CZ299175B6 - Method of determining enantiomeric purity of 2-(4-fluorphenyl)-?,delta-dihydroxy-5-(1-methylethyl)-3-phenyl-4[phenylamino)-carbonyl]-1H-pyrrole-1-heptanoic acid and salts thereof - Google Patents

Abstract

In the present invention, there is disclosed a method of determining enantiomeric purity of 2-(4-fluorphenyl)-?,delta-dihydroxy-5-(1-methylethyl)-3-phenyl-4[phenylamino)-carbonyl]-1H-pyrrole-1-heptanoic acid and salts thereof of the general formula I using chiral chromatography process. The invented determination method can be used when checking substance of a medicament of in a liquid and solid medicament forms.

Description

Technical field

The present invention relates to a method for determining the enantiomeric purity of 2- (4-fluorophenyl) -3,8-dihydroxy-5- (1-methylethyl) -3-phenyl-4 - [(phenylamino) carbonyl] -1H-pyrrole-1-heptanoic acid and its salts by chiral chromatography. The assay can be used in substance control or in liquid and solid dosage forms.

BACKGROUND OF THE INVENTION

2- (4-Fluorophenyl) -β, η 5 -dihydroxy-5- (1-methylethyl) -3-phenyl-4 - [(phenylamino) carbonyl] -1H-pyrrole-1-heptanoic acid compounds of formula I

COOM

wherein M represents hydrogen, lithium, sodium, potassium, calcium or magnesium are HMG CoA reductase inhibitors and have been described in U.S. Pat. No. 4,681,893, U.S. Pat. No. 5,273,995, U.S. Pat. No. 5,273,995.

In this series, the hemi-calcium salt of R- (R *, R *) - 7- (3-phenyl-4-phenylcarbamoyl-2- (4-fluorophenyl) -5-isopropylpyrrol-1-yl) -3,5-dihydroxyheptanoic acid Antihypercholesterolemic is known under the name atorvastatin calcium.

A process for preparing crystalline forms of this substance is disclosed in patents, eg, US 5,963,915, US 6,121,461. For atorvastatin hemi-calcium used as a drug substance, some manufacturers declare a specific optical rotation in the range of -6.5 to 8.8 ° (for determination of 1% solution in dimethylsulfoxide, 25 ° C). For chiral purity, the presence of the (-) enantiomer determined as the corresponding lactone is reported.

For example, chiral spectroscopic methods are used to determine chiral purity, but are limited in multi-component assays or require repeated sample preparation. GC analyzes of enantiomeric compounds are routinely used, the necessary derivatization with reagents modifies specific functional groups and produces diastereomers, so the use of derivatizing reagents with high enantiomeric purity is practically inevitable.

These and other separation techniques such as capillary electrophoresis are reviewed in Chiral

Chromatography, T.E. Beesley, R., P.W. Scott, Wiley, N.Y., 2002.

-1 CZ 299175 B6

However, the work on enantiomeric purity of atorvastatin or substances from the group of statins is not described. There are no published papers describing separation of atorvastatin enantiomer by chiral chromatography.

It has now been found that enantiomeric purity (by weight) of 2- (4-fluorophenyl) -3,5-dihydroxy-5 (1-methylethyl) -3-phenyl-4 - [(phenylamino) carbonyl] -1H-pyrrole The 1-heptanoic acid and its salts can be determined by the process of the invention by separating the individual enantiomers by high performance liquid chromatography in a chiral environment.

The invention therefore relates to a method for determining the enantiomeric purity of 2- (4-fluorophenyl) -β, 6-dihydroxy-5- (1-methylethyl) -3-phenyl-4 - [(phenylamino-carbonyl) -1H-pyrrole-1-heptanoic acid and its salts by chiral chromatography.

SUMMARY OF THE INVENTION

The present invention provides a method for determining the enantiomeric purity of 2- (4-fluorophenyl) -β, 6-dihydroxy-5- (1-methylethyl-3-phenyl) - [(phenylamino) carbonyl] -1H-pyrrole-1-heptanoic acid and its salts of formula I, coon

wherein M is hydrogen, lithium, sodium, potassium, calcium or magnesium, comprising: 2- (4-fluorophenyl) -P, 6-dihydroxy-5- (1-methylethyl) -3-phenyl-4; - [(phenylamino) carbonyl] 25 1 H-pyrrole-1-heptanoic acid of formula I wherein M represents lithium, potassium, sodium, calcium or magnesium in water and / or an organic solvent selected from the group of dipolar aprotic or polar solvents an organic acid having a pK value of <4.7 or sulfuric acid, hydrochloric acid, perchloric acid, phosphoric acid and its salts in a molar ratio of 0.9 to 10 moles per mole of the compound of formula I, where appropriate the precipitate formed is separated, I, where M represents a hydrogen atom, is fed to a chromatographic chiral column and after detection, the enantiomeric purity is determined based on the ratio of peak areas of the individual enantiomers in the chromatogram.

Solvents for solution preparation are selected from the group of solvents such as water, methanol, ethanol, acetonitrile, dioxane, tetrahydrofuran, dimethylformamide or mixtures thereof. Preferably, solvents used in the chromatography are also used as the mobile phase. This reaction is carried out at temperatures from 15 ° C to 35 ° C, preferably at room temperature, and the reaction time is from 1 hour to 5 hours.

Because of steric problems in the enantiomeric interactions with the stationary phase, especially in the relatively large dimensions of the salt molecule (e.g. calcium or magnesium salt), prior to separation

The enantiomer releases the corresponding acid. As acids with pK < 4.7, organic acids such as formic acid, acetic acid, benzenesulfonic acid, chloroacetic acid, trichloroacetic acid, oxalic acid, citric acid, tartaric acid and the like can be used. Solutions of inorganic acids such as sulfuric acid, acid can be used. hydrochloric acid, perchloric acid, phosphoric acid or a salt thereof, such as sodium or potassium dihydrogen phosphate. The acids are added directly to the sample I solution or dissolved in the solvent used to prepare the sample I solution in 0.9 to 10 times the equimolar amount per mole of the compound of formula I. The use of larger amounts of acid has an effect on stability and leads to undesirable products. However, it has been found that, under the conditions employed, during the reaction between an organic acid with a pK < 4.7 or dilute sulfuric acid, hydrochloric acid, perchloric acid, phosphoric acid or preferably a salt thereof and 2- (4-fluorophenyl) -p, 5-Dihydroxy-5- (1-methylethyl-3-phenyl-4 - [(phenylamino) carbonyl] -1H-pyrrole-1-heptanoic acid does not cause cracemization. determine the proportion even in the case of a non-quantitative course of the reaction at a defined reaction time.

Special chemically modified chiral stationary phases, eg cellulose derivatives, amylose, proteins (peptides), cyclodextrins, macrocyclic antibiotics, Pirkle type phases, polysiloxanes, chiral substituted polymers 20 helicidal polymethacrylates, immobilized proteins, immobilized proteins are used as stationary phase for enantiomer separation. albumin, α-glycoproteins (AGP), ovomucoid OVM, avidin or some synthetically prepared phases such as Whelk 01, macrocyclic ethers, metal complexes such as Cu complex with amino acids, immobilized L-proline, L-valine, D-acid tartaric and the like are particularly preferred. The stationary phase of TEICOPLANIN TAG, which has the character of a weak amphoteric ion exchanger, can be used for the separation of free acids. It will be appreciated that other columns having the indicated properties may also be used. Most separations are carried out at room temperature, but the column temperature can be adjusted to achieve higher efficiency. However, the mobile phase and column temperature must be kept constant during the analysis.

On the basis of chromatographic records, as documented by the DV spectra of the individual peaks of the enantiomers, separation of the enantiomers with a resolution value of R1 = 2.1 was achieved. The order of elution of the individual enantiomers can be determined, for example, by the addition of pure enantiomers.

Advantageous separation conditions are obtained using a weak amphoteric ion exchange type chiral column, e.g. TEICOPLANIN TAG, permethylated TEICOPLANIN, TEICOPLANIN,

RISTOCETIN, VANCOMYCIN with mobile phase methanol, methanol - water, acetonitrile, acetonitrile - water, ethanol, ethanol - water.

It is preferred to carry out the separation with the addition of a buffer to the mobile phase, such as acetic acid - diethylamine, propionic acid - diethylamine, formic acid - diethyl amine, other amines such as triethylamine or ammonia and salts thereof may also be used. Using the above mobile phases, there is no undesirable qualitative change in the separated enantiomers during the chromatographic process (the peak shape remained symmetrical and reproducible). By comparing the peak areas of the individual enantiomers from the chromatographic record, the enantiomeric purity is determined. Obviously, the content can be calculated using the calibration curves obtained with the calibration standards of the individual enantiomers. As a detector it is possible to use detection with diode array spectrophotometer, but also another detector.

The present invention provides a reliable method for determining the enantiomeric purity of 2- (4-fluorophenyl) -β, 6-dihydroxy-5- (1-methylethyl) -3-phenyl-4 - [(phenylamino) carbonyl] -1H-pyrrole-150 heptane of the acid and its salts by chiral chromatography. The invention is illustrated by the following non-limiting examples.

-3GB 299175 B6

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Determination of the enantiomeric purity of the hemicalcium salt of [R- (R * R *) -2- (4-fluorophenyl) -3,6-dihydroxy-5 (1-methylethyl) -3-phenyl] - [(phenylamino) carbonyl] - 1 H-pyrrole-1-heptanoic acid

A CHIROBIOTIC TAG chiral chromatography column (200 mm x 4.6 mm x 5 µm) was used. Mobile phase composition: methanol / acetic acid / diethylamine in a ratio of 100 / 0.1 / 0.05 (needles).

Methanolic solutions of enantiomers at a concentration of 0.5 mg / ml and with equimolar addition of oxalic acid were added to the chromatography column. Mobile phase flow rate 0.5 ml / min. The measurements were performed at 20 ° C using a LCT 5100 column thermostat.

The enantiomeric purity was determined by the area ratio of the individual enantiomers.

1 hour after addition of oxalic acid 2 hours after addition of oxalic acid 5 hours after addition of oxalic acid 20 98.5 ± 1.8 98.8 ± 2.0 98.3 ± 2.0

Example 2

Determination of enantiomeric composition of a sample of 2- (4-fluorophenyl) -P, iodihydroxy-5- (1-methylethyl) -3-phenyl-4 - [(phenylamino) -carbonyl] -1H-pyrrole-1-heptanoate hemi-calcium salt racemate acid

The same conditions as in Example 1 were used. Percentage of the sample was determined by the area ratio of the individual enantiomers.

Sample of the racemate 1

1 hour after addition of oxalic acid 2 hours after addition of oxalic acid 5 hours after addition of oxalic acid 35 44.7 ± 3: 55.3 ± 3 46.7 ± 3: 53.3 ± 3 49.4 ± 1.5: 50.6 ± 1.0 Sample of racemate 2 1 hour after addition of oxalic acid 2 hours after addition of oxalic acid 5 hours after addition of oxalic acid 40 42.6 ± 3: 57.3 ± 3 46.1 ± 2.5: 53.9 ± 3 48.2 ± 1.7: 51.8 ± 1.5

Example 3

Determination of the enantiomeric purity of the sodium salt of [R- (R * R *) -2- (4-fluorophenyl) -P, 6-dihydroxy-5- (1-methylethyl) -3-phenyl-4 - [(phenylamino) -carbonyl] -1H —Pyrrole-1 — heptanoic acid

The same conditions were used as in Example 1 except that an equimolar addition of 0.01 N perchloric acid solution was used. Based on the ratio of the areas of the individual enantiomers, the percentage in the sample was 98.3 ± 2.0%.

-4GB 299175 B6

Example 4

Determination of the enantiomeric purity of the hemicalcium salt of [R- (R *, R *) -2- (4-fluorophenyl) -3, b-dihydroxy-5 (1-methylethyl) -3-phenyl] - [(phenylamino) carbonyl] -1H -pyrrole-1-heptanoic acid

A CHIROBIOTIC TAG chiral chromatography column (250 mm x 4.6 mm x 5 µm) was used. Mobile phase composition: methanol / water / acetic acid / diethylamine in a ratio of 98/2 / 0.1 / 0.05 (v / v / v / v).

A methanolic sample solution of 0.5 mg / ml 10 with 10-fold molar addition of oxalic acid was added to the chromatography column. Mobile phase flow rate 0.5 ml / min. The measurements were performed at 20 ° C using a LCT 5100 column thermostat.

The enantiomeric purity was determined by the area ratio of the individual enantiomers.

1 hour after addition Oxalic acid 2 hours after addition of oxalic acid 5 hours after addition of oxalic acid 98.5 ± 1.8 98.8 ± 2.0 98.3 ± 2.0

Example 5

Determination of the enantiomeric purity of the hemicalcium salt of [R- (R *, R *)] - 2- (4-fluorophenyl) -3,8-dihydroxy-5 (1-methylethyl) -3-phenyl-4 - [(phenylamino) carbonyl] 1 H-pyrrole-1-heptanoic acid

A CHIROBIOTIC TAG chiral chromatography column (2S0 mm x 4.6 mm x 5 μιη) was used.

Mobile phase composition: methanol / acetic acid / diethylamine in a ratio of 100 / 0.1 / 0.05 (v / v / v).

Methanolic solutions (20 μΐ) of 0.5 mg / ml enantiomeric samples were added to the chromatography column with 10-fold molar addition of oxalic acid. Mobile phase flow rate 0.5 ml / min. Measurements were performed at 20 ° C using a LCT 5100 column thermostat using an ultraviolet detector at 220-400 nm.

Under these conditions, the retention time of the dextrorotatory enantiomers Id is 6.8 minutes and the retention time of the levorotatory enantiomers 11.6 minutes.

The precision of the optical purity determination of both enantiomers was determined for both the prepared enantiomers as such and also for the mixture with increasing amounts of one enantiomer. 2% by weight of the levorotatory enantiomers in the sample of the dextrorotatory enantiomers were determined.

Example 6

Determination of the enantiomeric purity of the hemicalcium salt of [R- (R *, R *)] - 2- (4-fluorophenyl) -3,5-dihydroxy-5 (1-methylethyl) -3-phenyl-4 - [(phenylamino) carbonyl] 1 H-pyrrole-1-heptanoic acid

A CHIROBIOTIC TAG chiral chromatography column (250 mm x 4.6 mm x 5 μηι) was used. Mobile phase composition: tetrahydrofuran / ammonium acetate (0.01 mol / l, water) ratio 99.95 / 0.05 (v / v).

Pipette 2 ml of atorvastatin Ca solution (c = 1.0 mg / ml, THF) into a 10.0 ml graduated flask and add 200 μ 200 dilute HCl (1: 100 in tetrahydrofuran). Mobile phase flow rate 0.5 ml / min. Measurements are made after 2 hours at 20 ° C using a LCT 5100 column thermostat using an ultraviolet detector at 220-400 nm. The enantiomeric purity was 98.5% based on the ratio of the areas of the individual enantiomers.

-5 CZ 299175 B6

Example 7

Determination of the enantiomeric purity of the hemicalcium salt of [R- (R *, R *)] - 2- (4-fluorophenyl) -3,5-dihydroxy-5 (1-methylethyl) -3-phenyl-4 - [(phenylamino) - carbonyl] -1H-pyrrole-1-heptanoic acid

A CHIROBIOTIC TAG chiral chromatography column (250 mm x 4.6 mm x 5 µm) was used.

The same conditions as in Example 6 were used except that methanol and ammonium acetate (0.01 mol / l, methanol) were used as the buffer solution. Pipette 2 mL of atorvastatin Ca solution (c = 1.0 mg / mL, methanol) into a 10.0 mL volumetric flask, add 200 µL dilute HCl 10 (1: 100 in methanol), detection 248 nm. The enantiomeric purity was 99.7% based on the area ratio of the individual enantiomers.

Claims (7)

PATENT CLAIMS A method for determining the enantiomeric purity of 2- (4-fluorophenyl) -3,6-dihydroxy-5- (1-methylethyl-20-3-phenyl-4 - [(phenylamino) carbonyl] -1H-pyrrole-1-heptanoic acid and its salts of formula I COOM wherein M represents hydrogen, lithium, sodium, potassium, calcium or magnesium, characterized in that to 2- (4-fluorophenyl) -P, 8-dihydroxy-5- (1-methylethyl) -3 -phenyl-1 - [(phenylamino) carbonyl] -1H-pyrrole-1-heptanoic acid of formula I wherein M represents lithium, potassium, sodium, calcium or magnesium in water and / or an organic solvent selected from the group of dipolar aprotic or polar solvents add an organic acid with pK <4.7 or sulfuric acid, hydrochloric acid, perchloric acid, acid The phosphoric acid or its salts in a molar ratio of 0.9 to 10 moles per mole of the compound of formula 1, or the resulting precipitate is separated, and the liberated acid of formula I, where M is hydrogen, is fed into a macrocyclic-type stationary phase chiral column. antibiotic or cyclodextrin type and after detection, the enantiomeric purity is determined by the ratio of peak areas of the individual enantiomers in the chromatogram. 2. A process according to claim 1, wherein M represents a calcium atom. Process according to claim 1 or 2, characterized in that hydrochloric acid is used as the inorganic acid. -6GB 299175 B6 Process according to claim 1 or 2, characterized in that oxalic acid is used as the organic acid. Method according to claims 1 to 4, characterized in that 5 - methanol - water or methanol is used as the mobile phase. Method according to one of Claims 1 to 5, characterized in that a spectrophotometer with a diode array in the region of 190 nm to 450 nm is used. ío Process according to one of Claims 1 to 6, characterized in that the mobile phase is treated by adding ammonia or a salt thereof.