FR2571372A1 - Process for the separation of the diastereiosomers of zearalanol - Google Patents

Abstract

The two isomers of - 7, 14, 16-trihydroxy-3-methyl-3,4,5,6,7,8,9,10,11,12-decahydro-1H-2-benzoxacy clotetradecin-1-one -have [3S-(3R<*>, 7S<*>)] and [3S-(3R<*>, 7R<*>)] configurations. They are separated by a system of reverse phase high performance chromotography coupling a column of high flowrate and average particle size with a second column of high resolution.

Description

The present invention relates to an industrial process for resolving a mixture of zearalanols into its diastereoisomers by a chromatographic technique. The term zearalanol conforms to the old nomenclature: 6- (6,10-dihydroxy undecyl) -ss-resorcylic acid-u-lactone.

en zéaralanol répondant à la formule

par réduction de la liaison oléfinique et du groupe cétone, il se forme un nélange de diastéréoisomères du zéaralanol, dont l'un présente un point de fusion plus élevé que l'autre Cette réduction est décrite dans le brevet
U.S. 3.239.345 publié le 8/3/1966.When converting the zearalenone corresponding to the formula

in zearalanol corresponding to the formula

by reduction of the olefinic bond and of the ketone group, a mixture of diastereoisomers of zearalanol is formed, one of which has a higher melting point than the other. This reduction is described in the patent
US 3,239,345 issued 3/8/1966.

The new chemical abstract nomenclature gives the chemical name -7,14,16-trihydroxy-3 methyl-decahydro-3,4,5,6,7,8,9,10,11,12-1H-2-benzoxacy clotetradecine - 1- one: with the configuration [3S- (3R *, 7S *) 1 for zearalanol a with the highest melting point and the configuration [3S- (3R *, 7R *)] for the zearalanol ss with the point of lower fusion.

These non-enantiomorphic stereoisomers are referred to as diastereomers. Diastereomers usually exhibit differences in their physicochemical properties, such as optical rotation, melting point, solubility, solid phase spectrometry.

The two diastereomers of zearalanol are useful as anabolic and estrogenic substances for administration to animals.

However, depending on the use for which it is intended, it may be preferable to use only one or the other of these diastereomers.

It then becomes necessary to separate the diastereoisomers into zearalanol with a high melting point (pF = 182 / 1840C) and zearalanol with a lower melting point (pF = 1550C).

The reduction of zearalenone according to the general technique described in US Pat. No. 3,239,345 results in a substantially 50:50 mixture of the two diastereomers.

According to this same patent, it is possible to separate, in the pure state, the high-melting point isomer by repeated crystallizations in the isopropanol / water mixture. 0.18 g of high melting point isomer is thus obtained from 1 g of mixture (Example 6).

The separation by this process is not, however, quite complete and the remaining zearalanol still contains a significant amount of each diastereomer.

The separation process can be improved, so the patent
US 3,687,982 claims a process for separating a mixture of diastereoisomers of zearalanol, comprising the following stages 1 - Dissolution of the mixture of diastereomers in acetic acid.

2 - Maintaining the solution obtained at a temperature sufficient to carry out the esterification and obtain the 6'-acetate derivatives of the diastereoisomers of zearalanol.

3 - Separation of the 6 ′ acetate esters from the diastereoisomers of zearalanol by fractional crystallization.

4 - Deesterification of the acetate groups resulting in the diastereoisomers separated from zearalanol.

This new process brings significant improvements compared to the original technique of direct crystallization thus making it possible to obtain, according to the example, 1.38 g of high melting point isomer from a mixture containing substantially 55% of isomer. high melting point and 45% low melting isomer.

However, this technique has drawbacks; thus, four stages are necessary comprising numerous manipulations and using various reagents, the recovery of which can be expensive.

On the other hand, the yield of each stage is not quantitative, thus the acetylation only makes it possible to recover 81 to 84% compared to the theory.

The present invention relates to an industrial process for the separation of diastereomers by chromatography on a high performance chromatographic column system (HPLC) using a reversed phase eluting with an aqueous solution of methanol or acetonitrile.

The present invention makes it possible to obtain 100% of each diastereoisomer in the pure state.
Silica gels are currently manufactured and marketed with a uniform particle diameter. The perfect sphericity of the silica grains ensures better chromatographic performances, the tightening of the particle size tolerance also increases these performances.

These silica gels are treated with an alkyl trichlorosilane to obtain grafted silica gels. The most commonly used hydro-carbon chains are the C18 groups. We use in our process this type of silica R.P, 18 containing about 20 8 carbon and commercially available.

The process consists in using a high capacity chromatographic column containing a reverse phase, for example a C18 grafted silica of medium particle size allowing a high nominal speed of the eluent and ensuring the separation of at least 80% of the mixture of diastereisomers.

This high capacity column is coupled with a medium capacity column containing a C18 grafted silica reverse phase of small particle size ensuring the separation of the remaining mixture of diastereomers.

The eluent is constituted by an aqueous solution of acetonitrile or of methanol making it possible to work under wide temperature conditions.

The mixture of diastereoisomers of zeranol containing substantially 50% of each diastereomer is dissolved at a ratio of 5 to 10% in a mixture of solvents substantially identical to that used for the elution. The solution to be injected can be maintained at a temperature sufficient to avoid recrystallization of the mixture of diastereomers.

This solution is injected onto the high-capacity HPLC column containing the grafted silica gel RP 18 of average particle size, for example 15 to 25 microns.

Then elution is carried out by an eluent which can be a mechanol system: water comprising from 60 μ of methanol and 40 μ of water to 80 μ of methanol and 20 μ of water, preferably 70 μ of methanol and 30 μ of water, or else an acetonitrile system: water comprising from 65 p of acetonitrile and 35 p of water to 40 p of acetonitrile and 60 p of water, preferably 50 p of acetonitrile and 50 p of water.

The fractions are collected or recycled automatically based on the response of the refractive index detector.

The fractions isolated from the first chromatographic peak (lowest retention volume) contain exclusively the low melting point isomer (A) and are collected.

The intermediate fraction, containing about 10% of unresolved isomers (C), is diverted to the next HPLC column and finally the remaining fractions containing the second pure diastereomer (B) (high melting point isomer) are collected again.

From an injection containing 50 μ of low melting point isomer and 50 μ of high melting point isomer, 45 μ of each isomer in the pure state are thus isolated at the outlet of the first column.

The intermediate collection fraction (C) containing substantially 5 p of each diastereoisomer is immediately injected into a column of smaller capacity containing a grafted silica RP 18 of fine particle size, for example 10 microns and eluted with the same eluent as above. The total resolution of the mixture is thus obtained, namely 5 p of each diastereoisomer.

The invention is illustrated by the following examples given without limitation.

EXAMPLE 1
Description of the chromatographic system 1 Glass column 300 mm x 500 mm
Filling 5 kg silica gel RP 18 - 15725 microns
Lichroprep (Merck)
Solvent acetonitrile 50 / water 50
Injection of 100 g of mixture of 50/50 isomers into
2.4 liters of eluting solvent
Flow rate 300 ml / mn Pressure <1.5 Bar
Refractive index detector - Jobin-Yvon; Iota
Fraction collector 2 Column Stainless steel 80 mm x 1000 mm
(prepamatic Jobin-Yvon)
Filling 1 kg spherical silica gel 10 microns
RP 18 Nucleosil (Macherey Nagel)
Solvent acetonitrile 50 / water 50
Injection 10 g of approximately 50/50 mixture of isomers
in solution in the eluting solvent
Flow rate 65 ml / min
Pressure 12/15 bar
Refractive index detector Jobin Yvon; Iota
Fraction reader collar
The solution containing the 100 g of mixture of isomers (C) of relative composition of approximately 50/50 is injected into the first column (1) (FIG. I), then the elution pump is started; the eluent is recycled automatically until the appearance of the first isomer detected by the change in refractive index. The collection of the first peak (A) (low melting point isomer) is started and maintained until the refractive index drops back to a value between 10 and 15% of the maximum value of the first peak (figure II). . This maximum value is reached after approximately 100 minutes. The collector then sends the partially mixed intermediate fraction (C) to the sample reserve of the second column.

When the second chromatographic peak appears and the refractive index value reaches 10% of the maximum reached on the first peak, the fraction collector (3) collects the eluate containing the second pure isomer (high melting point diastereomer ) (B) (figure II).

After evaporation, each collection contains 45 g of each of the two * pure isomers.

The intermediate fraction (C) is in turn injected into the second column and eluted with the same eluting mixture. The solvent is automatically recycled by the second collector (4) until the appearance of the first isomer detected by the change in refractive index (figure III), the collection of the first peak (A) isomer of low melting point is then started and maintained until the refractive index returns to its initial value, the separation being quantitative on this second column. The solvent is then recycled until the appearance of a second chromatographic peak representing the passage of the second isomer (B) which is collected.

After evaporation of fractions A and B, substantially 50 g of high melting point isomer of zeranol and 50 g of low melting point isomer of zeranol are recovered. the purity of each isomer is determined by analytical HPLC and is greater than 99%.

EXAMPLE 2
The whole of the technique described in Example 1 is used with the only difference that an elution solvent consisting of a 70 methanol / water 30 mixture is used.

The order of elution of the two isomers is identical but the elution times are slightly increased; the totality of each of the two diastereoisomers is recovered in the same way with a purity> 99%.

Claims (5)

i - Process for the industrial chromatographic separation of the diastereoisomers of zearalanol of formula 2 - Process according to claim 1, characterized in that the major part of the separation is carried out on a high flow rate column and in that the part which is not completely separated is derived on a high resolving power column. 3 - Process according to claims 1 and 2 where one uses as stationary phase a silica grafted type R.Po 18 of average particle size on the column at high flow rate and a spherical silica type R.P. 18 having a particle size of about 10 microns in the second column. 4 - Process according to claims 1 to 3 wherein the eluent is a mixture of acetonitrile / water, the ratio of which is between 65/35 and 40/60. 5 - Process according to claims 1 to 3 wherein the eluent is a mixture of methanol / water, the ratio of which is between 60/40 and 80/20. 6 - Apparatus for chromatographic separation according to claim 1 for coupling a high capacity column operating at low pressure and packed with a grafted silica gel type RP 18 ae average particle size to a medium capacity column operating at medium pressure and filled with a grafted silica gel of the RP 18 type of fine particle size and ensuring complete separation of the diastereoisomers in a single cycle. characterized in that the chromatographic separation is carried out using a solid phase consisting of a silica grafted C18 with an aqueous polar solvent, the system comprising a column of large diameter for coarse separation coupled with a column of smaller diameter having a high resolving power.

CLAIMS 1 - Process for the industrial chromatographic separation of the diastereoisomers of zearalanol of formula 2 - Process according to claim 1 where one uses as stationary phase a grafted silica of RP 18 type of average particle size on the high flow column and a grafted spherical silica of RP 18 type having a particle size of about 10 microns in the second column. 3 - Process according to claims 1 and 2 wherein the eluent is a mixture of acetonitrile / water, the ratio of which is between 65/35 and 40/60. 4 - Process according to claims 1 and 2 wherein the eluent is a mixture of methanol / water, the ratio of which is between 60/40 and 80/20. 5 - Apparatus for chromatographic separation according to claim 1 for coupling a high capacity column operating at low pressure and packed with a grafted silica gel type RP 18 of medium particle size to a medium capacity column operating at medium pressure and filled with a grafted silica gel of the RP 18 type of fine particle size and ensuring complete separation of the diastereoisomers in a single cycle.