus, in an embodiment of the invention wherein the solvent system comprises about 19% (V/V) MeOH in ACN, the concentration of the feed of tofisopam is limited to a concentration of less than 135.5 g/L. Preferably, the concentration of the feed of a mixture of enantiomers of tofisopam in the practice of the invention is from about 45 to about 120 g/L, most preferably about 100 g/L.

The solvent systems employed in the practice of the invention may be isocratic or may comprise a gradient from one solvent composition to another solvent composition over one or more time intervals during the chromatographic separation. Preferably the solvent system is isocratic.

Preparative Scale Resolutions

The isolation method of the present invention is particularly suited to preparative scale resolution of enantiomeric mixtures of tofisopam. The method of the invention is useful for resolving tofisopam under overloading conditions, i.e., amounts of tofisopam high enough to interact with all adsorption sites in the upstream portion of the separation medium, as well as with separations of dilute solutions, Le., non-overloading conditions, such as analytical scale separations of tofisopam. The temperature under which the chromatographic resolution is performed may conveniently be controlled by maintaining the separation medium in a controlled temperature environment and/or maintaining a controlled temperature for the solvent system flowing through the separation medium. The preferred temperature range for chromatographic resolutions is from about 0° C to about 60° C, preferably from about 25° C to about 45° C, most preferably about 40° C.

The solvent composition of the solvent system, the flow rate of the solvent system through the resolution medium, and the amount of the separation medium are factors which contribute to the time required for the resolution of a mixture of enantiomers of tofisopam in the practice of the method of the invention. Preferably, the chromatographic separation is completed in from about 15 to about 25 minutes, more preferably from about 18 to about 22 minutes.

In a non-simulated moving bed process, such as the process described in Examples 7 — 10 herein, no limitation on the number of configurations for a given number of columns exists. The potential for variation of the average length of given zones represented by column sections during the chromatographic separation can lead to an increase efficiency by using fewer columns, reducing the amount of the solid phase, reducing the amount of solvent(s), and increasing purity and yields. According to one non-simulated moving bed chromatography method of the invention, a VariCol™ process is used to isolate (R)-tofisopam, such as a five to six column, four variable zone VariCol™ system. The zones vary in length during operation, and have an average rather than fixed length in a given operation cycle. In another embodiment, a three column-four variable zone VariCol™ system is used. Such a system cannot be utilized in a SMB process, as at least four columns with four zones are necessary to simulate the true moving bed system in an SMB process. For the SMB process, the minimal number of columns required for the separation is equal to the number of zones of the system, whereas the number of columns can be smaller than the number of zones in the VariCol™ system.

The practice of the invention is illustrated by the following non-limiting examples. Examples 7-10 show representative parameters for the isolation of the (R)-enantiomer of tofisopam using a non-simulated moving bed process (VariCol™ process). Other column configurations can be utilized for the chiral separations described herein, using at least three columns.

Examples

Example 1: Comparison of Different Chiral Stationary Phases Using Dilute Solutions Of Racemic Tofisopam:

Chromatographic separations of racemic tofisopam in dilute solution were conducted using the chiral separation media listed in Table 1. AU of the separation media were packed in 4.6 mm (i.d.) x 250mm chromatography columns.

Table 2 below, lists the chiral separation media, the solvent systems, the temperature and the flow rate employed for each chiral separation medium tested. Solvent systems were prepared using HPLC grade solvents obtained from E.M. Science.

The Table also summarizes retention factors for all components resolved in each chromatographic separation.

Table 2

Of the tested chiral separation media, two materials resolved racemic tofϊsopam into four separate peaks. These materials were:

(S,S)-β-GEM, a commercially available material comprising a (2S)-2- {( 1 S)[(3,5-dinitrophenyl)carbonylamino]phenylmethyl} -3,3-dimethylbutanoate covalently bound to silica through an alkylene (C 13) ester linkage; and

a chiral separation medium comprising an amylose derivative of formula

Ia:

coated onto silica gel.

Separations were also observed for Chiralpak ^® AD ^® , Chiralcel ^® OD ^® , (R,R) Whelk-O ^® , and Shiseido RU-I. Chiralpak ^® AD ^® and Chiralcel ^® OD ^® exhibited poor resolution (R ₃ < 0.5). The separation media, (i?,iϋ) Whelk-O ^® , and Shiseido RU-I gave high retention constants that resulted in long retention times thus requiring substantially higher amounts of the solvent system.

Example 2: Determination of EIution order of tofisopam enantiomers on a chiral separation medium comprising an amylose derivative of formula Ia.

A 4.6 mm (i.d.) x 250mm chromatography column packed with a chiral separation medium, comprising an amylose derivative of formula Ia, was stabilized under conditions of: 100% ACN at a flow rate of 1.0 mL/min at 22° C. Racemic tofisopam (0. lmg) was injected into the column, and fractions of the effluent were collected every 0.5 minutes between 1.5 and 6.0 minutes following the injection. These fractions were analyzed for the amounts of each of the four isomers of tofisopam. The data is plotted in Fig. 1, was constructed to show the peak profile of the resolution in 100% ACN.

Tofisopam isomers were eluted in the following order: (iϊ)-(-); (5)-(-)/(JS)-(+); and (R)-(+). Due to the interconversion of the conformers during

the chromatographic method, the presence of both the (/?)-(-) and the (/?)-(+) conformers is observed throughout the elution of the (S)-enantiomer.

Example 3: Overload injection of racemic tofϊsopam on a chiral separation medium comprising an amylose derivative of formula Ia eluted with 100% ACN.

A 4.6 mm (i.d.) x 250mm chromatography column packed with a chiral separation medium comprising an amylose derivative of formula Ia was stabilized under conditions of: 100% ACN at a flow rate of 1.0 mL/min at 25° C. Racemic tofisopam (300μL at a concentration of 33 g/L) was injected into the column. Figure 2 shows the resulting separation (LJV detection at 345nm). The yield of isolated (i?)-tofisopam was approximately 60% by weight. The measured enantiomeric excess (e.e.) of the (λ)-toiϊsopam isolated in this separation was less than 95% e.e., due to the presence of the (S)-(+) enantiomer as a contaminant.

Example 4; Modification of the solvent system: Resolution of tofisopam on a chiral separation medium comprising an amylose derivative of formula Ia eluted with ACN/MeOH mixtures; Chromatographic separations of racemic tofisopam in dilute solution were conducted using a chiral separation medium comprising an amylose derivative of formula Ia, packed in a 4.6 mm (i.d.) x 250mm chromatography column. The solvent system was modified to compare resolution of tofisopam eluting with 100% ACN to resolution with ACN/MeOH mixtures wherein MeOH is present in a range of concentrations from about 5% to about 40%

A 4.6 mm (i.d.) x 250 mm, chromatography column, packed with a chiral separation medium comprising an amylose derivative of formula Ia, was stabilized under conditions of: 85/15 (WV) ACN/MeOH at a flow rate of 1.6 mL/min at 25° C. A dilute injection of racemic tofisopam was injected. Figure 3 shows the resulting separation.

Additional ACN/MeOH solvent compositions and the resulting resolution data are listed below in Table 3.

Table 3

Selectivity between the enantiomers and their respective conformers of tofisopam was increased by the addition of MeOH to the solvent system.

Example 5; Overload injection of racemic tofϊsopam on a chiral separation medium comprising an amylose derivative of formula Ia eluted with 85/15

ACN/MeOH (WV).

A 4.6 mm (i.d.) x 250mm, chromatography column, packed with a chiral separation medium comprising an amylose derivative of formula Ia, was stabilized under conditions of: 85/15 (V/V) ACN/MeOH at a flow rate of 1.0 mL/min at 25° C. Racemic tofϊsopam (9.9mg) was injected. Figure 4 shows the resulting separation (UV detection at 345nm). The vertical dotted line marks the cut point (11.6 min) where collection of (λ)-(+)-tofϊsopam was commenced.

Analysis of the fractions obtained from this overload experiment showed a yield

of greater than 83% by weight, and an enantiomeric excess greater than 98% for the (i?)-enantiomer.

Example 6: Overload injection of racemic tofisopam on a chiral separation medium comprising an amylose derivative of formula Ia eluted with 81/19 ACN/MeOH (V/V).

A 4.6 mm (i.d.) x 250mm, chromatography column packed with a chiral separation medium comprising an amylose derivative of formula Ia was stabilized under conditions of: 81/19 (V/V) ACN/MeOH at a flow rate of 1.17 mL/min at 25° C. Racemic tofisopam (500 μL of a 104 g/L concentration) was injected. Figure 5 shows the resulting separation (UV detection at 355nm). The vertical dotted line marks the cut point (8.0 min) where collection of (/?)-(+)- tofisopam was commenced. Analysis of the fractions obtained from this overload experiment showed a yield of approximately 73% by weight and an enantiomeric excess greater than 98% for the (λ)~enantiomer.

Higher concentrations of MeOH in the solvent system were observed to yield increased selectivity. However solubility of racemic tofisopam decreased with the higher concentrations of MeOH.

Examples 7-10. Isolation of (RVtofisopam from a mixture of tofisopam enantiomers in a non-simulated moving bed process (VariCol™ process).

Five 2.5 cm (i.d.) x 10.6 cm chromatography columns packed with a chiral separation medium comprising an amylose derivative of formula Ia, were connected in series in a loop. Multiple inlets and outlets were placed in the loop between the columns. After the system was stabilized by passing ACN through the columns, a solution of tofisopam enantiomers in ACN (concentration 48—50 g/L) was continuously injected into the system.

The separation was performed using the temperatures, feed concentrations and flow rates listed in Table 4 below.

The conditions of Table 4 resulted in a column configuration shown in Table 5.

The yield and product purity from the Example 7-10 purifications are set forth in Table 6. In evaluating the data, it should be appreciated that the maximum yield can only be approximately 80% because the minor (R)-(-)- isomer co-elutes with the (S)-isomers.

Example 7 demonstrates that (R)-tofisopam can be obtained from a mixture of tofϊsopam enantiomers with a purity of about 93%, at a yield of 36% (less than 50% of maximum theoretical yield). According to Example 8 (35°C), under a different column configuration than was used for Example 7, high purity (99.3%) (R)-tofϊsopam was obtained with a yield of about 72% by weight (approximately 90% of the maximum theoretical yield). At the 37°C temperature of Example 8, high purity (99.3%) (R)-tofϊsopam was obtained at a yield of about 75% by weight (approximately 94% of the maximum theoretical yield). Finally, Example 10 demonstrates that even higher purity (99.5%) (R)- tofisopam can be obtained at 40 ⁰ C at a yield of about 78% by weight (approximately 98% of the maximum theoretical yield).

While preferred embodiments of the present invention have been shown and described, it will be understood by one skilled in the art that various changes or modifications can be made without varying from the scope of the invention.

All references cited herein are incorporated by reference. The present invention may be embodied in other specific forms without departing from the spirit of essential attributes thereof and, accordingly, reference should be made to the appended claims, rather than to the foregoing specification, as indication of the scope of the invention.