Microbial transformation of simvastatin

Compounds of formula (I) and (II): ##STR1## are HMG-CoA reductase inhibitors.

BACKGROUND OF THE INVENTION 
Hypercholesterolemia is known to be one of the prime risk factors for 
atherosclerosis and coronary heart disease, the leading cause of death and 
disability in western countries. The bile acid sequestrants seem to be 
moderately effective as antihypercholesterolemic agents but they must be 
consumed in large quantities, i.e., several grams at a time, and they are 
not very palatable. 
MEVACOR.RTM. (lovastatin), now commercially available, is one of a group of 
very active antihypercholesterolemic agents that function by limiting 
cholesterol biosynthesis by inhibiting the enzyme, HMG-CoA reductase. In 
addition to the natural fermentation products, mevastatin and lovastatin, 
there are a variety of semi-synthetic and totally synthetic analogs 
thereof. For example, simvastatin wherein the 8-acyl moiety is 
2,2-dimethylbutyryl is an even more potent HMG-CoA reductase inhibitor 
than lovastatin. 
The naturally occurring compounds and their semi-synthetic analogs have the 
following general structural formulae: 
##STR2## 
wherein: Z is hydrogen, C.sub.1-5 alkyl or C.sub.1-5 alkyl substituted 
with a member of the group consisting of phenyl, dimethylamino, or 
acetylamino; and 
R* is: 
##STR3## 
wherein Q is 
##STR4## 
or R.sup.3 --CH; R.sup.3 is H or OH; M is 
##STR5## 
R.sup.4 is hydrogen or hydroxy; X is CR.sup.5 R.sup.6, O, S, or NH; 
R.sup.5 and R.sup.6 are H, OH, or OR.sup.7 where R.sup.7 represents a 
phosphoryl or acyl moiety; 
R.sup.2 is hydrogen or methyl; and a, b, c, and d represent single bonds, 
one of a, b, c or d represents a double bond, or both a and c or both b 
and d represent double bonds provided that when a is a double bond, Q is 
##STR6## 
and when d is a double bond, M is 
##STR7## 
and provided that when R.sup.5 or R.sup.6 is OH or OR.sup.7 or X is O, S, 
or NH, a, b, and c are single bonds. 
U.S. Pat. No. 4,517,373 discloses semi-synthetic hydroxy containing 
compounds represented by the above general formula wherein R* is 
##STR8## 
U.S. Pat. No. 4,537,859 U.S. Pat. No. 4,448,979 also disclose 
semi-synthetic hydroxy-containing compounds represented by the above 
general formula wherein R* is 
##STR9## 
These compounds are prepared by the action of certain microorganisms on the 
corresponding non-hydroxylated substrates. One such organism described in 
U.S. Pat. No. 4,537,859 is of the genus Nocardia. 
Copending U.S. patent application Ser. No. 213,010 filed June 29, 1988 
discloses 5-oxygenated compounds of the above formula wherein R* is: 
##STR10## 
wherein R.sub.5 and R.sub.6 independently are H, OH or an oxygenated 
derivative OR.sub.7 provided that one and only one of R.sub.5 and R.sub.6 
is OH or OR.sub.7. 
Copending U.S. patent application Ser. No. (250,646) filed Sept. 29, 1988 
discloses a chemical methodology to the 5-oxygenated compounds described 
above. 
British patent No. GB 2,075,013 discloses compounds of the above formula 
wherein R* is: 
##STR11## 
wherein A amongst other groups is C.dbd.O, B amongst other groups is 
--CHOR.sup.3 in which R.sup.3 represents an H or a acyl group, R.sup.1 is 
an H or a methyl group and R.sup.2 is an H or a acyl group. The 3-keto, 
5-hydroxy compound in this disclosure is formed in minor amounts from a 
chemical oxidation of the hexahydro starting material.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to compounds of formula (I) and (II): 
##STR12## 
wherein: Z is H, C.sub.1-5 alkyl, or C.sub.1-5 alkyl substituted with a 
member of the group consisting of phenyl, dimethylamino or acetylamino; 
and pharmaceutically acceptable salts of the compounds of formula (II) in 
which Z is hydrogen. 
Compound (I) is prepared in a microbial transformation from simvastatin 
employing a novel microorganism (MA 6559) tentatively identified as an 
Actinoplanacete sp. The process involves the bioconversion of substrate 
(II) with the microorganism MA 6559. 
##STR13## 
The acyl moiety 
##STR14## 
can be branched or straight, preferably it is 2-methylbutyryl or 
2,2-dimethylbutyryl, most preferably 2,2-dimethylbutyryl. 
The characteristics of microorganism MA 6559 tentatively identified as 
Actinoplanacete sp. are described below: 
Microscopic observations--Culture grows as branched filaments ranging of 
approximately 6 microns diameter. Spherical to ovoid sporangia are 
detected on glycerolasparagine agar, oatmeal agar, yeast-malt extract agar 
and inorganic salts-starch agar. Sporangia range in size from 2.5-44 
microns in diameter. 
Oat Meal Agar 
Vegetative Growth: Reverse is hyaline 
Aerial Mass: Moderate, off white, powdery 
Soluble Pigment: None 
Glycerol-Asparagine 
Vegetative Growth: Obverse is mahogany 
Aerial Mycelium: Off white and cottony at periphery turning to dusty rose 
and powdery at colony center 
Soluble Pigment: Very light brown 
Inorganic Salts-Starch Agar 
Vegetative Growth: Mahogany 
Aerial Mycelium: Off white and cottony at periphery turning to dustry rose 
and powdery at colony center 
Soluble Pigment: Areas of browning around the periphery of growth with 
slight clearing of starch 
Yeast Extract-Malt Extract Agar 
Vegetative Growth: Mahogany to brown black 
Aerial Mass: Isolated areas of white, cottony growth against a powdery 
dusty rose colored mycelial matte 
Soluble Pigment: Yellow-brown 
Egg Albumin Agar 
Vegetative Growth: Pale yellow, flat 
Aerial Mass: Sparse, white and cottony limited to periphery of growth 
Soluble Pigment: None 
Nutrient Tyrosine Agar 
Vegetative Growth: Transparent to pale yellow Aerial Mass: None 
Soluble Pigment: None 
Decomposition of tyrosine: Negative 
Skim Milk Agar 
Vegetative Growth: Leathery and yellow 
Aerial Mass: Sparse, off white and powdery 
Soluble Pigment: None 
Hydrolysis of casein: Positive 
Tomato Paste Oatmeal Agar 
Vegetative Growth: Orange-yellow, rugose 
Aerial Mass: Powdery, varying in color from off white to purple-brown 
Gelatin Stabs 
Vegetative Growth: Orange yellow 
Aerial Mass: None 
Soluble Pigment: None 
Liquification of gelatin: Positive 
Peptone-Iron-Yeast Extract Agar Slants 
Vegetative Growth: Colorless, leathery 
Aerial Mass: Moderate, off white, powdery 
Soluble Pigment: None 
Melanin: Negative 
H.sub.2 S: Negative 
Tryptone Yeast Extract Broth 
Soluble Pigment: None 
______________________________________ 
Carbohydrate utilization pattern 
______________________________________ 
d-glucose 
++ d-maltose + sucrose +/- 
d-arabinose 
++ d-mannitol 
++ d-xylose ++ 
l-arabinose 
++ d-mannose ++ l-xylose - 
d-fructose 
++ l-mannose - alpha d-lactose 
++ 
l-glucose 
+/- d-raffinose 
++ beta d-lactose 
++ 
inositol 
+ l-rhamnose 
- 
______________________________________ 
Carbon source utilization studies were carried out using Pridham and 
Gottlieb basal medium supplemented with 1% carbon source. Scoring was 
graded according to the methods described in "Methods for Characterization 
of Streptomyces species", IJSB 16: pps 313-340. 
Culture is tentatively identified as an Actinoplanaces sp. 
The compound of this invention is useful as an antihypercholesterolemic 
agent for the treatment of arteriosclerosis, hyperlipidemia, familial 
hypercholesterolemia and like diseases in humans. It may be administered 
orally or parenterally in the form of a capsule, a tablet, an injectable 
preparation or the like. It is usually desirable to use the oral route. 
Doses may be varied, depending on the age, severity, body weight and other 
conditions of human patients, but daily dosage for adults is within a 
range of from about 2 mg to 2000 mg (preferably 2 to 100 mg) which may be 
given in two to four divided doses. Higher doses may be favorably employed 
as required. 
The compound of this invention may also be coadministered with 
pharmaceutically acceptable nontoxic cationic polymers capable of binding 
bile acids in a non-reabsorbable form in the gastrointestinal tract. 
Examples of such polymers include cholestyramine, colestipol and 
poly[methyl(3-trimethylaminopropyl) iminotrimethylene dihalide]. The 
relative amounts of the compounds of this invention and these polymers is 
between 1:100 and 1:15,000. 
The intrinsic HMG-CoA reductase inhibition activity of the claimed 
compounds is measured in the in vitro protocol described in J. Med. Chem., 
1985, 28, page 347. 
The compound of the formula (II), wherein R is hydrogen as the potassium 
salt, exhibited an IC.sub.50 of 0.30 .mu.g/ml in the above-referenced 
protocol. 
Included within the scope of this invention is the method of treating 
arteriosclerosis, familal hypercholesterolemia or hyperlipidemia, which 
comprises administering to a subject in need of such treatment a nontoxic 
therapeutically effective amount of the compounds of formulae (I) or (II) 
or pharmaceutical compositions thereof. 
The compound (I) is prepared in the instant process from the sodium salt of 
simvastatin, lovastatin or an analog having a 6-methyl group by one of the 
following methods: 
(a) adding the substrate to a growing culture Actinoplanacete sp. for a 
suitable incubation period followed by isolation and derivatization, if 
desired; 
(b) collecting a culture of the bioconverting microorganism and contacting 
the collected cells with the substrate. 
Cultivation of the bioconverting microorganism MA 6559 tentatively 
identified as a Actinoplanacete sp. can be carried out by conventional 
means in a conventional culture medium containing nutrients well known for 
use with such microorganisms. Thus, as is well known, such culture media 
contain sources of assimilable carbon and of assimilable nitrogen and 
often inorganic salts. Examples of sources of assimilable carbon include 
glucose, sucrose, starch, glycerin, millet jelly, molasses and soybean 
oil. Examples of sources of assimilable nitrogen include soybean solids 
(including soybean meal and soybean flour), wheat germ, meat extracts, 
peptone, corn steep liquor, dried yeast and ammonium salts, such as 
ammonium sulphate. If required, inorganic salts, such as sodium chloride, 
potassium chloride, calcium carbonate or phosphates, may also be included. 
Also, if desired, other additives capable of promoting the production of 
hydroxylation enzymes may be employed in appropriate combinations. The 
particular cultivation technique is not critical to the process of the 
invention and any techniques conventionally used for the cultivation of 
microorganisms may be employed with the present invention. In general, of 
course, the techniques employed will be chosen having regard to industrial 
efficiency. Thus, liquid culture is generally preferred and the deep 
culture method is most convenient from the industrial point of view. 
Cultivation will normally be carried out under aerobic conditions and at a 
temperature within the range from 20.degree. to 37.degree. C., more 
preferably from 26.degree. to 28.degree. C. 
Method (a) is carried out by adding the substrate to the culture medium in 
the course of cultivation. The precise point during the cultivation at 
which the starting compound is added will vary depending upon the 
cultivation equipment, composition of the medium, temperature of the 
culture medium and other factors, but it is preferably at the time when 
the hydroxylation capacity of the microorganism begins to increase and 
this is usually 1 or 2 days after beginning cultivation of the 
microorganism. The amount of the substrate added is preferably from 0.01 
to 5.0% by weight of the medium, more preferably from 0.05 to 0.5%, e.g., 
from 0.05 to 0.1% by weight. After addition of the substrate, cultivation 
is continued aerobically, normally at a temperature within the ranges 
proposed above. Cultivation is normally continued for a period of from 1 
to 2 days after addition of the substrate. 
In method (b), cultivation of the microorganism is first carried out under 
conditions such as to achieve its maximum hydroxylation capacity; this 
capacity usually reaches a maximum between 4 and 5 days after beginning 
the cultivation, although this period is variable, depending upon the 
nature and temperature of the medium, the species of microorganism and 
other factors. The hydroxylation capacity of the culture can be monitored 
by taking samples of the culture at suitable intervals, determining the 
hydroxylation capacity of the samples by contacting them with a substrate 
under standard conditions and determining the quantity of product obtained 
and plotting this capacity against time as a graph. When the hydroxylation 
capacity has reached its maximum point, cultivation is stopped and the 
microbial cells are collected. This may be achieved by subjecting the 
culture to centrifugal separation, filtration or similar known separation 
methods. The whole cells of the cultivating microorganism thus collected, 
preferably, are then washed with a suitable washing liquid such as 
physiological saline or an appropriate buffer solution. 
Contact of the collected cells of the microorganism MA 6559 with the 
substrate is generally effected in an aqueous medium, for example, in a 
phosphate buffer solution at a pH value of from 5 to 9. The reaction 
temperature is preferably within the range from 20.degree. to 45.degree. 
C., more preferably from 25.degree. to 30.degree. C. The concentration of 
the substrate in the reaction medium is preferably within the range from 
0.01 to 5.0% by weight. The time allowed for the reaction is preferably 
from 1 to 5 days, although this may vary depending upon the concentration 
of the substrate in the reaction mixture, the reaction temperature, the 
hydroxylation capacity of the microorganism (which may, of course, vary 
from species to species and will also, as explained above, depend upon the 
cultivation time) and other factors. 
The microorganism useful in the novel process of this invention has been 
tentatively identified as Actinoplanacete sp. A sample of the culture 
designated ATCC 53771 is available in the permanent culture collection of 
the American Type Culture Collection at 12301 Parklawn Drive, Rockville, 
Md. 20852. 
After completion of the conversion reaction by any of the above methods, 
the desired compound can be directly isolated, separated or purified by 
conventional means. For example, separation and purification can be 
effected by filtering the reaction mixture, extracting the resulting 
filtrate with a water-immiscible organic solvent (such as ethyl acetate), 
distilling the solvent from the extract, subjecting the resulting crude 
compound to column chromatography (for example on silica gel or alumina) 
and eluting the column with an appropriate eluent, especially in an HPLC 
apparatus. 
The following examples illustrate the preparation of these compounds and, 
as such, are not to be construed as limiting the invention set forth in 
the claims appended hereto. 
The composition of media employed in the following examples are listed 
below. 
______________________________________ 
Media 
(g/L) 
______________________________________ 
Seed Medium A 
Dextrose 1.0 
Dextrin 10.0 
Beef Extract 3.0 
Ardamine pH 5.0 
NZ Amine Type E 5.0 
MgSO.sub.4.7H.sub.2 O 
0.05 
K.sub.2 HPO.sub.4 0.37 
Adjust pH to 7.0 
Add CaCO.sub.3 0.5 
Transformation Medium B 
Mannitol 5 
Glycerol 5 
Hycase SF 2 
Beef Extract 1 
Cornsteep Liquor 3 
Adjust pH to 7.0 
______________________________________ 
EXAMPLE 1 
Preparation of 6-[2-[2-methyl, 
6-hydroxymethyl-1,2-dihydronaphthyl]ethyl]-4-hydroxy-3,4,5,6-tetrahydro-2H 
-pyran-2-one 
A. Culture Conditions and Bioconversion 
Seed cultures were prepared in medium A (50 ml in a 250 ml 3-baffle 
Erlenmeyer flask). The seed flasks were incubated on a rotary shaker (220 
rpm) at 27.degree. C. for 24 hours. The transformation flasks (50 ml 
medium B in 250 ml Erlenmeyer flask) were inoculated with 2.5 ml of seed 
culture plus 1 mg simvastatin (10 mg/ml in H.sub.2 O) and incubated at 
27.degree. C. on a rotary shaker. After 48 hours the cells were separated 
by centrifugation, washed once with sterile saline and resuspended in 
sterile 100 mM phosphate buffer (pH 6.0) containing 1% glucose. Each flask 
was charged with 10 mg simvastatin (10 mg/ml in H.sub.2 O) and incubated 
for 48 hours. 
Following incubation, the supernatant was extracted as described in B 
below. 
B. Isolation and Purification 
The centrifuged broth (50 ml) at pH 3 was extracted with three 25 ml 
portions of ethyl acetate. The ethyl acetate layers were combined, dried 
over sodium sulfate, and evaporated to a brown oil. The oil was dissolved 
in 50 ml of CH.sub.2 Cl.sub.2, two drops of CF.sub.3 COOH added and 
incubated at 50.degree. for one hour. The reaction mixture was evaporated 
and the residue redissolved in acetonitrile. Further purification was 
obtained by HPLC on a Partisil 10 ODS-3 column developed with 45% aqueous 
acetonitrile. The fractions at retention time 6.13 minutes were pooled and 
evaporated to yield the titled compound. 
H'NMR .delta.1.02 (d), 2.42(ddd), 2.59(dd), 4.18(m), 4.52(s), 4.52(m), 
5.84(dd), 6.43(dd), 7.04(s)* 7.12(dd), 7.14(d). 
FNT Doublet structure not resolved under experimental conditions. 
EXAMPLE 2 
Preparation of Ammonium Salts of Compounds II 
To lactone (1.0 mmol) from Example 1 Step b, in ethanol solution, is added 
with stirring in 0.1N NaOH (1.1 mmol) at ambient temperature. The 
resulting solution is cooled and acidified by the dropwise addition of 1N 
HCl. The resulting mixture is extracted with diethyl ether and the extract 
washed with brine and dried (MgSO.sub.4). The MgSO.sub.4 is removed by 
filtration and the filtrate saturated with ammonia (gas) to give the 
ammonium salt. 
EXAMPLE 3 
Preparation of Alkali and Alkaline Earth Salts of Compounds II 
To a solution of 49 mg of lactone from Example 1 Step b in 2 ml of ethanol 
is added 1 ml of aqueous 0.1N NaOH. After one hour at room temperature, 
the mixture is taken to dryness in vacuo to yield the desired sodium salt. 
In like manner, the potassium salt is prepared using one equivalent of 
potassium hydroxide, and the calcium salt, using one equivalent of 
Ca(OH).sub.2. 
EXAMPLE 4 
Preparation of Ethylenediamine Salts of Compounds II 
To a solution of 0.50 g of the ammonium salt from Example 2 in 10 ml of 
methanol is added 0.06 ml of ethylenediamine. The methanol is stripped off 
under vacuum to obtain the desired ethylenediamine salt. 
EXAMPLE 5 
Preparation of Tris(hydroxymethyl)aminomethane Salts of Compounds II 
To a solution of 202 mg of the ammonium salt from Example 2 in 5 ml of 
methanol is added a solution of 50 mg of tris(hydroxymethyl)aminomethane 
in 5 ml of methanol. The solvent is removed in vacuo to afford the desired 
tris(hydroxymethyl)aminomethane salt. 
EXAMPLE 6 
Preparation of L-Lysine Salts of Compounds II 
A solution of 0.001 mole of L-lysine and 0.0011 mole of the ammonium salt 
from Example 2 in 15 ml of 85% ethanol is concentrated to dryness in vacuo 
to give the desired L-lysine salt. 
Similarly prepared are the L-arginine, L-ornithine, and the 
.alpha.,.beta.-diaminobutyric acid salts. 
EXAMPLE 7 
Preparation of Tetramethylammonium Salts of Compounds II 
A mixture of 68 mg of ammonium salt from Example 2 in 2 ml of methylene 
chloride and 0.08 ml of 24% tetramethylammonium hydroxide in methanol is 
concentrated to dryness to yield the desired tetramethylammonium salt. 
EXAMPLE 8 
Preparation of Methyl Esters of Compounds II 
To a solution of 400 mg of lactone from Example 1 Step b in 100 ml of 
absolute methanol is added 10 ml 0.1M sodium methoxide in absolute 
methanol. This solution is allowed to stand at room temperature for one 
hour, then is diluted with water and extracted twice with ethyl acetate. 
The organic phase is separated, dried (Na.sub.2 SO.sub.4), filtered and 
evaporated in vacuo to yield the desired methyl ester. 
In like manner, by the use of equivalent amounts of the alkoxides derived 
from propanol, butanol, isobutanol, t-butanol, amyl alcohol, isoamyl 
alcohol, 2-dimethylaminoethanol, benzyl alcohol, phenylethanol, 
2-acetamidoethanol and the like, and employing the corresponding alcohol 
as solvent the corresponding esters are obtained. 
EXAMPLE 9 
Preparation of Free Dihydroxy Acids 
The sodium salt of the compound II from Example 8 is dissolved in 2 ml of 
ethanol-water (1:1; v:v) and added to 10 ml of 0.1N hydrochloric acid from 
which the dihydroxy acid is extracted with ethyl acetate. The organic 
extract is washed once with water, dried (Na.sub.2 SO.sub.4), and 
evaporated in vacuo with a bath temperature not exceeding 30.degree. C. 
The dihydroxy acid derivative derived slowly reverts to the corresponding 
parent lactone on standing at room temperature. 
EXAMPLE 10 
As a specific embodiment of a composition of this invention, 20 mg of 
lactone from Example 1 Step b is formulated with sufficient finely divided 
lactose to provide a total amount of 580 to 590 mg to fill a size 0, 
hard-gelatin capsule.