CZ2004738A3 - 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

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

Description

METHOD FOR DETERMINING ENANTIOMERAL PURITY OF 2- (4-FLUORPHENYL) -β, 5-DIYDROXY-5- (1-METHYLETHYL) -3-PHENYL-4 - [(PHENYLAMINO) CARBONYL] -1HPYRROLY-1-HEPANA aaa aa aaaa aaa

Technical field

The present invention relates to a process for determining the enantiomeric purity of 2- (4-fluorophenyl) -8,8-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. Ό

BACKGROUND OF THE INVENTION

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

AND.

wherein M represents hydrogen, lithium, sodium, potassium, calcium or magnesium are HMG CoA reductase inhibitors and have been described in US 4681893, US 5273995, US 5273995.

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 is an antihypercholesterolemic agent known under the name atorvastatin calcium.

·· ·

A process for preparing crystalline forms of this substance is disclosed in patents, e.g., US 5969156, US 6121461. For atorvastatin hemi-calcium salt used as a drug substance, some manufacturers declare a specific optical rotation in the range of -6.5 to 8.8 ⁰ (to determine a 1% solution) in dimethylsulfoxide, 25 ° C). For chiral purity, the presence of the (-) enantiomers 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 derivatizing reagents modify specific functional groups and produce 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.

However, the work on enantiomeric purity of atorvastatin or substances from the group of statins is not described. There is also no paper describing the separation of atorvastatin enantiomers by chiral chromatography.

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

The invention therefore relates to a method for determining the enantiomeric purity of 2- (4-fluorophenyl) -β-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, 5-dihydroxy-5- (1-methylethyl-3-phenyl-4 - [(phenylamino) carbonyl] -1H-pyrrole-1-heptanoic acid, and its salts of the general formula I, 9 9 9 9 9 9 9 9 9

999 9999

9

99 99 9

COOM

I, 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] -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 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 the formula I, optionally the precipitate formed, and the liberated acid of the formula 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 peak area ratio 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 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.

Due to 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), the corresponding acid is liberated prior to separation of the enantiomers. 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 as acids with pK < 4.7. Solutions of inorganic acids such as acids can be used. 9

9 99 99 99 · · 9 9 9 999

9,999,999,999

9 99 99 999 99999

99,999,999

Sulfuric acid, hydrochloric acid, perchloric acid, phosphoric acid or its salts, 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 more acid has an effect on stability and leads to undesired products. However, it was found that under the conditions used during the reaction between the organic acid with pK &lt;

4,7 or with dilute sulfuric acid, hydrochloric acid, perchloric acid, phosphoric acid or, preferably, a salt thereof, and 2- (4-fluorophenyl) -?, 6-dihydroxy-5- (1-methylethyl-3-phenyl-4 - [(phenylamino) Because the individual enantiomers have the same response from a spectrophotometric detector or other detector, the proportion can be determined from the area ratio even in the case of a non-quantitative course of the reaction at a defined reaction time.

Special chemically modified chiral stationary phases, such as cellulose derivatives, amylose, proteins (peptides), cyclodextrins, macrocyclic antibiotics, Pirkle type phases, polysiloxanes, chiral substituted polymers - helical structure polymethacrylates, immobilized proteins, immobilized proteins - 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 particularly preferred is the stationary phase of TEICOPLANIN TAG, which has the character of a weak amphoteric ion exchanger, which can be used to separate 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 UV spectra of the individual peaks of the enantiomers, separation of enantiomers with a resolution value R ^ =

2.1. The order of elution of the individual enantiomers can be determined, for example, by the addition of pure enantiomers.

Preferred separation conditions are achieved using a weak amphoteric ion exchanger type chiral column such as TEICOPLANIN TAG, permethylated TEICOPLANIN, TEICOPLANIN, RISTOCETIN, VANCOMYCIN mobile phase methanol, methanol-water, acetonitrile, acetonitrile-water, ethanol, ethanol-water.

It is advantageous to carry out the separation with the addition of a buffer solution to the mobile phase, such as acetic acid - diethylamine, propionic acid - diethylamine, acid 9 9 9 9

9 9 9 9 9

Formic - diethylamine, other amines such as triethylamine or ammonia and its salts may also be used. Using the above mobile phases, no undesirable qualitative change of the separated enantiomers occurs 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 by diode array spectrophotometer, but also another detector.

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

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Determination of the enantiomeric purity of the hemicalcium salt of [R- (R * R *) - 2- (4-Phloro-phenyl) -P, 6-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. Composition of mobile phase: methanol / acetic acid / diethylamine in porn 100 / 0,1 /

0.05 (v / v / v).

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.

hours after adding oxalic acid:

hours after adding oxalic acid:

hours after adding oxalic acid:

98.5 ± 1.8

98.8 ± 2.0

98.3 ± 2.0 · · ,3 ,3 ,3 ,3 ,3 ,3 ,3 ,3 ,3 ,3 ,3 ,3 ,3

Example 2

Determination of enantiomeric composition of a sample of 2- (4-fluorophenyl) -β 1,8-dihydroxy-5- (1-methylethyl) -3-phenyl-4 - [(phenylamino) carbonyl] -1H-pyrrole-1-heptanoic acid hemi-calcium salt racemate acid

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

Sample of racemate 1 1 hour after addition of oxalic acid:

44.7 ± 3: 55.3 ± 3

Sample of racemate 2 1 h after addition of oxalic acid: 42.6 ± 3: 57.3 ± 3

Example 3 hours after addition of oxalic acid:

46.7 ± 3: 53.3 ± 3 hours after addition of oxalic acid: 46.1 ± 2.5: 53.9 ± 3 hours after addition of oxalic acid:

49.4 ± 1.5: 50.6 ± 1.0 hours after addition of oxalic acid: 48.2 ± 1.7: 51.8 ± 1.5

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%.

Example 4

Determination of the enantiomeric purity of the hemicalcium salt of [R- (R *, R *) -2- (4-fluorophenyl) -P, 8-dihydroxy-5- (1-methyl ethyl) -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 µm) was used. Mobile phase composition: methanol / water / acetic acid / diethylamine in porn 98/2 / 0.1 / 0.05 (v / v / v / v).

····

A methanolic solution of samples at a concentration was fed into the chromatography column

0.5 mg / ml with 10 times molar addition of oxalic acid. Mobile phase flow rate 0.5 ml / min. The measurements were performed at 20 ° C using a LCT 5100 column thermostat.

49 4

4 4 4 4 4

9 4 4 4 44

4 44 44 444 44494

4 4 4 4 4

444 4 44 44 94 3

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

hours after addition of oxalic acid: 98,5 ± 1,8 hours after addition of oxalic acid:

98.8 ± 2.0 hours after addition of oxalic acid: 98.3 ± 2.0

Example 5

Determination of the enantiomeric purity of the hemicalcium salt of [R- (R *, R *)] - 2- (4-fluorophenyl) -β, 6-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 µm) was used. Mobile phase composition: methanol / acetic acid / diethylamine in a ratio of 100 / 0.1 / 0.05 (νΝΝΓ).

Methanolic solutions (20 μΐ) of 0.5 mg / ml enantiomeric samples were added to the chromatography column with 10 times 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 determination of the optical purity 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.

·························· 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 «

999 99 99 99 99

Example 6

Determination of the enantiomeric purity of the hemicalcium salt of [R- (R *, R *)] - 2- (4-fluorophenyl) -P, 8-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 µm) 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 of 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.

Example 7

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 (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 graduated flask, add 200 μΐ dilute HCl (1: 100 in methanol), detection 248 nm. The enantiomeric purity was 99.7% based on the area ratio of the individual enantiomers.

Claims (8)

PATENT CLAIMS ΊΟΟΊ - '/ ΐΖ 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 of formula I COOM and. wherein M represents a hydrogen, lithium, sodium, potassium, calcium or magnesium atom, wherein 2- (4-fluorophenyl) -P, 6-dihydroxy-5- (1-methylethyl) 3-phenyl-4 - [(phenylamino) (carbonyl) -1H-pyrrole-1-heptanoic acid of formula I, wherein M represents lithium, potassium, sodium, calcium or magnesium, in an organic solvent selected from dipolar aprotic or polar solvents in water and / or organic solvent pK < 4.7 or sulfuric acid, hydrochloric acid, perchloric acid, phosphoric acid or its salts in a molar ratio of 0.9 to 10 moles per mole of the compound of formula I, optionally precipitate formed, and the liberated acid of formula I where M represents the 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. 2. The process of claim 1 wherein M is a calcium atom. Method according to claim 1 or 2, characterized in that chiral columns with a stationary phase of the macrocyclic antibiotic or cyclodextrin type are used. Process according to one of Claims 1 to 3, characterized in that hydrochloric acid is used as the inorganic acid. Process according to one of Claims 1 to 3, characterized in that oxalic acid is used as the organic acid. Method according to claims 1 to 5, characterized in that methanol-water or methanol is used as the mobile phase. Method according to one of claims 1 to 6, characterized in that a diode array spectrophotometer in the region of 190 nm to 450 nm is used for detection. Process according to one of Claims 1 to 7, characterized in that the mobile phase is treated by the addition of ammonia or a salt thereof.