WO2006134482A1

WO2006134482A1 - Process for producing atorvastatin, pharmaceutically acceptable salts thereof and intermediates thereof

Info

Publication number: WO2006134482A1
Application number: PCT/IB2006/001613
Authority: WO
Inventors: Shanghui Hu; Junhua Tao; Zhiyi Xie
Original assignee: Pfizer Inc.
Priority date: 2005-06-16
Filing date: 2006-06-06
Publication date: 2006-12-21
Also published as: WO2006134482A8; AR055975A1; JP2006345863A; CA2612293A1; EP1893767A1; TW200724688A

Abstract

A novel process for producing atrovastatin, pharmaceutically acceptable salts thereof, and intermediates thereof using an aldolase is described. Compounds so prepared are useful as inhibitors of the HMG-CoA reductase and may thus be used as hypolipidemic and hypocholesterolemic agents.

Description

PROCESS FOR PRODUCING ATORVASTATIN, PHARMACEUTICALLY ACCEPTABLE SALTS THEREOF AND INTERMEDIATES THEREOF

BACKGROUND OF THE INVENTION High levels of blood cholesterol and blood lipids are conditions involved in the onset of atherosclerosis. The conversion of HMG-CoA to mevalonate is an early and rate-limiting step in the cholesterol biosynthetic pathway. This step is catalyzed by the enzyme HMG-CoA reductase. It is known that inhibitors of HMG-CoA reductase are effective in lowering the blood plasma level of low density lipoprotein cholesterol (LDL-C), in man. (cf. M.S. Brown and J.L. Goldstein, New England Journal of Medicine, 305, No. 9, 515-517 (1981)). It has been established that lowering LDL-C levels affords protection from coronary heart disease {cf . Journal of the American Medical Association, 251 , No. 3, 351-374 (1984)).

To varying degrees, statins interfere with and/or inhibit HMG-CoA reductase from catalyzing the conversion of HMG-CoA to mevalonate. As such, statins are collectively potent lipid lowering agents. Thus, statins are the drugs of first choice for management of many lipid disorders. One representative statin is atorvastatin. Other representative statins include lovastatin, pravastatin, simvastatin and rosuvastatin.

Atorvastatin and pharmaceutically acceptable salts thereof are selective, competitive inhibitors of HMG-CoA reductase. Atorvastatin calcium is currently sold as Lipitor^® having the chemical name [R-(R^*, R^*)]-2-(4-fluorophenyl)-β,δ-dihydroxy-5-(1-methylethyl)-3-phenyl-4- [(phenylamino)carbonyl]-1 H-pyrrole-1-heptanoic acid calcium salt (2:1) trihydrate and the formula

As such, atorvastatin calcium is a potent lipid-lowering compound and is thus useful as a hypolipidemic and/or hypocholesterolemic agent. Atorvastatin calcium is also useful in the treatment of osteoporosis, benign prostatic hyperplasia (BPH) and Alzheimer's disease.

A number of patents and published International Patent Applications have issued describing atorvastatin, formulations of atorvastatin, as well as processes and key intermediates for preparing atorvastatin. These include: United States Patent Numbers 4,681,893; 5,003,080; 5,097,045; 5,103,024; 5,124,482; 5,149,837; 5,155,251 ; -5,216,174;

5,245,047; 5,248,793; 5,273,995; 5,280,126; 5,298,627; 5,342,952; 5,397,792; 5,446,054;

5,470,981 ; 5,489,690; 5,489,691 ; 5,510,488; 5,686,104; 5,969,156; 5,998;633; 6,087,511;

6,121 ,461 ; 6,126,971 ; 6,433,213; 6,476,235; 6,528,660; 6,600,051 ; 6,605,759; 6,613,916; 6,646,133; 6,730,797; 6,867,306; 6,891 ,047; WO99/32434; WO 01/36384; WO 02/41834; WO

02/43667; WO 02/43732; WO 02/051804; WO 02/057228; WO 02/057229; WO 02/057274;

WO 02/059087; WO 02/083637; WO 02/083638; WO 03/011826; WO 03/050085; WO

03/07072; and WO 04/022053.

The use of DERA (deoxyribose aldolase) family of aldolases in chemoenzymatic processes has been described. See US Pat. No. 5,795,749, WO 03/006656, WO

2004/027075, WO 2005/012246; Gijsen, H. J. M., el al. JACS, 1994, 116, 8422-8423; Gijsen,

H. J. M., et al., JACS, 1995, 117, 7585-7591; Greenberg, W. A., et al., PNAS₁2004, 101 , 5788-

5793. However, some of the processes provided poor overall yield as well as poor purity of product. In addition, the processes were limited to specific substrates. Accordingly, there exists a need in the art for a chemoenzymatic process that is effective and efficient for alternative substrates.

SUMMARY OF THE INVENTION

These and other needs are met by the present invention which is directed to a an efficient and cost-effective synthetic route for producing atorvastatin, pharmaceutically acceptable salts thereof (e.g. atorvastatin calcium) and intermediates thereof. The present invention provides a process comprising the step of reacting an aldehyde with an aminoaldehyde substrate, a N-protected aminoaldehyde substrate or a pyrrole aldehyde substrate under aldolase-catalyzed aldol condensation conditions to form the corresponding lactol.

The present invention also provides a process for producing atorvastatin or a pharmaceutically acceptable salt thereof comprising the steps of:

(a) reacting an aldehyde with an aminoaldehyde substrate, a N-protected aminoaldehyde substrate or a pyrrole aldehyde substrate under aldolase-catalyzed aldol condensation conditions to form the corresponding lactol;

(b) oxidizing said lactol to the corresponding lactone; and

(c) subjecting said lactone to conditions sufficient to produce atorvastatin or a pharmaceutically acceptable salt thereof.

(b) subjecting said lactol to catalytic dehydrogenation conditions followed by lactonization conditions to form the corresponding lactone; and (c) subjecting said lactone to conditions sufficient to produce atorvastatin or a pharmaceutically acceptable salt thereof.

The present invention also provides a process for producing atorvastatin or a pharmaceutically acceptable salt thereof comprising the steps of: (a) reacting an aldehyde with an aminoaldehyde substrate, a N-protected aminoaldehyde substrate or a pyrrole aldehyde substrate under aldolase-catalyzed aldol condensation conditions to form the corresponding lactol;

(b) ring-opening of said lactol to the corresponding carboxylic acid; and

(c) subjecting said carboxylic acid to conditions sufficient to produce atorvastatin or a pharmaceutically acceptable salt thereof.

Novel intermediates useful in the preparation of atorvastatin or a pharmaceutically acceptable salt thereof are also provided.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

Unless indicated otherwise, the following terms are defined as follows: The article "a" or "an" as used herein refers to both the singular and plural form of the object to which it refers.

The term "aldolase-catalyzed aldol condensation conditions" as used herein refers to any aldol condensation conditions known in the art that can be catalyzed by an aldolase, as described herein.

The term "conditions sufficient to produce atorvastatin or a pharmaceutically . acceptable salt thereof" as used herein refers to those means described in the art, including those means described herein. The term "a pharmaceutically acceptable salt" as used herein refers to the relatively non-toxic salts of atorvastatin which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use. These salts can be prepared by any means known in the art. Examples of a pharmaceutically acceptable salt of atorvastatin include, but are not limited to, all atorvastatin salts known in the art including, but not limited to, atorvastatin calcium and atorvastatin sodium. In a preferred embodiment, a pharmaceutically acceptable salt of atorvastatin is atorvastatin calcium.

The aldehyde for use in the present invention may be any aldehyde that will undergo an aldol condensation with a substrate, as described herein, in the presence of an aldolase, as described herein. An example of suitable aldehyde is, but is not limited to, acetaldehyde.

A substrate for use in the present invention may be any aminoaldehyde or N-protected aminoaldehyde. Such an aminoaldehyde or N-protected aminoaldehyde will react with an aldehyde under aldolase-catalyzed aldol condensation conditions, each as described herein. In one embodiment of the invention, a substrate for use in the invention is of the general formula (I): -A-

wherein: n = 1-4; preferably, n =1-3; more preferably, n = 1-2;

R' is hydrogen;

R" is hydrogen or a N-protecting group; or R' and R" taken together with nitrogen to which they are attached form a cyclic moiety.

Suitable N-protecting groups for the aminoaldehyde include, but are not limited to, benzyloxycarbonyl (CBz), butoxycarbonyl (Boc), 9- fluorenylmethoxycarbonyl (Fmoc), benzyl, and dibenzyl.

Examples of a suitable aminoaldehyde substrate include, but are not limited to:

propionaldehyde_j acetaldehyde_> N-CBz-3-Aminopropionaidehyde_j

Λ/-acetyl-3- Aminopropionaldehyde_j N-Fmoc-3-Aminopropionaldehyde_>

_ancl N-Fmoc-acetaldehyde

In one embodiment of the invention, the aminoaldehyde substrate is N-CBz-3- aminopropionaldehyde or N-Fmoc-3-aminopropionaldehyde. In one embodiment of the invention, the aminoaldehyde substrate is 3-amino-propionaldehyde. In one embodiment of the invention, the aminoaldehyde substrate is amino-acetaidehyde. In one embodiment of the invention, the aminoaldehyde substrate is N-CBz-3-aminopropionaldehyde (commercially available from Aldrich). In one embodiment of the invention, the aminoaldehyde substrate is N- acetyl-3-aminopropionaldehyde. In one embodiment of the invention, the aminoaldehyde substrate is N-Fmoc-3-aminopropionaldehyde.

Both N-Fmoc-aminoaldehydes were obtained via standard Dess-Martin oxidation of the corresponding N-Fmoc aminoalcohol.

The N-acetyl-3-aminopropionaldehyde was obtained from 3-amino-1-propanol by a two step procedure: N-acetylation of the 3-amino-1-propanol by methyl actetate followed by Dess- Martin oxidation to give the desired product with the correct ESI-MS [M+H]⁺ 116.25 and [M+Na]⁺ 138.20.

Alternatively, a substrate for use in the present invention may be a pyrrole aldehyde. In one embodiment, the pyrrole aldehyde has the following structure:

The pyrrole aldehyde was prepared from diketone 9 by a two-step procedure: Paal-Knorr reaction with 3-amino-1-propanol followed by Dess-Martin oxidation to give the desired product with correct ESI-MS [M+H]⁺ 455.4 and [M+Na]⁺ 477.3.

An aldolase for use in the present invention may be any compound that has aldolase activity towards an aminoaldehyde substrate, N-protected aminoaldehyde substrate, or pyrrole aldehyde substrate, each as described herein. In one embodiment of the invention, the aldolase is a 2-deoxyribose-5-phosphate aldolase (DERA). Examples of a suitable DERA • aldolase include, but are not limited to:

DERA 03 (E. CoIi) (commercially available from Sigma Aldrich, St. Louis, MO); DERA 04 (William A. Greenberg, et al., PNAS, (2004), Vol. 101 ,, No. 16, pp. 5788-

5793 or a modified version thereof);

DERA 06 (GenBank Accession NP_294929 or a modified version thereof);

DERA 08 (GenBank Accession NP_465519 or a modified version thereof);

DERA 11 (GenBank Accession NP_439273); DERA 12 (GenBank Accession NP_229359); or

DERA 15 (Haruhiko Sakuraba, et al., Journal of Biological Chemistry <2003), Vol. 278, No. 12, pp 10799-10806).

In one embodiment of the invention, the aldolase is an aldolase having an amino acid sequence identity of at least about 20% thereof; preferably, at least 70% thereof, to a DERA aldolase described herein. In one embodiment of the invention, the DERA aldolase is DERA 04 or DERA 06. In one embodiment of the invention, the DERA aldolase is DERA 06.

According to the invention, DERA 03, DERA 04, DERA 06, DERA 08, DERA 11 , DERA 12, and DERA 15 are identified by their nucleotide sequences and amino acid sequences set forth in Examples 10-23. More specifically, DERA 03 is an aldolase having a nucleotide sequence of SEQ ID

NO: 1 and an amino acid sequence of SEQ ID NO: 8.

DERA 04 is an aldolase having a nucleotide sequence of SEQ ID NO: 2 and an amino acid sequence of SEQ ID NO: 9.

DERA 06 is an aldolase having a nucleotide sequence of SEQ ID NO: 3 and an amino acid sequence of ^"SEQ ID NO: 10. OERA 08 is an aldolase having a nucleotide sequence of SEQ ID NO: 4 and an amino acid sequence of SEQ ID NO: 11.

DERA 11 is an aldolase having a nucleotide sequence of SEQ ID NO: 5 and an amino acid sequence of SEQ ID NO: 12.

DERA 12 is an aldolase having a nucleotide sequence of SEQ ID NO: 6 and an amino acid sequence of SEQ ID NO: 13.

DERA 15 is an aldolase having a nucleotide sequence of SEQ ID NO: 7 and an amino acid sequence of SEQ ID NO: 14.

The DERA aldolases described herein can be prepared by any means known in the art, including but not limited to standard protocols for protein expression in recombinant E. coli (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3^rd Ed., Cold Spring Harbor, NY 2001). As would be understood by one of skill in the art, modified versions of known DERA aldolases may be necessary or may result depending on cloning conditions and are encompassed by the present invention.

The following Schemes illustrate the present invention.

Scheme 1 General Process for forming Atorvastatin Calcium

oxidation

R= H, CBz, Boc, Fmoc, benzyl, or dibenzyl

R

Atorvastatin Calcium Scheme 1 describes in general a process encompassed by the present invention. As set forth in Scheme 1 , a DERA aldolase catalyzes an aldol condensation reaction between an aminoaldehyde or a N-protected aminoaldehyde and 2 mol of acetaldehyde to give the desired amino-lactol (A). Amino-lactol (A) can then be selectively oxidized to the corresponding lactone (B). The resulting lactone (B) can then be converted into atorvastatin lactone via deprotection followed by Parl-Knorr condensation with diketone (9):

diketone 9 to give the corresponding atorvastatin lactone. The opening of the atorvastatin lactone with Ca(OH)₂ will give atorvastatin calcium.

Alternatively, amino-lactol (A) can undergo catalytic (e.g. Pt/C, Pd/C) dehydrogenation to form carboxylic acid (C), which can then undergo lactonization to form (B) which in turn can then be converted into atorvastatin calcium as described above.

In another embodiment, carboxylic acid (C) is condensed with diketone (9) to give atorvastatin calcium.

According to the invention, as would be understood by one of skill in the art, the stereoselectivity of the enzymatic step can be confirmed via chemical preparation of racemic standards and the development of the related chiral chromatographic methods.

Oxidation of the lactol (A) to lactone (B) or carboxylic acid (C) can be performed by use of any oxidation means known in the art that will achieve the desired transformation. In one embodiment of the invention, selective oxidation can be achieved according to the methods described in Examples 2 and 3.

Any catalytic dehydrogenation means known in the art to convert (A) to (C) are encompassed by the present invention. Examples of suitable catalysts include, but are not limited to, Pt/C, Pd/C, Pt/Bi/C, Pd/Bi/C and any other dehydrogenation catalysts. In one embodiment of the invention, the catalytic dehydrogenation is performed at about pH 7 to about pH 10 using air or oxygen as terminant oxidant.

Any lactonization means known in the art to convert carboxylic acid (C) to lactone (B) are encompassed by the present invention including, but not limited to, the use of acid catalysts such as, but not limited to, HCI, H₂SO₄, Methanesulfonic acid (MSA), p-Toluenesulfonic acid (TSA) and any other lactonization acids known in the art.

Scheme 2

Scheme 2 illustrates three possible routes for the preparation of atorvastatin calcium from lactol (1). Lactol (1) can be prepared by a chemoeznymatic process described herein. Route 1 : Amino-lactone (2) can be prepared by acid-catalyzed lactonization of (3), followed by deprotection of benzyloxycarbonyl (CBz) of (2) by Pd/C catalyzed hydrogenation.

Route 2: ω-Aminoacid-diol (5) can be prepared via CBz deprotection of (3) by Pd/C catalyzed hydrogenation. Subsequent condensation of (5) with di-ketone (9):

diketone 9 gave atorvastatin calcium.

Route 3: Compound (8) (TBIA) can be prepared through acid-catalyzed diol protection of (3) to form (6); t-BuOH coupling of (6) to form (7); and CBz deprotection of (7) by Pd/C catalyzed hydrogenation to form (8). Compound (8) can then be taken on to form atorvastatin calcium by condensation with diketone (9), deprotection, lactonization and lactone ring opening by Ca(OH)₂.

In another embodiment, carboxylic acid (3) can be converted to tert-butyl-isopropylidine amine (8), followed by condensation with diketone (9), deprotection, lactonization and the opening of lactone to give atorvastatin calcium. Scheme 3

Scheme 3 describes another route for the formation of atorvastatin calcium from lactol (1) prepared by the process described herein. Protected amino-lactol (12) is prepared by conversion of lactol (1) to ethyl-protected lactol (11); followed by CBz deprotection of (11) via Pd/C catalyzed hydrogenation. Subsequent condensation of (12) with diketone (9) gives {13) which is then deprotected to give (14) which, in turn, is then oxidized to give atorvastatin lactone (10) which is then converted to atorvastatin calcium.

Scheme 4

Alternate Route for the Synthesis of Atorvastatin Calcium

In Scheme 4, atorvastatin calcium is prepared starting with diketone 9. Diketone 9 can be prepared according to procedures known in the art including, for example, Baumann, Kelvin L., Tetrahedron Letters (1992), 33(17), 2283-4. Diketone 9 is treated with pivalic acid to form the corresponding pyrrole alcohol which is then oxidized under Dess-Martin conditions to form the corresponding pyrrole aldehyde. The pyrrole aldehyde then undergoes an aldolase-catalyzed aldol condensation to form the corresponding pyrrole lactol. The resulting pyrrole lactol can then be converted to the pyrrole lactone by standard oxidation. The pyrrole lactone can then be converted to atorvastatin calcium by means known in the art.

The following non-limiting examples illustrate the invention.

Example 1 Preparation of 6R-r(Benzyloxycarbonyl)amino1ethyl-tetrahvdro-2H-pyran-2,4-diol (1)

¹

To a solution of DERA 04 (deoxyribose aldolase, 42,000 units, 14.0 mL) was added phosphate buffer (60 mL, pH 7.3, 0.05 M). [(Benzyloxycarbonyl)amino]propionaldehyde (10.0 g, 48.3 mmol, Aldrich) dissolved in dimethylsulfoxide (DMSO) (2.5 mL) and acetaldehyde (5.5 ml, 96.6 mmol, Aldrich) in phosphate buffer (5.5 mL, pH 7.3, 0.05 M) was continuously added to the above DERA solution by a programmed pump within 2 h. The conversion of the reaction was monitored by high performance liquid chromatography (HPLC). After reaching 95% of conversion in 20-24 h, the reaction mixture was extracted with ethyl acetate (100 mL). After the separation of two layers by centrifugation, the organic layer was dried and evaporated to give the crude lactol 1 (14.5 g) as an oil, which was directly submitted to next step. Pure 1 (10.8 g, 76%) was also obtained by silica-gel chromatography using heptane/ethyl acetate/methanol (1 :8:1). LC-MS m/z[M+Na]⁺318.2. ¹³C NMR (CDCI₃, 75 MHz) for the major anomer: δ 136.96, 157.00, 128.90, 128.61 , 93.07, 69.01 , 67.89, 67.10, 40.21 , 38.00, 35.50.

Example 2 Preparation of 6R-f1-(Benzyloxycarbonyl)amino1 ethyl-4R-hvdroxytetrahvdro-2-H-pyran-2-one

To a solution of lactol 1 (50 mg, 0.169 mmol; prepared according to Example 1) in acetonitrile (ACN) (1 mL) was added BaCO₃ (1.5 eq, 0.253 mmol, Aldrich). Bromine (1.5 eq, 0.253 mmol, Aldrich) was added dropwise to the above mixture under vigourously stirring. After 5 h, the filtration of residual solid gave a solution, which evaporated to afford crude lactone 2. LC- ESIMS: m/z [M+Na]⁺ m/z 316.1. The de and ee of the lactone were determined by chiral HPLC. Example 3

Preparation of SR.SR-Dihvdroxy-i-rbenzyloxycarbonvπamino^-heptanoic acid /3)

To a solution of crude lactol 1 (7.5 g; prepared according to Example 1) in t-butanol and H₂O (38 ml_/38 mL) was added Pt/C (3.0 g, 3% mol, Aldrich). The dehydrogenation was performed by bubbling air into the reaction mixture at 40 ⁰C. The pH of the reaction mixture was kept at 8.0 by continuous addition of saturated NaHCO₃ solution. After 24 h, the catalyst was filtered and the crude aqueous solution of 3 as a sodium salt was obtained after washing with EtOAc (70 mL). The aqueous solution was acidified to pH 4.0 with 10% HCI and extracted with EtOAc (2 x 70 mL). The organic layer was dried and concentrated to give the crude 3 as light yellow oil (4.0 g). LC-ESIMS m/z [M+Naf 334.1.

Example 4

Preparation of 6R-ri-(Benzyloxycarbonyl)amino1 ethyl-4R-hvdroxytetrahvdro-2-H-pyran-2-one (2)

To crude 3 (2.0 g; prepared according to Example 3) in tetrahydrofuran (THF) (50 mL) was added hydrochloric acid (HCI) in dioxane (100 uL, 4 M, Aldrich) and the reaction mixture was stirred at 23 ⁰C for 2-3 hours. After the evaporation of THF, the crude oil was dissolved in EtOAc (50 mL) and washed with water (pH 7.0, 50 mL). The organic layer was dried over Na₂SO₄ and after evaporation of the solvent, the residue formed long needles from heptane/acetone (4:1 , 50 mL) to give 2 as a white solid (1.5 g, -45% overall yield for 3 steps, de >97%, ee >99%). LC-ESIMS [M+Na]⁺m/z 316.1. ¹H NMR (CDCI₃, 300MHz): δ 7.23-7.50 (m, 5H), 5.19 (bra, 1H), 5.08 (s, 2H), 4.78 (m, 1H), 4.32 (s, 1H), 3.38 (m, 2H), 2.65 (m, 2H), 1.59- 2.02 (m, 4H). ¹³C NMR (CDCI₃, 75 MHz) δ 170.28, 156.71 , 136.49, 128.56, 128.17, 128.09, 73.82, 66.82, 62.59, 38.51 , 37.34, 35.81 , 35.64.

Example 5 Preparation of 6R-aminoethyl-4R-hvdroxytetrahvdro-2-H-pyran-2-one (4)

Lactone 2 (100 mg, 0.341 mmol; prepared according to Example 4) in THF (2 mL) was hydrogenated with Pd/C (5%, 100 mg, Aldrich) at 23 ⁰C for 1 h. After filtration of the catalyst, the free aminoethyl-lactone 4 was produced, which was directly used for the subsequent Paal- Knorr condensation (see Example 8). LC-ESIMS: m/z [M+THF]⁺ 232.3.

Example 6

Preparation of f6-(2-Benzyloxycarbonylamino-ethvO-2,2-dimethyl-f 1 ,31dioxan-4-vn-acetic acid tert-butyl ester (7)

To a crude acid 3 (1.0 g; prepared according to Example 3) in a mixture of acetone (2.0 mL) and dimethoxypropane (4.0 ml_, Aldrich) was added methylsuifonic acid (MSA) (15 μL, catalytic amount, Aldrich). The mixture was stirred at room temperature (rt) for 2-3 hours. After the evaporation of the solvents, to the crude residue in acetonitrile (ACN) (10 mL) was added DCC (1.0 eq, 720 mg, Aldrich) and t-butanol (2.0 eq, 660 μL, Aldrich). The mixture was stirred at rt for 2 hours (h). The crude product 7 is obtained after filtration of the formed solid and evaporation of the solvents, which is directly submitted to the next deprotection step. LC- ESIMS m/z [M+Na]⁺431.5.

Example 7 Preparation of tert-butyl-isopropylidine amine (TBIA) (8)

Crude 7 (2.3 g; prepared according to Example 6) in MeOH (20 mL) is hydrogenated with Pd/C (5%, 500 mg, Aldrich) at 23 ⁰C. After the filtration of the catalyst and the evaporation of MeOH, the crude product is dissolved in an acidic buffer (pH 4.0), which is washed with ethyl acetate (1 x 50 mL). The pH of the aqueous phase is adjusted to 10 by 1 N NaOH and is extracted with ethyl acetate (2 x 50 mL). The organic phase is dried and evaporated to afford 8 as an oil.

Example 8 Preparation of Atorvastatin lactone (10)

The mixture of amino-lactone 4 (77 mg, 0.486 mmol; prepared according to Example 5), diketone 9 (0.7 eq, 142 mg) and pivalic acid (1.0 eq, 34.7 mg, Fluka) in toluene/heptane (1 :1 , 3 mL) was refluxed at 110 ⁰C for 24 h. The LC-MS spectrum indicated that <5% of atorvastain lactone 10 was formed. LC-ESlMS: m/z [M+Na]⁺ 563.2.

Example 9 Preparation of N-CBz-3-amino-ethyl-lactol (11)

' 11

To crude lactol 1 (100 mg; prepared according to Example 1) in EtOH (1 mL) was added 2 mL of H₂SO₄. The mixure was stirred at rt for 1 h. After removal of the solvent, the crude protected lactol 11 was afforded with >95% conversion. LC-ESI-MS: m/z [M+Naf 346.3.

Example 10 SEQ ID NO: 1 - Nucleotide sequence of DERA03 atgactgatctgaaagcaagcagcctgcgtgcactgaaattgatggacctgaccaccctgaatgacgacgacaccgacgagaaa gtgatcgccctgtgtcatcaggccaaaactccggtcggcaataccgccgctatctgtatctatcctcgctttatcccgattgctcgcaaaa ctctgaaagagcagggcaccccggaaatccgtatcgctacggtaaccaacttcccacacggtaacgacgacatcgacategcgct ggcagaaacccgtgcggcaatcgcctacggtgctgatgaagttgacgttgtgttcccgtaccgcgcgctgatggcgggtaacgagc aggttggttttgacctggtgaaagcctgtaaagaggcttgcgcggcagcgaatgtactgctgaaagtgatcatcgaaaccggcgaac tgaaagacgaagcgctgatccgtaaagcgtctgaaatctccatcaaagcgggtgcggacttcatcaaaacctctaccggtaaagtg gctgtgaacgcgacgccggaaagcgcgcgcatcatgatggaagtgatccgtgatatgggcgtagaaaaaaccgttggtttcaaac cggcgggcggcgtgcgtactgcggaagatgcgcagaaatatctcgccattgcagatgaactgttcggtgctgactgggcagatgcg cgtcactaccgctttggcgcttccagcctgctggcaagcctgctgaaagcgctgggtcacggcgacggtaagagcgccagcagcta ctaa

Example 11

SEQ ID NO: 2 - Nucleotide Sequence of DERA04 atgggtaatatcgcgaaaatgattgatcacaccctcttaaaacccgaagcaaccgaacaacaaattgtacaattatgcacggaagc gaaacaatatggctttgcagcagtatgcgtaaatccgacatgggttaaaaccgccgcacgtgaattaagcgggacagacgttcgtgt gtgtactgtaattggatttcccttgggcgctacgactccagaaactaaagcattcgaaactactaacgcgattgaaaatggagcacgg gaagtagatatggtaattaatattggtgcattgaaatctggacaagatgaactggtggaacgtgatattcgtgccgttgttgaagctgca gcaggccgcgcgcttgtgaaagtaattgtagaaacagcccttcttactgatgaagaaaaagttcgcgcttgtcaattagcagtaaaag cgggtgccgattatgtgaagacgtcgacaggatttagcggtggtggtgcaacggtggaagatgtggctttaatgcggaaaacggttg gtgatcgtgcaggggtcaaagcaagcggcggagtacgtgactggaaaacagcagaagcaatgattaacgcaggagcaacgcg cattggcacaagttctggagtagcaatcgtaacaggtggaaccggccgggcagactattaa

Example 12

SEQ ID NO: 3 - Nucleotide Sequence of DERA06 atgggactcgcctcctacatcgaccacacgctgcttaaggccaccgccacgctcgccgacatccgcacgctgtgtgaggaagccc gcgagcactcgttctacgcggtgtgcatcaacccggtctttattccccacgcccgcgcctggctcgaaggcagcgacgtgaaggtcg ccaccgtctgcggctttcccctcggcgccatcagctccgagcagaaagctctggaagcccgcctgagcgccgaaacgggcgccg acgaaatcgatatggtcatccacatcggctcggcgcttgccggcgactgggacgcggtggaagccgacgtgcgggcagtgcgccg cgcggtgcccgagcaggtgctcaaggtgattatcgaaacctgctacctgaccgacgagcaaaagcgcttggcgactgaggtcgcc gtacagggcggcgccgacttcgtgaagacgagcacaggcttcggcaccggcggcgccaccgtggacgacgtgcgcctgatggc ggaagtgatcgggggccgcgccggactcaaggcggcgggcggcgtccgcactcctgccgacgcgcaagccatgatcgaggcg ggcgcgacccggctgggcacctcgggcggcgtgggtctggtgtcgggcggcgaaaacggagccggctactga

Example 13

SEQ ID NO: 4 - Nucleotide Sequence of DERA08 atgggaattgctaaaatgatcgatcacactgctttaaaaccagacacaacgaaagaacaaattttaacactaacaaaagaagcaa gagaatacggttttgcttccgtatgcgtaaatccaacttgggtaaaactatccgctgaacaacttgctggagcagaatctgtagtatgta ctgttatcggtttcccactaggagcgaatacccctgaagtaaaagcatttgaagtaaaagatgctatccaaaacggtgcaaaagaag tggatatggttattaatatcggcgcactaaaagacaaagacgacgaactagtagaacgtgatattcgcgctgtagtcgatgctgccaa aggaaaagcattagtaaaagtaattatcgaaacttgcctattaacagacgaagaaaaagttcgcgcatgtgaaatcgctgtaaaag cgggaacagacttcgttaaaacatccactggattctccacaggtggcgcaactgccgaagatatcgccttaatgcgtaaaactgtag gaccaaacatcggcgtaaaagcatctggtggggttcgtacgaaagaagacgtagaaaaaatgatcgaagcaggcgcaactcgta ttggcgcaagtgcaggtgtcgcaattgtttccggcgaaaaaccagccaaaccagataattactaa

Example 14

SEQ ID NO: 5 - Nucleotide Sequence of DERA11 atgacatcaaatcaacttgctcaatatatcgatcacaccgcacttaccgcagaaaaaaatgaacaagatatttcgacactctgtaatg aagcgattgaacacggattttattctgtatgtatcaattctgcttatattccactcgctaaagaaaaacttgctggctcaaatgtaaaaattt gcaccgtagttggattccctttgggggcgaatttaacctcagtcaaagcatttgaaacgcaagaatctattaaagcgggtgcaaatga aattgatatggtgattaatgtaggttggataaaatcgcaaaaatgggatgaagtaaaacaagatattcaagcggtatttaatgcttgtaa tggcacgccattaaaagtgattttagaaacttgtttgctcactaaagatgaaatagtgaaagcctgcgaaatttgtaaagaaatcggtgt agcttttgttaaaacatcaacaggctttaataaaggtggtgcgaccgtagaagatgttgcattgatgaaaaacacggtcggcaatattg gtgttaaagcatcaggtggtgtgcgtgatactgaaactgcacttgcaatgattaaggcgggtgcgactcgcattggtgcaagcgctgg cattgcgattattagcggtactcaagacactcaaagcacttactaa

Example 15 SEQ ID NO: 6 - Nucleotide Sequence of DERA12 atgatagagtacaggattgaggaggcagtagcgaagtacagagagttctacgaattcaagcccgtcagagaaagcgcaggtattg aagatgtgaaaagtgctatagagcacacgaatctgaaaccgtttgccacaccagacgatataaaaaaactctgtcttgaagcaagg gaaaatcgtttccatggagtctgtgtgaatccgtgttatgtgaaactggctcgtgaagaactcgaaggaaccgatgtgaaagtcgtcac cgttgttggttttccactgggagcgaacgaaactcggacgaaagcccatgaggcgattttcgctgttgagagtggagccgatgagatc gatatggtcatcaacgttggcatgctcaaggcaaaggagtgggagtacgtttacgaggatataagaagtgttgtcgaatcggtgaaa ggaaaagttgtgaaggtgatcatcgaaacgtgctatctggatacggaagagaagatagcggcgtgtgtcatttccaaacttgctgga gctcatttcgtgaagacttccacgggatttggaacaggaggggcgaccgcagaagacgttcatctcatgaaatggatcgtgggagat gagatgggtgtaaaagcttccggagggatcagaaccttcgaggacgctgttaaaatgatcatgtacggtgctgatagaataggaac gagttcgggagttaagatcgncaggggggagaagagagatatggaggnga Example 16

SEQ ID NO: 7 - Ncleotide Sequence of DERA15 atgccgtcggccagggatatactgcagcagggtctagacaggctagggagccctgaggacctcgcctcgaggatagactctacgc tactaagccctagggctacggaggaggacgttaggaatcttgtgagagaggcgtcggactacgggtttagatgcgcggttctgactc cagtgtacacagtaaagatttctgggctggctgagaagcttggtgtgaagctatgtagcgttataggctttcccctgggccaggccccg ctcgaggtaaagctagttgaggcacaaactgttttagaggctggggctactgagcttgatgttgtcccccatctctcactaggccccga agctgtttacagggaggtctcagggatagtgaagttggcgaaaagctatggagccgttgtgaaagtaatattagaagcgccactctg ggatgacaaaacgctctccctcctggtggactegtcgaggagggcgggggcggatatagtgaagacaagcaccggggtctataca aagggtggtgatccagtaacggtcttcaggctggccagtcttgccaagccccttggtatgggtgtaaaggcaagcggcggtataagg agtggcatcgacgccgtcctcgccgtaggagctggcgcggatatcatagggacaagcagtgctgtaaaggttttggagagcttcaa atccctagtctaa Example 17

SEQ ID NO: 8 - Amino Acid Sequence of DERA03 mtdlkasslralklmdlttlndddtdekvialchqaktpvgntaaiciyprfipiarktlkeqgtpeiriatvtnfphgnddidialaetraaiay gadevdwfpyralmagneqvgfdlvkackeacaaanvllkviietgelkdealirkaseisikagadfiktstgkvavnatpesarim mevirdmgvektvgfkpaggvrtaedaqkylaiadelfgadwadarhyrfgassllasllkalghgdgksassy Example 18

SEQ ID NO: 9 - Amino Acid Sequence of DERA04 mgniakmidhtllkpeateqqivqlcteakqygfaavcvnptwvktaarelsgtdvrvctvigfplgattpetkafettnaiengarevd mvinigalksgqdelverdiravveaaagralvkvivetalltdeekvracqlavkagadyvktstgfsgggatvedvalmrktvgdra gvkasggvrdwktaeaminagatrigtssgvaivtggtgrady Example 19

SEQ ID NO: 10 - Amino Acid Sequence of DERA06 mglasyidhtllkatatladirtlceearehsfyavcinpvfipharawlegsdvkvatvcgfplgaisseqkalearlsaetgadeidmvi higsalagdwdaveadvravrravpeqvlkviietcyltdeqkrlatevavqggadfvktstgfgtggatvddvrlmaeviggraglkaa ggvrtpadaqamieagatrlgtsggvglvsggengagy Example 20

SEQ ID NO: 11 - Amino Acid Sequence of DERA08 mgiakmidhtalkpdttkeqiltltkeareygfasvcvnptwvklsaeqlagaeswctvigfplgantpevkafevkdaiqngakevd mvinigalkdkddelverdirawdaakgkalvkviietclltdeekvraceiavkagtdfvktstgfstggataedialmrktvgpnigvk asggvrtkedvekmieagatrigasagvaivsgekpakpdny Example 21

SEQ ID NO: 12 - Amino Acid Sequence of DERA11 mtsnqlaqyidhtaltaekneqdistlcneaiehgfysvcinsayiplakeklagsnvkictwgfplganltsvkafetqesikaganei dmvinvgwiksqkwdevkqdiqavfnacngtplkviletclltkdeivkaceickeigvafvktstgfnkggatvedvalmkntvgnigv kasggvrdtetalamikagatrigasagiaiisgtqdtqsty Example 22

SEQ ID NO: 13 - Amino Acid Sequence of DERA12 mieyrieeavakyrefyefkpvresagiedvksaiehtnlkpfatpddikklclearenrfhgvcvnpcyvklareelegtdvkNMwgf plganetrtkaheaifavesgadeidmvinvgmlkakeweyvyedirswesvkgkwkviietcyldteekiaacvisklagahfvkt stgfgtggataedvhlmkwivgdemgvkasggirtfedavkmimygadrigtssgvkivqggeerygg Example 23

SEQ ID NO: 14 - Amino acid sequence of DERA15 mpsardilqqgldrlgspedlasridstllsprateedvrnivreasdygfrcavltpvytvkisglaeklgvklcsvigfplgqaplevklve aqtvleagateldvvphlslgpeavyrevsgivklaksygavvkvileaplwddktlslivdssrragadivktstgvytkggdpvtvfrla slakplgmgvkasggirsgidavlavgagadiigtssavkvlesfkslv . AII publications, including but not limited to, issued patents, patent applications, and journal articles, cited in this application are each herein incorporated by reference in their entirety.

Although the invention has been described above with reference to the disclosed embodiments, those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention. Accordingly, the invention is limited only by the following claims.

Claims

The claimed invention is:

1. A process comprising the step of reacting an aldehyde with an aminoaldehyde substrate, a N-protected aminoaldehyde substrate or a pyrrole aldehyde substrate under aldolase-catalyzed aldo! condensation condition to form the corresponding lactol.

2. A process for producing atorvastatin or a pharmaceutically acceptable salt thereof comprising the steps of:

(b) oxidizing said lactol to the corresponding lactone; and

3. The process of claim 2, wherein said aminoaldehyde or said N-protected aminoaldehyde is 3-aminopropionaldehyde, aminoacetaldehyde, N-CBz-3- aminopropionaldehyde, N-acetyl-3-aminopropionaldehyde, N-Fmoc-3-aminopropionaldehyde, or N-Fmoc-aminoacetaldehyde.

4. The process of claim 2, wherein said aminoaldehyde or said N-protected aminoaldehyde is N-CBz-3-aminopropionaldehyde or N-Fmoc-3-aminopropionaldehyde.

5. The process of claim 2, wherein said aminoaldehyde or said N-protected aminoaldehyde is N-CBz-3-aminopropionaldehyde.

6. The process of claim 2, wherein said aldolase is a≤-deoxyribose-'5-phosphate aldolase (DERA) aldolase.

7. The process of claim 2, wherein said aldolase is OERA 03 comprising a nucleotide sequence of SEQ ID NO: 1 or an amino acid sequence of SEQ ID NO: 8;

DERA 04 comprising a nucleotide sequence of SEQ ID NO: 2 or an amino acid sequence of SEQ ID NO: 9;

DERA 06 comprising a nucleotide sequence of SEQ ID NO: 3 or an amino acid sequence of SEQ ID NO: 10; DERA 08 comprising a nucleotide sequence of SEQ ID NO: 4 or an amino acid sequence of SEQ ID NO: 11 ;

DERA 11 comprising a nucleotide sequence of SEQ ID NO: 5 or an amino acid sequence of SEQ ID NO: 12;

DERA 12 comprising a nucleotide sequence of SEQ ID NO: 6 or an amino acid sequence of SEQ ID NO: 13; DERA 15 comprising a nucleotide sequence of "SEQ ID NO: 7 or an amino acid sequence of SEQ ID NO: 14; or an aldolase having an amino acid sequence identity of at least about20% thereof.

8. The process of claim 2, wherein said aldolase is DERA 04 comprising a nucleotide sequence of SEQ ID NO: 2 or an amino acid sequence of "SEQ ID NO: 9; or -DERA 06 comprising a nucleotide sequence of SEQ ID NO: 3 or an amino acid sequence of SEQ ID NO: 10.

9. The process of claim 2, wherein said aldolase is DERA 06 comprising a nucleotide sequence of SEQ ID NO: 3 or an amino acid sequence of SEQ ID NO: 10.

10. The process of claim 2, wherein said pharmaceutically acceptable salt of atorvastatin is atorvastatin calcium.

11. A process for producing atorvastatin or a pharmaceutically acceptable salt thereof comprising the steps of:

(a) reacting an aldehyde with an aminoaldehyde substrate, a N-protected aminoaldehyde substrate or a pyrrole aldehyde substrate under aldolase÷caialyzed aldol condensation conditions to form the corresponding lactol;

(b) subjecting said lactol to catalytic dehydrogenation conditions followed by lactonization conditions to form the corresponding lactone; and

12. The process of claim 11 , wherein said aminoaldehyde or=said N-protected aminoaldehyde is 3-aminopropionaldehyde, aminoacetaldehyde, N--CBZ-3- aminopropionaldehyde, N-acetyl-3-aminopropionaldehyde, N-Fmoc-3-aminopropionaldehyde, or N-Fmoc-aminoacetaldehyde.

13. The process of claim 11 , wherein said aminoaldehyde or said N-protected aminoaldehyde is N-CBz-3-aminopropionaldehyde or N-^*Fmoc-3-aminopropionaldehyde.

14. The process of claim 11 , wherein said aminoaldehyde or said N-protected aminoaldehyde is N-CBz-3-aminopropionaldehyde.

15. The process of claim 11 , wherein said aldolase is a 2-deoxyribose-5- phosphate aldolase (DERA) aldolase.

16. The process of claim 11 , wherein. said aldolase is DERA 03 comprising a nucleotide sequence of SEQ ID NO: 1 or an amino acid sequence of SEO IDWO: 8; DERA 04 comprising a nucleotide sequence of SEQ ID NO: 2 or an amino acid sequence of SEQ ID NO: 9;

DERA 11 comprising a nucleotide sequence of ¹SEQ ID NO: 5 or an amino acid sequence of SEQ ID NO: 12;

DERA 12 comprising a nucleotide sequence of SEQ ID NO: 6 or an amino acid sequence of SEQ ID NO: 13;

DERA 15 comprising a nucleotide sequence of SEQ ID NO: 7 or an amino acid sequence of SEQ ID NO: 14; or an aldolase having an amino acid sequence identity of at least about 20% thereof.

17. The process of claim 11 , wherein said aldolase is DERA 04 -comprising a nucleotide sequence of SEQ ID NO: 2 or an amino acid sequence of SEQ ID NO: 9; or DERA 06 comprising a nucleotide sequence of ¹SEQ ID NO: 3 or an amino acid sequence of SEQ ID NO: 10.

18. The process of claim 11 , wherein said aldolase is DERA 06 comprising a nucleotide sequence of SEQ ID NO: 3 or an amino acid sequence of SEQ ID NO: 10.

19. The process of claim 11 , wherein said pharmaceutically acceptable salt of atorvastatin is atorvastatin calcium.

20. A process for producing atorvastatin or a pharmaceutically acceptable salt thereof comprising the steps of:

(a) reacting an aldehyde with an aminoaldehyde substrate, a N-protected aminoaldehyde substrate or a pyrrole aldehyde substrate under aldolaseoatalyzed aldol condensation conditions to form the corresponding lactol;

(b) ring-opening of said lactol to the corresponding earboxylic acid; and

21. The process according to claim 20, wherein said pharmaceutically acceptable salt of atorvastatin is atorvastatin calcium.