Biotechnological process for preparing hydroxylated ML-236B...

Details Biotechnological process for preparing hydroxylated ML-236B... Biotechnological process for preparing hydroxylated ML-236B...

2000-12-27

2002-04-02

Lankford, Jr., Leon B. (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing oxygen-containing organic compound

C435S125000, C435S252100, C435S253600, C435S253500, C435S872000, C435S146000, C424S200100, C544S292000, C549S292000, C560S119000, C560S188000, C560S256000

Reexamination Certificate

active

06365382

ABSTRACT:

Conventionally, the preparation of pharmaceutically acceptable salts of the HMG-CoA reductase inhibitors, such as the M4 and M-4′ substances, is a fed-batch process. The first part comprises preparation of a ML-236B substance by fermentation with microorganisms of the genus Penicillium and its isolation using conventional isolation techniques and preparation of the sodium salt thereof as described in U.S. Pat. No. 4,137,322. The second part comprises cultivation of a microorganism of one of the aforementioned genera in the medium to which ML-236B, typically in the form of sodium salt is added, isolation of the M-4 and M-4′ substances and optionally preparation of pharmaceutically acceptable salts thereof as disclosed in U.S. Pat. No. 4,346,227 and U.S. Pat. No. 4,537,859.
TECHNICAL FIELD
The present invention relates to a novel process for preparing hydroxylated ML-236 B derivatives, known as M-4 and M-4′, and analogues thereof, in particular to an enzymatic hydroxylation by means of a microbiological process.
PRIOR ART
ML-236B and derivatives thereof as well as analogues are known as HMG-CoA reductase inhibitors and are disclosed in GB Pat. No. 1,555,831. They are produced by fermentation with various microorganisms of the genera Gilbertella, Streptomyces, Circinella, Monascus, Nocardia, Amycolata, Mucor or Penicillium (as disclosed in U.S. Pat. No. 4,346,227 and U.S. Pat. No. 4,537,859). U.S. Pat. No. 5,153,124 describes the hydroxylation of a ML-236 B derivative by employing microorganisms of the genus Streptomyces or Amycolata. In EP 0 649 907 A1 the use of various microorganisms is disclosed, including
Amycolatopsis mediterranei
(ATCC 21411) among many other microorganisms. The hydroxylated forms of ML-236B, its derivatives and analogues, especially those
DESCRIPTION OF THE INVENTION AND THE PREFERRED EMBODIMENTS
The object of the invention has been solved by the provision of a process for preparing a compound defined by the following formula (I)
wherein R
1
represents substituted or unsubstituted alkyl or substituted or unsubstituted aryl, and R
2
independently represents H, substituted or unsubstituted alkyl or a cation;
or the corresponding lactone (II)
wherein R
1
is as defined above; of formula (I) or the corresponding lactone (II) with the microorganism
Amycolatopsis orientalis
, especially the strain deposited as ATCC 19795, in a suitable fermentation medium.
A fermentation medium should preferably comprise a source of assimilable carbon, such as glucose, saccharose, dextrins, glycerol, starch, soybean oil and molasses, a source of assimilable nitrogen, such as soybean flour, meat and yeast extracts, peptones, ammonium salts and, if required, various inorganic salts, such as sodium chloride, potassium chloride, magnesium sulphate, calcium carbonate and phosphates. Most conventional culture media may be used that employ microorganisms of the genus Amycolatopsis for fermentation. Suitable inoculation and fermentation media are, for example, described by J. J. McIntyre et al. in Biotechnology and Bioengineering, vol. 49, pp. 412-420 (1996); L. D. Boeck et al. in The Journal of Antibiotics, Vol. XXXVII No. 5, pp. 446-453 (1984); G. J. Clark et al. in Microbiology, Vol. 141, Pt. 3, pp. 663-669 (1995); and in U.S. Pat. No. 4,547,488; all descriptions being incorporated herein by way of reference.
Fermentation is carried out under aerobic conditions and at a temperature within a suitable range, for example from 20° to 36° C., preferably from 24° to 30° C. The aforementioned substrate compound to be hydroxylated may be contacted with the microorganism, preferably in the form of sodium salt, at any time in the course of fermentation or after completion of the fermentation, but an addition at the vegetative part of fermentation, which is normally between 24 and 48 hours after beginning cultivation, is particularly preferred. The compound substrate is added to a total final concentration of 0.01 wt.-% to 5 wt.-%, preferably 0.05 wt.-% to 0.5 wt.-%, based on the total fermentation broth weight or the contacting liquid. Addition to a fermentation medium may be either batch, fed-batch or continuous. Fermentation is usually completed within 2 to 5 days after addition of the substrate compound. Then, the microorganism cells are separated, preferably by filtration, and the resulting filtrate is extracted with an organic solvent which is preferably water immiscible or has a limited miscibility with water, such as ethyl acetate, ether (for example diethyl ether) or chloroform. Subsequently, the organic solvent is removed, suitably by evaporation, and if desired the resulting crude product is subjected to further conventional purification and isolation processes, for example employing column chromatography, e.g. using silica gel column, and eluting the desired compound with and appropriate eluent. If required, the resulting product may be subjected to recrystallization, salification (if it is desired in the form of a salt), lactonization or esterification, with methods known to those skilled in the art.
The product obtained by the microbiological process is typically a mixture of both 6′-hydroxy &agr;- and &bgr;-configurations and can be used as such. If desired, the isomers with the respective &agr;- and &bgr;-configuration may be separated by an appropriate isomer resolution technique known to those skilled in the art.
The structural moieties of R, and R
2
can be selected from the definitions as specified above for obtaining HMG-CoA reductase inhibitors or intermediates thereof. Thus, R
1
may represent optionally substituted alkyl or optionally substituted aryl. The alkyl group include straight chain, branched or cyclic hydrocarbon optionally having unsaturated double bonds and optionally being substituted. The main chain of the alkyl group has 1 to 15, preferably 1 to 10 and more preferably 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, hexyl, the isomers and the branched derivatives thereof, and the like. The optional substituents include one or more of the group consisting of halogen such as chloro, amino, lower alkyl amino such as mono- or dimethylamino, hydroxy, alkoxy, cyano, nitro, and the like. A preferred substituent is hydroxy. If present, a branching hydrocarbon group preferably has 1 to 4 carbon atoms, such as methyl and ethyl. Possible aryl residues include substituted or unsubstituted phenyl, biphenyl and naphthyl,
or the corresponding lactone compound (IIa); wherein R
1
and R
2
are as defined above, and the 6′
OH—group represents an &agr;- or a &bgr;-configuration, or a mixture of both &agr;- and &bgr;-configurations. The compounds of formula (Ia) or (IIa) are obtained by starting with a 6′-H analogue having the corresponding configuration.
A particularly preferred example for R
1
is an alkyl residue having the following structure and configuration:
In a specifically preferred embodiment of the present invention, a compound having the following formula (III) or the salt thereof or the corresponding lactone is prepared in order to provide a particularly active and potent HMG-CoA reductase inhibitor or an intermediate thereof: for the prevention and/or treatment of diseases. The compounds obtained by the process according to the present invention, or the HMG-CoA reductase inhibitors derived therefrom, can be effectively used as an antihyper-cholesterolemic agent. The compounds can therefore be used for the preparation of a medicament for the control of cholesterol in the body of an individual. They can further be used for the prevention or the treatment of atherosclerosis. The obtained inhibitors and pharmaceuticals are particularly useful as preventives for reducing the risk of stroke, transient ischemic attack and myocardial infarction.


REFERENCES:
patent: 4346227 (1982-08-01), Terahara et al.
patent: 4410629 (1983-10-01), Terahara et al.
patent: 4537859 (1985-08-01), Terahara et al.
patent: 4547488 (1985-10-01), Merkel
patent: 5153124 (1992-10-01), Furuya et al.
pa

Affiliated with

Also associated with

Rating

Biotechnological process for preparing hydroxylated ML-236B... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Biotechnological process for preparing hydroxylated ML-236B..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Biotechnological process for preparing hydroxylated ML-236B... will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2898909