WO2014140006A1

WO2014140006A1 - Process for enantioselective synthesis of 3-hydroxy-glutaric acid monoesters and use thereof

Info

Publication number: WO2014140006A1
Application number: PCT/EP2014/054693
Authority: WO
Inventors: Burghard KÖNIG; Frank Wetterich; Harald GRÖGER; Richard METZNER
Original assignee: Sandoz Ag; Universität Bielefeld
Priority date: 2013-03-11
Filing date: 2014-03-11
Publication date: 2014-09-18
Also published as: EP2778229A1

Abstract

The present invention relates to a process for the enzymatic enantioselective synthesis of 3-hydroxy- glutaric acid esters of formula 1 with R ¹being alkyl, aryl, or substituted alkyl, as well as the use compounds of formula 1 in the synthesis.

Description

Process for enantioselective synthesis of 3-hydroxy-glutaric acid monoesters and use thereof

The present invention relates to a process for the enantioselective synthesis of 3-hydroxy- glutaric acid esters of formula 1

O OH O 1 with R¹ being alkyl, aryl, or substituted alkyl, as well as the use of compounds of formula 1 in the synthesis.

The compounds of type 1 are considered to be promising precursors in the synthesis of Rosuvastatin and its respective alkene 6 as a typically required intermediate (review of Rosuvastatin synthesis: Z. Casar, Curr. Org. Chem. 2010, 14, 816-845). The synthesis of this intermediate 6 starting from monoester 1 is outlined in the following figure 1 .

Figure 1. Use of monoester 1 in the synthesis of the phosphorus ylide 4 as intermediate for Rosuvastatin (7) Corresponding diester compounds of type 8 as well as their O-acylated derivatives of type 9

8 9

with R¹ being alkyl, aryl, or substituted alkyl, and R² being -H, alkyl, , aryl, substituted alkyl, aryloxy or benzyloxy; are considered to be easily accessible and accordingly economically extremely attractive starting compounds for the synthesis of target molecules of type 1

So far, compounds of type 1 are manufactured in a laborious way using stoichiometric amounts of chiral auxiliaries (e.g. (R)-mandelic acid benzyl ester, 11 ) and, in addition, with moderate space-time yields starting from 3-hydroxy-glutaric acid anhydride 10 (Figure 2). )[T. Konoike, Y. Araki, J. Org. Chem. 1994, 59, 7849-7854]. Another disadvantage of this existing multi-stage route is the need to first prepare the anhydride 10 from the simple and easily accessible 3-hydroxy-glutaric acid diesters of type 8 in multiple steps of synthesis. [C. H. Heathcock, C. R. Hadley, T. Rosen, P. D. Theisen, S. J. Hecker, J. Med. Chem. 1987, 30, 1858-1873]

a) Toluene

b) AcOEt/Toluene

Figure 2. Synthesis of monoester 1 from anhydride 10 using a chiral auxiliary

A direct use of easily accessible and economically highly attractive diester compounds 8 or 9 in an enantioselective catalytic synthesis process comprising only a few steps by a desymmetrizing enantioselective hydrolysis without a need for producing an anhydride as well as avoiding stoichiometric amounts of chiral auxiliaries would represent a substantially more attractive access to the synthesis of compounds of type 1.

For representative compounds of the general diester type 8, such a reaction using biocatalysts has already been reported, however, their desired direct hydrolytic conversion to monoesters of type 1 so far turned out to be difficult. Using esterases and lipases for non- O-acylated 3-hydroxy-glutaric acid diesters of type 2 (with R² = H), high enantioselectivities of up to 95 % ee were obtained, but for the "wrong" enantiomeric (S)-form. Using o chymotrypsin, the formation of the ("correct") required absolute configuration ((R)-form) is in fact observed, but here, lower, for technical purposes insufficient, enantioselectivities were achieved. So, the use of ochymotrypsin in combination with the respective dimethylesters only leads to an enantioselectivity of 76%. [E. E. Jacobsen, B. H. Hoff, A. R. Moen, T. Anthonsen, J. Mol. Cat. B Enzym. 2003, 21, 55-58] This low enantioselectivity was confirmed by our own experiments, in which, under similar reaction conditions starting from the corresponding diethyl ester as substrate, an ee-value of only about 60% ee was achieved with ochymotrypsin (comparative experiment, Figure 3).

approx. 60% ee

Figure 3. Hydrolysis of 3-hydroxy-glutaric acid diethyl ester with a-chymotrypsin

Accordingly, this synthetic access of a "direct hydrolysis" of diesters 8 has no relevance for the present-day technical manufacture of monoester compounds of type 1 despite the in principle apparent advantages of the route of synthesis.

For an alternative use of diesters of type 9 in the enzymatic hydrolysis using a-chymotrypsin instead, an efficient, fast and highly enantioselective hydrolysis forming the required ("correct") (R)-enantiomere has been described (Figure 4). [R. Ohrlein, G. Baisch, Adv. Synth. Catal. 2003, 345, 713-715 as well as EP 1 404 642] However, so far, no suitable catalyst as well as generally no process for the thereon following required reaction step of selective cleavage of the O-acyl group is known. The suitability of an enzyme as catalyst for this step seemed unlikely based on the expected lack of acceptance of charged substrates (carboxylates) of lipases and esterases as well as based on the expected side reactions of proteases. In detail, such side reactions comprise the hydrolytic cleavage of the -C(0)-OR¹ ester group of the molecule, which should be favored due to the small alcohol moiety. Consequently, the industrially developed solution, which, however, is suitable for another statin-based active compound, consists of a chemical chain extension of the obtained hydrolysis product of the diester (of general type 9) via the corresponding acid chloride into an non-polar compound, in which the polar carboxylate group is not present any more and accordingly, the conversion of this hydrophilic monoester by an esterase runs smoothly as expected, avoiding abovementioned disadvantages, and produces the target product (Figure 4).

(COCI)₂,

0 °c

O. OMe

o o o

100% yield

(CH₂CI)₂, Ethene,

AICI₃, 0 °C

O OMe

— ' cleavage of

O O O protection group O OH O

CI n OeEtt F Ecsttperraacspe ( (PP\L FE), Π CI' OEt pH 7

89% yield 76% yield

Figure 4. Efficient a-chymotrypsin-catalysed diester hydrolysis as well as preceding derivatisation of the reaction product before cleavage of the methoxy-acetyl functionality.

However, such an overall procedure cannot be transferred just like that to the synthesis of the intermediates needed for the statin Rosuvastatin. Accordingly, a synthesis process for the direct conversion of compounds of type 9 to compounds of type 1 remains desirable.

Accordingly, an object of the present invention is the provision of a synthesis of compounds of abovementioned general Formula 1

O OH O

HO'

1 which does not provide the abovementioned disadvantages and, in an efficient manner, is based on compounds of type 9

or of type 15

15

Furthermore, the necessity for producing an anhydride as well as the use of stoichiometric amounts of chiral auxiliaries, as described for these reactions in the state of the art, should be avoided.

The object was solved by a process according to the present invention, characterized in that the hydrolysis of the O-acyl bond of a compound of general formula 15,

15 wherein R¹ is alkyi, aryl, or a substituted alkyi; and R² is -H, alkyi, aryl, aryloxy or benzyloxy, is performed in the presence of an enzyme (enzyme component 2).

In another aspect of the present invention, the process further comprises the steps that in a first step a compound of general formula 9

9 wherein R¹ is alkyi, aryl, or a substituted alkyi; and R² is -H, alkyi, aryl, aryloxy or benzyloxy, is cleaved in the presence of an enzyme (enzyme component 1 ), preferably hydrolytically and enantioselectively, to the corresponding monoester of general formula 15

O

o o o

15

The compound 15 may optionally be isolated after this step.

This compound 15 may subsequently, as described above, be converted in the presence of a further enzyme (enzyme component 2) by a hydrolysis of the O-acyl group to a compound of general formula 1

1 with R¹ being alkyl, particularly aryl, substituted alkyl.

In this connection, the cleavage of the O-acetyl-group of this bi-enzymatic synthesis sequence may be considered as particularly surprising. So, the cleavage of an O-acyl-group, e.g. an O-acetyl group, in the presence of a simple ester-group using an enzyme was not to be expected in such a way (see also the above comments relating to Figure 4). Based on existing literature, it could rather be expected that, particularly in the presence of proteases as enzyme component, the cleavage occurs preferably at the remaining OEt-group instead of the O-acetyl-group. Proteases, which can accept charged compounds such as deprotonated carboxylic acids as substrates, have a high tendency of hydrolytic cleavage of simple carboxylic acids esters such as methylesters and ethylesters and would accordingly not attack, as desired, the O-acetyl-group of intermediate 15, but the simpler alkylester moiety

(particularly with R¹ = methyl, ethyl) which is also present in this molecule. At the same time, it would be expected that, in the presence of lipases and "classic" esterases such as porcine liver esterase as an alternative to the enzyme components belonging to the group of hydrolases, no further cleavage occurs since the substrate for the second hydrolysis step is a deprotonated carboxylic acid and thus, a charged compound. Esterases as well as lipases are typically "unreactive" for such substrates. This is evident, for example, for the use of porcine liver esterase for mono-hydrolysis of diesters in aqueous reaction medium. The reasons why in this case an enzymatic hydrolysis in the desired and at the same time surprising manner is possible, are still unclear and currently subject of intensive research.

In a preferred embodiment, a cephalosporin C acetylhydrolase (EC 3.1 .1 .41 ) is used herein as enzyme component 2. Cephalosporin C acetylhydrolases of Bacillus substilis [F. Knauseder, M. Schiestl, K. Schorgendorfer, US Patent US6465233, 2002.] have proven to be preferred. The cephalosporin C acetylhydrolase is preferably used in recombinant form. For the enzyme formulation, the use of Cephalosporin C-acetylhydrolase is possible in its "free" form as well as in immobilized form [W. Riethorst, A. Reichert, Chimia 1999, 600-607.] and alsoin form of a , optionally recombinant, whole-cell catalyst.

In another preferred embodiment of the introductory step of the conversion, catalyzed by enzyme component 1 , of the O-acylated diester 9 to the O-acylated monoester 15 (which then in turn represents the substrate for the above described hydrolysis in the presence of enzyme component 2 to the desired monoester of formula 1 , providing a free, non-acylated hydroxyl function in 3-position), a protease is used. In this connection, the use of o chymotrypsin, trypsin or mixtures thereof proved to be particularly preferred. These proteases may be used in any form, for example as proteins directly isolated from natural sources as well as in recombinant form. The use in "free" form as well as in immobilized form is also possible for enzyme component 1 of the enzyme formulation. Here, the biotransformation may for example be performed with purified proteins as well as with crude extracts in non-immobilized and immobilized form, respectively. The degree of purity of the used enzyme formulations is within the knowledge and discretion of the skilled person. Instead of the use of enzymes as "cell-free enzyme formulations", the use of enzymes in form of a, optionally recombinant, whole cell catalyst is also possible (which in turn also may be used in non-immobilized as well as immobilized form).

The compounds of type 1 produced by the process according to the present invention are particularly suitable for the synthesis of Rosuvastatin (7) or a salt thereof in the context of a multi-step synthesis. The individual reactions starting from a compound of type 2 to the desired Rosuvastin (7) have already been described in detail in the literature. [Review of Rosuvastatin synthesis: Z. Casar, Curr. Org. Chem. 2010, 14, 816-845]. Accordingly, after providing a compound 1 manufactured by means of the process according to the present invention, wherein R¹ is alkyl, aryl, or substituted aryl, at first a conversion from 1 to O- silylated compound 2

SiMe₂f-Bu

O o" O

2 is performed using a silylating agent of formula X-SiMe₂t-Bu, wherein X is CI, Br, followed by a transformation of compound 2 to anhydride 3

SiMe₂f-Bu

O O o" O

3 using a chloroformic acid methyl ester. The anhydride 3 then reacts with methyl- triphenylphosphonium bromide in a well-proven manner to ylide 4

SiMe₂f-Bu

Ph O o" O

Ph-,

Ph ^UK

4

Subsequently, a Wittig-reaction of compound 4 with aldehyde 5

provides compound 6

which is finally converted in an established manner according to methods known in the art via hydrolysis, reduction and neutralization, e.g. with a basic calcium salt to the desired Rosuvastatin (7) or salt thereof

Rosuvastatin (7) salt

The formed intermediates may be isolated and purified before the following reaction step, respectively. Alternatively, the isolation and purification of the intermediates may also be omitted and the formed reaction mixtures or the respective raw products obtained after extraction and optional removal of solvent components, comprising the desired intermediate, may be directly used in the subsequent reaction step.

A particular preferred embodiment of the invention comprises a process for the manufacture of Rosuvastatin (7), characterized in that a compound 1 , obtained according to the process as described above, is converted to Rosuvastatin (7).

In another particular preferred embodiment, the process for the synthesis of Rosuvastatin (7) comprises the following synthesis steps of:

a) providing a compound 1 , wherein R¹ is alkyl, aryl, or substituted alkyl, synthesized according to the process of the present invention, as described above, b) converting compound 1 using i-butyldimethylchorosilane (Me₂(i-Bu)SiCI) to silylated compound 2

SiMe₂f-Bu

O θ' O

2 c) converting compound 2 using chloroformic acid methyl ester to compound 3

SiMe₂f-Bu

O O o" O

3 d) subsequent converting compound 3 using methyltriphenylphosphonium halide, such as - bromide and/or -chloride, to compound 4

e) subsequent converting of compound 4 with compound 5

in a Wittig-reaction to compound 6

f) subsequent converting of compound 6 to Rosuvastatin (7) or a salt thereof

Rosuvastatin (7)

In a particular preferred embodiment, the formed intermediate compound 15 and/or 1 and/or 2 and/or 3 and/or 4 and/or 6 is/are not isolated but the reaction solution containing the respective compound 15 and/or 1 and/or 2 and/or 3 and/or 4 and/or 6 is/are further directly used in situ, without processing and purification, in the subsequent synthesis step.

The term "alkyl", as used herein, refers to a linear or branched hydrocarbon with 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms (-(Ci-Ci₀)alkyl). Examplary -(Ci-Ci₀)alkyls are methyl, ethyl, n- propyl, /^'-propyl, n-butyl, sec-butyl, ie f-butyl, 3-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl and n-decyl. In one embodiment, the alkyls are selected from linear or branched -(CrC₄)alkyls. Examplary -(Ci-C₄)alkyls are methyl, ethyl, n-propyl, /^'-propyl, n-butyl, sec-butyl, or ie f-butyl.

The term "aryl", as used herein, relates to -(C₆-Ci₄)aryls. Typical examples for -(C₆-Ci₄)aryls are phenyl, naphthalenyl, phenanthryl, indenyl or the like. (C₆-Ci₄)aryls are preferred. A particular preferred aryl is phenyl.

The term "substituted alkyl", as used herein, relates to a linear or branched hydrocarbon with 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms (-(Ci-Cio)alkyl), in which one or more hydrogen atoms of the hydrocarbon is/are replaced by other substituents. Representative substituents are halogen (i.e. -F, -CI, -Br, or -I), phenyl, -O(Ci-Ci₀)alkyl, or the like. Preferred substituted alkyls are, for example, chloromethyl, bromomethyl, iodomethyl, methoxymethyl, ethoxymethyl, or benzyl.

The term "benzyloxy", as used herein, means -0-CH₂-phenyl.

The term "aryloxy", as used herein, means that one of the abovementioned aryl-groups is bound via an oxygen atom, i.e. -O-aryl. A typical example for an aryloxy-group is phenoxy, i.e. -O-Ph.

The term that an enzyme is used "in free form" means in the context of the present invention that the enzyme is not immobilized. This also includes the use of the enzymes as whole cell catalysts, i.e. the use of cells that provide the desired enzymes.

The term "enantioselective" as used herein means that one of the two possible enantiomers is preferably formed by conversion of a prochiral reactant. The enantiomeric excess or "ee" indicates the degree of enantioselectivity: major enantiomer (mol) - minor enantiomer (mol)

% ee = x 100%

major enantiomer (mol) + minor enantiomer (mol) The illustration of compounds, such as compound 1 or 15, shows the respective preferred absolute configuration. The disclosure however also comprises the other possible configurations.

The term "salt", as used herein, relates preferably to a salt that may be formed by an acidic or basic functional group. Typical examples include sulfates, citrates, acetates, chlorides, bromides, iodides, nitrates, phosphates, sulfonates or the like. Further, salts of the compounds that provide an acidic functional group such as a carboxylic acid include salts with acceptable inorganic and organic bases. Suitable bases include, for example, hydroxides of alkaline metals such as sodium, potassium, cesium or lithium; hydroxides of alkaline earth metals such as calcium or magnesium or hydroxides of other metals.

Experimental examples:

acetylation of 3-hydroxy-glutaric acid diethylester

M = 204.22 g/mol Ci i Hi₈0₆

M = 246.26 g/mol

Zinc perchlorate-hexahydrate (78.2 mg, 0.21 mmol, 0.10 mol%) was added to acetic anhydride (19.9 mL, 210 mmol, 1 .05 eq.) and stirred for 10 minutes at room temperature. 3- Hydroxy-glutaric acid was added to the clear, colorless solution and stirred for further 4 hours at room temperature. Distilled water (50 mL) was added and the emulsion was stirred for another 15 minutes at room temperature. After addition of methyl ie f-butyl ether (MTBE, 100 mL), the phases were separated and the organic phase was washed thrice with saturated NaHC0₃-solution (100 mL each). The organic phase was dried over MgS0₄ and the solvent was completely removed in vacuo. 3-Acetoxy-glutaric acid diethylester (47.9 g, 195 mmol, 97.5%) was obtained as a colorless, viscous liquid. Example 2: Desymmetrization of prochiral 3-acetoxy-glutaric acid diethylester using a hydrolase (with isolation of the intermediate)

C-i -i H-isOe CgH-^Oe M = 246.26 g/mol M = 218.20 g/mol

3-Acetoxy-glutaric acid diethylester (24.6 g, 100.0 mmol) was emulsified in a phosphate buffer (15.0 ml_, 50mM, pH 8.0) by heavily stirring with a propeller mixer. An enzyme formulation of chymotrypsin/trypsin in a 1 :1 ratio (1 .5 g, Biozym CHY-04) was dissolved in phosphate buffer (10.0 ml_, 50mM, pH 7.0) and the pH-value was adjusted to pH 8.0 using 4.0 M NaOH. After addition of this enzyme solution to the substrate mixture, the resulting emulsion was stirred at room temperature for about 27 hours (time of the stoichiometric titer consumption) and the pH was maintained at pH 8.0 ± 0.3 using a pH-Stat (Titrino, 4.0 M NaOH). Here, the resulting carboxylic acid was neutralized with 4.0 M NaOH and the reaction process was monitored by the (base) consumption. Subsequently, the reaction solution was saturated with solid sodium chloride and adjusted to about pH 1 -2 with cone. HCI. The denatured hydrolase was separated over a fritted-glass filter and the filtrate was extracted twice with ethyl acetate (100 mL each). The organic phase was dried over MgS0₄ and the solvent was removed in a rotary evaporator. (3R)-acetoxy-5-ethoxy-5-oxy-valeric acid was obtained as a colorless, viscous liquid. The ee-value analysis over the (S)-1 - phenylethylamine-derivatives (Daicel ChiralPak OJ-H, SC-C0₂//^'-PrOH 95:5 (v/v), 0.8 mL/min, 10 MPa back pressure, 220 nm) showed a diastereomeric excess of 97.5% de, corresponding to an enantiomeric excess of 97.5% ee.

Example 3: Deacetylation of enantiomer-enriched Hydro-(3 ?)-acetoxy-glutaric acid ethylesters using a hydrolase (with isolation of the intermediate)

M = 218.20 g/mol (3R)-Acetoxy-5-ethoxy-5-oxy-valeric acid (20.7 g, 95.0 mmol) was emulsified in phosphate buffer (24 mL, 50 mM, pH 8.0) and the pH was adjusted to pH 8.0 with 4.0 M NaOH. After addition of immobilized cephalosporin C acetylhydrolase (1.0 g, obtained according to W. Riethorst, A. Reichert, Chimia 1999, 600-607), the solution was stirred at room temperature and the pH was maintained using a pH-Stat (Titrino, 4.0 M NaOH). Here, the resulting carboxylic acid was neutralized with 4.0 M NaOH and the reaction process was monitored by the (base) consumption. The reaction process showed a complete conversion after about 26 hours. Subsequently, the reaction solution was saturated with solid sodium chloride, the pH was adjusted to about pH 1 -2 with cone. HCI and extracted thrice with ethyl acetate (75 mL each). The combined organic phases were dried over MgS0₄ and the solvent was completely removed in a rotary evaporator. (3R)-5-Ethoxy-3-hydroxy-5-oxy-valeric acid (13.9 g, 78.9 mmol, 83.0% yield) was obtained as a colorless, viscous liquid.

Example 4: Bi-enzymatic, sequential one-pot process for the synthesis of (3 ?)-5- ethoxy-3-hydroxy-5-oxy-valeric acid

3-Acetoxy-glutaric acid diethylester (49.2 g, 200.0 mmol) was emulsified in phosphate buffer (30.0 mL, 50mM, pH 8.0) by heavily stirring with a propeller mixer. An enzyme formulation of chymotrypsin/trypsin in a ratio of 1 :1 (3.0 g, Biozym CHY-04) was dissolved in phosphate buffer (20.0 mL, 50mM, pH 7.0) and the pH was adjusted to pH 8.0 with 4.0 M NaOH. After addition of this enzyme solution to the substrate mixture, the resulting emulsion was stirred for about 27 hours (time of stoichiometric titer consumption) at room temperature and the pH was maintained at pH 8.0 ± 0.3 using a pH-Stat (Titrino, 4.0 M NaOH). Here, the resulting carboxylic acid was neutralized with 4.0 M NaOH and the reaction progress was monitored by the (base) consumption. Subsequently, the homogeneous reaction solution was transferred to an ultrafiltration stirring cell and separated from the homogeneously dissolved chymotrypsin/trypsin mixture at 800 rpm and 2.0 bar argon positive pressure using an ultrafiltration membrane (MWC 10,000 Da). Immobilized cephalosporin C acetylhydrolase (2.0 g) was given to the filtrate and the suspension was stirred at room temperature. The pH was maintained at pH 8.0 ± 0.3 using a pH-Stat (Titrino, 4.0 M NaOH). Here, the resulting carboxylic acid was neutralized with 4.0 M NaOH and the reaction progress was monitored by the (base) consumption. The reaction process showed a complete conversion after about 26 hours. Subsequently, the suspension was separated from the immobilized enzyme using a fritted-glass filter (Por 4). The filtrate was saturated with solid sodium chloride, adjusted to pH 1 -2 with cone. HCI and subsequently extracted thrice with ethylacetate (100 mL each). The combined organic phases were dried over MgS0₄ and the solvent was completely removed in a rotary evaporator. (3R)-5-Ethoxy-3-hydroxy-5-oxy-valeric acid (33.1 g, 188 mmol, 94% yield) was obtained as light-red, viscous liquid. A selectivity of 98.1 % ee (Daicel ChiralPak OJ-H, SC-C0₂/ -PrOH 95:5, 0.8 mL/min, 10 MPa back pressure, 220 nm) was determined by re-acetylation and derivaterization with (S)-l -phenylethylamine.

Claims

1 . A process for the synthesis of a compound of general formula 1

1 wherein R¹ is alkyi, aryl or substituted alkyi, comprising the reaction of a compound of general formula 15

15 wherein R¹ is alkyi, aryl or substituted alkyi; and R² is -H, alkyi, aryl, substituted alkyi, aryloxy or benzyloxy, characterized in that the hydrolysis of the O-acyl bond is performed in the presence of an enzyme (enzyme component 2).

2. Process according to claim 1 , further comprising the following steps: a) reacting the compound of formula 9

9 in the presence of an enzyme (enzyme component 1 ) hydrolytically to a monoester of general formula 15, wherein R¹ is alkyi, aryl or substituted alkyi; and R² is -H, alkyi, aryl, substituted alkyi, aryloxy or benzyloxy; b) optionally isolating compound 15.

3. Process according to claim 1 , wherein the enzyme component 2 comprises an acyl hydrolase.

4. Process according to claim 1 or 2, wherein acetyl hydrolase is used as enzyme component 2.

5. Process according to any one of claims 1 to 4, wherein a Cephalosporin C-acetyl hydrolase (EC 3.1.1.41 ) is used as enzyme component 2.

6. Process according to any one of claims 1 to 5, wherein a Cephalosporin C-acetyl hydrolase of Bacillus substilis is used as enzyme component.

7. Process according to any one of claims 1 to 6, wherein water, optionally with added buffer components or salts is used as reaction medium.

8. Process according to any one of claims 1 to 7, wherein the conversions of the overall process are performed with volumetric amounts of substrates (substrate concentrations) of >100 mM, preferably > 1 M and even more preferably >2M.

9. Process according to any one of claims 1 to 8, wherein the compound 9 and/or 15 as substrate component in the respective reaction step

(a) is added over a specified period;

(b) is provided in full from the beginning; or

(c) an amount is provided at the beginning and the remaining amount is subsequently added.

10. Process according to any one of claims 1 to 9, wherein a protease is used as enzyme component 1 .

1 1 . Process of claim 10, wherein the protease comprises a-Chymotrypsin, Trypsin or mixture thereof.

12. Process according to any one of claims 1 to 1 1 , wherein the enzymes are used in free form.

13. Process for the manufacture of Rosuvastatin (7), the process comprising the following steps a) providing a compound 1 obtained by a process of any one of claims 1 to 12; b) converting the compound 1 to Rosuvastatin (7) or a salt thereof.

14. Process according to any one of claims 1 to 13, wherein R¹ is methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, tert-butyl, phenyl or benzyl.

15. Process according to any one of claims 1 to 14, wherein R² is methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, tert-butyl, chlormethyl, methoxymethyl, phenyl or benzyl.