Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Cyclopentanohydrophenanthrene ring system doai
Reexamination Certificate
1998-08-07
2001-05-08
Shah, Mukund J. (Department: 1624)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Cyclopentanohydrophenanthrene ring system doai
C540S056000
Reexamination Certificate
active
06228851
ABSTRACT:
The present invention refers to a new process for the purification of the compound 11&bgr;-21-dihydroxy-2′-methyl-5′&bgr;H-pregna-1,4-dieno[17,16-d-]oxazoline-3,20-dione of formula I;
The above compound is related to deflazacort (INN—International Nonproprietary Name), in that the acetate moiety on the C-21 of deflazacort is substituted by a hydroxy moiety.
Deflazacort is a compound employed in therapy since some years as a calcium-sparing gluco-corticoid agent.
These compounds belong to the more general class of pregneno-oxazolines, for which anti-inflammatory, glucocorticoid and hormone-like pharmaceological activities are reported. Examples of compounds of the above class are disclosed in U.S. Pat. No. 3,413,286 and U.S. Pat. No. 4,440,764.
The preparation of the compound of formula I is disclosed in EP-B-322630, wherein said compound is referred to as 11&bgr;-21-dihydroxy-2′-methyl-5′&bgr;H-pregna-1,4-dieno[17,16-d]oxazoline-3,20-dione.
According to the fermentation process disclosed in the above cited EP-B-322630, 2′-methyl-4-pregnen-21-ol-[17a,16a-d]oxazolinyl-3,20-dione or 2′-methyl-4-pregnen-21-acetyloxy-[17a,16a-d]oxazolinyl-3,20-dione is contacted with a sequentially growing mixed culture of a Curvularia strain and an Arthrobacter strain. More in particular, according to a preferred embodiment, the above compound is added to a growing culture of
C. lunata
NRRL 2380 in a suitable fermentation medium after 12-24 hours from inoculum, and, after 48-72 hours from inoculum, a growing culture of
A. simplex
ATCC 6946 of 18-36 hours is added to the mixture and further cultivated for 40-55 hours; the fermentation is carried out under submerged conditions, temperature is kept between 27° C. and 32° C. and pH between 6 and 8; the fermentation product of formula I is then recovered by extracting the fermentation broth with an organic solvent (e.g. chloroform), concentrating the organic extracts and precipitating the compound by adding a non-solvent (e.g. petroleum ether).
As the concentration of the compound of formula I in the fermentation broth is very low, high amounts of organic solvent are necessary for completely extracting the compound. The use of high amounts of organic solvents, in particular halogenated solvents, may give rise to some problems, with respect to the safety, industrial hygiene and environment protection.
It has now been found that the compound of formula I can be conveniently recovered from an aqueous solution resulting from fermentation broths or process streams, by adsorbing the compound contained in said aqueous solution on an adsorbent polymeric resin having a styrenic or acrylic matrix and subsequently desorbing the said compound by eluting the resin with a suitable mixture of water with a water-miscible organic solvent.
Typical aqueous solutions containing the compound of formula I accompained by undesired products are the filtered fermentation broths from the suitable mycelia, optionally together with the aqueous washings of said mycelia, or partially purified process streams. Examples of the undesired accompanying products are colored impurities, side-products, unhexausted starting materials, salts and water soluble components of the fermentation media.
In particular, the purification process of the present invention may conveniently be applied for recovering the compound of formula I from the filtered fermentation broth obtained from the fermentation process disclosed in EP-B-322630.
Suitable resins for the present purification process will have an average particle size of about 20-50 mesh and the following average physical characteristics:
Porosity volume: about 30%-75%;
Surface Area: about 140-800 m
2
/g;
Skeletal Density: about 1.06-1.10 g/ml (styrenic resin)
about 1.20-1.26 g ml (acrylic resins);
Average pore diameter: about 20-100 Å.
Examples of adsorbent styrenic based polymeric resins suitable for the above recovering process are the commercially available resins such as Kastel® S/112 (Dow Chemical), Amberlite® XAD/2, XAD/4 or XAD/16 (Rohm & Haas), or the like. Examples of adsorbent acrylic based polymeric resins suitable for the above recovering process are the commercially available resins such as Kastel® S/221 or S/223 (Dow Chemical), Amberlite® XAD/7 or XAD/8 (Rohm & Haas), and the like.
In general for the process of the present invention, the acrylic based polymeric resins are preferably employed, particularly preferred being those having a particle size of about 20-50 mesh and the following average physical characteristics:
Porosity volume: about 30%-60%
Surface Area: about 350-550 m
2
/g;
Skeletal Density: about 1.24-1.25 g/ml;
Average pore Diameter: about 20-80 Å.
Examples of adsorbent acrylic based polymeric resins with the above characteristics which may be suitably employed are the previously mentioned Kastel® S/221 and Amberlite® XAD/7.
Particularly preferred for the present purification process is an acrylic based polymeric resins having a particle size of about 20-50 mesh and the following average physical characteristics:
Porosity volume: about 30%-60%
Surface Area: about 350-550 m
2
/g;
Skeletal Density: about 1.25 g/ml;
Average pore Diameter: about 20-40 Å.
For instance, the commercially available resin Kastel® S/221 may suitably be employed.
Suitable eluting mixtures are mixtures from 0% to 80% of water with a water miscible organic solvent such as lower ketones (e.g. acetone, ethylmethylketone); lower alcohols (e.g. methanol, ethanol, propanol, butanol); and the like.
Preferably, mixtures from 10% to 50% of water with one of the above organic solvents are employed, particularly preferred being a mixture containing about 30% of water Acetone is the preferred solvent.
When the process of the present invention is employed for the purification of the compound of formula I obtained according to the fermentation process as described in EP-B-322630, the following general procedure may conveniently be applied.
When the said fermentation process is completed, the fermentation mass is first filtered according to the known techniques. The mycelial cake is repeatedly washed with water and the washings are then combined with the filtered broth. Alternatively, the mycelium can be washed with an organic solvent selected from those previously listed, in order to recover the activity contained therein; in this case, the washings are combined with the filtered broth only after having removed the solvent, e.g. by stripping under vacuum.
The filtered broth combined with the washings is then applied on the top of a column containing the adsorbing resin; in general, the eluted solution contains only traces of product. The resin is then washed with water for eliminating salts and other water-soluble impurities (as above, also this washing water will generally contain only traces of the product).
The compound of formula I is then desorbed from the resin, by eluting with a mixture of water and an organic solvent as above defined, preferably a mixture 30/70 water/acetone. The eluate is concentrated and the product is recovered by filtration. With this procedure the major amount of product is recovered from the filtered broth; as a general indication, more than 90% of the total amount of the desired compound initially applied on the column is recovered with this first elution.
The remaining amount can be recovered by repeating the above procedure after having combined the mother liquors from the first elution and the washings with water. The total recovery yield is about 96%.
For better illustrating the invention, the following examples are given.
REFERENCES:
A. Santos-Montes, J. of Chromatography B : Biochemical Applications, vol. 652, No. 1, Jan. 14, 1994, pp 83-89.
E. Martinelli et al, Drug Metabolism and Disposition, vol. 7, No. 5 (9-10/79) pp 335-339, 1979.
A. Assandri et al, Xenobiotica, vol. 13, No. 3, Mar. 1983, pp 185-196.
A. Santos-Montes et al, J. of Chromatography B : Biochemical Applications, vol. 657, No. 1, Jul. 1, 1994, pp 248-253.
Coleman Brenda
Gruppo Lepetit S.p.A.
Gupta Balaram
Shah Mukund J.
LandOfFree
Process for the purification of... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Process for the purification of..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Process for the purification of... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2438340