HU188729B - Process for increasing the stereoselectivity of the attachment - Google Patents

Abstract

The present invention relates to a method for increasing the stereoselectivity of benzodiazepines. The method according to the invention is that the potency of binding of chiral benzodiazepine enantiomers to serum protein is selectively altered by the warfarin additive, the stereoisomers are separated and purified from the additive. The most important advantage of our method is that it greatly increases the stereoselectivity of the binding between the chiral adsorbent and the material to be resolved, which makes the process suitable for preparative purposes. -1-

Description

BACKGROUND OF THE INVENTION This invention relates to a process for increasing the stereoselectivity of benzodiazepine binding by resolution of chiral benzodiazepine racemates by chromatography or ultrafiltration or dialysis or centrifugation (separation into stereoisomers).

As is known, chiral compounds (eg, mirror-like asymmetries) that make up the body (e.g., amino acids, sugars, etc.) are optically active, so that only one of the two chemically possible stereoisomers is present in the body. Enzymes that determine the vital functions of life. receptors are also typically stereospecific. As a result, there is usually a significant order of magnitude difference between the two stereoisomers of synthesized chiral drugs. The identification of the more effective stereoisomer is of great importance because it allows the dose of the drug to be reduced and the undesirable side effects to be eliminated while maintaining the desired therapeutic effect. However, many drugs are marketed in racemic form. The reason for this is that the resolution of the racemate formed by chemical synthesis by classical chemical methods or the elaboration of stereospecific synthesis entails costly, high loss of material. Resolution is particularly difficult for radioactive preparations required for pharmacological studies. In many cases, the drug is not tested in the form of pure stereoisomers.

Chiral adsorbents, e.g. stereoselective binding to the surface of serum proteins allows the quantitative resolution of racemates. described such a solution e.g. Κ. K. Stewart and RF Doherty (Resolution of DL-tryptophan by affinity chromatography on bovine serum albumin - Agarose columns, Proc. Nat. Acad. Sci. USA 70, 2880 (1973)), and C. Lagercrantz, T. Larsson and I Denfors (Stereoselective binding of the enantiomers of warfarin and tryptophan to the serum albumin from someficient species studied by affinity chromatography on columns of immobilized serum albumin. Comp. Biochem. Physiol. 69C, 375 [1980]).

The disadvantage of these processes is that they have to be repeated many times in order to produce a small amount of material and thus a considerable amount of material. This is particularly disadvantageous if the binding selectivity is low.

It is an object of the present invention to overcome the shortcomings described. Our solution greatly increases the selectivity of binding between the serum protein and the chiral benzodiazepines to be resolved. This allows maximum utilization of the adsorbent capacity, which also makes the process suitable for preparative purposes.

The present invention is based on the discovery that the selectivity of the binding sites of serum proteins can be greatly enhanced by the use of an additive.

According to the present invention, in the light of the above discovery, the shortcomings of the prior art are overcome by a process of molecular linkage between the serum protein and the benzodiazepine racemate. For preparative use, particularly in the resolution of a radioactive racemate, it is desirable to separate the stereoisomers by chromatography, since this will minimize the amount of radioisotopic loss. The use of ultrafiltration, dialysis or centrifugation is advantageous when only one stereoisomer is required.

The following examples illustrate the application of the process, which of course is not limited to the invention.

/. example

For the resolution of the racemate of lorazepar acellate radioactive (7-chloro-5- (2-chlorophenyl) -1,3-dihydro-3-hydroxy-2H-1,4 benzodiazepin-2-one acetate), 1.8 x 26 cm an alphinite chromatography column was prepared. The column was loaded with 50 µl of Sepharose 4B containing 1% immobilized human serum albumin. 50 ml of 1 (Γ ⁴ M warfarin (3- (3-oxo-1-phenylbutyl) -4-hydroxy-humarin)) pH 7.4 phosphate buffer was passed through the column. 6 mg of radioactive i / Morazepam was added to the column. acetate is eluted with warfarin buffer, the eluate is collected in 10 mL fractions and the radioactivity in a 0.1 mL sample from each fraction is determined by liquid scintillation 50% of the radioactivity assigned to the / stereoisomer is ca. Elute at 100 mL, The J stereoisomer can be washed off the column by passing 100 mL of 1% albumin buffer, In the absence of additive (Warfarin), under certain conditions, the two enantiomers are only partially separated on the column which does not allow pure product, The eluate containing the two radioactive samples was lyophilized and the radioactive d- and llorazepam acetate stereoisomers were extracted from the residue with chloroform (3 x 30 ml) and evaporated. from rine by further preparative silica gel thin layer chromatography. The eluent used őszszetétele 60 ml of benzene, 40 ml of diethyl ether, 7 ml ELAN (Lorazepam acetate _Rf = 0.52, warfarin _Rf = 0.74). The resolved radioactive enantiomers are obtained in an amount of 2.0-2.5 mg. The column can be reused after regeneration. The resolved products are re-dispensed to the column eluting at a single peak in buffer. CD spectra ^confirm that the first eluted substance is Z-lorazepam acetate (Δε ”/ _Χ ^ηη, = -21.9 M“ 'cm'), whereas in the presence of warfarin only protein washable substance zilorazepam acetate (Δε ".// ^'11 = + 21.7 M' cm ^- ').

Example 2

For 200 ml f ⁴ M warfarin and 1% bovine serum albumin pH: 7.4 Phosphate buffer: 20 mg (9-lorazepam acetate) in 4 ml ethanol

188,729 beats. In an ultrafiltration cell, filter through an AMICON YM ~ 10 membrane. The filtrate was lyophilized, the chlorazepam acetate was extracted with chloroform (3 x 30 mL) and evaporated. Separation of warfarin from the preparative silica gel layer was carried out by thin layer chromatography (60 mL benzene, 40 mL diethyl ether, 5 mL mL ethanol). The product was 5 mg of Morazepam acetate. In the absence of the additive (warfarin), a mixture of enantiomers is obtained as in the example. 10

In addition to achieving the intended benefits, our process also has the unexpected, surprising advantage that, when lorazepam acetate is resolved, the stereoselectivity is increased hundreds of times compared to the known solutions, which makes the separation of stereoisomers free of cross-contamination.

The residue was extracted with chloroform (3 x 30 ml). The chloroform is evaporated and the isolated stereoisomers are separated from the inactive warfarin by preparative silica gel thin layer chromatography. The solvent mixture used was a mixture of 50 ml benzene, 50 ml ether and 7 ml ethanol. (Oxazepam acetate R _F = 0.63, warfarin R _F = 0.79). Resolved radioactive enantiomers are obtained in an amount of 2.0-2.5 mg. The CD spectra confirm the optical purity of the products. The first eluting substance was / oxazepam acetate (Ae ²²⁴ x ^nm = -46.3 M ^-1 cm -1), while the second peak (N-oxazepam acetate (Δε ²² /, ™ = + 45.9 M 'cm'). In the absence of an additive, the two stereoisomers are only partially separated and therefore a less pure product is obtained with a lower yield.

Example 3 20

An affinity chromatography column was prepared for resolution of the racemate of radioactive oxazepam acetate (7-chloro-1,3 dihydro-3-hydroxy-5-phenyl-24 -1,4-benzodiazepin-2-one acetate) ²⁵ (1.4 x 23 cm). The 40 m! Sepharose 4B gel containing 1% human serum proteins immobilized on 10 ~ ^4M warfarin containing pH 7.4 phosphate buffer was equilibrated. The column task 6mg radioactive (// -.. Oxazepam acetate, followed by elution with ³⁰ well puff ral radioactivity in the eluate measured by liquid scintillation warfari- The first peak (/ stereoisomer) eluted at about 80 ml volumes of (/.. - The stereoisomer leaves the column well separated, at a peak of about 220 ml. The eluate containing the two radioactive samples is lyophilized and the radioactive d- and / or oxazepam acetate

Claims (2)

1. A process for increasing the stereoselectivity of benzodiazepine binding, wherein a molecular link is established between the stereoisomers of serum protein, preferably human serum albumin, and chiral benzodiazepine racemates, characterized in that the butyl) - 4 - hydroxy coumarin), the stereoisomers are separated and purified from the additives. 2. The process of claim 1, wherein the stereoisomers are separated by chromatography, ultrafiltration, dialysis, or centrifugation. Without illustration