Enzymatic hydroxylation process for the preparation of HMG-CoA reductase inhibitors and intermediates thereof

An enzymatic hydroxylation process for the preparation of compounds useful as HMG-CoA reductase inhibitors and/or as intermediates in the preparation of HMG-CoA reductase inhibitors uses a microorganism or an enzyme derived from, or having the structure of an enzyme derived from, said microorganism, which is capable of catalyzing the hydroxylation process.

FIELD OF THE INVENTION 
The present invention relates to an enzymatic hydroxylation process for the 
preparation of compounds useful as HMG-CoA reductase inhibitors and/or as 
intermediates in the preparation of HMG-CoA reductase inhibitors. 
SUMMARY OF THE INVENTION 
The present invention provides a method for the preparation of a compound 
of the formula I: 
##STR1## 
its partially or completely hydrogenated analogs or a salt thereof, 
wherein 
R is alkyl or aryl; 
Z is the open chain moiety 
##STR2## 
the lactone 
##STR3## 
R.sup.2 is hydrogen, alkyl, ammonium, alkyl-ammonium or alkali metal; 
comprising the step of contacting a compound of the formula II: 
##STR4## 
a partially or completely hydrogenated analog or a salt thereof, wherein 
R, Z and R.sup.2 are as defined for formula I, with a microorganism, or 
with an enzyme derived from, or having the structure of an derived from 
said microorganism, which is capable of catalyzing the hydroxylation of 
said compound of the formula II to yield said compound of the formula I, 
and effecting said hydroxylation; 
where said microorganism is selected from the genera Nocardia, Amycolata, 
Saccharopolyspora, Streptomyces, Amycolatopsis, Saccharothrix or 
Gilbertella, provided that when the compound of formula II is compactin, 
the microorganism is not Amycolata, Nocardia or Streptomyces. 
The enzymatic hydroxylation process of the present invention provides an 
efficient means for obtaining compounds of the formula I, which may 
themselves exhibit HMG-CoA reductase inhibitory activity, and/or which may 
be used as intermediates in the preparation of other HMG-CoA reductase 
inhibitors. Reduction or elimination of byproducts may be achieved by 
employing the hydroxylation method of the present invention, which method 
may also be conducted under mild reaction conditions. 
DETAILED DESCRIPTION OF THE INVENTION 
The method of the present invention is described further as follows. 
Definitions 
The terms "enzymatic process" or "enzymatic method" as used herein denote a 
process or method employing an enzyme or microorganism. 
The term "alkyl" as used herein, alone or as part of another group, denotes 
both straight and branched chain, optionally substituted hydrocarbons 
containing 1 to 12 carbons in the normal chain, preferably 1 to 6 carbons, 
such as methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, 
pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 
2,2,4-trimethyl-pentyl, nonyl, decyl, undecyl, dodecyl, the various 
branched chain isomers thereof, and the like. Exemplary substituents may 
include one or more groups selected from the following: halo (especially 
chloro), trihalomethyl, alkoxy (for example, where two alkoxy substituents 
form an acetal), aryl such as unsubstituted aryl (e.g., phenyl), 
alkyl-aryl or haloaryl, cycloalkyl such as unsubstituted cycloalkyl or 
alkyl-cycloalkyl, hydroxy or protected hydroxy, carboxyl, 
alkyloxy-carbonyl, alkylamino, dialkylamino such as dimethylamino, 
alkylcarbonylamino such as acetylamino, amino, arylcarbonylamino, nitro, 
cyano, thiol, or alkylthio. Preferred alkyl substituents are hydroxy 
groups. 
The term "alkenyl" as employed herein, alone or as part of another group, 
denotes optionally substituted straight or branched chain hydrocarbon 
groups as described above for alkyl, further containing at least one 
carbon to carbon double bond. 
The term "cycloalkyl" as employed herein, alone or as part of another 
group, denotes an optionally substituted, saturated homocyclic carbon ring 
system, preferably containing from 1 to 3 rings and from 3 to 12, 
preferably from 3 to 8, carbons per homocyclic ring, such as cyclopropyl, 
cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, 
cyclododecyl, and adamantyl. Exemplary optional substituents include one 
or more alkyl groups as described above, or one or more of those groups 
described above as alkyl substituents. 
The term "aryl" as used herein denotes monocyclic or bicyclic substituted 
or unsubstituted aromatic groups containing from 6 to 12 carbons in the 
ring portion, such as phenyl, biphenyl, naphthyl, substituted phenyl, 
substituted biphenyl or substituted naphthyl. Exemplary substituents 
(preferably three or fewer) include one or more of the following groups: 
alkyl such as unsubstituted alkyl, haloalkyl or cycloalkylalkyl, halogen, 
alkoxy such as unsubstituted alkoxy or haloalkoxy, hydroxy, aryl such as 
phenyl or halophenyl, aryloxy such as phenoxy, alkylcarbonyloxy or 
aroyloxy, allyl, cycloalkyl, alkylamino, dialkylamino, amido such as 
alkylcarbonylamino or arylcarbonylamino, amino, nitro, cyano, alkenyl, 
thiol, alkylcarbonyl, or arylcarbonyl, or methylenedioxy where the 
methylene group may be substituted by lower alkyl group(s) (that is, alkyl 
groups as described above having 1 to 6 carbon atoms), arylalkenyl 
group(s), and/or alkylthio group(s). 
The terms "halo" or "halogen" as used herein denote chlorine, fluorine, 
bromine or iodine. 
The term "salt(s)" as employed herein refers to acidic and/or basic salts 
formed with inorganic and/or organic bases. The nontoxic, pharmaceutically 
acceptable salts are preferred. Exemplary pharmaceutically acceptable 
salts include those formed from cations such as sodium, potassium, 
aluminum, calcium, lithium, magnesium, zinc and tetramethylammonium as 
well as those salts formed from amines such as ammonia, ethylenediamine, 
N-methylglucamine, lysine, arginine, ornithine, choline, 
N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, 
N-benzylphenethylamine, 
1-p-chlorobenzyl-2pyrrolidine-1'-yl-methylbenzimidazole, diethylamine, 
piperazine and tris(hydroxymethyl)aminomethane. 
The term "pharmaceutically acceptable cation" denotes a positive counterion 
forming a pharmaceutically acceptable salt, such as those described above. 
The term "ATCC" as used herein refers to the accession number of the 
American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 
20852, an organism depository. 
Starting Materials 
Compounds of the formula II to be employed in the hydroxylation method of 
the present invention may be obtained by methods known to the skilled 
artisan. Such compounds are disclosed, for example, in U.S. Pat. No. 
4,450,171. 
Preferred Compounds 
Compounds of the formula I having the following formula Ia are preferred: 
##STR5## 
its partially or completely hydrogenated analogs or alkali metal salts 
thereof, wherein R is alkyl and R.sup.2 is as defined in formula I. 
A particularly preferred example is: 
##STR6## 
its partially or completely hydrogenated analogs or alkali metal salts 
thereof. 
Compounds of the formula II having the following formula IIa are preferred 
for use as the starting material: 
##STR7## 
its partially or completely hydrogenated analogs or alkali metal salts 
thereof, wherein R is alkyl and R.sup.2 is as defined in formula II. A 
particularly preferred example is 
##STR8## 
its partially or completely hydrogenated analogs or alkali metal salts 
thereof. 
Compounds of the formula I, or any compound described herein, in which one 
or more of the double bonds is absent, may be obtained by hydrogenating 
the corresponding compound in which such bonds are present according to 
methods known to the skilled artisan. 
Any of the products of the present method may be isolated and purified by 
known methodologies such as by filtering off cells or cellular materials 
where appropriate, extraction, crystallization, thin layer or column 
chromatography, high performance liquid chromatography and the like. 
The above structures shown for methyl compactin and methyl pravastatin are 
referred to herein as the acid form of these compounds. Where the carboxyl 
group of these compounds is in the alkali metal salt form, the compounds 
are referred to herein as the salt form. 
As discussed below, the use of an aqueous medium is preferred in conducting 
the hydroxylation method of the present invention. It is therefore 
preferred to prepare, or to employ as starting materials, those compounds 
in which Z is the open chain moiety as defined above since such compounds 
are relatively more water soluble than the corresponding compounds in 
which Z is a lactone moiety. Compounds of the formula II in which Z is a 
lactone moiety may, for example, be hydrolyzed to the open chain form 
prior to use in the process of the present invention. 
Enzymes and Microorganisms 
The enzyme or microorganism employed in the method of the present invention 
may be any enzyme or microorganism, regardless of origin or purity, having 
the ability to catalyze the conversion as described herein. Genera of 
microorganisms suitable as sources of catalyzing enzymes include Nocardia, 
Amycolata, Saccharopolyspora, Streptomyces, Amycolatopsis, Saccharothrix 
or Gilbertella. 
Exemplary species suitable for use in the present invention include 
Amycolata autotrophica such as ATCC 35204, Streptomyces californicus such 
as ATCC 15436, Amycolatopsis mediterranei such as ATCC 21411, 
Saccharothrix australensis such as ATCC 31497, Gilbertella persicaria such 
as ATCC 38591, Saccharopolyspora hirsuta such as ATCC 27875, 27876 or 
20501, Saccharopolyspora erythraea such as ATCC 11635, and the like. 
Particularly preferred are Amycolata autotrophica such as ATCC 35204 and 
Saccharopolyspora hirsuta such as ATCC 20501. 
With respect to the use of microorganisms, the method of the present 
invention may be carried out using any microbial cellular material having 
the ability to catalyze the conversion as described herein. The cells may 
be used in the form of intact wet cells or dried cells such as 
lyophilized, spray-dried or heat-dried cells. Cells may also be used in 
the form of treated cell material such as ruptured cells or cell extracts. 
The cells or cellular materials, such as isolated fungal mycelia, may be 
employed in the free state or immobilized on a support such as by physical 
adsorption or entrapment. One or more species of microorganism may be 
employed when carrying out the instant process. 
The method of the present invention may be carried out subsequent to the 
growth of the microorganism(s) employed, for example, by growing the 
microorganism(s) either in the presence or absence of a compound of the 
formula II starting material, harvesting and, preferably, washing (e.g., 
with water) the microbial materials, and then contacting the microbial 
materials obtained with the compound of the formula II starting material. 
The method of the present invention may also be carried out by in situ 
fermentation and reaction, that is, reaction in the presence of actively 
growing microorganisms. 
The reaction may be conducted under quiescent (static) conditions, or by 
employing agitation. Agitation, such as shake-flask culture or aeration 
and agitation, is preferably employed when the compound of the formula II 
starting material is added to actively growing cultures. In such cases, an 
anti-foaming agent may be employed. 
The growth of microorganisms may be achieved by the skilled artisan, for 
example, by the use of an appropriate medium containing nutrients such as 
carbon and nitrogen sources and trace elements. Exemplary assimilable 
carbon sources include glucose, glycerol, maltose, dextrin, starch, 
lactose, sucrose, molasses, soybean oil, cotton seed oil, etc. Exemplary 
assimilable nitrogen sources include soybean meal, peanut meal, cotton 
seed meal, fish meal, corn steep liquor, peptone, rice bran, meat extract, 
yeast, yeast extract, sodium nitrate, ammonium nitrate, ammonium sulfate, 
etc. Inorganic salts such as sodium chloride, phosphates, calcium 
carbonate, etc., may be added to the culture medium. A minor amount of a 
metal salt or heavy metal may also be added. 
The same or different media may be employed at various stages of the growth 
of the microorganisms. Preferred media for the growth of microorganisms 
are those described in the examples herein, which media may be employed 
for the growth of any microorganism employed in the method of the present 
invention. 
Enzymes, when employed, are preferably derived from the aforementioned 
microorganisms, or they may be synthetically or otherwise prepared. For 
example, they may be derived from genetically engineered host cells. The 
use of the genetically engineered host cells themselves, or cells which 
have otherwise been modified, is also contemplated where such cells are 
capable of producing enzymes having the structure of enzymes derived from 
the above recited genera of microorganisms. 
Reaction Conditions 
The method of the present invention may be conducted in an aqueous medium, 
such as a buffered aqueous medium. The aqueous phase is conveniently 
water, preferably deionized water, or a suitable aqueous buffer solution, 
especially a phosphate buffer solution. Use of an aqueous medium is 
preferred for the present hydroxylation method. 
The reaction in the present invention may also be conducted in an organic 
medium or in a medium which is a mixture of an organic medium and an 
aqueous medium. Use of an organic or organic/aqueous medium may enhance 
solubilization of the less water soluble compounds of the formula II 
starting materials, such as those where Z is a lactone moiety. Less water 
soluble starting materials may, for example, be dissolved in an organic 
solvent such as methyl or ethyl alcohol, and the solution added to an 
aqueous medium for conversion. Liquids forming such organic media may be 
immiscible in water or, preferably, may be miscible in water. Exemplary 
organic media include toluene, hexane, benzene, acetone, 
dimethylsulfoxide, cyclohexane, xylene, trichlorotrifluoroethane, alkanols 
such as methyl or ethyl alcohol or butanol, and the like. 
It is preferred that the starting material is dissolved, for example, in 
water or an alcohol, prior to addition to the reaction medium. 
The reaction medium preferably contains between about 0.5 to about 3 mg of 
a compound of the formula II starting compound per ml of liquid medium. 
The pH of the reaction medium is preferably between about 6.0 and about 
7.5. 
To carry out the hydroxylation reaction of the present invention, water or 
an organic alcohol, for example, an alkanol such as methyl or ethyl 
alcohol, may be added. It is preferred to employ these materials in an 
amount providing a molar excess, preferably a large molar excess, based on 
the compound of formula II starting material. 
The amount of microbial cells added, where employed in the present process, 
is preferably an amount ranging from about 10 to about 1000 mg per mg of 
the compound of formula II starting material. The amount of enzyme added, 
where employed in the present process, is preferably an amount ranging 
from about 1 to about 100 mg per mg of the compound of formula II starting 
material. 
The reaction medium is preferably held at a temperature between about 27 
and 40.degree. C., and is most preferably held between about 28 and about 
34.degree. C. The reaction time can be appropriately varied depending upon 
the amount of enzyme produced by the microbial cells, or used per se, and 
its specific activity. Typical reaction times are between about 2.5 hours 
and about 72 hours. Reaction times may be reduced by increasing the 
reaction temperature and/or increasing the amount of enzyme added to the 
reaction solution. 
Preparation of HMG-CoA Reductase Inhibitors 
HMG-CoA reductase (3-hydroxy-3-methylglutaryl coenzyme A reductase, EC 
1.1.1.34) is a key enzyme in cholesterol biosynthesis. Inhibitors of this 
enzyme find utility as anticholesterolemic agents, that is, in lowering or 
maintaining plasma cholesterol levels. In addition to the treatment and 
prevention of hypercholesterolemia, HMG-CoA reductase inhibitors find 
utility in the treatment and prevention of atherosclerosis, 
hyperlipoproteinaemia, and/or hyperlipidemia. 
Compounds of the formula II described above may themselves exhibit HMG-CoA 
reductase inhibiting activity (e.g., methyl compactin), and/or may be 
employed as intermediates in the preparation of other compounds having 
HMG-CoA reductase inhibiting activity. In the latter case, the present 
invention further provides a method wherein hydroxylation is conducted 
according to the above-described method of the present invention and, 
subsequently, the hydroxylated product so formed is employed in the 
preparation of an HMG-CoA reductase inhibitor (e.g., groups are 
deprotected, added or otherwise modified thereon). Preferably, the 
inhibitor so prepared has enhanced HMG-CoA reductase inhibiting activity 
relative to any such activity the hydroxylated product from which it is 
prepared may possess. 
HMG-CoA reductase inhibitors obtained according to the method of the 
present invention may, for example, be administered to mammals, 
particularly humans, by modes and in dosages selected according to methods 
known to the skilled artisan. 
A particularly preferred method for the preparation of an HMG-CoA reductase 
inhibitor of the present invention is that comprising the steps of: 
(A) hydroxylating a compound of the formula IIa, wherein R is 
##STR9## 
and R.sup.2 is H, or a salt thereof with Amycolata autotrophica (ATCC 
35204) or Saccharopolyspora hirsuta subspecies kobensis (ATCC 20501) to 
yield methyl pravastatin having the structure: 
##STR10## 
or the alkali metal, especially sodium or lithium, salt thereof.