Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing a...
Reexamination Certificate
2002-06-14
2004-12-07
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing a...
C435S052000, C435S055000, C435S058000, C435S059000, C435S061000
Reexamination Certificate
active
06828120
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is a process to transform medroxyprogesterone acetate (I) to 11&bgr;,17&agr;,21-trihydroxy-6&agr;-methylpregna-1,4-diene-3,20-dione 21-acetate (VI).
2. Description of the Related Art
The microbial enzymatic &Dgr;
1
-dehydrogenation of steroids is known to those skilled in the art.
Biotechnology and Bioengineering
, 37, 97-102 (1991) discloses the &Dgr;
1
-dehydrogenation of 6&agr;-methylhydrocortisone 21-acetate by
Arthrobacter simplex
in an organic solvent.
U.S. Pat. No. 4,684,610 discloses a process for converting 1,2-saturated steroids to 1,2-dehydro, steroids by contacting the 1,2-saturated steroid with
A. simplex
or
Bacillis cyclooxydans
in the presence of exogenous electron carrier and a water-immiscible aromatic hydrocarbon solvent.
U.S. Pat. No. 4,749,649 discloses the use of scavengers of toxic oxygen species in the microbial &Dgr;
1
-dehydrogenation of steroids.
The microbial enzymatic 11&bgr;-hydroxylation of steroids with microorganisms such as
Curvularia lunata
is known to those skilled in the art, see for example U.S. Pat. No. 3,419,470. The concentration of the substrate undergoing 11-&bgr; hydroxylation is quite low. For example, U.S. Pat. No. 3,530,038 discloses a maximum concentration of steroid substrates 17&agr;-acetoxypregn-4-en-21-ol-3,20-dione and 17&agr;,21-diacetoxypregn-4-ene-3,20-dione is 0.5 g/L. European Patent EP 0 042 451 A1 again discloses that the amount of steroid substrate concentration does not exceed 0.5 g/L. U.S. Pat. No. 4,353,985 does not give specific examples, and uses 17&agr;,21-orthoesters. U.S. Pat. No. 4,588,683 discloses that the steroid substrate concentration of 17&agr;,20&bgr;,21-trihydroxypregn-4-en-3-one is 0.5 g/L. U.S. Pat. No. 4,898,693 discloses that the steroid substrate concentration of 6&agr;-fluoro-17&agr;-hydroxy-16&agr;-methylpregn-4-en-3,20-dione is 0.4 g/L. All but the last patent listed above relate to eliminating unwanted 14&agr;-hydroxylation resulting in high yields of 11&bgr;-hydroxylated product (80-92%). However, the substrate charge to the fermentation does not exceed 0.5 g/L. Japanese Published Application 62-118898 discloses the use of
C. lunata
MCI1690 (registered at Microbial Industry Institute strain No. 8515) at a substrate concentration of 10 g/L but with substance S and analogs thereof, not the substrate of the present invention. The process of the present invention uses a different culture than that of the Japanese 62-118898 and provides high yields using high substrate concentrations.
The functionalization of the C
21
-methyl group of pregnanes followed by displacement with acetate to produce the corresponding 21-acetate is known to those skilled in the art. GB 2,318,790 discloses the transformation of the C
21
-methyl group of a &Dgr;
1-11
&bgr;-hydroxy steroid to the corresponding 21-hydroxy steroid by functionalization with one bromine atom followed by displacement with acetate. The process of the present invention does not use bromine.
GB 2,318,790 discloses the transformation of 17&agr;-hydroxy-6&agr;-methylpregn-4-ene-3,20-dione 17-acetate (I) to 11&bgr;,17&agr;-dihydroxy-21-diiodo-6&agr;-methylpregna-1,4-diene-3,20-dione (V) by microbial &Dgr;
1
-dehydrogenation by use of
Nocardia simplex
, microbial 11&bgr;-hydroxylation by use of
C. lunata
and 21-hydroxylation by use of bromine. The present invention transforms 17&agr;-hydroxy-6&agr;-methylpregn-4-ene-3,20-dione 17-acetate (I) to 11&bgr;,17&agr;-dihydroxy-21-diiodo-6&agr;-methylpregna-1,4-diene-3,20-dione (V) but does not use bromine.
SUMMARY OF INVENTION
Disclosed is a process for the preparation of 11&bgr;,17&agr;,21-trihydroxy-6&agr;-methylpregna-1,4-diene-3,20-dione 21-acetate (VI) which comprises:
(1) contacting 17&agr;-hydroxy-6&agr;-methylpregn-4-ene-3,20-dione 17-acetate (I) with a &Dgr;
1
-dehydrogenase to produce 17&agr;-hydroxy-6&agr;-methylpregna-1,4-diene-3,20-dione 17 acetate (II);
(2) contacting 17&agr;-hydroxy-6&agr;-methylpregna-1,4-diene-3,20-dione 17 acetate (II) with a 11&bgr;-hydroxylase to produce 11&bgr;,17&agr;-dihydroxy-6&agr;-methylpregna-1,4-diene-3,20-dione 17-acetate (III);
(3) hydrolyzing the 11&bgr;,17&agr;-dihydroxy-6&agr;-methylpregna-1,4-diene-3,20-dione 17-acetate (III) to producel 11&bgr;,17&agr;-dihydroxy-6&agr;-methylpregna-1,4-diene-3,20-dione (IV);
(4) contacting 11&bgr;,17&agr;-dihydroxy-6&agr;-methylpregna-1,4-diene-3,20-dione (IV) with iodine, a catalyst, a mild base to produce 11&bgr;,17&agr;-dihydroxy-21-diiodo-6&agr;-methylpregna-1,4-diene-3,20-dione (V) and
(5) contacting 11&bgr;,17&agr;-dihydroxy-21-diiodo-6&agr;-methylpregna-1,4-diene-3,20-dione (V) with a salt of acetic acid.
Also disclosed is a diiodo steroid of the formula:
Further disclosed is a process for the removal of residual unhydroxylated material which comprises:
(1) contacting 17&agr;-hydroxy-6&agr;-methylpregn-4-ene-3,20-dione 17-acetate (I) with a &Dgr;
1
-dehydrogenase to produce 17&agr;-hydroxy-6&agr;-methylpregna-1,4-diene-3,20-dione 17 acetate (II),
(2) crystallizing the 17&agr;-hydroxy-6&agr;-methylpregna-1,4-diene-3,20-dione 17 acetate (II) produced from a nonpolar solvent or solvent mixture.
Additionally disclosed is a process for the purification of 11&bgr;,17&agr;-dihydroxy-6&agr;-methylpregna-1,4-diene-3,20-dione 17-acetate (III) produced by a 11&bgr;-hydroxylase which comprises:
(1) crystallization from a solvent selected from the group consisting of toluene, benzene, xylene, n-butyl acetate and mixtures thereof with hydrocarbon solvents selected from the group consisting of hexane, heptane, isooctane, cyclohexane and methylcyclohexane.
REFERENCES:
patent: 3419470 (1968-12-01), Zaffaroni et al.
patent: 3530038 (1970-09-01), de Flines et al.
patent: 4353985 (1982-10-01), Petzoldt et al.
patent: 4588683 (1986-05-01), Goodhue et al.
patent: 4684610 (1987-08-01), Evans
patent: 4749649 (1988-06-01), Evans et al.
patent: 4898693 (1990-02-01), Hempel et al.
patent: 2318790 (1998-05-01), None
patent: 62-118898 (1987-05-01), None
Biotechnology and Bioengineering, 37, 97-102 (1991).
Japanese Published Application 62-118898 May 1989.
Gilbert Ivan G.
Pearlman Bruce Allen
White Michael J.
Engelmann John H.
Lilling Herbert J.
Pharmacia and Upjohn Company
Stein Bruce
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