EP2121631A2

EP2121631A2 - Triol form of rosuvastatin

Info

Publication number: EP2121631A2
Application number: EP08726880A
Authority: EP
Inventors: Valerie Niddam-Hildesheim; Anna Balanov; Irena Veinberg
Original assignee: Teva Pharmaceutical Industries Ltd
Current assignee: Teva Pharmaceutical Industries Ltd
Priority date: 2007-03-13
Filing date: 2008-03-13
Publication date: 2009-11-25
Also published as: JP5330225B2; KR100945760B1; WO2008112317A3; WO2008112317A2; KR20090010195A; BRPI0803085A2; CA2680604C; IL200449A0; MX2008014552A; JP2009519353A; CA2680604A1

Abstract

Provided is a rosuvastatin triol and its use as a reference standard for analysis of rosuvastatin. (I)

Description

TRIOL FORM OF ROSUV AST ATIN

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Nos. 60/906,914 filed on March 13, 2007 and 60/918,466 filed on March 15, 2007, the disclosures of which are incorporated by reference in their entirety herein.

FIELD OF THE INVENTION

The present invention relates to rosuvastatin triol and its use as a reference standard for analysis of rosuvastatin.

BACKGROUND OF THE INVENTION

Statins are currently the most therapeutically effective drugs available for reducing low-density lipoprotein (LDL) particle concentration in the blood stream of patients at risk for cardiovascular disease. Thus, statins are used in the treatment of hypercholesterolemia, hyperlipoproteinemia, and atherosclerosis. A high level of LDL in the bloodstream has been linked to the formation of coronary lesions that obstruct the flow of blood and can rupture and promote thrombosis. Goodman and Gilman, The Pharmacological Basis of Therapeutics, page 879 (9th Ed. 1996).

Statins inhibit cholesterol biosynthesis in humans by competitively inhibiting the 3-hydroxy-3-methyl-glutaryl-coenzyme A ("HMG-CoA") reductase enzyme. HMG-CoA reductase catalyzes the conversion of HMG to mevalonate, which is the rate-determining step in the biosynthesis of cholesterol. Decreased production of cholesterol causes an increase in the number of LDL receptors and corresponding reduction in the concentration of LDL particles in the bloodstream. Reduction in the LDL level in the bloodstream reduces the risk of coronary artery disease. J.A.M.A., 1984, 251, 351-74.

Currently available statins include lovastatin, simvastatin, pravastatin, fluvastatin, cerivastatin and atorvastatin. Lovastatin (disclosed in U.S. Pat. No. 4,231,938) and simvastatin (disclosed in U.S. Pat. No. 4,444,784) are administered in the lactone form. After absorption, the lactone ring is opened in the liver by chemical or enzymatic hydrolysis, and the active hydroxy acid is generated. Pravastatin (disclosed in U.S. Pat. No. 4,346,227) is administered as the sodium salt. Fluvastatin (disclosed in U.S. Pat. No. 4,739,073) and cerivastatin (disclosed in U.S. Pat. Nos. 5,006,530 and 5,177,080), also administered as the sodium salt, are entirely synthetic compounds that are in part structurally distinct from the fungal derivatives of this class that contain a hexahydronaphthalene ring. Atorvastatin and two new "superstates," rosuvastatin and pitavastatin, are administered as calcium salts.

Rosuvastatin calcium (monocalcium bis (+) 7-[4-(4-fluorophenyl)-6- isopropyl-2-(N-methyl-N-methylsulfonylaminopyrimidin)-5-yl]-(3R,5S)-dihydroxy- (E)-ό-heptenoate) is an HMG-CoA reductase inhibitor, developed by Shionogi for the once daily oral treatment of hyperlipidaemia (Ann Rep, Shionogi, 1996; Direct communications, Shionogi, 8 Feb 1999 & 25 Feb 2000). Rosuvastatin calcium has the following chemical formula:

Rosuvastatin calcium is marketed under the name CRESTOR for treatment of a mammal such as a human. According to the maker of CRESTOR^®, it is administered in a daily dose of from about 5 mg to about 40 mg. For patients requiring less aggressive LDL-C reductions or who have pre-disposing factors for myopathy, the 5 mg dose is recommended, while 10 mg dose is recommended for the average patient, 20 mg dose for patients with marked hypercholesterolemia and aggressive lipid targets (>190 mg/dL), and the 40 mg dose for patients who have not been responsive to lower doses.

U.S. Pat. No. 5,260,440 discloses and claims rosuvastatin, its calcium salt (2:1), and its lactone form. The process of the '440 patent prepares rosuvastatin by reacting 4-(4-fluorophenyl)-6-isopropyl-2-(N-methyl-N-methylsulfonylamino)-5- pyrimidinecarbaldehyde with methyl (3R)-3-(tert-butyldimethylsilyloxy)-5-oxo-6- triphenylphosphoranylidene hexanate in acetonitrile under reflux. The silyl group is then cleaved with hydrogen fluoride, followed by reduction with sodium borohydride (NaBH₄ ) and diethylmethoxyborane in tetrahydrofuran (THF) to obtain a methyl ester of rosuvastatin.

The ester is then hydrolyzed with sodium hydroxide (NaOH) in ethanol at room temperature, followed by removal of ethanol and addition of ether, to obtain the sodium salt of rosuvastatin. The sodium salt is then converted to the calcium salt. The sodium salt is dissolved in water and maintained under a nitrogen atmosphere. Calcium chloride is then added to the solution, resulting in precipitation of rosuvastatin calcium (2:1). The process for preparation of the intermediates disclosed in the '440 patent is incorporated herein by reference.

The product mixture of a reaction rarely is a single compound pure enough to comply with pharmaceutical standards. Side products and byproducts of the reaction and adjunct reagents used in the reaction will, in most cases, be present. At certain stages during processing of the rosuvastatin contained in the product mixture into an active pharmaceutical ingredient ("API"), the rosuvastatin must be analyzed for purity, typically by HPLC or GC analysis, to determine if it is suitable for continued processing or ultimately for use in a pharmaceutical product. The rosuvastatin does not need to be absolutely pure. Absolute purity is a theoretical ideal that is unattainable. Rather, there are purity standards intended to ensure that an API is not made less safe for clinical use because of the presence of impurities. In the United States, the Food and Drug Administration guidelines recommend that applicants limit some impurities to below 0.1%.

Generally, side products, byproducts and adjunct reagents (collectively "impurities") are identified spectroscopically and by other physical methods and then the impurities are associated with a peak position in a chromatogram (or a spot on a TLC plate). (Strobel p. 953) (Strobel, H.A.; Heineman, W.R., Chemical Instrumentation: A Systematic Approach, 3^rd dd. (Wiley & Sons: New York 1989)). Thereafter, the impurity can be identified by its position in the chromatogram, which is conventionally measured in minutes between injection of the sample on the column and elution of the particular component through the detector, known as the "retention time." This time period varies daily based upon the condition of the instrumentation and many other factors. To mitigate the effect that such variations have upon accurate identification of an impurity, practitioners use "relative retention time" ("RRT") to identify impurities. (Strobel p. 922). The RRT of an impurity is its retention time divided by the retention time of some reference marker. In theory, rosuvastatin itself could be used as the reference marker, but as a practical matter it is present in such overwhelming proportion in the mixture that it tends to saturate the column, leading to irreproducible retention times, i.e., the maximum of the peak corresponding to rosuvastatin tends to wander (Strobel Fig. 24.8(b) p. 879, contains an illustration of the sort of asymmetric peak that is observed when a column is overloaded). Thus, it is sometimes desirable to select an alternative compound that is added to, or is present in, the mixture in an amount significant enough to be detectable and sufficiently low as not to saturate the column and to use that compound as the reference marker.

A compound in a relatively pure state can be used as a "reference standard" (a "reference marker" is similar to a reference standard but it is used for qualitative analysis) to quantify the amount of the compound in an unknown mixture. When the compound is used as an "external standard," a solution of a known concentration of the compound is analyzed by the same technique as the unknown mixture. (Strobel p. 924, Snyder p. 549) (Snyder, L.R.; Kirkland, JJ. Introduction to Modern Liquid Chromatography, 2nd ed. (John Wiley & Sons: New York 1979)). The amount of the compound in the mixture can be determined by comparing the magnitude of the detector response. See also USP 6,333,198, incorporated herein by reference.

The reference standard compound also can be used to quantify the amount of another compound in the mixture if the "response factor," which compensates for differences in the sensitivity of the detector to the two compounds, has been predetermined. (Strobel p. 894). For this purpose, the reference standard compound may be added directly to the mixture, in which case it is called an "internal standard." (Strobel p. 925, Snyder p. 552).

The reference standard compound can even be used as an internal standard when the unknown mixture contains some of the reference standard compound by using a technique called "standard addition," wherein at least two samples are prepared by adding known and differing amounts of the internal standard. (Strobel pp. 391-393, Snyder pp. 571, 572). The proportion of detector response due to the reference standard compound that is originally in the mixture can be determined by extrapolation of a plot of detector response versus the amount of the reference standard compound that was added to each of the samples to zero. (e.g. Strobel, Fig. 11.4 p. 392).

The present invention provides compounds that can be used as a reference standard and reference marker for quantification and identification of rosuvastatin and impurities present in batches of rosuvastatin.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a rosuvastatin triol having the following structure:

Wherein X is a hydrogen, a C₁-C₄ alkyl group, or an alkali or alkaline earth metal cation, with the proviso that when X is an alkaline earth metal, two molecules of rosuvastatin are present to one of the metal cation.

In another embodiment, the present invention provides a rosuvastatin triol in acid form has the following structure:

In yet another embodiment, the present invention provides a rosuvastatin triol in ester form having the following structure:

Wherein R is a C₁-C₄ alkyl ester.

In one embodiment, the present invention provides a rosuvastatin triol in salt form having the following structure:

Wherein M is an alkali or alkaline earth metal cation, with the proviso that when X is an alkaline earth metal, two molecules of rosuvastatin are present to one of the metal cation.

In one embodiment, the present invention provides a rosuvastatin triol in lactone form having the following structure:

In another embodiment the present invention provides each of the above forms of the triol in isolated or purified form, substantially free of the corresponding rosuvastatin diol form. In yet another embodiment, the present invention provides a process for preparing a rosuvastatin triol Ci-C₄ ester comprising combining rosuvastatin C₁-C₄ ester with a solution of borane dimethylsulfide complex in a suitable organic solvent to obtain a reaction mixture, combining the resulting reaction mixture with a solution of NaOH in water, adding hydrogen peroxide (H₂O₂) and recovering the triol ester.

In another embodiment the present invention provides a process for preparing rosuvastatin triol Ci-C₄ ester comprising oxidizing rosuvastatin diol Ci to C₄ ester to obtain the rosuvastatin triol ester with a hydroxyl group at position 7.

In another embodiment the present invention provides a process comprising combining rosuvastatin C₁-C₄ ester with a solution of a borane in an organic solvent to obtain a reaction mixture, combining the resulting reaction mixture with a solution of an inorganic base in water, and adding peroxide and recovering the triol ester.

In one embodiment the present invention provides a process for reducing amount of impurities present in rosuvastatin calcium by measuring amount of rosuvastatin calcium triol in batches of rosuvastatin calcium, selecting batches of the rosuvastatin calcium with desirable level of the triol and preparing pharmaceutical compositions with the selected rosuvastatin calcium batch.

In another embodiment, the present invention provides a process for reducing amount of rosuvastatin triol calcium present in a mixture comprising rosuvastatin diol calcium and rosuvastatin triol calcium comprising measuring amount of rosuvastatin triol C₁-C₄ ester in batches of rosuvastatin diol Ci-C₄ ester, selecting batches of the rosuvastatin diol CpC₄ ester with of the triol Ci-C₄ ester and preparing pharmaceutical compositions of rosuvastatin diol calcium with the selected rosuvastatin diol Ci-C₄ ester batch.

In another embodiment, the present invention provides a method of determining the amount of an impurity in a sample of rosuvastatin ester (preferable t- butyl) comprising measuring by GC or HPLC the area under a peak corresponding to rosuvastatin triol ester in a reference standard comprising a known amount of rosuvastatin triol ester (preferably t-butyl); measuring by GC or HPLC the area under a peak corresponding to rosuvastatin triol ester in a sample comprising rosuvastatin triol and rosuvastatin diol esters (preferably t-butyl); and determining the amount of the rosuvastatin triol ester in the sample by comparing the area of reference standard with that of the test sample. In yet another embodiment, the present invention provides a method of determining the amount of an impurity in a sample of rosuvastatin calcium comprising measuring by GC or HPLC the area under a peak corresponding to rosuvastatin triol calcium in a reference standard comprising a known amount of rosuvastatin triol calcium; measuring by GC or HPLC the area under a peak corresponding to rosuvastatin triol calcium in a sample comprising rosuvastatin triol and rosuvastatin diol calcium salts; and determining the amount of the triol calcium in the sample by comparing the area of reference standard with that of the test sample. hi one embodiment, the present invention provides a method of identifying the relative retention time (RRT) of an impurity in a sample of rosuvastatin diol ester (preferably t-butyl) comprising measuring by GC or HPLC the relative retention time (RRT) corresponding to rosuvastatin triol ester in a reference marker sample; carrying out GC or HPLC with a test sample comprising of the rosuvastatin diol ester and rosuvastatin triol ester to obtain an GC or HPLC chromatogram with retention times; and determining the relative retention time (RRT) of the triol ester in the sample by comparing the relative retention time (RRT) of the reference marker to the relative retention time (RRT) of the test sample. hi another embodiment, the present invention provides a method of identifying the relative retention time (RRT) of an impurity in a sample of rosuvastatin diol calcium comprising measuring by GC or HPLC the relative retention time (RRT) corresponding to rosuvastatin triol calcium in a reference marker sample; carrying out GC or HPLC with a test sample comprising of the rosuvastatin diol and rosuvastatin triol calcium salts to obtain an GC or HPLC chromatogram with retention times; and determining the relative retention time (RRT) of the triol calcium in the sample by comparing the relative retention time (RRT) of the reference marker to the relative retention time (RRT) of the test sample. hi one embodiment the present invention provides a process for preparing rosuvastatin triol acid with the following structure: comprising hydrolyzing an ester of the following strcutre

and converting the hydro lyzed ester with an acid, wherein R is a C₁-C₄ group. hi one embodiment the present invention provides a process for preparing rosuvastatin triol lactone with the following structure:

comprising hydrolyzing an ester of the following structure

and converting the hydrolyzed ester to a lactone, wherein R is a Ci -C₄ ester. In one embodiment the present invention provides a process for preparing rosuvastatin triol acid with the following structure:

comprising hydrolyzing a lactone having the following structure

and converting the hydrolized lactone to an the salt, wherein M is an alkali metal or an alkaline earth metal with the proviso that if the metal cation is an alkaline earth metal, two molecules of rosuvastatin are present for each cation. .

In one embodiment the present invention provides a process for preparing rosuvastatin triol salt with the following structure:

Comprising contacting an acid with the following structure with a base, with the proviso that if the metal cation is an alkaline earth metal, two molecules of rosuvastatin are present for each cation.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is an NMR of TBRE (t-butyl Rosuvastatin Ester) triol. Figure 2 is an HPLC chromatogram illustrating use of rosuvastatin triol calcium as a reference standard (including a reference marker).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "diol" refers to the two hydroxyl groups present on rosuvastatin. Diol rosuvastatin is used herein synonymously as rosuvastatin.

As used herein the term "substantially free" refers to having less than about 30% of the corresponding compound (e.g., diol or diastereoisomer), more preferably less than about 20%, even more preferably less than about 10%, and most preferably less than about 5%, based on area percentage HPLC.

As used herein, the term "triol lactone" refers to the lactone of rosuvastatin triol.

As used herein, the term "reference standard" refers to a compound that may be used both for quantitative and qualitative analysis of an active pharmaceutical ingredient. For example, the retention time of the compound in HPLC allows for setting a relative retention time, thus making qualitative analysis possible. The concentration of the compound in solution before injection into an HPLC column allows for comparison of the areas under the peaks in an HPLC chromatogram, thus making quantitative analysis possible.

A "reference marker" is used in qualitative analysis to identify components of a mixture based upon their position, e.g. in a chromatogram or on a Thin Layer Chromatography (TLC) plate (Strobel pp. 921, 922, 953). For this purpose, the compound does not necessarily have to be added to the mixture if it is present in the mixture. A "reference marker" is used only for qualitative analysis, while a reference standard may be used for quantitative or qualitative analysis, or both. Hence, a reference marker is a subset of a reference standard, and is included within the definition of a reference standard.

Although some of the knowledge of those in the art regarding reference standards has been described in general terms up to this point, those skilled in the art also understand that the detector response can be, for example, the peak heights or integrated peak areas of a chromatogram obtained, e.g. by UV or refractive index detection, from the eluent of an HPLC system or, e.g. flame ionization detection or thermal conductivity detection, from the eluent of a gas chromatograph, or other detector response, e.g. the UV absorbance, of spots on a fluorescent TLC plate. The position of the reference standard may be used to calculate the relative retention time for rosuvastatin and other impurities.

The present invention provides a rosuvastatin triol having the following structure:

wherein X is a hydrogen, an alkali or alkaline earth metal or a C₁-C₄ alkyl group. Preferably X is hydrogen (i.e. rosuvastatin triol acid), calcium (Ca²⁺) (i.e. rosuvastatin triol calcium) or tert-butyl (i.e. rosuvastatin triol tert-butyl ester ("TBRE")). The present invention provides a rosuvastatin triol having the following structure: wherein X is a hydrogen, an alkali or alkaline earth metal or a C₁-C₄ alkyl group. Preferably X is hydrogen (i.e. rosuvastatin triol acid), calcium (Ca²⁺) (i.e. rosuvastatin triol calcium) or tert-butyl (i.e. rosuvastatin triol tert-butyl ester ("TBRE")) in its isolated form.

The present invention provides a rosuvastatin triol in an acid form having the following structure:

The present invention provides rosuvastatin triol in ester form having the following structure

wherein R is a Ci-C₄ alkyl group. Preferably, R is a t-butyl or methyl group. More preferably, the R is t-butyl. The present invention provides rosuvastatin triol in a salt form having the following structure:

wherein M is an alkali metal or alkaline earth metal cation. Preferably, M is calcium. One of ordinary skill of art would appreciate that when M is an alkaline earth metal cation, such as calcium, the salt would be a hemi calcium salt (2:1 ratio):

The present invention further provides rosuvastatin triol in lactone form has the following structure:

The present invention also provides each of the above forms of the rosuvastatin triol substantially free of the corresponding rosuvastatin diol form. Thus, the present invention provides: a) Rosuvastatin triol Ci-C₄ ester substantially free of rosuvastatin diol Ci- C₄ ester. Also provided is rosuvastatin triol t-butyl ester substantially free of rosuvastatin diol t-butyl ester; b) Rosuvastatin triol acid substantially free of rosuvastatin diol acid; c) Rosuvastatin triol salt (preferably calcium salt) substantially free of rosuvastatin diol salt (preferably calcium salt); and d) Rosuvastatin triol lactone substantially free of rosuvastatin diol lactone.

The present invention also provides each of the above forms of the rosuvastatin triol in racemic, (7S) and (7R) configuration. The (7S) and (7R) configurations are diastereoisomers.

Specifically, the present invention provides: a) Rosuvastatin triol Ci-C₄ ester, preferably t-butyl ester, in racemic, (7S) and (7R) forms. In one embodiment, the (7S) form is substantially free of the (7R) form. In one embodiment the (7R) form is substantially free of the (7S) form. b) Rosuvastatin triol acid in racemic, (7S) and (7R) forms. In one embodiment, the (7S) form is substantially free of the (7R) form. In one embodiment the (7R) form is substantially free of the (7S) form. c) Rosuvastatin triol salt (such as calcium), in racemic, (7S) and (7R) forms. In one embodiment, the (7S) form is substantially free of the (7R) form. In one embodiment the (7R) form is substantially free of the (7S) form. d) Rosuvastatin triol lactone in racemic, (7S) and (7R) forms. In one embodiment, the (7S) form is substantially free of the (7R) form, hi one embodiment, the (7R) form is substantially free of the (7S) form.

The present invention also provides a method for preparing rosuvastatin triol ester. The triol ester can be prepared by oxidizing rosuvastatin Ci-C₄ ester, particularly t-butyl ester. The oxidation of the ester can be carried out by combining rosuvastatin Ci -C₄ ester, particularly t-butyl ester, with borane (e.g. BH₃, B₂H₆). Complexes of borane, as well as various monoalkyl (Ci-Cg)- and dialkyl (Ci-C₈)- boranes may be used. Preferably, a solution of borane dimethylsulfide complex in a suitable organic solvent is combined with the ester. The reaction mixture can be stirred. A solution of an inorganic base, preferably NaOH, in water is then combined with the reaction mixture followed by addition of H₂O₂ (preferably about 30% in water). The H₂O₂ is preferably added dropwise. The temperature during H₂O₂ addition is preferably kept below about 5O⁰C.

In addition to H₂O₂, other oxidation reagents can be used. For example, any other peroxides can be used including t-Butyl Hydroperoxide (TBHP) and Magnesium monoperoxyphthalate hexahydrate (MMPP).

The inorganic base is preferably an alkali metal base, more preferably a hydroxide base, such as NaOH, KOH and LiOH. Another base that can be used is NH₄OH.

The organic phase can be separated and washed with water and/or brine to remove water miscible by-products such as borane by products (e.g.: H₃BO₃). It can also be washed with sodium sulphite to remove excess hydrogen peroxide. The organic phase can then be concentrated to obtain a residue. Concentration can be done by reducing the pressure to less than 1 atmosphere such as less than about lOOmmHg.

After the reaction, if desired, a precipitating agent, such as ammonium chloride or another salt can be added to precipitate impurities out of the reaction mixture. Ammonium chloride is used to remove H₃BO₃, the reaction-by-product. Instead of using ammonium chloride, an acid such as acetic acid or HCl can be used to neutralize the basic mixture. The H₃BO₃ can be removed by washing with water.

The rosuvastatin triol ester can then be purified and isolated from the corresponding rosuvastatin diol ester by chromatography.

The present invention provides rosuvastatin triol ester in its isolated form.

The triol ester can then be converted to the corresponding acid, salt or lactone.

The triol ester can be converted to the triol salt by hydrolysis of the ester and addition of a suitable source of ions. To obtain the calcium salt either a combination of sodium hydroxide and calcium chloride can be used, or calcium hydroxide can be used.

The rosuvastatin ester can be converted to the salt by suspending the ester in a mixture of an organic solvent and water mixture and combined with a base such as sodium hydroxide to obtain a solution. The organic solvent may be Ci-C₄ alcohol, preferably ethanol. The organic solvent is then evaporated under reduced pressure followed by addition of calcium chloride, which results in precipitation of the calcium salt of the triol. The precipitate can be recovered by conventional techniques such as filtration. The present invention provides rosuvastatin triol salt in its isolated form.

To obtain the rosuvastatin triol acid, the salt is combined with an acid, such as hydrochloric or sulfuric acid. In one embodiment Rosuvastatin triol calcium is suspended in an organic solvent such as dichloromethane, to which aqueous HCl is added. The rosuvastatin triol acid is then isolated from the reaction mixture, such as by separation of the organic phase followed by removal of organic solvent, such as by evaporation under reduced pressure.

The acid can also be obtained after hydrolysis of the ester, by acidification of the reaction mixture instead of addition of calcium chloride. Inorganic acids such as HCl and H₂SO₄ can be used.

The present invention provides rosuvastatin triol acid in its isolated form.

The rosuvastatin lactone can then be obtained from the acid under conditions that favor lactonization. hi one embodiment, rosuvastatin triol calcium is dissolved in in an organic solvent such as acetonitrile, to which aqueous HCl is added. The reaction mixture can then be stirred. The organic solvent and water can then be removed, such as by evaporation under reduced pressure to obtain the lactone.

As stated above, these compounds, namely rosuvastatin triol acid, salt, lactone and ester can be used as reference marker/standards. Figure 2 illustrates that the compounds of the present invention can used as reference standards to both quantify and identify amount of impurities present in a composition of rosuvastatin. Rosuvastatin triol calcium is close to rosuvastatin diol calcium on the column, yet does not overlap with the peak for rosuvastatin. This lack of overlap is ideal since it can make quantification easier.

The present invention provides rosuvastatin triol lactone in its isolated form.

The present invention provides a process for reducing amount of rosuvastatin triol calcium present in a mixture comprising rosuvastatin calcium and rosuvastatin triol calcium comprising measuring amount of rosuvastatin calcium triol in batches of rosuvastatin diol calcium, selecting batches of the rosuvastatin diol with desirable level of the triol and preparing pharmaceutical compositions with the selected rosuvastatin diol batch. Salts in general other than calcium can also be used in this process.

The present invention provides a process for reducing amount of rosuvastatin triol calcium present in a mixture comprising rosuvastatin diol calcium and rosuvastatin triol calcium comprising measuring amount of rosuvastatin triol Ci-C₄ ester in batches of rosuvastatin diol Ci-C₄ ester, selecting batches of the rosuvastatin diol C_J-C₄ ester with of the triol Ci-C₄ ester and preparing pharmaceutical compositions of rosuvastatin diol calcium with the selected rosuvastatin diol Ci-C₄ ester batch. The ester is preferably t-butyl.

The present invention provides a process for reducing amount of rosuvastatin triol calcium present in a mixture comprising rosuvastatin diol calcium and rosuvastatin triol calcium comprising measuring amount of rosuvastatin triol lactone in batches of rosuvastatin diol lactone, selecting batches of the rosuvastatin diol lactone with desirable level of the triol lactone and preparing pharmaceutical compositions of rosuvastatin diol calcium with the selected rosuvastatin diol lactone batch.

The present invention provides a process for reducing amount of rosuvastatin triol calcium present in a mixture comprising rosuvastatin diol calcium and rosuvastatin triol calcium comprising measuring amount of rosuvastatin triol acid in batches of rosuvastatin diol acid, selecting batches of the rosuvastatin diol acid with desirable level of the triol acid and preparing pharmaceutical compositions of rosuvastatin diol calcium with the selected rosuvastatin diol acid batch.

The present invention provides a method of determining the amount of an impurity in a sample of rosuvastatin diol ester (preferable t-butyl ester) comprising measuring by GC or HPLC the area under a peak corresponding to rosuvastatin triol ester in a reference standard comprising a known amount of rosuvastatin triol ester (preferably t-butyl); measuring by GC or HPLC the area under a peak corresponding to rosuvastatin triol ester in a test sample comprising rosuvastatin triol and rosuvastatin diol esters (preferably t-butyl); and determining the amount of the triol ester in the sample by comparing the area of reference standard with that of the test sample.

The present invention provides a method of determining the amount of an impurity in a sample of rosuvastatin calcium comprising measuring by GC or HPLC the area under a peak corresponding to rosuvastatin triol calcium in a reference standard comprising a known amount of rosuvastatin triol calcium; measuring by GC or HPLC the area under a peak corresponding to rosuvastatin triol calcium in a test sample comprising rosuvastatin triol and rosuvastatin diol calcium salts; and determining the amount of the triol calcium in the sample by comparing the area of reference standard with that of the test sample. Salts in general other than calcium can also be used in this process.

The present invention provides a method of determining the amount of an impurity in a sample of rosuvastatin acid comprising measuring by GC or HPLC the area under a peak corresponding to rosuvastatin triol acid in a reference standard comprising a known amount of rosuvastatin triol acid; measuring by GC or HPLC the area under a peak corresponding to rosuvastatin triol acid in a test sample comprising rosuvastatin acid and rosuvastatin diol acid; and determining the amount of the triol acid in the sample by comparing the area of reference standard with that of the test sample.

The present invention provides a method of determining the amount of an impurity in a sample of rosuvastatin lactone comprising measuring by GC or HPLC the area under a peak corresponding to rosuvastatin triol lactone in a reference standard comprising a known amount of rosuvastatin triol lactone; measuring by GC or HPLC the area under a peak corresponding to rosuvastatin triol lactone in test a sample comprising rosuvastatin triol lactone and rosuvastatin diol lactone; and determining the amount of the triol lactone in the sample by comparing the area of reference standard with that of the test sample.

The present invention provides a method of identifying the relative retention time (RRT) of an impurity in a sample of rosuvastatin diol ester (preferably t-butyl) comprising measuring by GC or HPLC the relative retention time (RRT) corresponding to rosuvastatin triol ester in a reference marker sample; carrying out GC or HPLC with a test sample comprising of the rosuvastatin ester and rosuvastatin triol ester to obtain an HPLC or GC chromatogram with retention times; and determining the relative retention time (RRT) of the triol ester in the sample by comparing the relative retention time (RRT) of the reference marker to the relative retention time (RRT) of the test sample. Preferably the rosuvastatin diol and rosuvastatin triol ester are tert-butyl esters.

Accordingly in another embodiment, the present invention provides a method of determining the relative retention time (RRT) of an impurity in a sample of rosuvastatin diol calcium comprising measuring by GC or HPLC the relative retention time (RRT) corresponding to rosuvastatin triol calcium in a reference marker sample; carrying out GC or HPLC with a test sample comprising of the rosuvastatin diol and rosuvastatin triol calcium salts to obtain an HPLC chromatogram with retention times; and determining the relative retention time (RRT) of the triol calcium in the sample by comparing the relative retention time (RRT) of the reference marker to the relative retention time (RRT) of the test sample. Salts in general other than calcium can also be used in this process.

Accordingly in another embodiment, the present invention provides a method of identifying the relative retention time (RRT) of an impurity in a sample of rosuvastatin diol acid comprising measuring by GC or HPLC the relative retention time (RRT) corresponding to rosuvastatin triol acid in a reference marker sample; carrying out GC or HPLC with a test sample comprising of the rosuvastatin diol acid and rosuvastatin triol acid to obtain an HPLC chromatogram with retention times; and determining the relative retention time (RRT) of the triol acid in the sample by comparing the relative retention time (RRT) of the reference marker to the relative retention time (RRT) of the test sample.

Accordingly in another embodiment, the present invention provides a method of determining the relative retention time (RRT) of an impurity in a sample of rosuvastatin diol lactone comprising measuring by GC or HPLC the relative retention time (RRT) corresponding to rosuvastatin triol lactone in a reference marker sample; carrying out GC or HPLC with a test sample comprising of the rosuvastatin diol acid and rosuvastatin triol acid to obtain an HPLC chromatogram with retention times; and determining the relative retention time (RRT) of the triol acid in the sample by comparing the relative retention time (RRT) of the reference marker to the relative retention time (RRT) of the test sample.

Having described the invention with reference to certain preferred embodiments, other embodiments will become apparent to one skilled in the art from consideration of the specification. The invention is further defined by reference to the following examples describing in detail the preparation of the composition and methods of use of the invention. It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practiced without departing from the scope of the invention.

EXAMPLES

Example 1 : Synthesis of Triol Ester Trio I ester

TBRE (10 g) was mixed with IM solution of borane dimethylsulfide complex in THF (56 ml) in an inert atmosphere. The mixture was stirred for 3 h at 2O⁰C. A solution of NaOH (74 g) in water (5 ml) was slowly added. H₂O₂ (30% in water, 15 ml) was added dropwise, so that the temperature of the mixture was kept below 5O⁰C. The mixture was stirred for 0.5 h. A concentrated solution of ammonium chloride (150 ml) was added, and the precipitate was filtered out. The phases were separated and organic phase was first washed with a concentrated solution of sodium sulfite (40 ml), then with a mixture of water (100 ml) and brine (100 ml), and finally with brine (150 ml). Then an organic solvent was removed at reduced pressure, giving a semi-solid residue, containing triol, non-reacted TBRE and some impurities.

Triol was isolated by two chromatographic separations. First separation was performed on an RP- 18 column (RediSep^® C- 18 Reversed Phase Column), using a gradient from 40% to 45% of EtOH in water. The second separation was performed on normal silica column (RediSep^® Normal Phase Disposable Column), using a gradient from 0% to 1% of EtOH in CH₂Cl₂ as an eluent. Purity 93%. MS (ES+): M+H-556 M+Na+= 578 RediSep^® is manufactured by Teledyne Isco, Inc (Nebraska).

Example 2: Synthesis of Rosu-Ca-triol

2g of material, consisting of TBRE and 2.85% triol-ester (3.7 mmole of the carboxylic group) was suspended in EtOH (10 mL)/water (6 mL) mixture. Saturated NaOH solution (0.35 g, 4.1 mmole) was added dropwise at room temperature and the mixture was stirred for 2 h. The solution was concentrated under vacuum to remove EtOH. Calcium salt was precipitated from the water solution by addition of CaCl₂ (0.41g, 3.7 mmole) at 4O⁰C upon stirring. Stirring was continued for 1 h at room temperature, and the precipitate was filtered, washed with water and dried. The material contained 2.82% Rosu-Ca-triol and 95% Rosu-Ca

Example 3: Synthesis of Rosuvastatin triol acid

Rosu triol Ca (0.5g) is suspended in dichloromethane (5 mL) and HCl (IN in water, 1 mL) is added. After stirring for 15 minutes phases are separated, and organic phase is concentrated in vacuum, giving the residue, which contains mainly the product. It may be additionally purified by column chromatography (silica gel, dichloromethane- methanol mixtures as eluent), giving pure Rosu triol acid.

Example 4: Synthesis of Rosuvastatin triol lactone

Rosu triol Ca (4 g) is dissolved in acetonitrile (40 mL) and HCl (IN in water, 40 mL) is added. The mixture is stirred at room temperature overnight. Acetonitrile and water are removed by distillation at reduced pressure. The residue, containing the product, may be additionally purified by flash chromatography (silica gel, hexane-ethyl acetate mixtures as eluent), giving pure rosu triol lactone.

Example 5: Synthesis of Rosuvastatin triol Calcium

2g of pure triol-ester (3.7 mmol) is suspended in EtOH (10 mL)/water (6 mL) mixture. Saturated NaOH solution (0.35 g, 4.1 mmole) is added dropwise at room temperature and the mixture is stirred for 2 h. The solution is concentrated under vacuum to remove EtOH. Calcium salt is precipitated from the water solution by addition Of CaCl₂ (0.41g, 3.7 mmole) at 4O⁰C upon stirring. Stirring is continued for 1 h at room temperature, and the precipitate is filtered, washed with water and dried, giving Rosu-Ca-triol. MS conditions

Instrument: Bruker Esquir

Source: Positive/ Neς

Target mass: 556 Da

Compound stability: 50 %

Trap drive: 100 %

Octopole RF: 195.3 Vpp

Capillary exit: 111.8 V

Drying gas flow rate: 10 Umin

Nebulizer: 60 psig

Drying gas temperature: 365 ⁰C

V cap: 4000 V

HPLC method for Impurity profile of TBRE

Column: Cl 8

Mobile phase: Gradient of Eluent A and Eluent B

Gradient: Time(min) Eluent A(%) Eluent B(%)

0 100 0

2 84 16

23 84 16

36 10 90

40 10 90

Eluent A: 60% 0.005M Ammonium Acetate 40% Acetonitrile:Ethanol=2:3

Eluent B: 100% Acetonitrile:Ethanol = 1 : 4

UV detection: 243nm

Run time: 55min

Flow rate: 0.6mL/min

Injection volume: 5μL

Column temperature: 5⁶C

Discard limit: Less than 0.02%

Sample preparation: 0.5mg/mL

RT of TBRE: about 24.5min RRT of Triol -TBRE impurity is 0.6 corresponding to the main peak of TBRE.

HPLC method for Impurity profile of ROSU

Column: Cl 8

Mobile phase: Gradient of Eluent A , Eluent B and Eluent C

Gradient: Time(mm) Eluent A(%) Eluent B(%) Eluent C(%)

0 100 0 0

15 0 100 0

20 0 93 7

30 0 78 22

40 0 5 95

45 0 5 95

Eluent A: 60% 0.05% Acetic acid glacial pH 3.5 with Ammonium hydroxide 35%

Acetonitrile 5%Ethanol

Eluent B: 55% 0.05% Acetic acid glacial pH 3.5 with Ammonium hydroxide 45%

Acetonitrile

Eluent C: 100% Ethanol

UV detection: 243nm

Run time: 45min

Flow rate: 0.5mL/min

Injection volume: 10μL

Column temperature: 20⁰C

Discard limit: Less than 0.02%

Sample preparation: 0.2mg/mL

RT of ROSU: about 19min

RRT of Triol -ROSU impurity is 0.7 corresponding to the main peak of ROSU.

Claims

What is claimed is:

1. A rosuvastatin triol having the following structure:

wherein X is a hydrogen, an alkali or alkaline earth metal, or a C₁-C₄ alkyl group.

2. The rosuvastatin triol of claim 1, wherein the rosuvastatin triol is isolated.

3. The rosuvastatin triol of claim 1 or 2, wherein the rosuvastatin triol is substantially free from corresponding rosuvastatin diol.

4. The rosuvastatin triol of claim 1 in acid form having the following structure:

5. The rosuvastatin triol of claim 4, wherein the rosuvastatin triol is isolated.

6. The rosuvastatin triol of claim 4 or 5, wherein the rosuvastatin triol is substantially free from corresponding rosuvastatin diol.

7. The rosuvastatin triol of claim 1 in ester form having the following structure:

wherein R represents a Ci_-4 alkyl.

8. The rosuvastatin triol of claim 7, wherein the rosuvastatin triol ester is isolated.

9. The rosuvastatin triol of claim 7 or 8, wherein the rosuvastatin triol ester is substantially free from corresponding rosuvastatin diol ester.

10. The rosuvastatin triol of claim 7, 8, or 9, wherein the ester is a tert-butyl ester.

11. The rosuvastatin triol of claim 1, in salt form having the following structure:

wherein M represents an alkali or alkaline earth metal cation,

12. The rosuvastatin triol of claim 11, wherein the metal cation is an alkaline earth metal with two molecules of rosuvastatin present for each cation.

13. The rosuvastatin triol of claim 12, wherein the metal cation is Ca2+.

14. The rosuvastatin triol of claim 11, 12, or 13, wherein the rosuvastatin triol is isolate d.

15. The rosuvastatin triol of claim 11, 12, 13 or 14, wherein the rosuvastatin triol is substantially free from corresponding rosuvastatin diol.

16. Rosuvastatin triol of claim 1 in lactone form having the following structure:

17. The rosuvastatin triol of claim 16, wherein the rosuvastatin triol lactone is isolated.

18. The rosuvastatin triol of claim 16 or 17, wherein the rosuvastatin triol lactone is substantially free from corresponding rosuvastatin diol.

19. The rosuvastatin triol of any one of claims 1-18, wherein the rosuvastatin triol is selected from the group consisting of: rosuvastatin triol in (7S) form; rosuvastatin triol in (7R) form; and rosuvastatin triol racemic.

20. A process for preparing the triol of any of claims 7-10 or 18-19 comprising oxidizing rosuvastatin diol C₁ to C₄ ester to obtain the rosuvastatin triol ester with a hydroxyl group at position 7.

21. The process of claim 20, wherein the process comprises combining rosuvastatin Ci-C₄ ester with a solution of a borane in an organic solvent to obtain a reaction mixture, combining the resulting reaction mixture with a solution of an inorganic base in water, and adding peroxide and recovering the triol ester.

22. The process of claim 20 or 21, wherein the borane is a borane complex of dimethylsulfide.

23. The process of claim 22, wherein the borane is a monoalkyl - or dialkyl — borane

24. The process of claim 21 or 22, wherein the peroxide is H₂O_2.

25. The process of claim 21 or 22, wherein the peroxide is t-Butyl Hydroperoxide (TBHP) or Magnesium monoperoxyphthalate hexahydrate (MMPP).

26. The process of claim 0, wherein the base is an inorganic base.

27. The process of claim 26, wherein inorganic base is an alkali metal base.

28. The process of claim 27, wherein the base is a hydroxide base.

29. The process of claim 28, wherein the hydroxide base is NaOH, KOH or LiOH

30. The process of claim 21, wherein the base is NH₄OH

31. The process of any one of claims 0-30, wherein the organic solvent is a C₃-C₈ ether.

32. The process of claim 31, wherein the organic solvent is tetrahydrofuran.

33. A process for preparing the triol salt of claim 11 comprising suspending the triol ester of the following formula:

wherein R is a Ci-C₄ ester, in a mixture of water and an organic solvent, and combining the suspension with a base and a source of ions.

34. The process of claim 33, wherein the organic solvent is a Ci-C₄ alcohol.

35. The process of claim 33, wherein the organic solvent is ethanol.

36. The process of claim 33, 34 or 35, wherein the source of ions is calcium chloride

37. A process for preparing the triol acid of claim 4 comprising contacting the rosuvastatin triol salt of the following formula:

with an acid, wherein M is an alkali or alkaline earth metal.

38. The process of claim 37, wherein the acid is hydrochloric or sulfuric acid.

39. The process of claim 37 or 38, wherein rosuvastatin triol salt is combined with an organic solvent.

40. The process of claim 39, wherein the organic solvent is dichloromethane.

41. A process for preparing rosuvastatin triol acid of claim 4 comprising hydrolyzing an ester of the following formula:

with an acid, wherein R is a Cj-C₄ ester.

42. The process of claim 41, wherein the acid is HCl or H₂SO_4.

43. A process for preparing the lactone of any one of claims claim 16-19, comprising hydrolyzing the triol ester of the following formula:

wherein R is a C]-C₄ ester, and converting the hydrolyzed ester to a lactone.

44. A process for preparing rosuvastatin triol salt of any one of claims 11-15 or 18-19 with the following structure:

comprising hydrolyzing a lactone having the following structure

and converting the hydrolyzed lactone to an the salt, wherein M is an alkali metal or an alkaline earth metal.

45. A process for preparing rosuvastatin triol salt of any one of claims 16-20 with the following structure:

comprising contacting an acid with the following structure with a base..

46. A process for reducing amount of impurities present in a pharmaceutical composition of rosuvastatin calcium comprising measuring amount of rosuvastatin triol calcium in batches of rosuvastatin diol calcium, selecting batches of the rosuvastatin diol calcium with desirable level of the rosuvastatin triol calcium and preparing pharmaceutical compositions with the selected rosuvastatin diol batch.

47. A process for reducing amount of rosuvastatin triol ester present in a mixture comprising rosuvastatin diol ester and rosuvastatin triol ester comprising measuring amount of rosuvastatin triol Cj-C₄ ester in batches of rosuvastatin diol Ci-C₄ ester, selecting batches of the rosuvastatin diol C₁-C₄ ester with of the triol Ci-C₄ ester and preparing pharmaceutical compositions of rosuvastatin diol calcium with the selected rosuvastatin diol Ci-C₄ ester batch.

48. A method of determining the amount of an impurity in a sample of rosuvastatin diol ester comprising measuring by GC or HPLC the area under a peak corresponding to rosuvastatin triol ester in a reference standard that comprises a known amount of rosuvastatin triol ester; measuring by HPLC or GC the area under a peak corresponding to rosuvastatin triol ester in a sample comprising rosuvastatin triol and rosuvastatin diol esters; and determining the amount of the triol ester in the sample by comparing the area of reference standard with that of the test sample.

49. The method of claim 48, wherein the triol ester is a t-butyl ester.

50. A method of determining the amount of an impurity in a sample of rosuvastatin calcium comprising measuring by GC or HPLC the area under a peak corresponding to rosuvastatin triol calcium in a reference standard comprising a known amount of rosuvastatin triol calcium; measuring by GC or HPLC the area under a peak corresponding to rosuvastatin triol calcium in a sample comprising rosuvastatin triol and rosuvastatin diol calcium salts; and determining the amount of the triol calcium in the sample by comparing the area of reference standard with that of the test sample.

51. A method of identifying the relative retention time (RRT) of an impurity in a sample of rosuvastatin diol ester comprising measuring by GC or HPLC the relative retention time (RRT) corresponding to rosuvastatin triol ester in a reference marker sample; carrying out GC or HPLC with a test sample comprising of the rosuvastatin diol ester and rosuvastatin triol ester to obtain an GC or HPLC chromatogram with retention times; and identifying the relative retention time (RRT) of the triol ester in the sample by comparing the relative retention time (RRT) of the reference marker to the relative retention time (RRT) of the test sample.

52. The method of claim 51, wherein the triol ester is a t-butyl ester.

53. A method of identifying the relative retention time (RRT) of an impurity in a sample of rosuvastatin diol calcium comprising measuring by GC or HPLC the relative retention time (RRT) corresponding to rosuvastatin triol calcium in a reference marker sample; carrying out GC or HPLC with a test sample comprising of the rosuvastatin diol and rosuvastatin triol calcium salts to obtain an HPLC chromatogram with retention times; and identifying the relative retention time (RRT) of the triol calcium in the sample by comparing the relative retention time (RRT) of the reference marker to the relative retention time (RRT) of the test sample.

54. Use of a compound according to any of Claims 1-19 as a reference standard or reference marker for determining the purity of rosuvastatin acid, rosuvastatin ester, rosuvastatin salt (preferably the calcium salt), and rosuvastatin lactone.