U.S. patent application number 11/389937 was filed with the patent office on 2008-01-31 for processes for preparations of 9,11-epoxy steroids and intermediates useful therein.
Invention is credited to Dennis K. Anderson, Julio A. Baez, Bernhard Erb, Thomas R. Kowar, Sastry A. Kunda, Jon P. Lawson, Leo J. Letendre, Chin Liu, John S. Ng, Mark J. Pozzo, Yuen-Lung L. Sing, Ping T. Wang, Richard M. Weier, Joseph Wieczorek, Edward E. Yonan.
Application Number | 20080027237 11/389937 |
Document ID | / |
Family ID | 34527657 |
Filed Date | 2008-01-31 |
United States Patent
Application |
20080027237 |
Kind Code |
A1 |
Ng; John S. ; et
al. |
January 31, 2008 |
Processes for preparations of 9,11-epoxy steroids and intermediates
useful therein
Abstract
Multiple novel reaction schemes, novel process steps and novel
intermediates are provided for the synthesis of 9,11-epoxy
steroids.
Inventors: |
Ng; John S.; (Chicago,
IL) ; Liu; Chin; (Vernon Hills, IL) ;
Anderson; Dennis K.; (St. Charles, MO) ; Lawson; Jon
P.; (Glencoe, MO) ; Wieczorek; Joseph; (Cary,
IL) ; Kunda; Sastry A.; (Chesterfield, MO) ;
Letendre; Leo J.; (Manchester, MO) ; Pozzo; Mark
J.; (Chesterfield, MO) ; Sing; Yuen-Lung L.;
(St. Louis, MO) ; Wang; Ping T.; (Ballwin, MO)
; Yonan; Edward E.; (Carol Stream, IL) ; Weier;
Richard M.; (Lake Bluff, IL) ; Kowar; Thomas R.;
(Mt. Prospect, IL) ; Baez; Julio A.; (San Diego,
CA) ; Erb; Bernhard; (Gipf-Oberfrick, CH) |
Correspondence
Address: |
PHARMACIA CORPORATION;GLOBAL PATENT DEPARTMENT
POST OFFICE BOX 1027
ST. LOUIS
MO
63006
US
|
Family ID: |
34527657 |
Appl. No.: |
11/389937 |
Filed: |
March 27, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11052549 |
Feb 7, 2005 |
7112669 |
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11389937 |
Mar 27, 2006 |
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10647866 |
Aug 25, 2003 |
6887991 |
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11052549 |
Feb 7, 2005 |
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10112355 |
Mar 29, 2002 |
6610844 |
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10647866 |
Aug 25, 2003 |
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09319673 |
Dec 13, 1999 |
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PCT/US97/23090 |
Dec 11, 1997 |
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10112355 |
Mar 29, 2002 |
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60049388 |
Jun 11, 1997 |
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60033315 |
Dec 11, 1996 |
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Current U.S.
Class: |
552/502 |
Current CPC
Class: |
C07J 1/0059 20130101;
C07J 71/0015 20130101; C07J 41/0094 20130101; C07J 75/00 20130101;
C07D 301/12 20130101; C12P 33/10 20130101; C07J 71/00 20130101;
C07J 71/0005 20130101; C07J 21/00 20130101; C07J 53/002 20130101;
C07J 31/006 20130101; C12P 33/005 20130101; C07J 21/003
20130101 |
Class at
Publication: |
552/502 |
International
Class: |
C07J 75/00 20060101
C07J075/00 |
Claims
1. A process for the preparation of a compound of Formula II:
##STR313## wherein -A-A- represents the group
--CHR.sup.4--CHR.sup.5-- or --CR.sup.4.dbd.CR.sup.5--; R.sup.3,
R.sup.4 and R.sup.5 are independently selected from the group
consisting of hydrogen, halo, hydroxy, lower alkyl, lower alkoxy,
hydroxyalkyl, alkoxyalkyl, hydroxy carbonyl, cyano and aryloxy;
R.sup.1 represents an alpha-oriented lower alkoxycarbonyl or
hydroxycarbonyl radical; --B--B-- represents the group
--CHR.sup.6--CHR.sup.7-- or an alpha- or beta-oriented group:
##STR314## where R.sup.6 and R.sup.7 are independently selected
from the group consisting of hydrogen, halo, lower alkoxy, acyl,
hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl,
acyloxyalkyl, cyano and aryloxy; and R.sup.8 and R.sup.9 are
independently selected from the group consisting of hydrogen,
hydroxy, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,
hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and
aryloxy, or R.sup.8 and R.sup.9 together comprise a carbocyclic or
heterocyclic ring structure, or R.sup.8 or R.sup.9 together with
R.sup.6 or R.sup.7 comprise a carbocyclic or heterocyclic ring
structure fused to the pentacyclic D ring; the process comprising:
removing an II.alpha.-leaving group from a compound of Formula IV:
##STR315## wherein -A-A-, --B--B--, R.sup.1, R.sup.3, R.sup.8, and
R.sup.9 are as defined above, and R.sup.2 is a leaving group the
abstraction of which is effective for generating a double bond
between the 9- and 11-carbon atoms.
2. A process as set forth in claim 1 wherein said compound of
Formula II corresponds to Formula IIAA: ##STR316## wherein: -A-A-
represents the group --CH.sub.2--CH.sub.2-- or --CH.dbd.CH--;
--B--B-- represents the group --CH.sub.2--CH.sub.2-- or an alpha-
or beta-oriented group of Formula IIIA: ##STR317## R.sup.1
represents an alpha-oriented lower alkoxycarbonyl radical; X
represents two hydrogen atoms or oxo; Y.sup.1 and Y.sup.2 together
represent the oxygen bridge -0-, or Y.sup.1 represents hydroxy, and
Y.sup.2 represents hydroxy, lower alkoxy or, if X represents
H.sub.2, also lower alkanoyloxy; and salts of compounds in which X
represents oxo and Y.sup.2 represents hydroxy, the process
comprising: contacting a solution comprising a lower alkanoic acid
and a salt of a lower alkanoic acid with a compound corresponding
to Formula IVAA: ##STR318## wherein -A-A-, --B--B--, R.sup.1, X,
y.sup.1 and Y.sup.2 are as defined in Formula IIAA, and R.sup.2 is
lower alkylsulfonyloxy or acyloxy.
3. A process as set forth in claim 1 wherein said compound of
Formula IV is Methyl Hydrogen
17.alpha.-Hydroxy-11.alpha.-(methylsulfonyl)oxy-3-oxopregn-4-ene-7.alpha.-
,21-dicarboxylate, .gamma.-Lactone and said compound of Formula II
is Methyl Hydrogen
17.alpha.-Hydroxy-3-oxopregna-4,9(11)-diene-7.alpha.,21-dicarboxylate,
.gamma.-Lactone.
4. A process for the preparation of a compound of Formula IV:
##STR319## wherein -A-A- represents the group
--CHR.sup.4--CHR.sup.5-- or --CR.sup.4.dbd.CR.sup.5--; R.sup.3,
R.sup.4 and R.sup.5 are independently selected from the group
consisting of hydrogen, halo, hydroxy, lower alkyl, lower alkoxy,
hydroxyalkyl, alkoxyalkyl, hydroxy carbonyl, cyano and aryloxy;
R.sup.1 represents an alpha-oriented lower alkoxycarbonyl or
hydroxycarbonyl radical; --B--B-- represents the group
--CHR.sup.6--CHR.sup.7-- or an alpha or beta-oriented group:
##STR320## where R.sup.6 and R.sup.7 independently selected from
the group consisting of hydrogen, halo, lower alkoxy, acyl,
hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl,
acyloxyalkyl, cyano and aryloxy; and R.sup.8 and R.sup.9 are
independently selected from the group consisting of hydrogen,
hydroxy, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,
hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and
aryloxy, or R.sup.8 and R.sup.9 together comprise a carbocyclic or
heterocyclic ring structure, or R.sup.8 or R.sup.9 together with
R.sup.6 or R.sup.7 comprise a carbocyclic or heterocyclic ring
structure fused to the pentacyclic D ring; and R.sup.2 is lower
alkylsulfonyloxy or acyloxy or a halide; the process comprising:
reacting a lower alkylsulfonylating or acylating reagent or a
halide generating agent such as thionyl halide, sulfuryl halide, or
oxalyl halide with a compound of Formula V ##STR321## wherein
-A-A-, --B--B--, R.sup.1, R.sup.3, R.sup.8, and R.sup.9 are as
defined above.
5. A process as set forth in claim 4 wherein said compound of
Formula IV corresponds to Formula IVAA: ##STR322## wherein: -A-A-
represents the group --CH.sub.2--CH.sub.1-- or --CH.dbd.CH--;
R.sup.1 represents an alpha-oriented lower alkoxycarbonyl radical;
R.sup.2 represents lower alkylsulfonyloxy or acyloxy; B--B--
represents the group --CH.sub.2--CH.sub.2-- or an alpha- or
beta-oriented group: ##STR323## X represents two hydrogen atoms or
oxo; Y.sup.1 and Y.sup.2 together represent the oxygen bridge -0-,
or Y.sup.1 represents hydroxy, and Y.sup.2 represents hydroxy,
lower alkoxy or, if X represents H.sup.2 also lower alkanoyloxy;
and salts of compounds in which X represents oxo and Y.sup.2
represents hydroxy, the process comprising: reacting a lower
alkylsulfonyl or acyl halide in the presence of a hydrogen halide
scavenger with a compound corresponding to the formula: ##STR324##
wherein -A-A-, --B--B--, R.sup.1, X, Y.sup.1 and Y.sup.2 are as
defined in Formula IVAA.
6. A process as set forth in claim 4 wherein said compound of
Formula IV is Methyl Hydrogen
17.alpha.-Hydroxy-11.alpha.-(methylsulfonyl)oxy-3-oxopregn-4-ene-7.alpha.-
,21-dicarboxylate, .gamma.-Lactone and said compound of Formula V
is Methyl Hydrogen
11.alpha.,17.alpha.-Dihydroxy-3-oxopregn-4-ene-7.alpha.,21-dicarboxylate,
.gamma.-Lactone.
7. A process for the preparation of a compound of Formula V:
##STR325## wherein -A-A- represents the group
--CHR.sup.4--CHR.sup.5-- or --CR.sup.4.dbd.CR.sup.5--; R.sup.3,
R.sup.4 and R.sup.5 are independently selected from the group
consisting of hydrogen, halo, hydroxy, lower alkyl, lower alkoxy,
hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano and aryloxy;
R.sup.1 represents an alpha-oriented lower alkoxycarbonyl or
hydroxycarbonyl radical; --B--B-- represents the group
--CHR.sup.5--CHR.sup.7-- or an alpha or beta-oriented group:
##STR326## where R.sup.6 and R.sup.7 are independently selected
from the group consisting of hydrogen, halo, lower alkoxy, acyl,
hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl,
acyloxyalkyl, cyano and aryloxy; and R.sup.8 and R.sup.9 are
independently selected from the group consisting of hydrogen,
hydroxy, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,
hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and
aryloxy, or R.sup.8 and R.sup.9 together comprise a carbocyclic or
heterocyclic ring structure, or R.sup.8 or R.sup.9 together with
R.sup.6 or R.sup.7 comprise a carbocyclic or heterocyclic ring
structure fused to the pentacyclic D ring; the process comprising:
reacting a compound of Formula VI with an alkali metal alkoxide
corresponding to the formula R.sup.10OM wherein M is alkali metal
and R.sup.10OM- corresponds to the alkoxy substituent of R1, said
compound of Formula VI having the structure: ##STR327## wherein
-A-A-, --B--B--, R.sup.3, R.sup.8, and R.sup.9 are as defined
above.
8. A process as set forth in claim 7 wherein the compound of
Formula VA corresponds to the formula: ##STR328## wherein -A-A-
represents the group --CH.sup.2--CH.sup.2-- or --CH.dbd.CH--;
R.sup.1 represents an alpha-oriented lower alkoxycarbonyl radical;
--B--B-- represents the group --CH.sup.2--CH.sup.2-- or an alpha-
or beta-oriented group: ##STR329## X represents two hydrogen atoms
or oxo; Y.sup.1 and Y.sup.2 together represent the oxygen bridge
-0-, or Y.sup.1 represents hydroxy, and Y.sup.2 represents hydroxy,
lower alkoxy or, if X represents H.sub.2, also lower alkanoyloxy;
and salts of compounds in which X represents oxo and Y.sup.2
represents hydroxy, the process comprising: reacting a compound of
Formula VIM with an alkali metal alkoxide corresponding to the
formula R.sup.10OM in the presence of an alcohol having the formula
R.sup.10OH, wherein M is alkali metal and R.sup.10O-- corresponds
to the alkoxy substituent of R.sup.1, said compound of Formula VIAA
having the structure: ##STR330## wherein -A-A-, --B--B--, Y.sup.1,
Y.sup.2 and X are as defined in Formula VAA.
9. A process as set forth in claim 7 wherein the compound of
Formula V is Methyl Hydrogen IIa,17a
Dihydroxy-3-oxopregn-4-ene-7.alpha.,21-dicarboxylate,
.gamma.-Lactone and the compound of Formula VI is
4'S(4'.alpha.),7'.alpha.
Hexadecahyadro-11'.alpha.-hydroxy-10'.beta.,13'.beta.-dimethyl-3',5,20'-t-
rioxospiro[furan-1(3H),
17'.beta.-[4,7]methano[17H]cyclopenta[a]phenanthrene]-5'.beta.(2'H)-carbo-
nitrile.
10. A process as set forth in claim 7 wherein cyanide ion is formed
as a by-product of the reaction, the process further comprising
removal of cyanide ion from the reaction zone during the reaction
to reduce the extent of any reaction of cyanide ion with the
product of Formula V.
11. A process as set forth in claim 10 wherein cyanide ion is
removed from the reaction by precipitation with a precipitating
agent.
12. A process as set forth in claim 11 wherein said reaction is
carried out in a solvent medium, and said precipitating agent
comprises a salt comprising a cation which forms a cyanide compound
of lower solubility in said medium than the solubility of the
precipitating agent therein.
13. A process as set forth in claim 12 wherein said cation is
selected from the group consisting of alkaline earth metal ions and
transition metal ions.
14. A process for the preparation of a compound of Formula VI:
##STR331## wherein -A-A- represents the group
--CHR.sup.4--CHR.sup.5-- or --CR.sup.4.dbd.CR.sup.5--; R.sup.3,
R.sup.4 and R.sup.5 are independently selected from the group
consisting of hydrogen, halo, hydroxy, lower alkyl, lower alkoxy,
hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano and aryloxy;
--B--B-- represents the group --CHR.sup.6--CHR.sup.7-- or an alpha
or beta-oriented group: ##STR332## where R.sup.6 and R.sup.7 are
independently selected from the group consisting of hydrogen, halo,
lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl,
alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy; and R.sup.8
and R.sup.9 are independently selected from the group consisting of
hydrogen, hydroxy, halo, lower alkoxy, acyl, hydroxyalkyl,
alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl,
cyano and aryloxy, or R.sup.8 and R.sup.9 together comprise a
carbocyclic or heterocyclic ring structure, or R.sup.8 or R.sup.9
together with R.sup.6 or R.sup.7 comprise a carbocyclic or
heterocyclic ring structure fused to the pentacyclic D ring; the
process comprising: hydrolyzing a compound corresponding to Formula
VIII: ##STR333## wherein -A-A-, --B--B--, R.sup.3, R.sup.8, and
R.sup.9 are as defined above.
15. A process as set forth in claim 14 wherein said compound of
Formula VI corresponds to the formula: ##STR334## wherein: -A-A-
represents the group --CH.sub.2--CH.sub.2-- or --CH.dbd.CH--;
--B--B-- represents the group --CH.sub.2--CH.sub.2-- or an alpha-
or beta-oriented group: ##STR335## X represents two hydrogen atoms
or oxo; Y.sup.1 and Y.sup.2 together represent the oxygen bridge
-0-, or Y.sup.1 represents hydroxy, and Y.sup.2 represents hydroxy,
lower alkoxy or, if X represents H.sup.2, also lower alkanoyloxy;
and salts of compounds in which X represents oxo and Y.sup.2
represents hydroxy, the process comprising: hydrolyzing a compound
of Formula VIIAA in the presence of an acid and an organic solvent
and/or water, said compound of Formula VIIAA having the structure:
##STR336## wherein -A-A-, --B--B--, Y.sup.1 Y.sup.2 and X are as
defined in Formula VIAA.
16. A process as set forth in claim 14 wherein said compound of
Formula VI is 4'S(4'.alpha.),7'.alpha.a-Hexadecahydro
11'.alpha.-hydroxy-10'.beta.,13'.beta.-dimethyl-3',5,20'-trioxospiro[fura-
n-2(3H),17'.beta.-[4,7]methano[17H]cyclopenta[a]phenanthrene]-5'.beta.(2'H-
)-carbonitrile and said compound of Formula VII is
5'R(5'.alpha.),7'.beta.-20'-Aminohexadecahydro-11'.beta.-hydroxy-10'.alph-
a.,13'.alpha.-dimethyl-3',5-dioxospiro[furan-2(3H),17'.alpha.(S'H)-[7,4]me-
theno[4H]cyclopenta[a]phenanthrene]-5'-carbonitrile.
17. A process for the preparation of a compound of Formula VII:
##STR337## wherein -A-A- represents the group
--CHR.sup.4--CHR.sup.5-- or --CR.sup.4.dbd.CR.sup.5--; R.sup.3,
R.sup.4 and R.sup.5 are independently selected from the group
consisting of hydrogen, halo, hydroxy, lower alkyl, lower alkoxy,
hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano and aryloxy;
--B--B-- represents the group --CHR6-CHR7- or an alpha- or
beta-oriented group: ##STR338## where R.sup.6 and R.sup.7 are
independently selected from the group consisting of hydrogen, halo,
lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl,
alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy; and R.sup.8
and R.sup.9 are independently selected from the group consisting of
hydrogen, hydroxy, halo, lower alkoxy, acyl, hydroxyalkyl,
alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl,
cyano and aryloxy, or R.sup.8 and R.sup.9 together comprise a
carbocyclic or heterocyclic ring structure, or R.sup.8 or R.sup.9
together with R.sup.6 or R.sup.7 comprise a carbocyclic or
heterocyclic ring structure fused to the pentacyclic D ring; the
process comprising: reacting a compound of Formula VIII with a
source of cyanide ion in the presence of an alkali metal salt, said
compound of Formula VIII having the structure: ##STR339## wherein
-A-A-, --B--B--, R.sup.3, R.sup.8, and R.sup.9 are as defined
above.
18. A process as set forth in claim 17 wherein said compound of
Formula VII corresponds to Formula VIIAA: ##STR340## wherein: -A-A-
represents the group --CH.sub.2--CH.sub.2-- or --CH.dbd.CH--;
--B--B-- represents the group --CH.sub.2--CH.sub.2-- or an alpha-
or beta-oriented group: ##STR341## X represents two hydrogen atoms
or oxo; Y.sup.1 and Y.sup.2 together represent the oxygen bridge
-0-, or Y.sup.1 represents hydroxy, and Y.sup.2 represents hydroxy,
lower alkoxy or, if X represents H.sub.2, also lower alkanoyloxy;
and salts of compounds in which X represents oxo and Y.sup.2
represents hydroxy, the process comprising: reacting a cyanide
source such as ketone cyanohydrin in the presence of an alkali
metal salt such as LiCl and in the presence of a base with an
11a-hydroxy compound corresponding to the formula: ##STR342##
wherein -A-A-, --B--B--, Y.sup.1, Y.sup.2, and X are as defined in
Formula VIIAA.
19. A process as set forth in claim 17 wherein said compound of
Formula VII is
5'R(5'.alpha.),7'.beta.-20'-Aminohexadecahydro-11'.beta.-hydroxy-1-
0'.alpha.,13'.alpha.-dimethyl-3',5-dioxospiro[furan-2(3H),17'.alpha.(5'H)--
[7,4]metheno[4H]cyclopenta[a]phenanthrene]-5'-carbonitrile and said
compound of Formula VIII is
II.alpha.,I7.alpha.-Dihydroxy-3-oxopregna-4,6-diene-21-carboxylic
Acid, .gamma.-Lactone.
20. A process as set forth in claim 17 wherein said source of
cyanide ion comprises an alkali metal cyanide, the reaction between
said compound of Formula VIII and cyanide ion being carried out in
the presence of an acid and water.
21.-152. (canceled)
Description
BACKGROUND OF THE INVENTION
[0001] This invention relates to the novel processes for the
preparation of 9,11-epoxy steroid compounds, especially those of
the 20-spiroxane series and their analogs, novel intermediates
useful in the preparation of steroid compounds, and processes for
the preparation of such novel intermediates. Most particularly, the
invention is directed to novel and advantageous methods for the
preparation of methyl hydrogen
9,11.alpha.-epoxy-17.alpha.-hydroxy-3-oxopregn-4-ene-7.alpha.,21-dicarbox-
ylate, .gamma.-lactone (also referred to as eplerenone or
epoxymexrenone).
[0002] Methods for the preparation of 20-spiroxane series is
compounds are described in U.S. Pat. No. 4,559,332. The compounds
produced in accordance with the process of the '332 patent have an
open oxygen containing ring E of the general formula: ##STR1## in
which
[0003] -A-A- represents the group --CH.sub.2--CH.sub.2-- or
--CH.dbd.CH--;
[0004] R.sup.1 represents an .alpha.-oriented lower alkoxycarbonyl
or hydroxycarbonyl radical;
[0005] --B--B-- represents the group --CH.sub.2--CH.sub.2-- or an
.alpha.- or .beta.-oriented group; ##STR2##
[0006] R.sup.6 and R.sup.7 being hydrogen;
[0007] X represents two hydrogen-atoms or oxo;
[0008] Y.sup.1 and Y.sup.2 together represent the oxygen bridge
--O--, or
[0009] Y.sup.1 represents hydroxy, and
[0010] Y.sup.2 represents hydroxy, lower alkoxy or, if X represents
H.sub.2, also lower alkanoyloxy;
[0011] and salts of such compounds in which X represents oxo and
Y.sup.2 represents hydroxy, that is to say of corresponding
17.beta.-hydroxy-21-carboxylic acids.
[0012] U.S. Pat. No. 4,559,332 describes a number of methods for
the preparation of epoxymexrenone and related compounds of Formula
IA. The advent of new and expanded clinical uses for epoxymexrenone
create a need for improved processes for the manufacture of this
and other is related steroids.
SUMMARY OF THE INVENTION
[0013] The primary object of the present invention is the provision
of improved processes for the preparation of epoxymexrenone, other
20-spiroxanes and other steroids having common structural features.
Among the particular objects of the invention are: to provide an
improved process that produces products of Formula IA and other
related compounds in high yield; the provision of such a process
which involves a minimum of isolation steps; and the provision of
such a process which may be implemented with reasonable capital
expense and operated at reasonable conversion cost.
[0014] Accordingly, the present invention is directed to a series
of synthesis schemes for epoxymexrenone; intermediates useful in
the manufacture of epoxymexrenone; and syntheses for such novel
intermediates.
[0015] The novel synthesis schemes are described in detail in the
Description of Preferred Embodiments. Among the novel intermediates
of this invention are those described immediately below.
[0016] A compound of Formula IV corresponds to the structure:
##STR3## wherein: [0017] -A-A- represents the group
--CHR.sup.4--CHR.sup.5-- or --CR.sup.4.dbd.CR.sup.5--; [0018]
R.sup.3, R.sup.4 and R.sup.5 are independently selected from the
group consisting of hydrogen, halo, hydroxy, lower alkyl, lower
alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxy carbonyl, cyano and
aryloxy; [0019] R.sup.1 represents an alpha-oriented lower
alkoxycarbonyl or hydroxycarbonyl radical; [0020] R.sup.2 is an
11.alpha.-leaving group the abstraction of which is effective for
generating a double bond between the 9- and 11-carbon atoms;
[0021] --B--B-- represents the group --CHR.sup.6--CHR.sup.7-- or an
alpha- or beta-oriented group: ##STR4## [0022] where R.sup.6 and
R.sup.7 are independently selected from the group consisting of
hydrogen, halo, lower alkoxy, acyl, hydroxalkyl, alkoxyalkyl,
hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and
aryloxy; and [0023] R.sup.8 and R.sup.9 are independently selected
from the group consisting of hydrogen, hydroxy, halo, lower alkoxy,
acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl,
alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy, or R.sup.8 and
R.sup.9 together comprise a carbocyclic or heterocyclic ring
structure, or R.sup.8 or R.sup.9 together with R.sup.6 or R.sup.7
comprise a carbocyclic or heterocyclic ring structure fused to the
pentacyclic D ring.
[0024] A compound of Formula IVA corresponds to Formula IV wherein
R.sup.8 and R.sup.9 together with the ring carbon to which they are
attached form the structure: ##STR5## where X, Y.sup.1, Y.sup.2 and
C(17) are as defined above.
[0025] A compound of Formula IVB corresponds to Formula IV wherein
R.sup.8 and R.sup.9 together form the structure of Formula XXXIII:
##STR6##
[0026] Compounds of Formulae IVC, IVD and IVE, respectively,
correspond to any of Formula IV, IVA, or IVB wherein each of -A-A-
and --B--B-- is --CH.sub.2--CH.sub.2--, R.sup.3 is hydrogen, and
R.sup.1 is alkoxycarbonyl, preferably methoxycarbonyl. Compounds
within the scope of Formula IV may be prepared by reacting a lower
alkylsulfonylating or acylating reagent, or a halide generating
agent, with a corresponding compound within the scope of Formula
V.
[0027] A compound of Formula V corresponds to the structure:
##STR7## wherein -A-A-, --B--B--, R.sup.1, R.sup.3, R.sup.8 and
R.sup.9 are as defined in Formula IV.
[0028] A compound of Formula VA corresponds to Formula V wherein
R.sup.8 and R.sup.9 with the ring carbon to which they are attached
together form the structure: ##STR8## where X, Y.sup.1, Y.sup.2 and
C(17) are as defined above.
[0029] A compound of Formula VB corresponds to Formula V wherein
R.sup.8 and R.sup.9 together form the structure of Formula XXXIII:
##STR9##
[0030] Compounds of Formulae VC, VD and VE, respectively,
correspond to any of Formula V, VA, or VB wherein each of -A-A- and
--B--B-- is --CH.sub.2--CH.sub.2--, R.sup.3 is hydrogen, and
R.sup.1 is alkoxycarbonyl, preferably methoxycarbonyl. Compounds
within the scope of Formula V may be prepared by reacting an alkali
metal alkoxide with a corresponding compound of Formula VI.
[0031] A compound of Formula VI corresponds to the structure:
##STR10## wherein -A-A-, --B--B--, R.sup.3, R.sup.8 and R.sup.9 are
as defined in Formula IV.
[0032] A compound of Formula VIA corresponds to Formula VI wherein
R.sup.8 and R.sup.9 together with the ring carbon to which they are
attached form the structure: ##STR11## where X, Y.sup.1, Y.sup.2
and C(17) are as defined above.
[0033] A compound of Formula VIB corresponds to Formula VI wherein
R.sup.8 and R.sup.9 together form the structure of Formula XXXIII:
##STR12##
[0034] Compounds of Formulae VIC, VID and VIE, respectively,
correspond to any of Formula VI, VIA, or VIB wherein each of -A-A-
and --B--B-- is --CH.sub.2--CH.sub.2--, and R.sup.3 is hydrogen.
Compounds of Formula VI, VIA, VIB and VIC are prepared by
hydrolyzing a compound corresponding to Formula VII, VIIA, VIIB or
VIIC, respectively.
[0035] A compound of Formula VII corresponds to the structure:
##STR13## wherein -A-A-, --B--B--, R.sup.3, R.sup.8 and R.sup.9 are
as defined in Formula IV.
[0036] A compound of Formula VIIA corresponds to Formula VII
wherein R.sup.8 and R.sup.9 together with the ring carbon to which
they are attached form the structure: ##STR14## where X, Y.sup.1,
Y.sup.2 and C(17) are as defined above.
[0037] A compound of Formula VIIB corresponds to Formula VII
wherein R.sup.8 and R.sup.9 together form the structure of Formula
XXXIII: ##STR15##
[0038] Compounds of Formulae VIIC, VIID and VIIE, respectively,
correspond to any of Formula VII, VIIA, or VIIB wherein each of
-A-A- and --B--B-- is --CH.sub.2--CH.sub.2--, and R.sup.3 is
hydrogen. A compound within the scope of Formula VII may be
prepared by cyanidation of a compound within the scope of Formula
VIII.
[0039] A compound of Formula VIII corresponds to the structure:
##STR16## wherein -A-A-, --B--B--, R.sup.3, R.sup.8 and R.sup.9 are
as defined in Formula IV.
[0040] A compound of Formula VIIIA corresponds to Formula VIII
wherein R.sup.8 and R.sup.9 together with the ring carbon to which
they are attached form the structure: ##STR17## where X, Y.sup.1,
Y.sup.2 and C(17) are as defined above.
[0041] A compound of Formula VIIIB corresponds to Formula VIII
wherein R.sup.8 and R.sup.9 together form the structure of Formula
XXXIII: ##STR18##
[0042] Compounds of Formulae VIIIC, VIIID and VIIIE, respectively,
correspond to any of Formula VIII, VIIIA, or VIIIB wherein each of
-A-A- and --B--B-- is --CH.sub.2--CH.sub.2--, and R.sup.3 is
hydrogen. Compounds within the scope of Formula VIII are prepared
by oxidizing a substrate comprising a compound of Formula XXX as
described hereinbelow by fermentation effective for introducing an
11-hydroxy group into the substrate in .alpha.-orientation.
[0043] A compound of Formula IX corresponds to the structure:
##STR19## where -A-A-, --B--B--, R.sup.3, R.sup.8 and R.sup.9 are
as defined in Formula IV, and R.sup.1 is as defined in Formula
V.
[0044] A compound of Formula IXA corresponds to Formula IX wherein
R.sup.8 and R.sup.9 together with the ring carbon to which they are
attached form the structure: ##STR20## where X, Y.sup.1, Y.sup.2
and C(17) are as defined above.
[0045] A compound of Formula IXB corresponds to Formula IX wherein
R.sup.8 and R.sup.9 together with the ring carbon to which they are
attached form the structure of Formula XXXIII: ##STR21##
[0046] Compounds of Formulae IXC, IXD and IXE, respectively,
correspond to any of Formula IX, IXA, or IXB wherein each of -A-A-
and --B--B-- is --CH.sub.2--CH.sub.2--, and R.sup.3 is hydrogen.
Compounds within the scope of Formula IX can be prepared by
bioconversion of a corresponding compound within the scope of
Formula X.
[0047] A compound of Formula XIV corresponds to the structure:
##STR22## wherein -A-A-, --B--B--, R.sup.3, R.sup.8 and R.sup.9 are
as defined in Formula IV.
[0048] A compound of Formula XIVA corresponds to Formula XIV
wherein R.sup.8 and R.sup.9 together with the ring carbon to which
they are attached form the structure: ##STR23## where X, Y.sup.1,
Y.sup.2 and C(17) are as defined above.
[0049] A compound of Formula XIVB corresponds to Formula XIV
wherein R.sup.8 and R.sup.9 together with the ring carbon to which
they are attached form the structure of Formula XXXIII:
##STR24##
[0050] Compounds of Formulae XIVC, XIVD and XIVE, respectively,
correspond to any of Formula XIV, XIVA, or XIVB wherein each of
-A-A- and --B--B-- is --CH.sub.2--CH.sub.2--, and R.sup.3 is
hydrogen. Compounds within the scope of Formula XIV can be prepared
by hydrolysis of a corresponding compound within the scope of
Formula XV.
[0051] A compound of Formula XV corresponds to the structure:
##STR25## wherein -A-A-, --B--B--, R.sup.3, R.sup.8 and R.sup.9 are
as defined in Formula IV.
[0052] A compound of Formula XVA corresponds to Formula XV wherein
R.sup.8 and R.sup.9 together with the ring carbon to which they are
attached form the structure: ##STR26## where X, Y.sup.1, Y.sup.2
and C(17) are as defined above.
[0053] A compound of Formula XVB corresponds to Formula XV wherein
R.sup.8 and R.sup.9 together with the ring carbon to which they are
attached form the structure of Formula XXXIII: ##STR27##
[0054] Compounds of Formulae XVC, XVD and XVE, respectively,
correspond to any of Formula XV, XVA, or XVB wherein each of -A-A-
and --B--B-- is --CH.sub.2--CH.sub.2--, and R.sup.3 is hydrogen.
Compounds within the scope of Formula XV can be prepared by
cyanidation of a corresponding compound within the scope of Formula
XVI.
[0055] A compound of Formula XXI corresponds to the structure:
##STR28## wherein -A-A-, --B--B--, R.sup.3, R.sup.8 and R.sup.9 are
as defined in Formula IV.
[0056] A compound of Formula XXIA corresponds to Formula XXI
wherein R.sup.8 and R.sup.9 together with the ring carbon to which
they are attached form the structure: ##STR29## where X, Y.sup.1,
Y.sup.2 and C(17) are as defined above.
[0057] A compound of Formula XXIB corresponds to Formula XXI
wherein R.sup.8 and R.sup.9 together form the structure of Formula
XXXIII: ##STR30##
[0058] Compounds of Formulae XXIC, XXID and XXIE, respectively,
correspond to any of Formula XXI, XXIA, or XXIB wherein each of
-A-A- and --B--B-- is --CH.sub.2--CH.sub.2--, and R.sup.3 is
hydrogen. Compounds within the scope of Formula XXI may be prepared
by hydrolyzing a corresponding compound within the scope of Formula
XXII.
[0059] A compound of Formula XXII corresponds to the structure:
##STR31## wherein -A-A-, --B--B--, R.sup.3, R.sup.8 and R.sup.9 are
as defined in Formula IV.
[0060] A compound of Formula XXIIA corresponds to Formula XXII
wherein R.sup.8 and R.sup.9 together with the ring carbon to which
they are attached form the structure: ##STR32## where X, Y.sup.1,
Y.sup.2 and C(17) are as defined above.
[0061] A compound of Formula XXIIB corresponds to Formula XXII
wherein R.sup.8 and R.sup.9 together form the structure of Formula
XXXIII: ##STR33##
[0062] Compounds of Formulae XXIIC, XXIID and XXIIE, respectively,
correspond to any of Formula XXII, XXIIA, or XXIIB wherein each of
-A-A- and --B--B-- is --CH.sub.2--CH.sub.2--, and R.sup.3 is
hydrogen. Compounds within the scope of Formula XXII may be
prepared by cyanidation of a compound within the scope of Formula
XXIII.
[0063] A compound of Formula XXIII corresponds to the structure:
##STR34## wherein -A-A-, --B--B--, R.sup.3, R.sup.8 and R.sup.9 are
as defined in Formula IV.
[0064] A compound of Formula XXIIIA corresponds to Formula XXIII
wherein R.sup.8 and R.sup.9 together with the ring carbon to which
they are attached form the structure: ##STR35## where X, Y.sup.1,
Y.sup.2 and C(17) are as defined above.
[0065] A compound of Formula XXIIIB corresponds to Formula XXIII
wherein R.sup.8 and R.sup.9 together form the structure of Formula
XXXIII: ##STR36##
[0066] Compounds of Formulae XXIIIC, XXIIID and XXIIIE,
respectively, correspond to any of Formula XXIII, XXIIIA, or XXIIIB
wherein each of -A-A- and --B--B-- is --CH.sub.2--CH.sub.2--, and
R.sup.3 is hydrogen. Compounds within the scope of Formula XXIII
can be prepared by oxidation of a compound of Formula XXIV, as
described hereinbelow.
[0067] A compound of Formula XXVI corresponds to the structure:
##STR37## wherein -A-A-, --B--B--, R.sup.3, R.sup.8 and R.sup.9 are
as defined in Formula IV.
[0068] A compound of Formula XXVIA corresponds to Formula XXVI
wherein each of -A-A- and --B--B-- is --CH.sub.2--CH.sub.2-- and
R.sup.3 is hydrogen. Compounds within the scope of Formula XXVI can
be prepared by oxidation of a compound of Formula XXVII.
[0069] A compound of Formula XXV corresponding to the structure:
##STR38## wherein -A-A-, --B--B--, R.sup.3, R.sup.8 and R.sup.9 are
as defined in Formula IV.
[0070] A compound of Formula XXVA corresponds to Formula XXV
wherein each of -A-A- and --B--B-- is --CH.sub.2--CH.sub.2--, and
R.sup.3 is hydrogen. Compounds within the scope of Formula XXV can
be prepared by cyanidation of a compound of Formula XXVI.
[0071] A compound of Formula 104 corresponds to the structure:
##STR39## wherein -A-A-, --B--B-- and R.sup.3 are as defined in
Formula IV, and R.sup.11 is C.sub.1 to C.sub.4 alkyl.
[0072] A compound of Formula 104A corresponds to Formula 104
wherein each of -A-A- and --B--B-- is --CH.sub.2--CH.sub.2--, and
R.sup.3 is hydrogen. Compounds within the scope of Formula 104 may
be prepared by thermal decomposition of a compound of Formula
103.
[0073] A compound of Formula 103 corresponds to the structure:
##STR40## wherein -A-A-, --B--B--, R.sup.3 and R.sup.11 are as
defined in Formula 104, and R.sub.12 is a C.sub.1 to C.sub.4
alkyl.
[0074] A compound of Formula 103A corresponds to Formula 103
wherein each of -A-A- and --B--B-- is --CH.sub.2--CH.sub.2--, and
R.sup.3 is hydrogen. Compounds within the scope of Formula 103 may
be prepared by reaction of a corresponding compound of Formula 102
with a dialkyl malonate in the presence of a base such as an alkali
metal alkoxide.
[0075] A compound of Formula 102 corresponds to the structure:
##STR41## wherein -A-A-, --B--B--, R.sup.3 and R.sup.11 are as
defined in Formula 104.
[0076] A compound of Formula 102A corresponds to Formula 102
wherein each of -A-A- and --B--B-- is --CH.sub.2--CH.sub.2--, and
R.sup.3 is hydrogen. Compounds within the scope of Formula 102 may
be prepared by reaction of a corresponding compound of Formula 101
with a trialkyl sulfonium compound in the presence of a base.
[0077] A compound of Formula 101 corresponds to the structure:
##STR42## wherein -A-A-, --B--B--, R.sup.3 and R.sup.11 are as
defined in Formula 104.
[0078] A compound of Formula 101A corresponds to Formula 101
wherein each of -A-A- and --B--B-- is --CH.sub.2--CH.sub.2--, and
R.sup.3 is hydrogen. Compounds within the scope of Formula 101 may
be prepared by reaction of 11.alpha.-hydroxyandrostene-3,17-dione
or other compound of Formula XXXVI with a trialkyl orthoformate in
the presence of an acid.
[0079] A compound of Formula XL corresponds to the Formula:
##STR43## wherein -E-E- is selected from among: ##STR44## R.sup.21,
R.sup.22 and R.sup.23 are independently selected from among
hydrogen, alkyl, halo, nitro, and cyano; R.sup.24 is selected from
among hydrogen and lower alkyl; R.sup.80 and R.sup.90 are
independently selected from keto and the substituents that may
constitute R.sup.8 and R.sup.9 (as defined hereinabove with
reference to Formula IV); and -A-A-, --B--B-- and R.sup.3 are as
defined in Formula IV.
[0080] A compound of Formula XLA corresponds to Formula XL wherein
R.sup.21, R.sup.22 and R.sup.23 are independently selected from
among hydrogen, halogen and lower alkyl.
[0081] A compound of Formula XLB corresponds to Formula XLA wherein
-E-E- corresponds to Formula XLIII, XLIV, XLV or XLVII. A compound
of Formula XLC corresponds to Formula XLB wherein -E-E- corresponds
to Formula XLV. A compound is of XLD corresponds to Formula XLB
wherein -E-E- corresponds to Formula XLVII.
[0082] A compound of Formula XLE corresponds to Formula XL wherein
R.sup.80 and R.sup.90 together with the ring carbon atom to which
they are attached comprise keto or: ##STR45## where X, Y.sup.1,
Y.sup.2 and C(17) are as defined above, or ##STR46##
[0083] Compounds of Formula XLIE correspond to Formula XL in which
R.sup.80 and R.sup.90 together form keto.
[0084] Compounds of Formulae XLF, XLG, XLH, XLJ, XLM, and XLN
correspond to Formula XL, XLA, XLB, XLC, XLD and XLE, respectively,
in which -A-A-, --B--B-- and R.sup.3 are as defined above.
[0085] A compound of Formula XLI corresponds to the Formula:
##STR47## wherein -E-E- is selected from among: ##STR48## R.sup.18
is C.sub.1 to C.sub.4 alkyl or the R.sup.18O-- groups together form
an O,O-oxyalkylene bridge; R.sup.21, R.sup.22 and R.sup.23 are
independently selected from among hydrogen, alkyl, halo, nitro, and
among hydrogen and lower alkyl; R.sup.80 and R.sup.90 are
independently selected from keto and the substituents that may
constitute R.sup.8 and R.sup.9; and -A-A-, --B--B-- and R.sup.3 are
as defined in Formula IV.
[0086] A compound of Formula XLIA- corresponds to Formula XLI
wherein R.sup.21, R.sup.22 and R.sup.23 are independently selected
from among hydrogen, halogen, and lower alkyl.
[0087] A compound of Formula XLIB corresponds to Formula XLIA
wherein -E-E- corresponds to Formula XLIII, XLIV, XLV or XLVII.
[0088] A compound of Formula XLIC corresponds to Formula XLI
wherein R.sup.80 and R.sup.90 together with the ring carbon atom to
which they are attached comprise keto or: ##STR49## where X,
Y.sup.1, Y.sup.2 and C(17) are as defined above.
[0089] Compounds of Formulae XLID correspond to Formula. XLI in
which the substituent XXXIV corresponds to the structure XXXIII
##STR50##
[0090] Compounds of Formula XLIE correspond to Formula XL in which
R.sup.80 and R.sup.90 together form keto.
[0091] Compounds of Formulae XLIF, XLIG, XLIH, XLIJ, XLIM, and XLIN
correspond to Formula XLI, XLIA, XLIB, XLIC, XLID and XLIE,
respectively, in which -A-A-, --B--B-- and R.sup.3 are as defined
above. Compounds within the scope of Formula XLI are prepared by
hydrolysis of corresponding compounds of Formula XL as defined
hereinbelow.
[0092] A compound of Formula XLII corresponds to the Formula:
wherein -E-E- is selected from among: ##STR51## ##STR52## R.sup.21,
R.sup.22 and R.sup.23 are independently selected from among
hydrogen, alkyl, halo, nitro, and cyano; R.sup.24 is selected from
among hydrogen and lower alkyl; R.sup.80 and R.sup.90 are
independently selected from keto and the substituents that may
constitute R.sup.8 and R.sup.9; and -A-A-, --B--B-- and R.sup.3 are
as defined in Formula IV.
[0093] A compound of Formula XLIIA corresponds to Formula XLII
wherein R.sup.21, R.sup.22 and R.sup.23 are independently selected
from among hydrogen, halogen and lower alkyl.
[0094] A compound of Formula XLIIB corresponds to Formula XLIIA
wherein -E-E- corresponds to Formula XLIII, XLIV, XLV or XLVII.
[0095] A compound of Formula XLIIC corresponds to Formula XLII
wherein R.sup.80 and R.sup.90 together with the ring carbon to
which they are attached comprise keto or: ##STR53## where X,
Y.sup.1, Y.sup.2 and C(17) are as defined above.
[0096] Compounds of Formulae XLIID correspond to Formula XLII in
which the substituent XXXIV corresponds to the structure XXXIII
##STR54##
[0097] Compounds of Formula XLIIE correspond to Formula XLII in
which R.sup.80 and R.sup.90 together form keto. Compounds of
Formulae XLIIF, XLIIG, XLIIH, XLIIJ, XLIIM and XLIIN correspond to
Formulae XLII, XLIIA, XLIIB, XLIIC, XLIID and XLIIE, respectively,
in which -A-A- and --B--B-- are --CH.sub.2--CH.sub.2 and R.sup.3 is
hydrogen. Compounds within the scope of Formula XLII are prepared
by deprotecting a corresponding compound of Formula XLI.
[0098] A compound of the Formula XLIX corresponds to the structure:
##STR55## wherein -E-E- is as defined in Formula XL, and -A-A-,
--B--B--, R.sup.1, R.sup.3, R.sup.8 and R.sup.9 are as defined in
Formula IV.
[0099] A compound of Formula XLIXA corresponds to Formula XLIX
wherein R.sup.8 and R.sup.9 with the ring carbon to which they are
attached together form the structure: ##STR56## where X, Y.sup.1,
Y.sup.2 and C(17) are as defined above.
[0100] A compound of Formula XLIXB corresponds to Formula XLIX
wherein R.sup.8 and R.sup.9 together form the structure of Formula
XXXIII: ##STR57## Compounds of Formulae XLIXC, XLIXD, XLIXE,
respectively, correspond to any of Formula XLIX, XLIXA or XLIXB
wherein each of -A-A- and --B--B-- is --CH.sub.2--CH.sub.2--,
R.sup.3 is hydrogen and R.sup.1 is alkoxycarbonyl, preferably
methoxycarbonyl. Compounds within the scope of Formula XLIX may be
prepared by reacting an alcoholic or aqueous solvent with a
corresponding compound Formula VI in the presence of a suitable
base.
[0101] A compound of Formula A203 corresponds to the structure:
##STR58## wherein -E-E- is selected from among: ##STR59## R.sup.18
is selected from among C.sub.1 to C.sub.4 alkyl; R.sup.21, R.sup.22
and R.sup.23 are independently selected from among hydrogen, alkyl,
halo, nitro, and cyano; R.sup.24 is selected from among hydrogen
and lower alkyl; and -A-A-, --B--B-- and R.sup.3 are as defined in
Formula IV.
[0102] A compound of Formula A203A corresponds to Formula A203
wherein R.sup.21, R.sup.22 and R.sup.23 are independently selected
from among hydrogen, halogen, and lower alkyl.
[0103] A compound of Formula A203B corresponds to Formula A203A
wherein -E-E- corresponds to Formula XLIII, XLIV, XLV or XLVII.
[0104] Compounds of Formulae A203C, A203D, and A203E respectively
correspond to Formula A203, A203A and A203B wherein each of -A-A-
and --B--B-- is --CH.sub.2--CH.sub.2--, and R.sup.3 is hydrogen.
Compounds within the scope of Formula A203 are prepared by reducing
a compound of Formula A202 as defined hereinbelow.
[0105] A compound of Formula A204 corresponds to the structure:
##STR60## wherein R.sup.19 is C.sub.1 to C.sub.4 alkyl, and -E-E-,
-A-A-, --B--B-- and R.sup.3 are as defined in Formula 203.
[0106] A compound of Formula A204A corresponds to Formula A204
wherein R.sup.21, R.sup.22 and R.sup.23 are independently selected
from among hydrogen, halogen, and lower alkyl.
[0107] A compound of Formula A204B corresponds to Formula A204A
wherein -E-E- corresponds to Formula XLIII, XLIV, XLV or XLVII.
[0108] Compounds of Formulae A204C, A204D, and A204E respectively
correspond to Formulae A204, A204A, and A204B wherein each of -A-A-
and --B--B-- is --CH.sub.2--CH.sub.2--, and R.sup.3 is hydrogen.
Compounds within the scope of Formula A204 are prepared by
hydrolysis of corresponding compounds of Formula A203.
[0109] A compound of Formula A205 corresponds to the structure:
##STR61## wherein R.sup.20 is C.sub.1 to C.sub.4 alkyl, and -E-E-,
R.sup.19, -A-A-, --B--B-- and R.sup.3 are as defined in Formula
204.
[0110] A compound of Formula A205A corresponds to Formula A205
wherein R.sup.21, R.sup.22 and R.sup.23 are independently selected
from among hydrogen, halogen, and lower alkyl.
[0111] A compound of Formula A205B corresponds to Formula A205A
wherein -E-E- corresponds to Formula XLIII, XLIV, XLV or XLVII.
[0112] Compounds of Formulae A205C, A205D and A205E respectively
correspond to Formula A205, A205A and A205B wherein each of -A-A-
and --B--B-- is --CH.sub.2--CH.sub.2--, and R.sup.3 is hydrogen.
Compounds within the scope of Formula A205 may be prepared by
reacting a corresponding compound of Formula A204 with an alkanol
and acid.
[0113] A compound of Formula A206 corresponds to the structure:
##STR62## wherein R.sup.19, R.sup.20, -E-E-, -A-A-, --B--B-- and
R.sup.3 are as defined in Formula 205.
[0114] A compound of Formula A206A corresponds to Formula A206
wherein R.sup.21, R.sup.22 and R.sup.23 are independently selected
from among hydrogen, halogen, and lower alkyl.
[0115] A compound of Formula A206B corresponds to Formula A206A
wherein -E-E- corresponds to Formula XLIII, XLIV, XLV or XLVII.
[0116] Compounds of Formulae A206C, A206D and A206E respectively
correspond to Formula A206, A206A, and A206B wherein each of -A-A-
and --B--B-- is --CH.sub.2--CH.sub.2--, and R.sup.3 is hydrogen.
Compounds within the scope of Formula A206 may be prepared by
reacting a corresponding compound within the scope of Formula A205
with a trialkyl sulfonium halide.
[0117] A compound of Formula A207 corresponds to the structure:
##STR63## wherein R.sup.25 is C.sub.1 to C.sub.4 alkyl, and -E-E-,
R.sup.19, R.sup.20, -A-A-, --B--B-- and R.sup.3 are as defined in
Formula A205.
[0118] A compound of Formula A207A corresponds to Formula A207
wherein R.sup.21, R.sup.22 and R.sup.23 are independently selected
from among hydrogen, halogen, and lower alkyl.
[0119] A compound of Formula A207B corresponds to Formula A207A
wherein -E-E- corresponds to Formula XLIII, XLIV, XLV or XLVII.
[0120] Compounds of Formulae A207C, A207D and A207E respectively
correspond to Formula A207, A207A and A207B wherein each of -A-A-
and --B--B-- is --CH.sub.2--CH.sub.2--, and R.sup.3 is hydrogen.
Compounds of Formula A207 can be prepared by reaction of compounds
of Formula A206 with a dialkyl malonate.
[0121] A compound of Formula A208 corresponds to the structure:
##STR64## wherein -E-E-, R.sup.80 and R.sup.90 are as defined in
Formula XLII; -A-A-, --B--B-- and R.sup.3 are as defined in Formula
104; and R.sup.19, R.sup.20, -A-A-, --B--B--, and R.sup.3 are as
defined in Formula 205.
[0122] A compound of Formula A208A corresponds to Formula A208
wherein R.sup.21 and R.sup.22 are independently selected from among
hydrogen, halogen, and lower alkyl.
[0123] A compound of Formula A208B corresponds to Formula A208A
wherein -E-E- corresponds to Formula XLIII, XLIV, XLV or XLVII.
[0124] A compound of Formula A208C corresponds to Formula A208
wherein R.sup.80 and R.sup.90 together with the ring carbon to
which they are attached comprise keto or: ##STR65## where X,
Y.sup.1, Y.sup.2 and C(17) are as defined above.
[0125] Compounds of Formulae 208D correspond to Formula 208C in
which the substituent XXXIV corresponds to the structure XXXIII
##STR66##
[0126] Compounds of Formulae A208E, A208F, A208G, A208H and A208J
respectively correspond to Formula A208, A208A, A208B, A208C and
A208D wherein each of -A-A- and --B--B-- is --CH.sub.2--CH.sub.2--,
and R.sup.3 is hydrogen. Compounds within the scope of Formula A208
can be prepared by thermal decomposition of corresponding compounds
of Formula A207.
[0127] A compound of Formula A209 corresponds to the structure:
##STR67## wherein R.sup.80 and R.sup.90 are as defined in Formula
XLI, and -E-E- and -A-A-, --B--B--, and R.sup.1 are as defined in
Formula 205.
[0128] A compound of Formula A209A corresponds to Formula A209
wherein R.sup.21 and R.sup.22 are independently selected from among
hydrogen, halogen, and lower alkyl.
[0129] A compound of Formula A209B corresponds to Formula A209A
wherein -E-E- corresponds to Formula XLIII, XLIV, XLV or XLVII.
[0130] A compound of Formula A209C corresponds to Formula A209B
wherein -E-E- corresponds to Formula XLIV.
[0131] A compound of Formula A209D corresponds to Formula A208
wherein R.sup.80 and R.sup.90 together with the ring carbon to
which they are attached comprise keto or: ##STR68## where X,
Y.sup.1, Y.sup.2 and C(17) are as defined above.
[0132] Compounds of Formulae 209E correspond to Formula A209D in
which the substituent XXXIV corresponds to the structure XXXIII
##STR69##
[0133] Compounds of Formulae A209F, A209G, A209H, A209J, A209L, and
A209M respectively correspond to Formula A209, A209A, A209B, A209C,
A209D and A209E wherein each of -A-A- and --B--B-- is
--CH.sub.2--CH.sub.2--, and R.sup.3 is hydrogen. Compounds within
the scope of Formula A209 may be prepared by hydrolysis of a
corresponding compound of Formula A208.
[0134] A compound of Formula A210 corresponds to the structure:
##STR70## wherein R.sup.80 and R.sup.90 are as defined in Formula
XLI, and the substituents -A-A-, --B--B-- and R.sup.3 are as
defined in Formula IV.
[0135] A compound of Formula A210A corresponds to Formula A210
wherein R.sup.80 and R.sup.90 together with the ring carbon to
which they are attached comprise keto or: ##STR71## wherein X,
Y.sup.1, Y.sup.2 and C(17) are as defined above.
[0136] Compounds of Formulae A210B correspond to Formula A210A in
which the substituent XXXIV corresponds to the structure XXXIII
##STR72##
[0137] Compounds of Formula A210C correspond to Formula A210A in
which R.sup.80 and R.sup.90 together form keto.
[0138] Compounds of Formulae A210D, A210E, A210F and A210G
respectively correspond to Formula A210, A210A, A210B and A210C
wherein each of -A-A- and --B--B-- is --CH.sub.2--CH.sub.2-- and
R.sup.1 is hydrogen. Compounds within the scope of Formula 210 can
be prepared by epoxidation of a compound of Formula 209 in which
-E-E- is ##STR73## C.dbd.CH.
[0139] A compound of Formula A211 corresponds to the Formula
##STR74## where -A-A-, --B--B-- and R.sup.3 are as described
above.
[0140] A compound of Formula A211A corresponds to Formula A211
wherein R.sup.80 and R.sup.90 together comprise keto or: ##STR75##
wherein X, Y.sup.1, Y.sup.2 and C(17) are as defined above.
[0141] Compounds of Formulae A211B correspond to Formula A211A in
which the substituent XXXIV corresponds to the structure XXXIII
##STR76##
[0142] Compounds of Formula A211C correspond to Formula A210A in
which R.sup.80 and R.sup.90 together form keto.
[0143] Compounds of Formulae A211D, A211E, A211F, and A211G,
respectively correspond to Formula A211, A211A, A211B and A211C
wherein each of -A-A- and --B--B-- is --CH.sub.2--CH.sub.2--, and
R.sup.3 is hydrogen. Compounds within the scope of Formula A211 can
be prepared by oxidation of a corresponding compound of Formula
A210, or in the course of epoxidation of the corresponding compound
of Formula A209 where -E-E- is ##STR77## C.dbd.CH. Compounds of
Formula A211 may be converted to compounds of Formula I in the
manner described hereinbelow.
[0144] A compound of Formula L corresponds to the structure:
##STR78## wherein R.sup.11 is C.sub.1 to C.sub.4 alkyl, and -A-A-,
--B--B--, R.sup.1, R.sup.2, R.sup.3, R.sup.8 and R.sup.9 are as
defined above.
[0145] Compounds of Formula LA correspond to Formula L wherein
R.sup.8 and R.sup.9 together with the carbon atom to which they are
attached comprises ##STR79## wherein X, Y.sup.1 and Y.sup.2 are as
defined above.
[0146] Compounds of Formula LB correspond to Formula L wherein
R.sup.8 and R.sup.9 correspond to Formula XXXIII ##STR80##
[0147] Compounds of Formulae LC, LD, LE correspond to Formulae L,
LA and LB, respectively, where -A-A- and --B--B-- are each
--CH.sub.2--CH.sub.2-- and R.sup.3 is hydrogen.
[0148] Based on the disclosure of specific reaction schemes as set
out hereinbelow, it will be apparent which of these compounds have
the greatest utility relative to a particular reaction scheme. The
compounds of this invention are useful as intermediates for
epoxymexrenone and other steroids.
[0149] Other objects and features will be in part apparent and in
part pointed out hereinafter.
BRIEF DESCRIPTION OF THE DRAWINGS
[0150] FIG. 1 is a schematic flow sheet of a process for the
bioconversion of canrenone or a canrenone derivative to the
corresponding 11.alpha.-hydroxy compound;
[0151] FIG. 2 is a schematic flow sheet of a preferred process for
the bioconversion/11-.alpha.-hydroxylation of canrenone and
canrenone derivatives;
[0152] FIG. 3 is a schematic flow sheet of a particularly preferred
process for the bioconversion/11-.alpha.-hydroxylation of canrenone
and canrenone derivatives;
[0153] FIG. 4 shows the particle size distribution for canrenone as
prepared in accordance with the process of FIG. 2; and
[0154] FIG. 5 shows the particle size distribution for canrenone as
sterilized in the transformation fermenter in accordance with the
process of FIG. 3.
[0155] Corresponding reference characters indicate corresponding
parts throughout the drawings.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0156] In accordance with the present invention, various novel
process schemes have been devised for the preparation of
epoxymexrenone and other compounds corresponding Formula I:
##STR81## wherein: [0157] -A-A- represents the group
--CHR.sup.4--CHR.sup.5-- or --CR.sup.4.dbd.CR.sup.5--; [0158] is
R.sup.3, R.sup.4 and R.sup.5 are independently selected from the
group consisting of hydrogen, halo, hydroxy, lower alkyl, lower
alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano and
aryloxy; [0159] R.sup.1 represents an alpha-oriented lower
alkoxycarbonyl or hydroxyalkyl radical; and [0160] --B--B--
represents the group --CHR.sup.6--CHR.sup.7-- or an alpha- or
beta-oriented group: ##STR82## [0161] where R.sup.6 and R.sup.7 are
independently selected from the group consisting of hydrogen, halo,
lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl,
alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy; and [0162]
R.sup.8 and R.sup.9 are independently selected from the group
consisting of hydrogen, hydroxy, halo, lower alkoxy, acyl,
hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl,
acyloxyalkyl, cyano and aryloxy, or R.sup.8 and R.sup.9 together
comprise a carbocyclic or heterocyclic ring structure, or R.sup.8
or R.sup.9 together with R.sup.6 or R.sup.7 comprise a carbocyclic
or heterocyclic ring structure fused to the pentacyclic D ring.
[0163] Unless stated otherwise, organic radicals referred to as
"lower" in the present disclosure contain at most 7, and preferably
from 1 to 4, carbon atoms.
[0164] A lower alkoxycarbonyl radical is preferably one derived
from an alkyl radical having from 1 to 4 carbon atoms, such as
methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec.-butyl and
tert.-butyl; especially preferred are methoxycarbonyl,
ethoxycarbonyl and isopropoxycarbonyl. A lower alkoxy radical is
preferably one derived from one of the above-mentioned
C.sub.1-C.sub.4 alkyl radicals, especially from a primary
C.sub.1-C.sub.4 alkyl radical; especially preferred is methoxy. A
lower alkanoyl radical is preferably one derived from a
straight-chain alkyl having from 1 to 7 carbon atoms; especially
preferred are formyl and acetyl.
[0165] A methylene bridge in the 15,16-position is preferably
.beta.-oriented.
[0166] A preferred class of compounds that may be produced in
accordance with the methods of the invention are the 20-spiroxane
compounds described in U.S. Pat. No. 4,559,332, i.e., those
corresponding to Formula IA: ##STR83## where: [0167] -A-A-
represents the group --CH.sub.2--CH.sub.2-- or --CH.dbd.CH--;
[0168] --B--B-- represents the group --CH.sub.2--CH.sub.2-- or an
alpha- or beta-oriented group of Formula IIIA: ##STR84## [0169]
R.sup.1 represents an alpha-oriented lower alkoxycarbonyl or
hydroxycarbonyl radical; [0170] X represents two hydrogen atoms,
oxo or .dbd.S; [0171] Y.sup.1 and Y.sup.2 together represent the
oxygen bridge --O--, or [0172] Y.sup.1 represents hydroxy, and
[0173] Y.sup.2 represents hydroxy, lower alkoxy or, if X represents
H.sub.2, also lower alkanoyloxy.
[0174] Preferably, 20-spiroxane compounds produced by the novel
methods of the invention are those of Formula I in which Y.sup.1
and Y.sup.2 together represent the oxygen bridge Especially
preferred compounds of the formula I are those in which X
represents oxo. Of compounds of the 20-spiroxane compounds of
Formula IA in which X represents oxo, there are most especially
preferred those in which Y.sup.1 together with Y.sup.2 represents
the oxygen bridge --O--.
[0175] As already mentioned, 17.beta.-hydroxy-21-carboxylic acid
may also be in the form of their salts. There come into
consideration especially metal and ammonium salts, such as alkali
metal and alkaline earth metal salts, for example sodium, calcium,
magnesium and, preferably, potassium salts, and ammonium salts
derived from ammonia or a suitable, preferably physiologically
tolerable, organic nitrogen-containing base. As bases there come
into consideration not only amines, for example lower alkylamines
(such as triethylamine), hydroxy-lower alkylamines (such as
2-hydroxyethylamine, di-(2-hydroxyethyl)-amine or
tri-(2-hydroxyethyl)-amine) cycloalkylamines (such as
dicyclohexylamine) or benzylamines (such as benzylamine and
N,N'-dibenzylethylenediamine), but also nitrogen-containing
heterocyclic compounds, for example those of aromatic character
(such as pyridine or quinoline) or those having an at least
partially saturated heterocyclic ring (such as N-ethylpiperidine,
morpholine, piperazine or N,N'-dimethylpiperazine).
[0176] Also included amongst preferred compounds are alkali metal
salts, especially potassium salts, of compounds of the formula IA
in which R.sup.1 represents alkoxycarbonyl, with X representing oxo
and each of Y.sup.1 and Y.sup.2 representing hydroxy.
[0177] Especially preferred compounds of the formula I and IA are,
for example, the following: [0178]
9.alpha.,11.alpha.-epoxy-7.alpha.-methoxycarbonyl-20-spirox-4-ene-3,21-di-
one, [0179]
9.alpha.,11.alpha.-epoxy-7.alpha.-ethoxycarbonyl-20-spirox-4-ene-3,21-dio-
ne, [0180]
9.alpha.,11.alpha.-epoxy-7.alpha.-isopropoxycarbonyl-20-spirox-4-ene-3,21-
-dione,
[0181] and the 1,2-dehydro analogue of each of the compounds;
[0182]
9.alpha.,11.alpha.-epoxy-6.alpha.,7.alpha.-methylene-20-spirox-4-ene-3,21-
-dione, [0183]
9.alpha.,11.alpha.-epoxy-6.beta.,7.beta.-methylene-20-spirox-4-ene-3,21-d-
ione, [0184]
9.alpha.,11.alpha.-epoxy-6.beta.,7.beta.;15.beta.,16.beta.-bismethylene-2-
0-spirox-4-ene-3,21-dione,
[0185] and the 1,2-dehydro analogue of each of these compounds;
[0186]
9.alpha.,11.alpha.-epoxy-7.alpha.-methoxycarbonyl-17.beta.-hydroxy-3-oxo--
pregn-4-ene-21-carboxylic acid, [0187]
9.alpha.,11.alpha.-epoxy-7.alpha.-ethoxycarbonyl-17.beta.-hydroxy-3-oxo-p-
regn-4-ene-21-carboxylic acid, [0188]
9.alpha.,11.alpha.-epoxy-7.alpha.-isopropoxycarbonyl-17.beta.-hydroxy-3-o-
xo-pregn-4-ene-21-carboxylic acid, [0189]
9.alpha.,11.alpha.-epoxy-17.beta.-hydroxy-6.alpha.,7.alpha.-methylene-3-o-
xo-pregn-4-ene-21-carboxylic acid, [0190]
9.alpha.,11.alpha.-epoxy-17.beta.-hydroxy-6.beta.,7.beta.-methylene-3-oxo-
-pregn-4-ene-21-carboxylic acid, [0191]
9.alpha.,11.alpha.-epoxy-17.beta.-hydroxy-6.alpha.,7.alpha.;15.beta.,16.b-
eta.-bismethylene-3-oxo-pregn-4-ene-21-carboxylic acid,
[0192] and alkali metal salts, especially the potassium salt or
ammonium of each of these acids, and also a corresponding
1,2-dehydro analogue of each of the mentioned carboxylic acids or
of a salt thereof; [0193]
9.alpha.,11.alpha.-epoxy-15.beta.,16.beta.-methylene-3,21-dioxo-20-spirox-
-4-ene-7.alpha.-carboxylic acid methyl ester, ethyl ester and
isopropyl ester, [0194]
9.alpha.,11.alpha.-epoxy-1565.beta.,16.beta.-methylene-3,21-dioxo-20-spir-
oxa-1,4-diene-7.alpha.-carboxylic acid methyl ester, ethyl ester
and isopropyl ester, [0195]
9.alpha.,11.alpha.-epoxy-3-oxo-20-spirox-4-ene-7.alpha.-carboxylic
acid methyl ester, ethyl ester and isopropyl ester,
9.alpha.,11.alpha.-epoxy-6.beta.,6.beta.-methylene-20-spirox-4-en-3-one,
[0196]
9.alpha.,11.alpha.-epoxy-6.beta.,7.beta.;15.beta.,16.beta.-bismet-
hylene-20-spirox-4-en-3-one, [0197]
9.alpha.,11.alpha.-epoxy,17.beta.-hydroxy-17.alpha.(3-hydroxy-propyl)-3-o-
xo-androst-4-ene-7.alpha.-carboxylic acid methyl ester, ethyl ester
and isopropyl ester, [0198]
9.alpha.,11.alpha.-epoxy,17.beta.-hydroxy-17.alpha.-(3-hydroxypropyl)-6.a-
lpha.,7.alpha.-methylene-androst-4-en-3-one, [0199]
9.alpha.,11.alpha.-epoxy-17.beta.-hydroxy-17.alpha.-(3-hydroxypropyl)-6.b-
eta.,7.beta.-methylene-androst-4-en-3-one, [0200]
9.alpha.,11.alpha.-epoxy-17.beta.-hydroxy-17.alpha.-(3-hydroxypropyl)-6.b-
eta.,7.beta.;15.beta.,16.beta.-bismethylene-androst-4-en-3-one,
[0201] including 17.alpha.-(3-acetoxypropyl) and
17.alpha.-(3-fromyloxypropyl) analogues of the mentioned androstane
compounds,
[0202] and also 1,2-dehydro analogues of all the mentioned
compounds of the androst-4-en-3-one and 20-spirox-4-en-3-one
series.
[0203] The chemical names of the compounds of the Formulae I and
IA, and of analogue compounds having the same characteristic
structural features, are derived according to current nomenclature
in the following manner: for compounds in which Y.sup.1 together
with Y.sup.2 represents --O--, from 20-spiroxane (for example a
compound of the formula IA in which X represents oxo and Y.sup.1
together with Y.sup.2 represents --O-- is derived from
20-spiroxan-21-one); for those in which each of Y.sup.1 and Y.sup.2
represents hydroxy and X represents oxo, from
17.beta.-hydroxy-17.alpha.-pregnene-21-carboxylic acid; and for
those in which each of Y.sup.1 and Y.sup.2 represents hydroxy and X
represents two hydrogen atoms, from
17.beta.-hydroxy-17.alpha.-(3-hydroxypropyl)-androstane. Since the
cyclic and open-chain forms, that is to say lactones and
17.beta.-hydroxy-21-carboxylic acids and their salts, respectively,
are so closely related to each other that the latter may be
considered merely as a hydrated form of the former, there is to be
understood hereinbefore and hereinafter, unless specifically stated
otherwise, both in end products of the formula I and in starting
materials and intermediates of analogous structure, in each case
all the mentioned forms together.
[0204] In accordance with the invention, several separate process
schemes have been devised for the preparation of compounds of
Formula I in high yield and at reasonable cost. Each of the
synthesis schemes proceeds through the preparation of a series of
intermediates. A number of these intermediates are novel compounds,
and the methods of preparation of these intermediates are novel
processes.
Scheme 1 (Starting with Canrenone or Related Material)
[0205] One preferred process scheme for the preparation of
compounds of Formula I advantageously begins with canrenone or a
related starting material corresponding to Formula XIII (or,
alternatively, the process can begin with androstendione or a
related starting material) ##STR85## wherein [0206] -A-A-
represents the group --CHR.sup.4--CHR.sup.3-- or
--CR.sup.4.dbd.CR.sup.5--; [0207] R.sup.3, R.sup.4 and R.sup.5 are
independently selected from the group consisting of hydrogen, halo,
hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl,
hydroxycarbonyl, cyano and aryloxy; [0208] --B--B-- represents the
group --CHR.sup.6--CHR.sup.7-- or an alpha- or beta-oriented group:
##STR86## [0209] where R.sup.6 and R.sup.7 are independently
selected from the group consisting of hydrogen, halo, lower alkoxy,
acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl,
alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy; and [0210] R.sup.8
and R.sup.9 are independently selected from the group consisting of
hydrogen, hydroxy, halo, lower alkoxy, acyl, hydroxyalkyl,
alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl,
cyano and aryloxy, or R.sup.8 and R.sup.9 together comprise a keto,
carbocyclic or heterocyclic ring structure, or R.sup.8 and R.sup.9
together with R.sup.6 or R.sup.7 comprise a carbocyclic or
heterocyclic ring structure fused to the pentacyclic D ring.
[0211] Using a bioconversion process of the type illustrated in
FIGS. 1 and 2, an 11-hydroxy group of .alpha.-orientation is
introduced in the compound of Formula XIII, thereby producing a
compound of Formula VIII: ##STR87## where -A-A-, --B--B--, R.sup.3,
R.sup.8 and R.sup.9 are as defined in Formula XIII. Preferably, the
compound of Formula XIII has the structure ##STR88## and the
11.alpha.-hydroxy product has the structure ##STR89## in each of
which [0212] -A-A- represents the group --CH.sub.2--CH.sub.2-- or
--CH.dbd.CH--; [0213] --B--B-- represents the group
--CH.sub.2--CH.sub.2-- or an alpha- or beta-oriented group:
##STR90## [0214] R.sup.3 is hydrogen, lower alkyl or lower alkoxy;
[0215] X represents two hydrogen atoms, oxo or .dbd.S; [0216]
Y.sup.1 and Y.sup.2 together represent the oxygen bridge --O--, or
[0217] Y.sup.1 represents hydroxy, and [0218] Y.sup.2 represents
hydroxy, lower alkoxy or, if X represents H.sub.2, also lower
alkanoyloxy; and salts of compounds in which X represents oxo and
Y.sup.2 represents hydroxy. More preferably, the compound of
Formula VIIIA produced in the reaction corresponds to a compound of
Formula VIIIA wherein -A-A- and --B--B-- are each
--CH.sub.2--CH.sub.2--; R.sup.3 is hydrogen; Y.sup.1, Y.sup.2, and
X are as defined in Formula XIIIA; and R.sup.1 and R.sup.9 together
form the 20-spiroxane structure: ##STR91##
[0219] Among the preferred organisms that can be used in this
hydroxylation step are Aspergillus ochraceus NRRL 405, Aspergillus
ochraceus ATCC 18500, Aspergillus niger ATCC 16888 and ATCC 26693,
Aspergillus nidulans ATCC 11267, Rhizopus oryzae ATCC 11145,
Rhizopus stolonifer ATCC 6227b, Streptomyces fradiae ATCC 10745,
Bacillus megaterium ATCC 14945, Pseudomonas cruciviae ATCC 13262,
and Trichothecium roseum ATCC 12543. Other preferred organisms
include Fusarium oxysnorum f.so.cepae ATCC 11171 and Rhizopus
arrhizus ATCC 11145.
[0220] Other organisms that have exhibited activity for this
reaction include Absidia coerula ATCC 6647, Absidia glauca ATCC
22752, Actinomucor elegans ATCC 6476, Aspergillus flavipes ATCC
1030, Aspergillus fumigatus ATCC 26934, Beauveria bassiana ATCC
7159 and ATCC 13144, Botrvosthaeria obtusa IMI 038560, Calonectria
decora ATCC 14767, Chaetomium cochliodes ATCC 10195, Corynespora
cassiicola ATCC 16718, Cunninghamella blakesleeana ATCC 8688a,
Cunninghamella echinulata ATCC 3655, Cunninghamella elegans ATCC
9245, Curvularia clavata ATCC 22921, Curvularia lunata ACTT 12017,
Cylindrocarpon radicicola ATCC 1011, Epicoccum humicola ATCC 12722,
Gongronella butleri ATCC 22822, Hypomyces chrysospermus ATCC IMI
109891, Mortierella isabellina ATCC 42613, Mucor mucedo ATCC 4605,
Mucor griseo-cyanus ATCC 1207A, Myrothecium verrucaria ATCC 9095,
Nocardia corallina ATCC 19070, Paecilomyces carneus ATCC 46579,
Penicillum patulum ATCC 24550, Pithomyces atro-olivaceus IFO 6651,
Pithomyces cynodontis ATCC 26150, Pycnosporium sp. ATCC 12231,
Saccharopolyspora erythrae ATCC 11635, Sepedonium chrysospermum
ATCC 13378, Stachylidium bicolor ATCC 12672, Streptomyces
hycroscopicus ATCC 27438, Streptomyces purpurascens ATCC 25489,
Syncephalastrum racemosum ATCC 18192, Thamnostylum piriforme ATCC
8992, Thielavia terricola ATCC 13807, and Verticillium theobromae
ATCC 12474.
[0221] Additional organisms that may be expected to show activity
for the 11.alpha.-hydroxylation include Cephalosporium aphidicola
(Phytochemistry (1996), 42 (2), 411-415, Cochliobolus lunatas (J.
Biotechnol. (1995), 42(2), 145-150), Tieghemella orchidis
(Khim.-Farm.Zh. (1986), 20(7), 871-876), Tieghemella hyalospora
(Khim.-Farm.Zh. (1986), 20(7), 871-876), Monosporium olivaceum
(Acta Microbiol. Pol., Ser. B. (1973), 5(2), 103-110), Aspergillus
ustus (Acta Microbiol. Pol., Ser. B. (1973), 5(2), 103-110),
Fusarium graminearum (Acta Microbiol. Pol., Ser. B. (1973), 5(2),
103-110), Verticillium glaucum (Acta Microbiol. Pol., Ser. B.
(1973), 5(2), 103-110), and Rhizopus nigricans (J. Steroid Biochem.
(1987), 28(2), 197-201).
[0222] The 11.beta.-hydroxy derivatives of androstendione and
mexrenone can be prepared according to the bioconversion processes
set forth in Examples 19A and 19B, respectively. The inventors
hypothesize by analogy that the corresponding .beta.-hydroxy isomer
of the compound of Formula VIII having a C11 .beta.-hydroxy
substituent instead of a C11 .alpha.-hydroxy substituent can also
be prepared using a similar bioconversion process employing
suitable microorganisms capable of carrying out the
11.beta.-hydroxylation, such as one or more of the microorganisms
disclosed herein.
[0223] Preparatory to production scale fermentation for
hydroxylation of canrenone or other substrates of Formula XIII, an
inoculum of cells is prepared in a seed fermentation system
comprising a seed fermenter, or a series of two or more seed
fermenters. A working stock spore suspension is introduced into the
first seed fermenter, together with a nutrient solution for growth
of cells. If the volume of inoculum desired or needed for
production exceeds that produced in the first seed fermenter, the
inoculum volume may be progressively and geometrically amplified by
progression through the remaining fermenters in the seed
fermentation train. Preferably, the inoculum produced in the seed
fermentation system is of sufficient volume and viable cells for
achieving rapid initiation of reaction in the production fermenter,
relatively short production batch cycles, and high production
fermenter activity. Whatever the number of vessels in a train of
seed fermenters, the second and subsequent seed fermenters are
preferably sized so that the extent of dilution at each step in the
train is essentially the same. The initial dilution of inoculum in
each seed fermenter can be approximately the same as the dilution
in the production fermenter. Canrenone or other Formula XIII
substrate is charged to the production fermenter along with
inoculum and nutrient solution, and the hydroxylation reaction
conducted there.
[0224] The spore suspension charged to the seed fermentation system
is from a vial of working stock spore suspension taken from a
plurality of vials constituting a working stock cell bank that is
stored under cryogenic conditions prior to use. The working stock
cell bank is in turn derived from a master stock cell bank that has
been prepared in the following manner. A spore specimen obtained
from an appropriate source, e.g., ATCC, is initially suspended in
an aqueous medium such as, for example, saline solution, nutrient
solution or a surfactant solution, (e.g., a nonionic surfactant
such as Tween 20 at a concentration of about 0.001% by weight), and
the suspension distributed among culture plates, each plate bearing
a solid nutrient mixture, typically based on a non-digestible
polysaccharide such as agar, where the spores are propagated. The
solid nutrient mixture preferably contains between about 0.5% and
about 5% by weight glucose, between about 0.05% and about 5% by
weight of a nitrogen source, e.g., peptone, between about 0.05% and
about 0.5% by weight of a phosphorus source, e.g., an ammonium or
alkali metal phosphate such as dipotassium hydrogen phosphate,
between about 0.25% and about 2.5% by weight yeast lysate or
extract (or other amino acid source such as meat extract or brain
heart infusion), between about 1% and about 2% by weight agar or
other non-digestible polysaccharide. Optionally, the solid nutrient
mixture may further comprise and/or contain between about 0.1% and
about 5% by weight malt extract. The pH of the solid nutrient
mixture is preferably between about 5.0 and about 7.0, adjusted as
required by alkali metal hydroxide or orthophosphoric acid. Among
useful solid growth media are the following: TABLE-US-00001 1.
Solid Medium #1: 1% glucose, 0.25% yeast extract, 0.3%
K.sub.2HPO.sub.4, and 2% agar (Bacto); pH adjusted to 6.5 with 20%
NaOH. 2. Solid Medium #2: 2% peptone (Bacto), 1% yeast extract
(Bacto), 2% glucose, and 2% agar (Bacto); pH adjusted to 5 with 10%
H.sub.3PO.sub.4. 3. Solid Medium #3: 0.1% peptone (Bacto), 2% malt
extract (Bacto), 2% glucose, and 2% agar (Bacto); pH as is 5.3. 4.
Liquid Medium: 5% blackstrap molasses, 0.5% cornsteep liquor, 0.25%
glucose, 0.25% NaCl, and 0.5% KH.sub.2PO.sub.4, pH adjusted to 5.8.
5. Difco Mycological agar (low pH).
[0225] The number of agar plates used in the development of a
master stock cell bank can be selected with a view to future
demands for master stock, but typically about 15 to about 30 plates
are so prepared. After a suitable period of growth, e.g., 7 to 10
days, the plates are scraped in the presence of an aqueous vehicle,
typically saline or buffer, for harvesting the spores, and the
resulting master stock suspension is divided among small vials,
e.g., one ml. in each of a plurality of 1.5 ml vials. To prepare a
working stock spore suspension for use in research or production
fermentation operations, the contents of one or more of these
second generation master stock vials can be distributed among and
incubated on agar plates in the manner described above for the
preparation of master stock spore suspension. Where routine
manufacturing operations are contemplated, as many as 100 to 400
plates may be used to generate second generation working stock.
Each plate is scraped into a separate working stock vial, each vial
typically containing one ml of the inoculum produced. For permanent
preservation, both the master stock suspension and the second
generation production inoculum are advantageously stored in the
vapor space of a cryogenic storage vessel containing liquid N.sub.2
or other cryogenic liquid.
[0226] In the process illustrated in FIG. 1, aqueous growth medium
is prepared which includes a nitrogen source such as peptone, a
yeast derivative or equivalent, glucose, and a source of phosphorus
such as a phosphate salt. Spores of the microorganism are cultured
in this medium in the seed fermentation system. The preferred
microorganism is Aspergillus ochraceus NRRL 405 (ATCC 18500). The
seed stock so produced is then introduced into the production
fermenter together with the substrate of Formula XIII. The
fermentation broth is agitated and aerated for a time sufficient
for the reaction to proceed to the desired degree of
completion.
[0227] The medium for the seed fermenter preferably comprises an
aqueous mixture which contains: between about 0.5% and about 5% by
weight glucose, between about 0.05% and about 5% by weight of a
nitrogen source, e.g., peptone, between about 0.05% and about 0.5%
by weight of a phosphorus source, e.g., an ammonium or alkali metal
phosphate such as ammonium phosphate monobasic or dipotassium
hydrogen phosphate, between about 0.25% and about 2.5% by weight
yeast lysate or extract (or other amino acid source such as
distiller's solubles), between about 1% and about 2% by weight agar
or other non-digestible polysaccharide. A particularly preferred
seed growth medium contains about 0.05% and about 5% by weight of a
nitrogen source such as peptone, between about 0.25% and about 2.5%
by weight of autolyzed yeast or yeast extract, between about 0.5%
and about 5% by weight glucose, and between about 0.05% by weight
and about 0.5% by weight of a phosphorus source such as ammonium
phosphate monobasic. Especially economical process operations are
afforded by the use of another preferred seed culture which
contains between about 0.5% and about 5% by weight corn steep
liquor, between about 0.25% and about 2.5% autolyzed yeast or yeast
extract, between about 0.5% and about 5% by weight glucose and
about 0.05% and about 0.5% by weight ammonium phosphate monobasic.
Corn steep liquor is a particularly economical source of proteins,
peptides, carbohydrates, organic acids, vitamins, metal ions, trace
matters and phosphates. Mash liquors from other grains may be used
in place of, or in addition to, corn steep liquor. The pH of the
medium is preferably adjusted within the range of between about 5.0
and about 7.0, e.g., by addition of an alkali metal hydroxide or
orthophosphoric acid. Where corn steep liquor serves as the source
of nitrogen and carbon, the pH is preferably adjusted within the
range of about 6.2 to about 6.8. The medium comprising peptone and
glucose is preferably adjusted to a pH between about 5.4 and about
6.2. Among useful growth media for use in seed fermentation:
TABLE-US-00002 1. Medium #1: 2% peptone, 2% yeast autolyzed (or
yeast extract), and 2% glucose; pH adjusted to 5.8 with 20% NaOH.
2. Medium #2: 3% corn steep liquor, 1.5% yeast extract, 0.3%
ammonium phosphate monobasic, and 3% glucose; pH adjusted to 6.5
with 20% NaOH.
[0228] Spores of the microorganism are introduced into this medium
from a vial typically containing in the neighborhood of 10.sup.9
spores per ml. of suspension. Optimal productivity of seed
generation is realized where dilution with growth medium at the
beginning of a seed culture does not reduce the spore population
density below about 10.sup.7 per ml. Preferably, the spores are
cultured in the seed fermentation system until the packed mycelial
volume (PMV) in the seed fermenter is at least about 20%,
preferably about 35% to about 45%. Since the cycle in the seed
fermentation vessel (or any vessel of a plurality which comprise a
seed fermentation train) depends on the initial concentration in
that vessel, it may be desirable to provide two or three seed
fermentation stages to accelerate the overall process. However, it
is preferable to avoid the use of significantly more than three
seed fermenters in series, since activity may be compromised if
seed fermentation is carried through an excessive number of stages.
The seed culture fermentation is conducted under agitation at a
temperature in the range of about 23.degree. to about 37.degree.
C., preferably in range of between about 24.degree. and about
28.degree. C.
[0229] Culture from the seed fermentation system is introduced into
a production fermenter, together with a production growth medium.
In one embodiment of the invention, non-sterile canrenone or other
substrate of Formula XIII serves as the substrate for the reaction.
Preferably, the substrate is added to the production fermenter in
the form of a 10% to 30% by weight slurry in growth medium. To
increase the surface area available for 11.alpha.-hydroxylation
reaction, the particle size of the Formula XIII substrate is
reduced by passing the substrate through an off line micronizer
prior to introduction into the fermenter. A sterile nutrient feed
stock containing glucose, and a second sterile nutrient solution
containing a yeast derivative such as autolyzed yeast (or
equivalent amino acid formulation based on alternative sources such
as distiller's solubles), are also separately introduced. The
medium comprises an aqueous mixture containing: between about 0.5%
and about 5% by weight glucose, between about 0.05% and about 5% by
weight of a nitrogen source, e.g., peptone, between about 0.05% and
about 0.5% by weight of a phosphorus source, e.g., an ammonium or
alkali metal phosphate such as dipotassium hydrogen phosphate,
between about 0.25% and about 2.5% by weight yeast lysate or
extract (or other amino acid source such as distiller's solubles),
between about 1% and about 2% by weight agar or other
non-digestible polysaccharide. A particularly preferred production
growth medium contains about 0.05% and about 5% by weight of a
nitrogen source such as peptone, between about 0.25% and about 2.5%
by weight of autolyzed yeast or yeast extract, between about 0.5%
and about 5% by weight glucose, and between about 0.05% and about
0.5% by weight of a phosphorus source such as ammonium phosphate
monobasic. Another preferred production medium contains between
about 0.5% and about 5% by weight corn steep liquor, between about
0.25% and about 2.5% autolyzed yeast or yeast extract, between
about 0.5% and about 5% by weight glucose and about 0.05% and about
0.5% by weight ammonium phosphate monobasic. The pH of the
production fermentation medium is preferably adjusted in the manner
described above for the seed fermentation medium, with the same
preferred ranges for the pH of peptone/glucose based media and corn
steep liquor based media, respectively. Useful bioconversion growth
media are set forth below: TABLE-US-00003 1. Medium #1: 2% peptone,
2% yeast autolyzed (or yeast extract), and 2% glucose; pH adjusted
to 5.8 with 20% NaOH. 2. Medium #2: 1% peptone, 1% yeast autolyzed
(or yeast extract), and 2% glucose; pH adjusted to 5.8 with 20%
NaOH. 3. Medium #3: 0.5% peptone, 0.5% yeast autolyzed (or yeast
extract), and 0.5% glucose; pH adjusted to 5.8 with 20% NaOH. 4.
Medium #4: 3% corn steep liquor, 1.5% yeast extract, 0.3% ammonium
phosphate monobasic, and 3% glucose; pH adjusted to 6.5 with 20%
NaOH. 5. Medium #5: 2.55% corn steep liquor, 1.275% yeast extract,
0.255% ammonium phosphate monobasic, and 3% glucose; pH adjusted to
6.5 with 20% NaOH. 6. Medium #6: 2.1% corn steep liquor, 1.05%
yeast extract, 0.21% ammonium phosphate monobasic, and 3% glucose;
pH adjusted to 6.5 with 20% NaOH.
[0230] Non-sterile canrenone and sterile nutrient solutions are
chain fed to the production fermenter in about five to about
twenty, preferably about ten to about fifteen, preferably
substantially equal, portions each over the production batch cycle.
Advantageously, the substrate is initially introduced in an amount
sufficient to establish a concentration of between about 0.1% by
weight and about 3% by weight, preferably between about 0.5% and
about 2% by weight, before inoculation with seed fermentation
broth, then added periodically, conveniently every 8 to 24 hours,
to a cumulative proportion of between about 1% and about 8% by
weight. Where additional substrate is added every 8 hour shift,
total addition may be slightly lower, e.g., 0.25% to 2.5% by
weight, than in the case where substrate is added only on a daily
basis. In the latter instance cumulative canrenone addition may
need to be in the range 2% to about 8% by weight. The supplemental
nutrient mixture fed during the fermentation reaction is preferably
a concentrate, for example, a mixture containing between about 40%
and about 60% by weight sterile glucose, and between about 16% and
about 32% by weight sterile yeast extract or other sterile source
of yeast derivative (or other amino acid source). Since the
substrate fed to the production fermenter of FIG. 1 is non-sterile,
antibiotics are periodically added to the fermentation broth to
control the growth of undesired organisms. Antibiotics such as
kanamycin, tetracycline, and cefalexin can be added without
disadvantageously affecting growth and bioconversion. Preferably,
these are introduced into the fermentation broth in a
concentration, e.g., of between about 0.0004% and about 0.002%
based on the total amount of the broth, comprising, e.g., between
about 0.0002% and about 0.0006% kanamicyn sulfate, between about
0.0002% and about 0.006% tetracycline HCl and/or between about
0.001% and about 0.003% cefalexin, again based on the total amount
of broth.
[0231] Typically, the production fermentation batch cycle is in the
neighborhood of about 80-160 hours. Thus, portions of each of the
Formula XIII substrates and nutrient solutions are typically added
about every 2 to 10 hours, preferably about every 4 to 6 hours.
Advantageously, an antifoam is also incorporated in the seed
fermentation system, and in the production fermenter.
[0232] Preferably, in the process of FIG. 1, the inoculum charge to
the production fermenter is about 0.5% to about 7%, more preferably
about 1% to about 2%, by volume based on the total mixture in the
fermenter, and the glucose concentration is maintained between
about 0.01% and about 1.0%, preferably between about 0.025% and
about 0.5%, more preferably between about 0.05% and about 0.25% by
weight with periodic additions that are preferably in portions of
about 0.05% to about 0.25% by weight, based on the total batch
charge. The fermentation temperature is conveniently controlled
within a range of about 20.degree. to about 37.degree. C.,
preferably about 24.degree. C. to about 28.degree. C., but it may
be desirable to step down the temperature during the reaction,
e.g., in 2.degree. C. increments, to maintain the packed mycelium
volume (PMV) below about 60%, more preferably below about 50%, and
thereby prevent the viscosity of the fermentation broth from
interfering with satisfactory mixing. If the biomass growth extends
above the liquid surface, substrate retained within the biomass may
be carried out of the reaction zone and become unavailable for the
hydroxylation reaction. For productivity, it is desirable to reach
a PMV in the range of 30 to 50%, preferably 35% to 45%, within the
first 24 hours of the fermentation reaction, but thereafter
conditions are preferably managed to control further growth within
the limits stated above. During reaction, the pH of the
fermentation medium is controlled at between about 5.0 and about
6.5, preferably between about 5.2 and about 5.8, and the fermenter
is agitated at a rate of between about 400 and about 800 rpm. A
dissolved oxygen level of at least about 10% of saturation is
achieved by aerating the batch at between about 0.2 and about 1.0
vvm, and maintaining the pressure in the head space of the
fermenter at between about atmospheric and about 1.0 bar gauge,
most preferably in the neighborhood of about 0.7 bar gauge.
Agitation rate may also be increased as necessary to maintain
minimum dissolved oxygen levels. Advantageously, the dissolved
oxygen is maintained at well above about 10%, in fact as high as
about 50% to promote conversion of substrate. Maintaining the pH in
the range of 5.5.+-.0.2 is also optimal for bioconversion. Foaming
is controlled as necessary by addition of a common antifoaming
agent. After all substrate has been added, reaction is preferably
continued until the molar ratio of Formula VIII product to
remaining unreacted Formula XIII substrate is at least about 9 to
1. Such conversion may be achieve within the 80-160 hour batch
cycle indicated above.
[0233] It has been found that high conversions are associated with
depletion of initial nutrient levels below the initial charge
level, and by controlling aeration rate and agitation rate to avoid
splashing of substrate out of the liquid broth. In the process of
FIG. 1, the nutrient level was depleted to and then maintained at
no greater than about 60%, preferably about 50%, of the initial
charge level; while in the processes of FIGS. 2 and 3, the nutrient
level was reduced to and maintained at no greater than about 80%,
preferably about 70%, of the initial charge level. Aeration rate is
preferably no greater than one vvm, more preferably in the range of
about 0.5 vvm; while agitation rate is preferably not greater than
600 rpm.
[0234] A particularly preferred process for preparation of a
compound of Formula VIII is illustrated in FIG. 2. A preferred
microorganism for the 11.alpha.-hydroxylation of a compound of
Formula XIII (for example, canrenone) is Aspergillus ochraceus NRRL
405 (ATCC 18500). In this process, growth medium preferably
comprises between about 0.5% and about 5% by weight corn steep
liquor, between about 0.5% and about 5% by weight glucose, between
about 0.1% and about 3% by weight yeast extract, and between about
0.05% and about 0.5% by weight ammonium phosphate. However, other
production growth media as described herein may also be used. The
seed culture is prepared essentially in the manner described for
the process of FIG. 1, using any of the seed fermentation media
described herein. A suspension of non-micronized canrenone or other
Formula XIII substrate in the growth medium is prepared aseptically
in a blender, preferably at a relatively high concentration of
between about 10% and about 30% by weight substrate. Preferably,
aseptic preparation may comprise sterilization or pasteurization of
the suspension after mixing. The entire amount of sterile substrate
suspension required for a production batch is introduced into the
production fermenter at the beginning of the batch, or by
periodical chain feeding. The particle size of the substrate is
reduced by wet milling in an on-line shear pump which transfers the
slurry to the production fermenter, thus obviating the need for use
of an off-line micronizer. Where aseptic conditions are achieved by
pasteurization rather than sterilization, the extent of
agglomeration may be insignificant, but the use of a shear pump may
be desirable to provide positive control of particle size. Sterile
growth medium and glucose solution are introduced into the
production fermenter essentially in the same manner as described
above. All feed components to the production fermenter are
sterilized before introduction, so that no antibiotics are
required.
[0235] Preferably, in operation of the process of FIG. 2, the
inoculum is introduced into the production fermenter in a
proportion of between about 0.5% and about 7%, the fermentation
temperature is between about 20.degree. and about 37.degree. C.,
preferably between about 24.degree. C. and about 28.degree. C., and
the pH is controlled between about 4.4 and about 6.5, preferably
between about 5.3 and about 5.5, e.g., by introduction of gaseous
ammonia, aqueous ammonium hydroxide, aqueous alkali metal
hydroxide, or orthophosphoric acid. As in the process of FIG. 1,
the temperature is preferably trimmed to control growth of the
biomass so that PMV does not exceed 55-60%. The initial glucose
charge is preferably between about 1% and about 4% by weight, most
preferably 2.5% to 3.5% by weight, but is preferably allowed to
drift below about 1.0% by weight during fermentation. Supplemental
glucose is fed periodically in portions of between about 0.2% and
about 1.0% by weight based on the total batch charge, so as to
maintain the glucose concentration in the fermentation zone within
a range of between about 0.1% and about 1.5% by weight, preferably
between about 0.25% and about 0.5% by weight. Optionally, nitrogen
and phosphorus sources may be supplemented along with glucose.
However, because the entire canrenone charge is made at the
beginning of the batch cycle, the requisite supply of nitrogen and
phosphorus bearing nutrients can also be introduced at that time,
allowing the use of only a glucose solution for supplementation
during the reaction. The rate and nature of agitation is a
significant variable. Moderately vigorous agitation promotes mass
transfer between the solid substrate and the aqueous phase.
However, a low shear impeller should be used to prevent degradation
of the myelin of the microorganisms. Optimal agitation velocity
varies within the range of 200 to 800 rpm, depending on culture
broth viscosity, oxygen concentration, and mixing conditions as
affected by vessel, baffle and impeller configuration. Ordinarily,
a preferred agitation rate is in the range of 350-600 rpm.
Preferably the agitation impeller provides a downward axially
pumping function so as to assist in good mixing of the fermented
biomass. The batch is preferably aerated at a rate of between about
0.3 and about 1.0 vvm, preferably 0.4 to 0.8 vvm, and the pressure
in the head space of the fermenter is preferably between about 0.5
and about 1.0 bar gauge. Temperature, agitation, aeration and back
pressure are preferably controlled to maintain dissolved oxygen in
the range of at least about 10% by volume during the bioconversion.
Total batch cycle is typically between about 100 and about 140
hours.
[0236] Although the principle of operation for the process of FIG.
2 is based on early introduction of substantially the entire
canrenone charge, it will be understood that growth of the
fermentation broth may be carried out before the bulk of the
canrenone is charged. Optionally, some portion of the canrenone can
also be added later in the batch. Generally, however, at least
about 75% of the sterile canrenone charge should be introduced into
the transformation fermenter within 48 hours after initiation of
fermentation. Moreover, it is desirable to introduce at least about
25% by weight canrenone at the beginning of the fermentation, or at
least within the first 24 hours in order to promote generation of
the bioconversion enzyme(s).
[0237] In a further preferred process as illustrated in FIG. 3, the
entire batch charge and nutrient solution are sterilized in the
production fermentation vessel prior to the introduction of
inoculum. The nutrient solutions that may be used, as well as the
preferences among them, are essentially the same as in the process
of FIG. 2. In this embodiment of the invention, the shearing action
of the agitator impeller breaks down the substrate agglomerates
that otherwise tend to form upon sterilization. It has been found
that the reaction proceeds satisfactorily if the mean particle size
of the canrenone is less than about 300.mu. and at least 75% by
weight of the particles are smaller than 240.mu.. The use of a
suitable impeller, e.g., a disk turbine impeller, at an adequate
velocity in the range of 200 to 800 rpm, with a tip speed of at
least about 400 cm/sec., has been found to provide a shear rate
sufficient to maintain such particle size characteristics despite
the agglomeration that tends to occur upon sterilization within the
production fermenter. The remaining operation of the process of
FIG. 3 is essentially the same as the process of FIG. 2. The
processes of FIGS. 2 and 3 offer several distinct advantages over
the process of FIG. 1. A particular advantage is the amenability to
use of a low cost nutrient base such as corn steep liquor. But
further advantages are realized in eliminating the need of
antibiotics, simplifying feeding procedures, and allowing for batch
sterilization of canrenone or other Formula XIII substrate. Another
particular advantage is the ability to use a simple glucose
solution rather than a complex nutrient solution for
supplementation during the reaction cycle.
[0238] In processes depicted in FIGS. 1 to 3, the product of
Formula VIII is a crystalline solid which, together with the
biomass, may be separated from the reaction broth by filtration or
low speed centrifugation. Alternatively, the product can be
extracted from the entire reaction broth with organic solvents.
Product of Formula VIII is recovered by solvent extraction. For is
maximum recovery, both the liquid phase filtrate and the biomass
filter or centrifuge cake are treated with extraction solvent, but
usually .gtoreq.95% of the product is associated with the biomass.
Typically, hydrocarbon, ester, chlorinated hydrocarbon, and ketone
solvents may be used for extraction. A preferred solvent is ethyl
acetate. Other typically suitable solvents include toluene and
methyl isobutyl ketone. For extraction from the liquid phase, it
may be convenient to use a volume of solvent approximately equal to
the volume of reaction solution which it contacts. To recover
product the from the biomass, the latter is suspended in the
solvent, preferably in large excess relative to the initial charge
of substrate, e.g., 50 to 100 ml. solvent per gram of initial
canrenone charge, and the resulting suspension preferably refluxed
for a period of about 20 minutes to several hours to assure
transfer of product to the solvent phase from recesses and pores of
the biomass. Thereafter, the biomass is removed by filtration or
centrifugation, and the filter cake preferably washed with both
fresh solvent and deionized water. Aqueous and solvent washes are
then combined and the phases allowed crystallization from the
solution. To maximize yield, the mycelium is contacted twice with
fresh solvent. After settling to allow complete separation of the
aqueous phase, product is recovered from the solvent phase. Most
preferably, the solvent is removed under vacuum until
crystallization begins, then the concentrated extract is cooled to
a temperature of about 0.degree. to about 20.degree. C., preferably
about 10.degree. to about 15.degree. C. for a time sufficient for
crystal precipitation and growth, typically about 8 to about 12
hours.
[0239] The processes of FIG. 2, and especially that of FIG. 3, are
particularly preferred. These processes operate at low viscosity,
and are amenable to close is control of process parameters such as
pH, temperature and dissolved oxygen. Moreover, sterile conditions
are readily preserved without resort to antibiotics.
[0240] The bioconversion process is exothermic, so that heat should
be removed, using a jacketed fermenter or a cooling coil within the
production fermenter. Alternatively, the reaction broth may be
circulated through an external heat exchanger. Dissolved oxygen is
preferably maintained at a level of at least about 5%, preferably
at least about 10%, by volume, sufficient to provide energy for the
reaction and assure conversion of the glucose to C0.sub.2 and
H.sub.2O, by regulating the rate of air introduced into the reactor
in response to measurement of oxygen potential in the broth. The pH
is preferably controlled at between about 4.5 and about 6.5.
[0241] In each of the alternative processes for 11-hydroxylation of
the substrate of Formula XIII, productivity is limited by mass
transfer from the solid substrate to the aqueous phase, or the
phase interface, where reaction is understood to occur. As
indicated above, productivity is not significantly limited by mass
transfer rates so long as the particle mean particle size of the
substrate is reduced to less than about 300.mu., and at least 75%
by weight of the particles are smaller than 240.mu.. However,
productivity of these processes may be further enhanced in certain
alternative embodiments which provide a substantial charge of
canrenone or other Formula XIII substrate to the production
fermenter in an organic solvent. According to one option, the
substrate is dissolved in a water-immiscible solvent and mixed with
the aqueous growth medium inoculum and a surfactant. Useful
water-immiscible solvents include, for example, DMF, DMSO,
C.sub.6-C.sub.12 fatty acids, C.sub.6-C.sub.12 n-alkanes, vegetable
oils, sorbitans, and aqueous surfactant solutions. Agitation of
this charge generates an emulsion reaction system having an
extended interfacial area for mass transfer of substrate from the
organic liquid phase to the reaction sites.
[0242] A second option is to initially dissolve the substrate in a
water miscible solvent such as acetone, methylethyl ketone,
methanol, ethanol, or glycerol in a concentration substantially
greater than its solubility in water. By preparing the initial
substrate solution at elevated temperature, solubility is
increased, thereby further increasing the amount of solution form
substrate introduced into the reactor and ultimately enhancing the
reactor payload. The warm substrate solution is charged to the
production fermentation reactor along with the relatively cool
aqueous charge comprising growth medium and inoculum. When the
substrate solution is mixed with the aqueous medium, precipitation
of the substrate occurs. However, under conditions of substantial
supersaturation and moderately vigorous agitation, nucleation is
favored over crystal growth, and very fine particles of high
surface area are formed. The high surface area promotes mass
transfer between the liquid phase and the solid substrate.
Moreover, the equilibrium concentration of substrate in the aqueous
liquid phase is also enhanced in the presence of a water-miscible
solvent. Accordingly, productivity is promoted.
[0243] Although the microorganism may not necessarily tolerate a
high concentration of organic solvent in the aqueous phase, a
concentration of ethanol, e.g., in the range of about 3% to about
5% by weight, can be used to advantage.
[0244] A third option is to solubilize the substrate in an aqueous
cyclodextrin solution. Illustrative cyclodextrins include
hydroxypropyl-.beta.-cyclodextrin and methyl-.beta.-cyclodextrin.
The molar ratio of substrate:cyclodextrin can be about 1:0.5 to
about 1:1.5, more preferably about 1:0.8 to about 1:1. The
substrate:cyclodextrin mixture can then be added aseptically to the
bioconversion reactor.
[0245] 11.alpha.-Hydroxycanrenone and other products of the
11.alpha.-hydroxylation process (Formulae VIII and VIIIA) are novel
compounds which may be isolated by filtering the reaction medium
and extracting the product from the biomass collected on the
filtration medium. Conventional organic solvents, e.g., ethyl
acetate, acetone, toluene, chlorinated hydrocarbons, and methyl
isobutyl ketone may be used for the extraction. The product of
Formula VIII may then be recrystallized from an organic solvent of
the same type. The compounds of Formula VIII have substantial value
as intermediates for the preparation of compounds of Formula I, and
especially of Formula IA.
[0246] Preferably, the compounds of Formula VIII correspond to
Formula VIIIA in which -A-A- and --B--B-- are
--CH.sub.2--CH.sub.2--, R.sup.3 is hydrogen, lower alkyl or lower
alkoxy, and R.sup.8 and R.sup.9 together constitute the
20-spiroxane ring: ##STR92##
[0247] Further in accordance with the process of Scheme 1, the
compound of Formula VIII is reacted under alkaline conditions with
a source of cyanide ion to produce an enamine compound of Formula
VII ##STR93## wherein -A-A-, --B--B--, R.sup.3, R.sup.8 and R.sup.9
are as defined above. Where the substrate corresponds to Formula
VIIIA, the product is of Formula VIIA ##STR94## wherein -A-A-,
--B--B--, R.sup.3, Y.sup.1, Y.sup.2, and X are as defined in
Formula XIIIA. Preferably, R.sup.3 is hydrogen.
[0248] Cyanidation of the 11.alpha.-hydroxyl substrate of Formula
VIII may be carried out by reacting it with a cyanide ion source
such as a ketone cyanohydrin, most preferably acetone cyanohydrin,
in the presence of a base and an alkali metal salt, most preferably
LiCl. Alternatively, cyanidation can be effected without a
cyanohydrin by using an alkali metal cyanide in the presence of an
acid.
[0249] In the ketone cyanohydrin process, the reaction is conducted
in solution, preferably using an aprotic polar solvent such as
dimethylformamide or dimethyl sulfoxide. Formation of the enamine
requires at least two moles of cyanide ion source per mole of
substrate, and preferably a slight excess of the cyanide source is
used. The base is preferably a nitrogenous base such as a
dialkylamine, trialkylamine, alkanolamine, pyridine or the like.
However, inorganic bases such as alkali metal carbonates or alkali
metal hydroxides can also be used. Preferably, the substrate of
Formula VIII is initially present in a proportion of between about
20 and about 50% by weight and the base is present in a proportion
of between 0.5 to two equivalents per equivalent of substrate. The
temperature of the reaction is not critical, but productivity is
enhanced by operation at elevated temperature. Thus, for example,
where triethylamine is used as the base, the reaction is
advantageously conducted in the range of about 80.degree. C. to
about 90.degree. C. At such temperatures, the reaction proceeds to
completion in about 5 to about 20 hours. When diisopropylethyl
amine is used as the base and the reaction is conducted at
105.degree. C., the reaction is completed at 8 hours. At the end of
the reaction period, the solvent is removed under vacuum and the
residual oil dissolved in water and neutralized to pH 7 with dilute
acid, preferably hydrochloric. The product precipitates from this
solution, and is thereafter washed with distilled water and air
dried. Liberated HCN may be stripped with an inert gas and quenched
in an alkaline solution. The dried precipitate is taken up in
chloroform or other suitable solvent, then extracted with
concentrated acid, e.g., 6N HCl. The extract is neutralized to pH 7
by addition of an inorganic base, preferably an alkali metal
hydroxide, and cooled to a temperature in the range of 0.degree. C.
The resulting precipitate is washed and dried, then recrystallized
from a suitable solvent, e.g., acetone, to produce a product of
Formula VII suitable for use in the next step of the process.
[0250] Alternatively, the reaction may be conducted in an aqueous
solvent system comprising water-miscible organic solvent such as
methanol or in a biphasic system comprising water and an organic
solvent such as ethyl acetate. In this alternative, product may be
recovered by diluting the reaction solution with water, and
thereafter extracting the product using an organic solvent such as
methylene chloride or chloroform, and then back extracting from the
organic extract using concentrated mineral acid, e.g., 2N HCl. See
U.S. Pat. No. 3,200,113.
[0251] According to a still further alternative, the reaction may
be conducted in a water-miscible solvent such as dimethylformamide,
dimethylacetamide, N-methyl, pyrolidone or dimethyl sulfoxide,
after which the reaction product solution is diluted with water and
rendered alkaline, e.g., by addition of an alkali metal carbonate,
then cooled to 0.degree. to 10.degree. C., thereby causing the
product to precipitate. Preferably, the system is quenched with an
alkali metal hypohalite or other reagent effective to prevent
evolution of cyanide. After filtration and washing with water, the
precipitated product is suitable for use in the next step of the
process.
[0252] According to a still further alternative, the enamine
product of Formula VII may be produced by reaction of a substrate
of Formula VIII in the presence of a proton source, with an excess
of alkali metal cyanide, preferably NaCN, in an aqueous solvent
comprising an aprotic water-miscible polar solvent such as
dimethylformamide or dimethylacetamide. The proton source is
preferably a mineral acid or C.sub.1 to C.sub.5 carboxylic acid,
sulfuric acid being particularly preferred. Anomalously, no
discrete proton source need be added where the cyanidation reagent
is commercial LiCN in DMF.
[0253] A source of cyanide ion such as an alkali metal salt is
preferably charged to the reactor in a proportion of between about
2.05 and about 5 molar equivalents per equivalent of substrate. The
mineral acid or other proton source is believed to promote addition
of HCN across the 4,5 and 6,7 double bonds, and is preferably
present in a proportion of at least one mole equivalent per mole
equivalent substrate; but the reaction system should remain basic
by maintaining an excess of alkali metal cyanide over acid present.
Reaction is preferably carried out at a temperature of at least
about 75.degree. C., typically 60.degree. C. to 100.degree. C., for
a period of about 1 to about 8 hours, preferably about 1.5 to about
3 hours. At the end of the reaction period, the reaction mixture is
cooled, preferably to about room temperature; and the product
enamine is precipitated by acidifying the reaction mixture and
mixing it with cold water, preferably at about ice bath
temperature. Acidification is believed to close the 17-lactone,
which tends to open under the basic conditions prevailing in the
cyanidation. The reaction mixture is conveniently acidified using
the same acid that is present during the reaction, preferably
sulfuric acid. Water is preferably added in a proportion of between
about 10 and about 50 mole equivalents per mole of product.
[0254] The compounds of Formula VII are novel compounds and have
substantial value as intermediates for the preparation of compounds
of Formula I, and especially of Formula IA. Preferably, the
compounds of Formula VII correspond to Formula VIIA in which -A-A-
and --B--B-- are --CH.sub.2--CH.sub.2--, R.sup.3 is hydrogen, lower
alkyl or lower alkoxy, and R.sup.8 and R.sup.9 together constitute
the 20-spiroxane ring: ##STR95##
[0255] Most preferably the compound of Formula VII is
5'R(5'.alpha.),7'.beta.-20'-Aminohexadecahydro-11'.beta.-hydroxy-10'.alph-
a.,13'.alpha.-dimethyl-3',5-dioxospiro[furan-2(3H),17'.alpha.(5'H)-[7,4]me-
theno[4H]cyclopenta[a]phenanthrene]-5'-carbonitrile.
[0256] In the conversion of the compound of Formula VIII to the
enamine of Formula VII, the 7-cyano derivative of the compound of
Formula VIII has been observed by chromatography in the crude
product. It is hypothesized that the 7-cyano compound is an
intermediate in the conversion process. It is further hypothesized
that the 7-cyano intermediate itself reacts to form a second
intermediate, the 5,7-dicyano derivative of the compound of Formula
VIII, which in turn reacts to form the enester. See, e.g., R.
Christiansen et al., The Reaction of Steroidal 4,6-Dien-3-Ones With
Cyanide, Steroids, Vol. 1, June 1963, which is incorporated herein
by reference. These novel compounds also have utility as
chromatographic markers as well as being synthetic intermediates.
In a preferred embodiment of this step of the overall Scheme 1
synthesis process, these intermediates are
7.alpha.-cyano-11.alpha.,17-dihydroxy-3-oxo-17.alpha.-pregn-4-ene-21-dica-
rboxylic acid, .gamma.-lactone, and
5.beta.,7.alpha.-dicyano-11.alpha.,17-dihydroxy-3-oxo-17.alpha.-pregnane--
21-dicarboxylic acid, .gamma.-lactone.
[0257] In the next step of the Scheme 1 synthesis, the enamine of
Formula VII is hydrolyzed to produce a diketone compound of Formula
VI ##STR96## where -A-A-, --B--B--, R.sup.3, R.sup.8 and R.sup.9
are as defined in Formula XIII. Any aqueous organic or mineral acid
can be used for the hydrolysis. Hydrochloric acid is preferred. To
enhance productivity, a water-miscible organic solvent, such as
dimethylacetamide or a lower alkanol, is preferably used as a
cosolvent. More preferably, dimethylacetamide is the solvent. The
acid should be present in proportion of at least one equivalent per
equivalent of Formula VII substrate. In an aqueous system, the
enamine substrate VII can be substantially converted to the
diketone of Formula VI in a period of about 5 hours at about
80.degree. C. Operation at elevated temperature increases
productivity, but temperature is not critical. Suitable
temperatures are selected based on the volatility of the solvent
system and acid.
[0258] Preferably, the enamine substrate of Formula VII corresponds
to Formula VIIA ##STR97## and the diketone product corresponds to
Formula VIA ##STR98## in each of which -A-A-, --B--B--, R.sup.3,
Y.sup.1, Y.sup.2 and X are as defined in Formula XIIIA. Preferably,
R.sup.3 is hydrogen.
[0259] At the end of the reaction period, the solution is cooled to
between about 0.degree. to 25.degree. C. to crystallize the
product. The product crystals may be recrystallized from a suitable
solvent such as isopropanol or methanol to produce a product of
Formula VI suitable for use in the next step of the process; but
recrystallization is usually not necessary. The products of Formula
VI are novel compounds which have substantial value as
intermediates for the preparation of compounds of Formula I, and
especially of Formula IA. Preferably, the compounds of Formula VI
correspond to Formula VIA in which -A-A- and --B--B-- are
--CH.sub.2--CH.sub.2--, R.sup.3 is hydrogen, lower alkyl or lower
alkoxy, and R.sup.8 and R.sup.9 together constitute the
20-spiroxane ring: ##STR99## Most preferably, the compound of
Formula VI is
4'S(4'.alpha.),7'.alpha.-Hexadecahydro-11'.alpha.-hydroxy-10'.beta.,13'.b-
eta.-dimethyl-3',5,20'-trioxospiro[furan-2(3H),17'.beta.-[4,7]methano[17H]-
cyclopenta[a]phenanthrene]-5'.beta.(2'H)-carbonitrile.
[0260] In a particularly, preferred embodiment of the invention,
the product enamine of Formula VII is produced from the compound of
Formula VIII in the manner described above, and converted in situ
to the diketone of Formula VI. In this embodiment of the invention,
a formula VIII substrate is reacted with an excess of alkali metal
cyanide in an aqueous solvent containing a proton source, or
optionally an excess of ketone cyanohydrin in the presence of a
base and LiCl, as described hereinabove. However, instead of
cooling the reaction mixture, acidifying, and adding water in
proportions calculated to cause precipitation of the enamine,
substantial cooling of the reaction mixture is preferably avoided.
Water and an acid, preferably a mineral acid such as sulfuric, are
instead added to the mixture at the end of the cyanidation
reaction. The proportion of acid added is sufficient to neutralize
excess alkali metal cyanide, which ordinarily requires introduction
of at least one molar equivalent acid per mole of Formula VIII
substrate, preferably between about 2 and about 5 mole equivalents
per equivalent substrate. However, the temperature is maintained at
high enough, and the dilution great enough, so that substantial
precipitation is avoided and hydrolysis of the enamine to the
diketone is allowed to proceed in the liquid phase. Thus, the
process proceeds with minimum interruption and high productivity.
Hydrolysis is preferably conducted at a temperature of at least
80.degree. C., more preferably in the range of about 90.degree. C.
to about 100.degree. C., for a period of typically about 1 to about
10 hours, more preferably about 2 to about 5 hours. Then the
reaction mixture is cooled, preferably to a temperature of between
about 0.degree. C. and about 15.degree. C., advantageously in an
ice bath to about 5.degree. C. to about 10.degree. C., for
precipitation of the product diketone of Formula VI. The solid
product may be recovered, as by filtration, and impurities reduced
by washing with water.
[0261] In the next step of the Scheme 1 synthesis, the diketone
compound of Formula VI is reacted with a metal alkoxide to open up
the ketone bridge between the 4 and 7 positions via cleavage of the
bond between the carbonyl group and the 4-carbon, form an
.alpha.-oriented alkoxycarbonyl substituent at the 7 position, and
eliminate cyanide at the 5-carbon. The product of this reaction is
a hydroxyester compound corresponding to Formula V ##STR100## where
-A-A-, --B--B--, R.sup.3, R.sup.8 and R.sup.9 are as defined in
Formula XIII, and R.sup.1 is lower alkoxycarbonyl or
hydroxycarbonyl. The metal alkoxide used in the reaction
corresponds to the formula R.sup.10OM where M is alkali metal and
R.sup.10O-- corresponds to the alkoxy substituent of R.sup.1.
Yields of this reaction are most satisfactory when the metal
alkoxide is potassium methoxide or sodium methoxide, but other
lower alkoxides can be used. A potassium alkoxide is particularly
preferred. Phenoxides, other aryloxides may also be used, as well
as arylsulfides. The reaction is conveniently carried out in the
presence of an alcohol corresponding to the formula R.sup.10OH
where R.sup.10 is as defined above. Other conventional solvents may
be used. Preferably, the Formula VI substrate is present in a
proportion of between about 2% and about 12% by weight, more
preferably at least about 6% by weight. Preferably, R.sup.10OM is
present in a proportion of between about 0.5 and about 4 moles per
mole of substrate, more preferably between about 1 and about 2
moles per mole of substrate, and still more preferably about 1.6
mole per mole of substrate. Temperature is not critical but
elevated temperature enhances productivity. Reaction time is
typically between about 4 and about 24 hours, preferably about 4 to
16 hours. Conveniently, the reaction is carried out at atmospheric
reflux temperature depending on the solvent used.
[0262] The time required for the reaction to reach equilibrium is
affected by the amount of alkoxide that is added to the reaction
mixture and the manner in which the alkoxide is added. The alkoxide
may be added in a single portion or in multiple portions or it may
be added continuously. When alkoxide is added in multiple portions,
it is preferable that about 1.6 equivalents of potassium methoxide
be added in two steps. In this two-step addition, 1 equivalent of
potassium methoxide is initially added to the reaction mixture
followed by the addition of 0.6 equivalents of potassium methoxide
about 90 minutes later. This two-step addition shortens the time to
reach equilibrium relative to a single portion addition of 1.6
equivalents of potassium methoxide.
[0263] Because the equilibrium is more favorable for the production
of the hydroxyester at low concentrations of the diketone, the
reaction is preferably run at rather high dilution, e.g., as high
as 40:1 for reaction with sodium methoxide. It has been found that
significantly higher productivity can be realized by use of
potassium methoxide rather than sodium methoxide, because a
dilution in the range of about 20:1 is generally sufficient to
minimize the extent of reverse cyanidation where potassium
methoxide is the reagent.
[0264] In accordance with the invention, it has been further
discovered that the reverse cyanidation reaction may be inhibited
by taking appropriate chemical or physical measures to remove
by-product cyanide ion from the reaction zone. Thus, in a further
embodiment of the invention, the reaction of the diketone with
alkali metal alkoxide may be carried out in the presence of a
precipitating agent for cyanide ion such as, for example, a salt
comprising a cation which forms an insoluble cyanide compound. Such
salts may, for example, include zinc iodide, ferric sulfate, or
essentially any halide, sulfate or other salt of an alkaline earth
or transition metal that is more soluble than the corresponding
cyanide. If zinc iodide is present in proportions in the range of
about one equivalent per equivalent diketone substrate, it has been
observed that the productivity of the reaction is increased
substantially as compared to the process as conducted in the
absence of an alkali metal halide.
[0265] Even where a precipitating agent is used for removal of
cyanide ion, it remains preferable to run at fairly high dilution,
but by use of a precipitating agent the solvent to diketone
substrate molar-ratio may be reduced significantly compared to
reactions in the absence of such agent. Recovery of the
hydroxyester of Formula V can be carried out according to either
the extractive or non-extractive procedures described below.
[0266] The equilibrium of the reaction also can be controlled to
favor the production of the hydroxyester of Formula V by removing
this hydroxyester from the reaction mixture after it is
synthesized. The removal of the hydroxyester can proceed either
stepwise or continuously through means such as filtration. The
removal of the hydroxyester can be used to control the equilibrium
either alone or in combination with the chemical or physical
removal of cyanide from the reaction mixture. Heating of the
resulting filtrate then drives the reaction equilibrium to favor of
the conversion of the remaining diketone of Formula VI to the
hydroxyester of V.
[0267] In the conversion of the diketone of Formula VI to the
hydroxyester of Formula V the 5-cyano hydroxyester has been
observed in the crude product in small amounts, typically less than
about 5% by weight. It is hypothesized that the 5-cyano
hydroxyester is an equilibrium intermediate between the diketone of
Formula VI and the hydroxyester of Formula V. It is further
hypothesized that this equilibrium intermediate is formed from the
diketone through methoxide attack on the 5,7-oxo group and
protonation of the enolate, and from the hydroxyester through a
Michael addition of by-product cyanide ion to the
3-keto-.DELTA..sup.4,5 function of the hydroxyester.
[0268] In addition, the 5-cyano-7-acid and the 17-alkoxide of the
hydroxyester of Formula V have been observed by chromatography in
the crude product. It is hypothesized that the 5-cyano hydroxyester
intermediate reacts with by-product cyanide ion (present as a
result of the decyanation which introduces the .DELTA..sup.4,5
double bond) to produce the 5-cyano-7-acid. It is hypothesized that
the action of the cyanide ion dealkylates the 7-ester group of the
5-cyano hydroxyester to yield the 5-cyano-7-acid and the
corresponding alkylnitrile.
[0269] It is further hypothesized that transient intermediate
17-alkoxide is formed from the attack of the methoxide on the
17-spirolactone of the hydroxyester (or a preceding intermediate
which subsequently converts into the hydroxyester). The 17-alkoxide
readily converts into the hydroxyester upon treatment with an acid.
Therefore, it generally is not observed in the product matrix.
[0270] The 5-cyano hydroxyester, the 5-cyano-7-acid, and the
17-alkoxide are novel compounds which are useful as chromatographic
markers and as intermediates in the preparation of the
hydroxyester. They can be isolated from the crude product of this
step of the Scheme 1 synthesis. Alternatively, they can be
synthesized directly for use as markers or intermediates. The
5-cyano hydroxyester can be synthesized by reacting a solution of
the isolated diketone of Formula VI with a base, such as an
alkoxide or an amine, and isolating the resulting precipitate. The
compound prepared preferably is 7-methyl hydrogen
5.beta.-cyano-11.alpha.,17-dihydroxy-3-oxo-17.alpha.-pregnane-7.alpha.,21-
-dicarboxylate, .gamma.-lactone.
[0271] The 5-cyano-7-carboxylic acid can be synthesized directly by
reacting the diketone of Formula VI with a weak aqueous base, such
as sodium acetate or sodium bicarbonate, and isolating the
resulting precipitate. The compound prepared preferably is
5-.beta.-cyano-11-.alpha.,17-dihydroxy-3-oxo-17.alpha.-pregnane-7.alpha.,-
21-dicarboxylic acid, .gamma.-lactone.
[0272] The 17-alkoxide can be synthesized directly by reacting a
solution of the hydroxyester of Formula V with an alkoxide to yield
a mixture of the 17-alkoxide and the corresponding hydroxyester.
The compound prepared preferably is dimethyl
11.alpha.,17-dihydroxy-3-oxo-17.alpha.-pregn-4-ene-7.alpha.,21-dicarboxyl-
ate, .gamma.-lactone.
[0273] Preferably, the diketone substrate of Formula VI corresponds
to Formula VIA ##STR101## and the hydroxyester product corresponds
to Formula VA ##STR102## in each of which -A-A-, --B--B--, R.sup.3,
Y.sup.1, Y.sup.2, and X are as defined in Formula XIIIA and R.sup.3
is as defined in Formula V. Preferably, R.sup.3 is hydrogen.
[0274] The products of Formula V are novel compounds which have
substantial value as intermediates for the preparation of compounds
of Formula I, and especially of Formula IA. Preferably, the
compounds of Formula V correspond to Formula VA in which -A-A- and
--B--B-- are --CH.sub.2--CH.sub.2--, R.sup.3 is hydrogen, lower
alkyl or lower alkoxy, and R.sup.8 and R.sup.9 together constitute
the 20-spiroxane ring: ##STR103## Most preferably, the compound of
Formula V is Methyl Hydrogen
11.alpha.,17.alpha.-Dihydroxy-3-oxopregn-4-ene-7.alpha.,21-dicar-
boxylate, .gamma.-Lactone.
[0275] The compound of Formula V may be isolated by filtration or
by acidifying the reaction solution, e.g., with a mineral acid such
as aqueous HCl or sulfuric acid, cooling to ambient temperature,
and extracting the product with an organic solvent such as
methylene chloride or ethyl acetate. The extract is washed with an
aqueous alkaline wash solution, dried and filtered, after which the
solvent is removed. Alternatively, the reaction solution containing
the product of Formula V may be quenched with concentrated acid.
The product solution is concentrated, cooled to between about
0.degree. to 25.degree. C. and the product solid is isolated by
filtration.
[0276] In a preferred embodiment, methanol and HCN are removed by
distillation after the conclusion of the reaction period, with
mineral acid (such as hydrochloric acid or sulfuric acid) being
added before the distillation and water being added after the
distillation. The mineral acid can be added in a single step, in
multiple steps or continuously. In a preferred embodiment, mineral
acid is continuously added over a period of about 10 to about 40
minutes, more preferably about 15 to about 30 minutes. Likewise,
water can be added to the still bottoms in a single step, in
multiple steps or continuously. In a preferred embodiment, the
concentrated reaction mixture is cooled from reflux temperature
prior to addition of water. Preferably, the mixture is cooled to a
temperature between about 50.degree. C. to about 70.degree. C.,
preferably between about 60.degree. C. to about 70.degree. C., and
more preferably about 65.degree. C., prior to addition of the
water. Water is then added, preferably continuously over a period
of about 15 minutes to about 3 hours, and more preferably over
about 60 minutes to about 90 minutes, while maintaining the
temperature approximately constant. Product of Formula V begins to
crystallize from the still bottoms as the water addition proceeds.
After the water has been added to the mixture, the diluted reaction
mixture is maintained at about the same temperature for about 1
hour and then cooled to about 15.degree. C. over an additional
period of about 4 to about 5 hours. The mixture is maintained at
about 15.degree. C. for a period of about 1 to 2 hours. A longer
holding period at 15.degree. C. increases the yield of the
cyanoester in the mixture. This mode of recovery provides a high
quality crystalline product without extraction operations.
[0277] According to another preferred mode of recovery of the
product of Formula V, methanol and HCN are removed by distillation
after the conclusion of the reaction period, with water and acid
being added before or during the distillation. Addition of water
before the distillation simplifies operations, but progressive
addition during the distillation allows the volume in the still to
be maintained substantially constant. Product of Formula V
crystallizes from the still bottoms as the distillation proceeds.
This mode of recovery provides a high quality crystalline product
without extraction operations.
[0278] In accordance with yet a further alternative, the reaction
solution containing the product of Formula V may be quenched with
mineral acid, e.g., 4N HCl, after which the solvent is removed by
distillation. Removal of the solvent is also effective for removing
residual HCN from the reaction product. It has been found that is
multiple solvent extractions for purification of the compound of
Formula V are not necessary where the compound of Formula V serves
as an intermediate in a process for the preparation of
epoxymexrenone, as described herein. In fact, such extractions can
often be entirely eliminated. Where solvent extraction is used for
product purification, it is desirable to supplement the solvent
washes with brine and caustic washes. But where the solvent
extractions are eliminated, the brine and caustic washes are too.
Eliminating the extractions and washes significantly enhances the
productivity of the process, without sacrificing yield or product
quality, and also eliminates the need for drying of the washed
solution with a dessicant such as sodium sulfate.
[0279] The crude 11.alpha.-hydroxy-7.alpha.-alkoxycarbonyl product
is taken up again in the solvent for the next reaction step of the
process, which is the conversion of the 11-hydroxy group to a
leaving group at the 11 position thereby producing a compound of
Formula IV: ##STR104## where -A-A-, R.sup.3, --B--B--, R.sup.8 and
R.sup.9 are as defined in Formula XIII, R.sup.1 is as defined in
Formula V, and R.sup.2 is lower arylsulfonyloxy, alkylsulfonyloxy,
acyloxy or halide. Preferably, the 11.alpha.-hydroxy is esterified
by reaction with a lower alkylsulfonyl halide, an acyl halide or an
acid anhydride which is added to the solution containing the
intermediate product of Formula V. Lower acid anhdyrides such as
acetic anhydride and trihalogenated acid anhydrides such as
trifluoroacetic anhydride can be used to prepare suitable acyloxy
leaving groups. Lower alkylsulfonyl halides, and especially
methanesulfonyl chloride, however, are preferred. Alternatively,
the 11-.alpha. hydroxy group could be converted to a halide by
reaction of a suitable reagent such as thionyl bromide, thionyl
chloride, sulfuryl chloride or oxalyl chloride. Other reagents for
forming 11.alpha.-sulfonic acid esters include tosyl chloride,
benzenesulfonyl chloride and trifluoromethanesulfonic anhydride.
The reaction is conducted in a solvent containing a hydrogen halide
scavenger such as triethylamine or pyridine. Inorganic bases such
as potassium carbonate or sodium carbonate can also be used. The
initial concentration of the hydroxyester of Formula V is
preferably between about 5% and about 50% by weight. The
esterification reagent is preferably present in slight excess.
Methylene chloride is a particularly suitable solvent for the
reaction, but other solvents such as dichloroethane, pyridine,
chloroform, methyl ethyl ketone, dimethoxyethane, methyl isobutyl
ketone, acetone, other ketones, ethers, acetonitrile, toluene, and
tetrahydrofuran can also be employed. The reaction temperature is
governed primarily by the volatility of the solvent. In methylene
chloride, the reaction temperature is preferably in the range of
between about -10.degree. C. and about 10.degree. C.
[0280] Preferably, the hydroxyester substrate of Formula V
corresponds to Formula VA ##STR105## and the product corresponds to
Formula IVA ##STR106## in each of which -A-A-, --B--B--, R.sup.3,
Y.sup.1, Y.sup.2, and X are as defined in Formula XIIIA, R.sup.1 is
lower alkoxycarbonyl or hydroxycarbonyl, and R.sup.2 is as defined
in Formula IV. Preferably, R.sup.3 is hydrogen.
[0281] The products of Formula IV are novel compounds which have
substantial value as intermediates for the preparation of compounds
of Formula I, and especially of Formula IA. Preferably, the
compounds of Formula IVA correspond to Formula VA in which -A-A-
and --B--B-- are --CH.sub.2--CH.sub.2--, R.sup.3 is hydrogen, lower
alkyl or lower alkoxy, and R.sup.8 and R.sup.9 together constitute
the 20-spiroxane ring: ##STR107## Most preferably, the compound of
Formula IV is Methyl Hydrogen
17.alpha.-Hydroxy-11.alpha.-(methylsulfonyl)oxy-3-oxopregn-4-ene-
-7.alpha.,21-dicarboxylate, .gamma.-Lactone. Where an acyloxy
leaving group is desired, the compound of Formula IV is preferably
7-methyl hydrogen
17-hydroxy-3-oxo-11.alpha.-(2,2,2-trifluoro-1-oxoethoxy)-17.alph-
a.-pregn-4-ene-7.alpha.,21-dicarboxylate, .gamma.-lactone; or
7-methyl
11.alpha.-(acetyloxy)-17-hydroxy-3-oxo-17.alpha.-pregn-4-ene-7.alpha.,21--
dicarboxylate, .gamma.-lactone.
[0282] If desired, the compound of Formula IV may be isolated by
removal of the solvent. Preferably, the reaction solution is first
washed with an aqueous alkaline wash solution, e.g., 0.5-2N NaOH,
followed by an acid wash, e.g., 0.5-2N HCl. After removal of the
reaction solvent, the product is recrystallized, e.g., by taking
the product up in methylene chloride and then adding another
solvent such as ethyl ether which lowers the solubility of the
product of Formula IV, causing it to precipitate in crystalline
form.
[0283] In the recovery of the product of Formula IV, or in
preparation of the reaction solution for conversion of the Formula
IV intermediate to the intermediate of Formula II as is further
described hereinbelow, all extractions and/or washing steps may be
dispensed with if the solution is instead treated with ion exchange
resins for removal of acidic and basic impurities. The solution is
treated first with an anion exchange resin, then with a cation
exchange resin. Alternatively, the reaction solution may first be
treated with inorganic adsorbents such as basic alumina or basic
silica, followed by a dilute acid wash. Basic silica or basic
alumina may typically be mixed with the reaction solution in a
proportion of between about 5 and about 50 g per kg of product,
preferably between about 15 and about 20 g per kg product. Whether
ion exchange resins or inorganic adsorbents are used, the treatment
can be carried out by simply slurrying the resin or inorganic
adsorbent with the reaction solution under agitation at ambient
temperature, then removing the resin or inorganic adsorbent by
filtration.
[0284] In an alternative and preferred embodiment of the invention,
the product compound of Formula IV is recovered in crude form as a
concentrated solution by removal of a portion of the solvent. This
concentrated solution is used directly in the following step of the
process, which is removal of the 11.alpha.-leaving group from the
compound of Formula IV, thereby producing an enester of Formula II:
##STR108## where -A-A-, --B--B--, R.sup.3, R.sup.8 and R.sup.9 are
as defined in Formula XIII, and R.sup.1 is as defined in Formula V.
For purposes of this reaction, the R.sup.2 substituent of the
compound of Formula IV may be any leaving group the abstraction of
which is effective for generating a double bond between the 9- and
11-carbons. Preferably, the leaving group is a lower
alkylsulfonyloxy or acyloxy substituent which is removed by
reaction with an acid and an alkali metal salt. Mineral acids can
be used, but lower alkanoic acids are preferred. Advantageously,
the reagent for the reaction further includes an alkali metal salt
of the alkanoic acid utilized. It is particularly preferred that
the leaving group comprise mesyloxy and the reagent for the
reaction comprise formic acid or acetic acid and an alkali metal
salt of one of these acids or another lower alkanoic acid. Where
the leaving group is mesyloxy and the removal reagent is either
acetic acid and sodium acetate or formic acid and potassium
formate, a relatively high ratio of 9,11-olefin to 11,12-olefin is
observed. If free water is present during removal of the leaving
group, impurities tend to be formed, particularly a 7,9-lactone
##STR109## where -A-A-, --B--B--, R.sup.8 and R.sup.9 are as
defined in Formula XIII, which is difficult to remove from the
final product. Hence, acetic anhydride or other drying agent is
used to remove the water present in formic acid. The free water
content of the reaction mixture before reaction should be
maintained at a level below about 0.5%, preferably below about 0.1%
by weight, as measured by Karl Fischer analysis for water, based on
total reaction solution. Although it is preferred that the reaction
mixture be kept as dry as practicable, satisfactory results have
been realized with 0.3% by weight water. Preferably, the reaction
charge mixture contains between about 4% and about 50% by weight of
the substrate of Formula IV in the alkanoic acid. Between about 4%
and about 20% by weight of the alkali metal salt of the acid is
preferably included. Where acetic anhydride is used as the drying
agent, it is preferably present in a proportion of between about
0.05 moles and about 0.2 moles per mole of alkanoic acid.
[0285] It has been found that proportions of by-product 7,9-lactone
and 11,12-olefin in the reaction mixture is relatively low where
the elimination reagent comprises a combination of trifluoroacetic
acid, trifluoroacetic anhydride and potassium acetate as the
reagent for elimination of the leaving group and formation of the
enester (9,11-olefin). Trifluoroacetic anhydride serves as the
drying agent, and should be present in a proportion of at least
about 3% by weight, more preferably at least about 15% by weight,
most preferably about 20% by weight, based on the trifluoroacetic
acid eliminating reagent.
[0286] In addition to the 7,9-lactone, other impurities and
by-products which are useful as synthetic intermediates and
chromatographic markers have been observed in this step of the
Scheme 1 synthesis. The novel 4,9,13-triene of the enester of
Formula II (for example, 7-methyl hydrogen
17-methyl-3-oxo-18-norpregna-4,9(11),13-triene-7.alpha.,21-dicar-
boxylate) has been isolated chromatographically from the product
solution. The amount of this compound produced appears to increase
with an increase in reaction time for this step of the synthesis.
It is hypothesized that the compound is formed when the lactone is
protonated and the resulting C17 carbonium ion facilitates the
migration of the angular methyl group from the C13 position.
Deprotonation of this intermediate yields the 4,9,13-triene.
[0287] The novel 5-cyano-.DELTA..sup.11,12 of the enester of
Formula II (for example, 7-methyl hydrogen
5.beta.-cyano-17-hydroxy-3-oxo-17.alpha.-pregn-11-ene-7.alpha.,21-dicarbo-
xylate, .gamma.-lactone) and the novel 5-cyano of the enester of
Formula II (for example, 7-methyl hydrogen
5-cyano-17-hydroxy-3-oxo-17.alpha.-pregn-11-ene-7.alpha.,21-dicarboxylate-
, .gamma.-lactone) also have been isolated chromatographically from
the crude product. It is hypothesized that these compounds are
formed via dehydration of the residual 5-cyano-7-acid and 5-cyano
hydroxyester, respectively, which are present in the crude product
solution as a result of the third step of the Scheme 1
synthesis.
[0288] The novel C17 epimer of the enester of Formula II (for
example, 7-methyl hydrogen
17-hydroxy-3-oxo-17.alpha.-pregna-4,9(11)-diene-7,21-dicarboxylate,
.gamma.-lactone) also has been isolated chromatographically from
the crude product. It is hypothesized that the acidic conditions of
the elimination reaction can result in racemization of the C17
chiral center to yield the 17-epimer of the enester. The 17-epimer
can be synthesized directly by reacting a compound of Formula IV
with a solution of potassium formate, formic acid and acetic
anhydride and isolating the 17-epimer.
[0289] Although not observed as an impurity in the crude product
solution, the 11-ketone of the hydroxyester of formula V can be
prepared by oxidizing the 11-hydroxy of the corresponding
hydroxyester with a suitable oxidizing agent such as a Jones
Reagent. The 11-ketone prepared preferably is 7-methyl hydrogen
17-hydroxy-3,11-dioxo-17.alpha.-pregna-4-ene-7.alpha.,21-dicarboxylate,
.gamma.-lactone.
[0290] Alternatively, the 11.alpha.-leaving groups from the
compound of Formula IV, may be eliminated to produce an enester of
Formula II by heating a solution of Formula IV in an organic
solvent such as DMSO, DMF or DMA.
[0291] Further in accordance with the invention, the compound of
Formula IV is reacted initially with an alkenyl alkanoate such as
isopropenyl acetate in the presence of an acid such as toluene
sulfonic acid or an anhydrous mineral acid such as sulfuric acid to
form the 3-enol ester: ##STR110## of the compound of Formula IV.
Alternatively, the 3-enol ester can be formed by treatment of the
compound of Formula IV with an acid anhydride and base such as
acetic acid and sodium acetate. Further alternatives include
treatment of the compound of Formula IV with ketene in the presence
of an acid to produce the compound of Formula IV(Z). The
intermediate of Formula IV(Z) is thereafter reacted with an alkali
metal formate or acetate in the presence of formic or acetic acid
to produce the .DELTA..sup.9,11 enol acetate of Formula IV(Y):
##STR111## which can then be converted to the enester of Formula II
in an organic solvent, preferably an alcohol such as methanol, by
either thermal decomposition of the enol acetate or reaction
thereof with an alkali metal alkoxide. The elimination reaction is
highly selective to the enester of Formula II in preference to the
11,12-olefin and 7,9-lactone, and this selectivity is preserved
through conversion of the enol acetate to the enone.
[0292] Preferably, the substrate of Formula IV corresponds to
Formula IVA ##STR112## and the enester product corresponds to
Formula IIA ##STR113## in each of which -A-A-, --B--B--, R.sup.3,
Y.sup.1, Y.sup.2, and X are as defined in Formula XIIIA, and
R.sup.1 is as defined in Formula V. Preferably, R.sub.3 is
hydrogen.
[0293] If desired, the compound of Formula II may be isolated by
removing the solvent, taking up the solid product in cold water,
and extracting with an organic solvent, such as ethyl acetate.
After appropriate washing and drying steps, the product is
recovered by removing the extraction solvent. The enester is then
dissolved in a solvent appropriate for the conversion to the
product of Formula I. Alternatively, the enester can be isolated by
adding water to the concentrated product solution and filtering the
solid product, thereby preferentially removing the 7,9-lactone.
Conversion of the substrate of Formula II to the product of Formula
IA may be conducted in the manner described in U.S. Pat. No.
4,559,332 which is expressly incorporated herein by reference, or
more preferably by the novel reaction using a haloacetamide
promoter as described below.
[0294] In another embodiment of the invention, the hydroxyester of
Formula V may be converted to the enester of Formula II without
isolation of the intermediate compound of Formula IV. In this
method, the hydroxyester is taken up in an organic solvent, such as
methylene chloride; and either an acylating agent, e.g.,
methanesulfonyl chloride, or halogenating reagent, e.g., sulfuryl
chloride, is added to the solution. The mixture is agitated and,
where halogenation is involved, an HCl scavenger such as imidazole
is added. This reaction is highly exothermic, and should therefore
be conducted at a controlled rate with full cooling. After the base
addition, the resulting mixture is warmed to moderate temperature,
e.g., about 0.degree. C. to room temperature or slightly above, and
reacted for a period of typically about 1 to about 4 hours. After
reaction is complete, the solvent is stripped, preferably under
high vacuum (e.g., about 24'' to about 28'' Hg) conditions at about
-10.degree. to about +15.degree. C., more preferably about
0.degree. to about 5.degree. C., to concentrate the solution and
remove excess base. The substrate is then redissolved in an organic
solvent, preferably a halogenated solvent such as methylene
chloride for conversion to the enester.
[0295] The leaving group elimination reagent is preferably prepared
by mixing an organic acid, an organic acid salt and a drying agent,
preferably formic acid, alkali metal formate and acetic anhydride,
respectively, in a dry reactor. Addition of acetic anhydride is
exothermic and results in release of CO, so the addition rate must
be controlled accordingly. To promote the removal of water, the
temperature of this reaction is preferably maintained in the range
of about 60.degree. to about 90.degree. C., most preferably about
65.degree. to about 75.degree. C. This reagent is then added to the
product solution of the compound of Formula IV to effect the
elimination reaction. After about 4 to about 8 hours, the reaction
mixture is preferably heated to a temperature of at least about
85.degree. C., but preferably not above about 95.degree. C. until
all volatile distillate has been removed, and then for an
additional period to complete the reaction, typically about 1 to
about 4 hours. The reaction mixture is cooled, and after recovery
by standard extraction techniques, the enester may be recovered as
desired by evaporating the solvent.
[0296] It has further been found that the enester of Formula II may
be recovered from the reaction solution by an alternative procedure
which avoids the need for extraction steps following the
elimination reaction, thereby providing savings in cost,
improvement in yield and/or improvement in productivity. In this
process, the enester product is precipitated by dilution of the
reaction mixture with water after removal of formic acid. The
product is then isolated by filtration. No extractions are
required.
[0297] According to a further alternative for conversion of the
hydroxyester of Formula V to the enester of Formula II without
isolation of the compound of Formula IV, the 11.alpha.-hydroxy
group of the Formula V hydroxyester is replaced by halogen, and the
Formula II enester is then formed in situ by thermal
dehydrohalogenation. Replacement of the hydroxy group by halogen is
effected by reaction with sulfuryl halide, preferably sulfuryl
chloride, in the cold in the presence of a hydrogen halide
scavenger such as imidazole. The hydroxyester is dissolved in a
solvent such as tetrahydrofuran and cooled to about 0.degree. C. to
about -70.degree. C. The sulfuryl halide is added and the reaction
mixture is warmed to moderate temperature, e.g., room temperature,
for a time sufficient to complete the elimination reaction,
typically about 1 to about 4 hours. The process of this embodiment
not only combines two steps into one, but eliminates the use of: a
halogenated reaction solvent; an acid (such as acetic acid); and a
drying reagent (such as acetic anhydride or sodium sulfate).
Moreover, the reaction does not require refluxing conditions, and
avoids the generation of by-product CO which results when acetic
acid is used as a drying reagent.
[0298] In accordance with a particularly preferred embodiment of
the invention, the diketone compound of Formula VI can be converted
to epoxymexrenone or other compound of Formula I without isolating
any intermediate in purified form. In accordance with this
preferred process, the reaction solution containing the
hydroxyester is quenched with a strong acid solution, cooled to
ambient temperature and then extracted with an appropriate
extraction solvent. Advantageously, an aqueous solution of
inorganic salt, e.g., about 10% by is weight saline solution, is
added to the reaction mixture prior to the extraction. The extract
is washed and dried by azeotropic distillation for removal of the
methanol solvent remaining from the ketone cleavage reaction.
[0299] The resulting concentrated solution containing between about
5% and about 50% by weight compound of Formula V is then contacted
in the cold with an acylating or alkylsulfonylating reagent to form
the sulfonic ester or dicarboxylic acid ester. After the
alkylsulfonation or carboxylation reaction is complete, the
reaction solution is passed over an acidic and then a basic
exchange resin column for the removal of basic and acidic
impurities. After each pass, the column is washed with an
appropriate solvent, e.g., methylene chloride, for the recovery of
residual sulfonic or dicarboxylic ester therefrom. The combined
eluate and wash fractions are combined and reduced, preferably
under vacuum, to produce a concentrated solution containing the
sulfonic ester or dicarboxylic ester of Formula IV. This
concentrated solution is then contacted with a dry reagent
comprising an agent effect for removal of the 11.alpha.-ester
leaving group and abstraction of hydrogen to form a 9,11 double
bond. Preferably, the reagent for removal of the leaving group
comprises the formic acid/alkali metal formate/acetic anhydride dry
reagent solution described above. After reaction is complete, the
reaction mixture is cooled and formic acid and/or other volatile
components are removed under vacuum. The residue is cooled to
ambient temperature, subjected to appropriate washing steps, and
then dried to give a concentrated solution containing the enester
of Formula II. This enester may then be converted to epoxymexrenone
or other compound of Formula I using the method described herein,
or in U.S. Pat. No. 4,559,332.
[0300] In an especially preferred embodiment of the invention, the
solvent is removed from the reaction is solution under vacuum, and
the product of Formula IV is partitioned between water and an
appropriate organic solvent, e.g., ethyl acetate. The aqueous layer
is then back extracted with the organic solvent, and the back
extract washed with an alkaline solution, preferably a solution of
an alkali metal hydroxide containing an alkali metal halide. The
organic phase is concentrated, preferably under vacuum, to yield
the enester product of Formula II. The product of Formula II may
then be taken up in an organic solvent, e.g., methylene chloride,
and further reacted in the manner described in the '332 patent to
produce the product of Formula I.
[0301] Where trihaloacetonitrile is used in the epoxidation
reaction, it has been found that the selection of solvent is
important, with halogenated solvents being highly preferred, and
methylene chloride being especially preferred. Solvents such as
dichloroethane and chlorobenzene give reasonably satisfactory
yields, but yields are generally better in a methylene chloride
reaction medium. Solvents such as acetonitrile and ethyl acetate
generally give poor yields, while reaction in solvents such as
methanol or water/tetrahydrofuran give little of the desired
product.
[0302] Further in accordance with the present invention, it has
been discovered that numerous improvements in the synthesis of
epoxymexrenone can be realized by use of a trihaloacetamide rather
than a trihaloacetonitrile as a peroxide activator for the
epoxidation reaction. In accordance with a particularly preferred
process, the epoxidation is carried out by reaction of the
substrate of Formula IIA with hydrogen peroxide in the presence of
trichloroacetamide and an appropriate buffer. Preferably, the
reaction is conducted in a pH in the range of about 3 to about 7,
most preferably between about 5 and about 7. However, despite these
considerations, successful reaction has been realized outside the
preferred pH ranges.
[0303] Especially favorable results are obtained with a buffer
comprising dipotassium hydrogen phosphate, and/or with a buffer
comprising a combination of dipotassium hydrogenphosphate and
potassium dihydrogen phosphate in relative proportions of between
about 1:4 and about 2:1, most preferably in the range of about 2:3.
Borate buffers can also be used, but generally give slower
conversions than dipotassium phosphate or
K.sub.2HPO.sub.4/KH.sub.2PO.sub.4 mixtures. Whatever the makeup of
the buffer, it should provide a pH in the range indicated above.
Aside from the overall composition of the buffer or the precise pH
it may impart, it has been observed that the reaction proceeds much
more effectively if at least a portion of the buffer is comprised
of dibasic hydrogenphosphate ion. It is believed that this ion may
participate essentially as a homogeneous catalyst in the formation
of an adduct or complex comprising the promoter and hydroperoxide
ion, the generation of which may in turn be essential to the
overall epoxidation reaction mechanism. Thus, the quantitative
requirement for dibasic hydrogenphosphate (preferably from
K.sub.2HPO.sub.4) may be only a small catalytic concentration.
Generally, it is preferred that K.sub.2HPO.sub.4 be present in a
proportion of at least about 0.1 equivalents, e.g., between about
0.1 and about 0.3 equivalents, per equivalent substrate.
[0304] The reaction is carried out in a suitable solvent,
preferably methylene chloride, but alternatively other halogenated
solvents such as chlorobenzene or dichloroethane can be used.
Toluene and mixtures of toluene and acetonitrile have also been
found satisfactory. Without committing to a particular theory, it
is posited that the reaction proceeds most effectively in a two
phase system in which a hydroperoxide intermediate is formed and
distributes to the organic phase of low water content, and reacts
with the substrate in the organic phase. Thus the preferred
solvents are those in which water solubility is low. Effective
recovery from toluene is promoted by inclusion of another solvent
such as acetonitrile.
[0305] In the conversion of substrates of Formula II to products of
Formula I, toluene provides a process advantage since the
substrates are freely soluble in toluene and the products are not.
Thus, the product precipitates during the reaction when conversions
reach the 40-50% range, producing a three phase mixture from which
the product can be conveniently separated by filtration. Methanol,
ethyl acetate, acetonitrile alone, THF and THF/water have not
proved to be as effective as the halogenated solvents or toluene in
carrying out the conversion of this step of the process.
[0306] While trichloroacetamide is a highly preferred reagent,
other trihaloacetamides such as trifluoroacetamide and
chlorodifluoroacetamide can also be used. Trihalomethylbenzamide,
and other compounds having an arylene, alkenyl or alkynyl moiety
(or other group which allows the transfer of the electron
withdrawing effect of the electron withdrawing group to the amide
carbonyl) between the electron withdrawing trihalomethyl group and
the carbonyl of the amide, may also be useful.
Heptafluorobutyramides may also be used, but with less favorable
results. Generically, the peroxide activator may correspond to the
formula: R.sup.oC(O)NH.sub.2 where R.sup.o is a group having an
electron withdrawing strength (as measured by sigma constant) at
least as high as that of the monochloromethyl group. The electron
withdrawing group preferably is attached directly to the amide
carbonyl for maximum effectiveness. More particularly, the peroxide
activator may correspond to the formula: ##STR114## where R.sup.p
is a group which allows the transfer of the electron withdrawing
effect of an electron withdrawing group to the amide carbonyl, and
preferably is selected from among arylene, alkenyl, alkynyl and
--(CX.sup.4X.sup.5).sub.n-- moieties; X.sup.1, X.sup.2, X.sup.3,
X.sup.4 and X.sup.5 are independently selected from among halo,
hydrogen, alkyl, haloalkyl and cyano and cyanoalkyl; and n is 0, 1
or 2; provided that when n is 0, then at least one of X.sup.1,
X.sup.2 and X.sup.3 is halo; and when R.sup.p is
--(CX.sup.4X.sup.5).sub.n-- and n is 1 or 2, then at least one of
X.sup.4 and X.sup.5 is halo. Where any of X.sup.1, X.sup.2,
X.sup.3, X.sup.4 or X.sup.5 is not halo, it is preferably
haloalkyl, most preferably perhaloalkyl. Particularly preferred
activators include those in which n is 0 and at least two of
X.sup.1, X.sup.2 and X.sup.3 are halo; or those in which R.sup.p is
--(CX.sup.4X.sup.5).sub.n--, n is 1 or 2, at least one of X.sup.4
and X.sup.5 is halo, the other of X.sup.4 and X.sup.5 is halo or
perhaloalkyl, and X.sup.1, X.sup.2 and X.sup.3 are halo or
perhaloalkyl. Each of X.sup.1, X.sup.2, X.sup.3, X.sup.4 and
X.sup.5 is preferably Cl or F, most preferably Cl, though mixed
halides may also be suitable, as may perchloralkyl or perbromoalkyl
and combinations thereof, provided that the carbon directly
attached to the amide carbonyl is substituted with at least one
halo group.
[0307] Preferably, the peroxide activator is present in a
proportion of at least about 1 equivalent, more preferably between
about 1.5 and about 2 equivalents, per equivalent of substrate
initially present. Hydrogen peroxide should be charged to the
reaction in at least modest excess, or added progressively as the
epoxidation reaction proceeds. Although the reaction consumes only
one to two equivalents of hydrogen peroxide per mole of substrate,
hydrogen peroxide is preferably charged in substantial excess
relative to substrate and activator initially present. Without
limiting the invention to a particular theory, it is believed that
the reaction mechanism involves formation of an adduct of the
activator and the peroxide anion, that the formation of this
reaction is reversible with the equilibrium favoring the reverse
reaction, and that a substantial initial excess of hydrogen
peroxide is therefore necessary in order to drive the reaction in
the forward direction. Temperature of the reaction is not narrowly
critical, and may be effectively carried out within the range of
about 0.degree. to about 100.degree. C. The optimum temperature
depends on the selection of solvent. Generally, the preferred
temperature is between about 20.degree. C. and about 30.degree. C.,
but in certain solvents, e.g., toluene the reaction may be
advantageously conducted in the range of about 60.degree. to about
70.degree. C. At about 25.degree. C., reaction typically requires
less than about 10 hours, typically about 3 to about 6 hours. If
needed, additional activator and hydrogen peroxide may be added at
the end of the reaction cycle to achieve complete conversion of the
substrate.
[0308] At the end of the reaction cycle, the aqueous phase is
removed, the organic reaction solution is preferably washed for
removal of water soluble impurities, after which the product may be
recovered by removal of the solvent. Before removal of solvent, the
reaction solution should be washed with at least a mild to
moderately alkaline wash, e.g., sodium carbonate. Preferably, the
reaction mixture is washed successively with: a mild reducing
solution such as a weak (e.g. about 3% by weight) solution of
sodium sulfite in water; an alkaline solution, e.g., NaOH or KOH
(preferably about 0.5N); an acid solution such as HCl (preferably
about 1N); and a final neutral wash comprising water or brine,
preferably saturated brine to minimize product losses. Prior to
removal of the reaction solvent, another solvent such as an organic
solvent, preferably ethanol may be advantageously added, so that
the product may be recovered by crystallization after distillation
for removal of the more volatile reaction solvent.
[0309] It should be understood that the novel epoxidation method
utilizing trichloroacetamide or other novel peroxide activator has
application well beyond the various schemes for the preparation of
epoxymexrenone, and in fact may be used for the formation of
epoxides across olefinic double bonds in a wide variety of
substrates subject to reaction in the liquid phase. The reaction is
particularly effective in unsaturated compounds in which the
olefins are tetrasubstituted and trisubstituted, i.e.,
R.sup.aR.sup.bC.dbd.CR.sup.cR.sup.d and
R.sup.aR.sup.bC.dbd.CR.sup.cH where R.sup.a to R.sup.d represent
substituents other than hydrogen. The reaction proceeds most
rapidly and completely where the substrate is a cyclic compound
with a trisubstituted double bond, or either a cyclic or acyclic
compound with a tetrasubstituted double bond. Exemplary substrates
for the epoxidation reaction include .DELTA..sup.9,11-canrenone,
and the following substrates: ##STR115##
[0310] Because the reaction proceeds more rapidly and completely
with trisubstituted and tetrasubstituted double bonds, it is
especially effective for selective epoxidation across such double
bonds in compounds that may include other double bonds where the
olefinic carbons are monosubstituted, or even disubstituted.
[0311] Other non-limiting examples illustrating the generic
epoxidation reaction include the following epoxidation reactions:
##STR116##
[0312] It should be further understood that the reaction may be
used to advantage in the epoxidation of monosubstituted or even
disubstituted double bonds, such as the 11,12-olefin in various
steroid substrates. However, because it preferentially epoxidizes
the more highly substituted double bonds, e.g., the 9,11-olefin,
with high selectivity, the process of this invention is especially
effective for achieving high yields and productivity in the
epoxidation steps of the various reaction schemes described
elsewhere herein.
[0313] The improved process has been shown to be a particularly
advantageous application to the preparation of: ##STR117## by
epoxidation of: ##STR118## the preparation of: ##STR119## by
epoxidation of: ##STR120##
[0314] Multiple advantages have been demonstrated for the process
of the invention in which trichloroacetamide is used in place of
trichloroacetonitrile as the oxygen transfer reagent for the
epoxidation reaction. The trichloroacetamide reagent system has a
low affinity for electronically deficient olefins such as
.alpha.,.beta.-unsaturated ketones. This allows for selective
epoxidation of a non-conjugated olefin in substrates containing
both types of double bonds. Additionally, in complex substrates
such as steroids, disubstituted and trisubstituted olefins can be
differentiated by reaction. Thus, good selectivity is observed in
the epoxidation of the isomeric .DELTA.-9,11 and .DELTA.-11,12
compounds. In this case, the 9,11 epoxide is formed with minimal
reaction of the isomer containing the .DELTA.-11,12 double bond.
Accordingly, reaction yield, product profile and final purity are
substantially enhanced in comparison to reactions in which a
trihaloacetonitrile is used. It has further been discovered that
the substantial excess oxygen generation observed with the use of
trihaloacetonitrile is minimized with trichloroacetamide, imparting
improved safety to the epoxidation process. Further in contrast to
the trichloroacetonitrile promoted reaction, the trichloroacetamide
reaction exhibits minimum exothermic effects, thus facilitating
control of the thermal profile of the reaction. Agitation effects
are observed to be minimal and reactor performance more consistent,
a further advantage over the trichloroacetonitrile process. The
reaction is more amenable to scaleup than the trichloroacetonitrile
promoted reaction. Product isolation and purification is simple.
There is no observable Bayer-Villager oxidation of carbonyl
function (peroxide promoted conversion of ketone to ester) as
experienced when using m-chloroperoxybenzoic acid or other
peracids. The reagent is inexpensive, readily available, and easily
handled.
[0315] In addition, the following compounds have been observed by
chromatography in the crude product from the step of the Scheme 1
synthesis in which the enester of Formula II is converted to the
compound of Formula I:
[0316] (1) the novel 11.alpha.,12.alpha. epoxide of the enester of
formula II, for example, 7-methyl hydrogen
11.alpha.,12.alpha.-epoxy-17-hydroxy-3-oxo-17.alpha.-pregn-4-ene-7.alpha.-
,21-dicarboxylate, .gamma.-lactone;
[0317] (2) the novel 4,5:9,11-diepoxide of the enester of formula
II, for example 7-methyl hydrogen
4.alpha.,5.alpha.:9.alpha.,11.alpha.-diepoxy-17-hydroxy-3-oxo-17.alpha.-p-
regnane-7.alpha.,21-dicarboxylate, .gamma.-lactone;
[0318] (3) the novel 12-ketone of the enester of formula II, for
example 7-methyl hydrogen
17-hydroxy-3,12-dioxo-17.alpha.-pregna-4,9(11)-diene-7.alpha.,21-dicarbox-
ylate, .gamma.-lactone;
[0319] (4) the novel 9,11-dihydroxy of the enester of formula II,
for example 7-methyl hydrogen
9.alpha.,11.beta.,17-trihydroxy-3-oxo-17.alpha.-pregna-4-ene-7.alpha.,21--
dicarboxylate, .gamma.-lactone;
[0320] (5) the novel 12-hydroxy analog of the enester of formula
II, for example 7-methyl hydrogen
12.alpha.,17-dihydroxy-3-oxo-17.alpha.-pregna-4,9(11)-diene-7.alpha.,21-d-
icarboxylate, .gamma.-lactone; and
[0321] (6) the novel 7-acid of the compound of Formula I, for
example
9,11.alpha.-epoxy-17-hydroxy-3-oxo-17.alpha.-pregn-4-ene-7.alpha.,21-dica-
rboxylic acid, .gamma.-lactone.
[0322] These compounds have utility as synthetic intermediates
and/or chromatographic markers in the preparation of the compound
of Formula I, particularly epoxymexrenone.
[0323] The 11.alpha.,12.alpha.-epoxide of the enester of formula II
is hypothesized to form via an impurity produced during the
previous step in which a compound of formula IV is converted to the
enester of formula II. This impurity was chromatographically
isolated and is the .DELTA..sup.11,12 enester. It typically is
produced with the .DELTA..sup.9,11 enester in a ratio of about
90:10 (.DELTA..sup.9,11 enester:.DELTA..sup.11,12 enester),
although this ratio can vary. Oxidation of the .DELTA..sup.11,12
enester during the conversion of the enester of formula II to the
compound of Formula I yields the 11.alpha.,12.alpha.-epoxide.
[0324] The 4,5:9,11-diepoxide of the enester of Formula I was
chromatographically isolated. It is hypothesized to result from
over-epoxidation of the enester. It typically is observed in the
crude product at levels of about 5% by weight or less, although
this amount can vary.
[0325] The 12-ketone of the enester of Formula II was
chromatographically isolated. It is hypothesized to result from
allylic oxidation of the enester. It typically is observed in the
crude product at levels of about 5% by weight or less, although
this amount can vary. The level of 12-ketone detected in the crude
product when trichloroacetonitrile was used as the hydrogen
peroxide activator was higher than the level detected when
trichloroacetamide was used as the activator.
[0326] The 9,11-dihydroxy of the enester of Formula II was
chromatographically isolated. It typically is observed in the crude
product at levels of about 5% by weight or less, although this
amount can vary. It is hypothesized to result from hydrolysis of
the epoxide of Formula I.
[0327] The 12-hydroxy of the enester of formula II was
chromatographically isolated. It typically is observed in the crude
product at levels of about 5% by weight or less, although this
amount can vary. It is hypothesized to result from hydrolysis of
the 11,12 epoxide with subsequent elimination of the
lip-hydroxy.
[0328] In addition, the compounds of Formula I prepared in
accordance with this disclosure can be further modified to provide
a metabolite, derivative, prodrug or the like with improved
properties (such as improved solubility and absorption) which
facilitate the administration and/or efficacy of epoxymexrenone.
The 6-hydroxy of the compound of Formula I (for example, 7-methyl
hydrogen
6.beta.,17-dihydroxy-9,11.alpha.-epoxy-3-oxo-17.alpha.-pregn-4-ene-7.alph-
a.,21-dicarboxylate, .gamma.-lactone) is a novel compound which has
been identified as a possible metabolite in the rat. The 6-hydroxy
metabolite can be prepared from the corresponding ethyl enol ether
(for example, 7-methyl hydrogen
9,11.alpha.-epoxy-3-ethoxy-17-hydroxy-17.alpha.-pregn-4-ene-7.alpha.,21-d-
icarboxylate, .gamma.-lactone). The ethyl enol ether of the
compound of Formula I can be prepared in accordance with the
procedure set forth in R. M. Weier and L. M. Hofmann (J. Med Chem
1977, 1304) which is incorporated by reference herein. The ethyl
enol ether is then reacted with m-chloroperbenzoic acid to yield
the corresponding 6-hydroxy of the compound of Formula I.
[0329] It is further hypothesized that the monocarboxylic salts of
epoxymexrenone, particularly the potassium and sodium salts, are
suitable alternatives to facilitate administration of a compound of
Formula I to an individual for whom administration of an
aldosterone antagonist is indicated. Under mild basic conditions it
is possible to selectively open the spirolactone of the compounds
of Formula I without hydrolyzing the C7 ester substituent to give
the corresponding 17.beta.-hydroxy-17.alpha.-(3-propionic acid)
analog. These open chain analogs are more polar than their lactone
counterparts and have shorter retention times when analyzed by
reverse phase HPLC. Acidic conditions generally cause the
regeneration of the lactone ring.
[0330] Under more forcing conditions, the spirolactone is opened
and the C7 ester is hydrolyzed to give the corresponding
by-products, 17.beta.-hydroxy-17.alpha.-(3-propionic acid)-7-acid
analogs of the compounds of Formula I. These dicarboxylic acids
have shorter retention times than the monocarboxylic acids when
analyzed by reverse phase HPLC. Acidic conditions (e.g., treatment
with a dilute acid such as 0.1-4 M hydrochloric acid) generally
cause the regeneration of the lactone ring of the dicarboxylic
acid.
[0331] The novel epoxidation method of the invention is highly
useful as the concluding step of the synthesis of Scheme 1. In a
particularly preferred embodiment, the overall process of Scheme 1
proceeds as follows: ##STR121##
[0332] The second of the novel reaction schemes (Scheme 2) of this
invention starts with canrenone or other substrate corresponding to
Formula XIII ##STR122## where -A-A-, --B--B--, R.sup.3, R.sup.8 and
R.sup.9 are as defined in Formula XIII. In the step of this
process, the substrate of Formula XIII is converted to a product of
Formula XII ##STR123## using a cyanidation reaction scheme
substantially the same as that described above for conversion of
the substrate of Formula VIII to the intermediate of Formula VII.
Preferably, the substrate of Formula XIII corresponds to Formula
XIIIA ##STR124## and the enamine product corresponds to Formula
XIIA ##STR125## in each of which -A-A-, --B--B--, R.sup.3, Y.sup.1,
Y.sup.2, and X are as defined in Formula XIIIA. Preferably, R.sup.3
is hydrogen.
[0333] In the second step of Scheme 2, the enamine of Formula XII
is hydrolyzed to an intermediate diketone product of Formula XI
##STR126## where -A-A-, --B--B--, R.sup.3, R.sup.8 and R.sup.9 are
as defined in Formula XIII, using a reaction scheme substantially
the same as that described above for conversion of the substrate of
Formula VIII to the intermediate of Formula VII. Preferably, the
substrate of Formula XII corresponds to Formula XIIA ##STR127## and
the diketone product corresponds to Formula XIA ##STR128## in each
of which -A-A-, --B--B--, R.sup.3, Y.sup.1, Y.sup.2, and X are as
defined in Formula XIIIA. Preferably, R.sup.3 is hydrogen.
[0334] Further in accordance with reaction scheme 2, the diketone
of Formula XI is reacted with an alkali metal alkoxide to form
mexrenone or other product corresponding to Formula X, ##STR129##
in each of which -A-A-, --B--B--, R.sup.3, R.sup.8 and R.sup.9 are
as defined in Formula XIII, and R.sup.1 is as defined in Formula V.
The process is carried out using substantially the same reaction
scheme that is described above for the conversion of the compounds
of Formula VI to those of Formula V. Preferably, the substrate of
Formula XI corresponds to Formula XIA ##STR130## and the
intermediate product corresponds to Formula XA ##STR131## in each
of which -A-A-, --B--B--, R.sup.3, Y.sup.1, Y.sup.2, and X are as
defined in Formula XIIIA, and R.sup.1 is as defined in Formula V.
Preferably, R.sup.3 is hydrogen.
[0335] Mexrenone and other compounds of Formula X are next
9.alpha.-hydroxylated by a novel bioconversion process to yield
products of Formula IX ##STR132## where -A-A-, --B--B--, R.sup.3,
R.sup.8 and R.sup.9 are as defined in Formula XIII, and R.sup.1 is
as defined in Formula V. Among the organisms that can be used in
this hydroxylation step are Nocardia conicruria ATCC 31548,
Nocardia aurentia ATCC 12674, Corynespora cassiicola ATCC 16718,
Streotomyces hydroscopicus ATCC 27438, Mortierella isabellina ATCC
42613, Beauvria bassiana ATCC 7519, Penicillum purpurogenum ATCC
46581, Hypomyces chrysospermus IMI 109891, Thamnostylum piriforme
ATCC 8992, Cunninghamella blakesleeana ATCC 8688a, Cunninghamella
echinulata ATCC 3655, Cunninghamella elegans ATCC 9245,
Trichothecium roseum ATCC 12543, Epicoccum humicola ATCC 12722,
Saccharopolyspora eythrae ATCC 11635, Beauvria bassiana ATCC 13144,
Arthrobacter simplex, Bacterium cyclooxydans ATCC 12673,
Cylindrocarpon radicicola ATCC 11011, Nocardia aurentia ATCC 12674,
Norcardia restrictus ATCC 14887, Pseudomonas testosteroni ATCC
11996, Rhodococcus equi ATCC 21329, Mycobacterium fortuitum NRRL
B8119, and Rhodococcus rhodochrous ATCC 19150. The reaction is
carried out substantially in the manner described above in
connection with FIGS. 1 and 2. The process of FIG. 1 is
particularly preferred.
[0336] Growth media useful in the bioconversions preferably contain
between about 0.05% and about 5% by weight available nitrogen;
between about 0.5% and about 5% by weight glucose; between about
0.25% and about 2.5% by weight of a yeast derivative; and between
about 0.05% and about 0.5% by weight available phosphorus.
Particularly preferred growth media include the following:
[0337] soybean meal: between about 0.5% and about 3% by weight
glucose; between about 0.1% and about 1% by weight soybean meal;
between about 0.05% and about 0.5% by weight alkali metal halide;
between about 0.05% and about 0.5% by weight of a yeast derivative
such as autolyzed yeast or yeast extract; between about 0.05% and
about 0.5% by weight of a phosphate salt such as K.sub.2HPO.sub.4;
pH=7;
peptone-yeast extract-glucose: between about 0.2% and about 2% by
weight peptone; between about 0.05% and about 0.5% by weight yeast
extract; and between about 2% and about 5% by weight glucose;
Mueller-Hinton: between about 10% and about 40% by weight beef
infusion; between about 0.35% and about 8.75% by weight casamino
acids; between about 0.15% and about 0.7% by weight starch.
[0338] Fungi can be grown in soybean meal or peptone nutrients,
while actinomycetes and eubacteria can be grown in soybean meal
(plus 0.5% to 1% by weight carboxylic acid salt such as sodium
formate for is biotransformations) or in Mueller-Hinton broth.
[0339] The production of 11.beta.-hydroxymexrenone from mexrenone
by fermentation is discussed in Example 19B. Similar bioconversion
processes can be used to prepare other starting materials and
intermediates. Example 19A discloses the bioconversion of
androstendione to 11.beta.-hydroxyandrostendione. Example 19C
discloses the bioconversion of mexrenone to
11.alpha.-hydroxymexrenone, .DELTA..sup.1,2-mexrenone,
6.beta.-hydroxymexrenone, 12.beta.-hydroxymexrenone, and
9.alpha.-hydroxymexrenone. Example 19D discloses the bioconversion
of canrenone to .DELTA..sup.9,11-canrenone.
[0340] The products of Formula IX are novel compounds, which may be
separated by filtration, washed with a suitable organic solvent,
e.g., ethyl acetate, and recrystallized from the same or a similar
solvent. They have substantial value as intermediates for the
preparation of compounds of Formula I, and especially of Formula
IA. Preferably, the compounds of Formula IX correspond to Formula
IX in which -A-A- and --B--B-- are --CH.sub.2--CH.sub.2--, R.sup.3
is hydrogen, lower alkyl or lower alkoxy, and R.sup.13 and R.sup.9
together constitute the 20-spiroxane ring: ##STR133##
[0341] In the next step of the Scheme 2 synthesis, the product of
Formula IX is reacted with a dehydration reagent (suitable
dehydration reagents such as PhSOCl or ClSO.sub.3H are known to
persons skilled in the art) to produce a compound of Formula II
##STR134## wherein -A-A-, --B--B--, R.sup.3, R.sup.8 and R.sup.9
are as defined in Formula XIII, and R.sup.1 is as defined in
Formula V. Preferably, the substrate of Formula IX corresponds to
Formula IXA ##STR135## and the intermediate product corresponds to
Formula IIA ##STR136## in each of which -A-A-, --B--B--, R.sup.3,
Y.sup.1, Y.sup.2, and X are as defined in Formula XIIIA, and
R.sup.1 is as defined in Formula V. Preferably, R.sup.3 is
hydrogen.
[0342] In the final step of this synthesis scheme, the product of
Formula II is converted to that of Formula I by epoxidation in
accordance with the method described in U.S. Pat. No. 4,559,332; or
preferably by the novel epoxidation method of the invention as
described hereinabove.
[0343] In a particularly preferred embodiment, the overall process
of Scheme 2 proceeds as follows: ##STR137##
[0344] The synthesis in this case begins with a substrate
corresponding to Formula XX ##STR138## where -A-A- and R.sup.3 are
as defined in Formula XIII, --B--B-- is as defined in Formula XIII
except that neither R.sup.6 nor R.sup.7 is part of a ring fused to
the D ring at the 16,17 positions, and R.sup.26 is lower alkyl,
preferably methyl. Preferably, R.sup.3 is hydrogen. Reaction of the
substrate of Formula XX with a sulfonium ylide produces the epoxide
intermediate corresponding to Formula XIX ##STR139## wherein -A-A-,
--B--B--, R.sup.3 and R.sup.26 are as defined in Formula XX.
Preferably, R.sup.3 is hydrogen.
[0345] In the next step of synthesis scheme 3, the intermediate of
Formula XIX is converted to a further intermediate of Formula XVIII
##STR140## wherein -A-A-, --B--B-- and R.sup.3 are as defined in
Formula XX. Preferably, R.sup.3 is hydrogen. In this step, Formula
XIX is substrate is converted to Formula XVIII intermediate by
reaction with NaCH(COOEt).sub.2 in the presence of a base in a
solvent.
[0346] Exposure of the compound of Formula XVIII to heat, water and
an alkali halide produces a decarboxylated intermediate compound
corresponding to Formula XVII ##STR141## wherein -A-A-, --B--B--
and R.sup.3 are as defined in Formula XX. Preferably, R.sup.3 is
hydrogen. The process for conversion of the compound of Formula XX
to the compound of Formula XVII corresponds essentially to that
described in U.S. Pat. Nos. 3,897,417, 3,413,288 and 3,300,489,
which are expressly incorporated herein by reference. While the
substrates differ, the reagents, mechanisms and conditions for
introduction of the 17-spirolactone moiety are essentially the
same.
[0347] Reaction of the intermediate of Formula XVII with a
dehydrogenation reagent yields the further intermediate of Formula
XVI. ##STR142## where -A-A-, --B--B-- and R.sup.3 are as defined in
Formula XX. Preferably, R.sup.3 is hydrogen.
[0348] Typically useful dehydrogenation reagents include
dichlorodicyanobenzoquinone (DDQ) and chloranil
(2,3,5,6-tetrachloro-p-benzoquinone). Alternatively, the
dehydrogenation could be achieved by a sequential halogenation at
the 6-position carbon followed by dehydrohalogenation reaction.
[0349] The intermediate of Formula XVI is next converted to the
enamine of Formula XVB ##STR143## wherein -A-A-, --B--B-- and
R.sup.3 are as defined in Formula XX. Preferably, R.sup.3 is
hydrogen. Conversion is by cyanidation essentially in the manner
described above for the conversion of the 11.alpha.-hydroxy
compound of Formula VIII to the enamine of Formula VII. Typically,
the cyanide ion source may be an alkali metal cyanide. The base is
preferably pyrrolidine and/or tetramethylguanidine. A methanol
solvent may be used.
[0350] The products of Formula XVB are novel compounds, which may
be isolated by chromatography. These and other novel compounds of
Formula XV have substantial value as intermediates for the
preparation of compounds of Formula I, and especially of Formula
IA. Compounds of Formula XV correspond to the structure ##STR144##
where -A-A-, --B--B--, R.sup.3, R.sup.8 and R.sup.9 are as defined
in Formula XIII. In the most preferred compounds of Formula XV and
Formula XVB, -A-A- and --B--B-- are --CH.sub.2--CH.sub.2--, and
R.sup.2 is hydrogen.
[0351] In accordance with the hydrolysis described above for
producing the diketone compounds of Formula VI, the enamines of
Formula XVB may be converted to the diketones of Formula XIVB
##STR145## wherein -A-A-, --B--B-- and R.sup.3 are as defined in
Formula XX. Preferably, R.sup.3 is hydrogen. Particularly preferred
for the synthesis of epoxymexrenone are those compounds of Formula
XIV which also fall within the scope of Formula XIVB as defined
below.
[0352] The products of Formula XIVB are novel compounds, which may
be isolated by precipitation. These and other novel compounds of
Formula XIV have substantial value as intermediates for the
preparation of compounds of Formula I, and especially of Formula
IA. Compounds of Formula XIV correspond to the structure ##STR146##
where -A-A-, --B--B--, R.sup.3, R.sup.8 and R.sup.9 are as defined
in Formula XIII. In the most preferred compounds of Formula XIV and
XIVB, -A-A- and --B--B-- are --CH.sub.2--CH.sub.2--, and R.sup.3 is
hydrogen.
[0353] The compounds of Formula XIVB are further converted to
compounds of Formula XXXI using essentially the process described
above for converting the diketone of Formula VI to the hydroxyester
of Formula V. In this instance, it is necessary to isolate the
intermediate ##STR147## before further conversion to a product of
Formula XXXII ##STR148## wherein -A-A-, --B--B-- and R.sup.3 are as
defined in Formula XX, and R.sup.1 is as defined in Formula V.
Preferably, R.sup.3 is hydrogen. Preferred compounds of Formula
XXXI are those which fall within Formula IIA. The compounds of
Formula XXXI are converted to compounds of Formula XXXII using the
method described hereinabove or in U.S. Pat. No. 4,559,332.
[0354] Preferably, the compound of Formula XIV is
4'S(4'.alpha.),7'.alpha.-1',2',3',4,4',5,5',6',7',8',10',12',13',14',15',-
16'-hexadecahydro-1.beta.-,13'.beta.-dimethyl-3',5,20'-trioxospiro[furan-2-
(3H),17'.beta.-[4,7]methano[17H]-cyclopenta[a]phenanthrene]5'-carbonitrile-
; and the compound of Formula XV is
5'R(5'.alpha.),7'.beta.-20'-amino-1',2',3',4,5,6',7',8',10',12',13',14',1-
5',16'-tetradecahydro-10'.alpha.,13'.alpha.-dimethyl-3',5-dioxospiro[furan-
-2(3H),17'.alpha.(5'H)-[7,4]metheno[4H]-cyclopenta[a]phenanthrene]-5'-carb-
onitrile. In a particularly preferred embodiment, the overall
process of Scheme 3 proceeds as follows: ##STR149##
[0355] The first three steps of Scheme 4 are the same as those of
Scheme 3, i.e., preparation of an intermediate of Formula XVII
starting with a compound corresponding to Formula XX.
[0356] Thereafter, the intermediate of Formula XVII is epoxidized,
for example, using the process of U.S. Pat. No. 4,559,332 to
produce the compound of Formula XXIV ##STR150## wherein -A-A-,
--B--B-- and R.sup.3 are as defined in Formula XX. However, in a
particularly preferred embodiment of the invention, the substrate
of Formula XVII is epoxidized across the 9,11-double bond using an
oxidation reagent comprising an amide type peroxide activator, most
preferably trichloroacetamide, according to the process as
described above in Scheme 1 for the conversion of the enester of
Formula II to the product of Formula I. The conditions and
proportions of reagents for this reaction are substantially as
described for the conversion of the Formula II enester to
epoxymexrenone. Particularly preferred compounds of Formula XXIV
are those in which -A-A- and --B--B-- are as defined in Formula
XIII and R.sup.3 is hydrogen.
[0357] It has been found that the epoxidation of the substrate of
Formula XVII can also be effected in very good yield using a
peracid such as, for example, m-chloroperoxybenzoic acid. However,
the trichloroacetamide reagent provides superior results in
minimizing the formation of Bayer-Villager oxidation by-product.
The latter by-product can be removed, but this requires trituration
from a solvent such as ethyl acetate, followed by crystallization
from another solvent such as methylene chloride. The epoxy compound
of Formula XXIV is dehydrogenated to produce a double bond between
the 6- and 7-carbons by reaction with a dehydrogenation (oxidizing)
agent such as DDQ or chloranil, or using the
bromination/dehydrobromination (or other
halogenation/dehydrohalogenation) sequence, to produce another
novel intermediate of Formula XXIII ##STR151## wherein -A-A-,
--B--B--, R.sup.3, R.sup.8 and R.sup.9 are as defined in Formula
XX.
[0358] Particularly preferred compounds of Formula XXIII are those
in which -A-A- and --B--B-- are as defined in Formula XIII and
R.sup.3 is hydrogen.
[0359] While direct oxidation is effective for the formation of the
product of Formula XXIII, the yields are generally low. Preferably,
therefore, the oxidation is carried out in two steps, first
halogenating the substrate of Formula XXIV at the C-6 position,
then dehydrohalogenating to the 6,7-olefin. Halogenation is is
preferably effected with an N-halo organic reagent such as, for
example, N-bromosuccinamide. Bromination is carried out in a
suitable solvent such as, for example, acetonitrile, in the
presence of halogenation promoter such as benzoyl peroxide. The
reaction proceeds effectively at a temperature in the range of
about 50.degree. to about 100.degree. C., conveniently at
atmospheric reflux temperature in a solvent such as carbon
tetrachloride, acetonitrile or mixture thereof. However, reaction
from 4 to 10 hours is typically required for completion of the
reaction. The reaction solvent is stripped off, and the residue
taken up in a water-immiscible solvent, e.g., ethyl acetate. The
resulting solution is washed sequentially with a mild alkaline
solution (such as an alkali metal bicarbonate) and water, or
preferably saturated brine to minimize product losses, after which
the solvent is stripped and a the residue taken up in another
solvent (such as dimethylformamide) that is suitable for the
dehydrohalogenation reaction.
[0360] A suitable dehydrohalogenation reagent, e.g.,
1,4-diazabicyclo[2,2,2]octane (DABCO) is added to the solution,
along with an alkali metal halide such as LiBr, the solution heated
to a suitable reaction temperature, e.g., 60.degree. to 80.degree.
C., and reaction continued for several hours, typically 4 to 15
hours, to complete the dehydrobromination. Additional
dehydrobromination reagent may be added as necessary during the
reaction cycle, to drive the reaction to completion. The product of
Formula XXIII may then be recovered, e.g., by adding water to
precipitate the product which is then separated by filtration and
preferably washed with additional amounts of water. The product is
preferably recrystallized, for example from dimethylformamide.
[0361] The products of Formula XXIII, such as 9,11-epoxycanrenone,
are novel compounds, which may be isolated by
extraction/crystallization. They have substantial value as
intermediates for the preparation of compounds of Formula I, and
especially of Formula IA. For example, they may be used as
substrates for the preparation of compounds of Formula XXII.
[0362] Using substantially the process described above for the
preparation of compounds of Formula VII, the compounds of Formula
XXIII are reacted with cyanide ion to produce novel epoxyenamine
compounds corresponding to Formula XXII ##STR152## wherein -A-A-,
--B--B--, R.sup.3, R.sup.8 and R.sup.9 are as defined in Formula
XX. Particularly preferred compounds of Formula XXII are those in
which -A-A- and --B--B-- are as defined in Formula XIII and R.sup.3
is hydrogen.
[0363] The products of Formula XXII are novel compounds, which may
be isolated by precipitation and filtration. They have substantial
value as intermediates for the preparation of compounds of Formula
I, and especially of Formula IA. In the most preferred compounds of
Formula XXII, -A-A- and --B--B-- are --CH.sub.2--CH.sub.2--, and
R.sup.3 is hydrogen.
[0364] Using substantially the process described above for
preparation of compounds of Formula VI, the epoxyenamine compounds
of Formula XXII are converted to novel epoxydiketone compounds of
Formula XXI ##STR153## wherein -A-A-, --B--B--, R.sup.3, R.sup.8
and R.sup.9 are as defined in Formula XIII. In the most preferred
compounds of Formula XXI, -A-A- and --B--B-- are
--CH.sub.2--CH.sub.2-- and R.sup.3 is hydrogen.
[0365] The products of Formula XXI are novel compounds, which may
be isolated by precipitation and filtration. They have substantial
value as intermediates for the preparation of compounds of Formula
I, and especially of Formula IA. Particularly preferred compounds
of Formula XXI are those in which -A-A- and --B--B-- are as defined
in Formula XIII. In the most preferred compounds of Formula XXI,
-A-A- and --B--B-- are --CH.sub.2--CH.sub.2-- and R.sub.3 is
hydrogen.
[0366] Using substantially the process described above for
preparation of the hydroxyester compounds of Formula V from the
diketone compounds of Formula VI, the epoxydiketone compounds of
Formula XXI are converted to compounds of Formula XXXII ##STR154##
wherein -A-A-, --B--B-- and R.sup.3 are as defined in Formula XX,
and R.sup.1 is as defined in Formula V.
[0367] As in the conversion of the diketone of formula V to the
hydroxyester of formula VI, a 5-.beta.-cyano-7-ester intermediate
is also formed in the conversion of the epoxydiketone of formula
XXI to compounds of formula XXXII. The 5-.beta.-cyano-7-ester
intermediates in both series can be isolated by treatment of the
corresponding diketone with an alcohol such as methanol in the
presence of a base such as triethylamine. Preferably, the
intermediates are prepared by refluxing a mixture of the diketone
in an alcohol such as methanol containing about 0.1 to about 2
equivalents of triethylamine per mole of diketone for about 4 to
about 16 hours. The products are isolated in pure form by cooling
the mixture to about 25 degrees followed by filtration. The
isolated intermediates can be converted to the compounds of Formula
XXXII on treatment with a base such an alkali metal alkoxide in a
solvent, preferably an alcohol such as methanol. Use of an alkoxide
in an alcohol establishes an equilibrium mixture similar to that
formed when the corresponding diketone of Formula XXI is treated
under the same conditions.
[0368] In addition, the 7.beta.-ester of the compound of Formula
XXXII (for example 7-methyl hydrogen
9,11.alpha.-epoxy-17-hydroxy-3-oxo-17.alpha.-pregn-4-ene-7.beta.,21-dicar-
boxylate, .gamma.-lactone) has been observed by chromatography in
the crude product of the final step of the process of Scheme 4.
Alkoxide and/or cyanide in the solution reacts with and converts
the 7.alpha.-ester into an epimeric-mixture of the 7.alpha.-ester
and its 7.beta.-ester epimer. The pure 7.beta.-ester can be
isolated from the epimeric mixture by selective
crystallization.
[0369] Preferably, the compound of Formula XXI is
4'S(4'.alpha.),7'.alpha.-9',11.alpha.-epoxyhexadecahydro-10.beta.-,13'.be-
ta.-dimethyl-3'5,20'-trioxospiro[furan-2(3H),17'.beta.-[4,7]methano[17H]-c-
yclopenta[a]phenanthrene-5'-carbonitrile; the compound of Formula
XXII is
5'R(5'.alpha.),7'.beta.-20'-amino-9,11.beta.-epoxyhexadecahydro-10',13'-d-
imethyl-3',5-dioxospiro[furan-2(3H),17'a(5'H)-[7,4]methene[4H]cyclopenta[a-
]phenanthrene-5'-carbonitrile; and the compound of Formula XXIII is
9,11.alpha.-epoxy-17.alpha.-hydroxy-3-oxopregna-4,6-diene-21-carboxylic
acid, .gamma.-lactone.
[0370] In a particularly preferred embodiment, the overall process
of Scheme 4 proceeds as follows: ##STR155##
[0371] The process of scheme 5 begins with a substrate
corresponding to Formula XXIX ##STR156## wherein -A-A-, --B--B--
and R.sup.3 are as defined in Formula XX.
[0372] The following microorganisms are capable of carrying out the
9.alpha.-hydroxylation of a compound of Formula XXXV (such as
androstendione) ##STR157## wherein -A-A-, --B--B-- and R.sup.3 are
as defined in Formula XIII, to a compound of Formula XXIX under
conditions similar to those described in Example 19B:
[0373] Asperigillus niger ATCC 16888 and 26693, Corynespora
cassiicola ATCC 16718, Curvularia clavata ATCC 22921, Mycobacterium
fortuitum NRRL B8119, Nocardia canicruria ATCC 31548, Pycnosporium
spp. ATCC 12231, Stysanus microsporus ATCC 2833, Syncenhalastrum
racemosum ATCC 18192, and Thamnostylum piriforme ATCC 8992.
[0374] The substrate corresponding to Formula XXIX is converted to
a product of Formula XXVIII ##STR158## by reaction with
trimethylorthoformate, wherein -A-A-, --B--B-- and R.sup.3 are as
defined in Formula XX. Following the formation of the compounds of
Formula XXVIII, those compounds are converted to the compounds of
Formula XXVII using the method described above for conversion of
the substrate of Formula XX to Formula XVII. Compounds of Formula
XXVII have the structure: ##STR159## wherein -A-A-, --B--B-- and
R.sup.3 are as defined in Formula XX, and R.sup.x is any of the
common hydroxy protecting groups. Alternatively, the C9
.alpha.-hydroxy group can be protected at an earlier step in this
synthesis scheme if protection at that step is desired, i.e., the
C9 hydroxy of the compound of Formula XXVIII or the C9 hydroxy of
the compound of Formula XXIX can be protected with any of the
common hydroxy protecting groups.
[0375] Using the method described above for the preparation of
compounds of Formula XVI, compounds of Formula XXVII are oxidized
to yield novel compounds corresponding to Formula XXVI ##STR160##
wherein -A-A-, --B--B-- and R.sup.3 are as defined in Formula XX.
Particularly preferred compounds of Formulae XXIX, XXVIII, XXVII
and XXVI are those in which -A-A- and --B--B-- are as defined in
Formula XIII, and R.sup.3 is hydrogen.
[0376] The products of Formula XXVI are novel compounds, which may
be isolated by precipitation/filtration. They have substantial
value as intermediates for the preparation of compounds of Formula
I, and especially of Formula IA. Particularly preferred compounds
of Formula XXVI are those in which -A-A- and --B--B-- are as
defined in Formula XIII, and R.sup.3 is hydrogen. In the most
preferred compounds of Formula XXVI, and -A-A- and --B--B-- are
--CH, --CH.sub.2--, and R.sup.3 is hydrogen.
[0377] Using the method defined above for cyanidation of compounds
of Formula VIII, the novel intermediates of Formula XXVI are
converted to the novel 9-hydroxyenamine intermediates of Formula
XXV ##STR161## wherein -A-A-, --B--B-- and R.sup.3 are as defined
in Formula XX.
[0378] The products of Formula XXV are novel compounds, which may
be isolated by precipitation/filtration. They have substantial
value as intermediates for the preparation of compounds of Formula
I, and especially of Formula IA. Particularly preferred compounds
of Formula XXV are those in which -A-A- and --B--B-- are as defined
in Formula XIII, and R.sup.3 is hydrogen. In the most preferred
compounds of Formula XXVI, and -A-A- and --B--B-- are
--CH.sub.2--CH.sub.2--, and R.sup.3 is hydrogen.
[0379] Using essentially the conditions described above for the
preparation of the diketone compounds of Formula VI, the
9-hydroxyenamine intermediates of Formula XXV are converted to the
diketone compounds of Formula XIVB. Note that in this instance the
reaction is effective for simultaneous hydrolysis of the enamine
structure and dehydration at the 9,11 positions to introduce the
9,11 double bond. The compound of Formula XIV is then converted to
the compound of Formula XXXI, and thence to the compound of Formula
XIII, using the same steps that are described above in scheme
3.
[0380] Preferably, the compound of Formula XIV is
4'S(4'.alpha.),7'.alpha.-1',2',3',4,4',5,5',6',7',8',10',12',13',14',15',-
16'-hexadecahydro-10.beta.-,13'.beta.-dimethyl-3',5,20'-trioxospiro[furan--
2(3H),17'.beta.-[4,7]methano[17H]-cyclopenta[a]phenanthrene]5'-carbonitril-
e; the compound of Formula XXV is
5'R(5'.alpha.),7'.beta.-20'-aminohexadecahydro-9'.beta.-hydroxy-10'a,13'.-
alpha.-dimethyl-3',5-dioxospiro[furan-2(3H),17'.alpha.(5'H)-[7,4]metheno[4-
H]cyclopenta[a]phenanthrene]-5'-carbonitrile; the compound of
Formula XXVI is
9.alpha.,17.alpha.-dihydroxy-3-oxopregna-4,6-diene-21-carboxylic
acid, .gamma.-lactone; and the compound of Formula XXVII is
9.alpha.,17.alpha.-dihydroxy-3-oxopregn-4-ene-21-carboxylic acid,
.gamma.-lactone.
[0381] In a particularly preferred embodiment, the overall process
of Scheme 5 proceeds as follows: ##STR162##
[0382] Scheme 6 provides an advantageous method for the preparation
of epoxymexrenone and other compounds corresponding to Formula I,
starting with 11.alpha. or 11.beta.-hydroxylation of androstendione
or other compound of Formula XXXV ##STR163## wherein -A-A-,
--B--B-- and R.sup.3 are as defined in Formula XIII, producing an
intermediate corresponding to the Formula XXXVI or its
corresponding 11.beta.-hydroxy isomer ##STR164## where -A-A-,
--B--B-- and R.sup.3 are as defined in Formula XIII. Except for the
selection of substrate, the process for conducting the
11.alpha.-hydroxylation is essentially as described hereinabove for
Scheme 1. The following microorganisms are capable of carrying out
the 11.alpha.-hydroxylation of androstendione or other compound of
Formula XXXV:
[0383] Absidia glauca ATCC 22752, Aspergillus flavipes ATCC 1030,
Aspergillus foetidus ATCC 10254, Aspergillus fumigatus ATCC 26934,
Aspergillus ochraceus NRRL 405 (ATCC 18500), Aspergillus niger ATCC
11394, Aspergillus nidulans ATCC 11267, Beauveria bassiana ATCC
7159, Fusarium oxysporum ATCC 7601, Fusarium oxysporum cepae ATCC
11171, Fusarium lini ATCC IFO 7156, Gibberella fujikori ATCC 14842,
Hypomyces chyrsospermus IMI 109891, Mycobaterium fortuitum NRRL
B8119, Penicillum patulum ATCC 24550, Pycnosporium spp. ATCC 12231,
Rhizopus arrhizus ATCC 11145, Saccharopolyspora erythraea ATCC
11635, Thamnostylum piriforme ATCC 8992, Rhizopus oryzae ATCC
11145, Rhizopus stolonifer ATCC 6227b, and Trichothecium roseum
ATCC 12519 and ATCC 8685.
[0384] The following microorganisms are capable of carrying out the
11.beta.-hydroxylation of androstendione or other compound of
Formula XXXV:
[0385] Aspergillus fumigatus ATCC 26934, Aspergillus niger ATCC
16888 and ATCC 26693, Epicoccum oryzae ATCC 7156, Curvularia lunata
ATCC 12017, Cunninghamella blakesleeana ATCC 8688a, and Pithomyces
atro-olivaceous IFO 6651.
[0386] 11.alpha.-Hydroxyandrost-4-ene-3,17-dione, or other compound
of Formula XXXVI, is next converted to 11.alpha.-hydroxy-3,4-enol
ether of Formula (101): ##STR165## where -A-A-, --B--B-- and
R.sup.3 are as defined in Formula XIII and R.sup.11 is methyl or
other lower alkyl (C.sub.1 to C.sub.4), by reaction with an
etherifying reagent such as trialkyl orthoformate in the presence
of an acid catalyst. To carry out this conversion, the
11.alpha.-hydroxy substrate is acidified by mixing with an acid
such as, e.g., benzene sulfonic acid hydrate or toluene sulfonic
acid hydrate and dissolved in a lower alcohol solvent, preferably
ethanol. A trialkyl orthoformate, preferably triethyl orthoformate
is introduced gradually over a period of 5 to 40 minutes while
maintaining the mixture in the cold, preferably at about 0.degree.
C. to about 15.degree. C. The mixture is then warmed and the
reaction carried out at a temperature of between 20.degree. C. and
about 60.degree. C. Preferably the reaction is carried out at
30.degree. to 50.degree. C. for 1 to 3 hours, then heated to reflux
for an additional period, typically 2 to 6 hours, to complete the
reaction. Reaction mixture is cooled, preferably to 0.degree. to
15.degree., preferably about 5.degree. C., and the solvent removed
under vacuum.
[0387] Using the same reaction scheme as described in Scheme 3,
above, for the conversion of the compound of Formula XX to the
compound of Formula XVII, a 17-spirolactone moiety of Formula
XXXIII is introduced into the compound of Formula 101. For example,
the Formula 101 substrate may be reacted with a sulfonium ylide in
the presence of a base such as an alkali metal hydroxide in a
suitable solvent such as DMSO, to produce an intermediate
corresponding to Formula 102: ##STR166## where -A-A-, R.sup.3,
R.sup.11, and --B--B-- are as defined in Formula 101. The
intermediate of Formula 102 is then reacted with a malonic acid
diester in the presence of an alkali metal alkoxide to form the
five membered spirolactone ring and produce the intermediate of
Formula 103 ##STR167## where -A-A-, R.sup.3, R.sup.11 and --B--B--
are as defined in Formula 102, and R.sup.12 is a C1 to C.sub.4
alkyl, preferably ethyl. Finally, the compound of Formula 103 in a
suitable solvent, such as dimethylformamide, is subjected to heat
in the presence of an alkali metal halide, splitting off the
alkoxycarbonyl moiety and producing the intermediate of Formula
104: ##STR168## where again -A-A-, R.sup.3, R.sup.11 and --B--B--
are as defined in Formula 102.
[0388] Next the 3,4-enol ether compound 104 is converted to the
compound of Formula XXIII, i.e., the compound of Formula VIII in
which R.sup.8 and R.sup.9 together form the moiety of Formula
XXXIII. This oxidation step is carried out in essentially the same
manner as the oxidation step for conversion of the compound of
Formula XXIV to the intermediate of Formula XXIII in the synthesis
of Scheme 4. Direct oxidation can be effected using a reagent such
as 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) or
tetrachlorobenzoquinone (chloranil), or preferably a two stage
oxidation is effected by first brominating, e.g., with an N-halo
brominating agent such as N-bromosuccinamide (NBS) or
1,3-dibromo-5,5-dimethyl hydantoin (DBDMH) and then
dehydrobrominating with a base, for example with DABCO in the
presence of LiBr and heat. Where NBS is used for bromination, an
acid must also be employed to convert 3-enol ether to the enone.
DBDMH, an ionic rather than free radical bromination reagent, is
effective by itself for bromination and conversion of the enol
ether to the enone.
[0389] The compound of Formula VIII is then converted to
epoxymexrenone or other compound of Formula I by the steps
described hereinabove for Scheme 1.
[0390] Each of the intermediates of Formulae 101, 102, 103 and 104
is a novel compound having substantial value as an intermediate for
epoxymexrenone or other compounds of Formulae IA and I. In each of
the compounds of Formulae 101, 102, 103 and 104, -A-A- and --B--B--
are preferably --CH.sub.2--CH.sub.2-- and R.sup.3 is hydrogen,
lower alkyl or lower alkoxy. Preferably, R.sup.3 is hydrogen. Most
preferably, the compound of Formula 101 is
3-ethoxy-11.alpha.-hydroxyandrost-3,5-dien-17-one, the compound of
Formula 102 is
3-ethoxyspiro[androst-3,5-diene-17.beta.,2'-oxiran]-11.alpha.-ol,
the compound of Formula 103 is ethyl hydrogen
3-ethoxy-11.alpha.-17.alpha.-dihydroxypregna-3,5-diene-21,21-dicarboxylat-
e, gamma-lactone, and the compound of Formula 104 is
3-ethoxy-11.alpha.-17.alpha.-dihydroxypregna-3,5-diene-21-carboxylic
acid, gamma-lactone.
[0391] In a particularly preferred embodiment, the overall process
of Scheme 6 proceeds as follows: ##STR169##
[0392] It is hypothesized that epoxymexrenone and other compounds
corresponding to Formula I likewise can be prepared from
11.beta.-hydroxyandrostendione or other compounds of Formula XXXV
which have been 11.beta.-hydroxylated. In other words,
epoxymexrenone and other compounds corresponding to Formula I can
be prepared in accordance with the general process set forth in
Scheme 6 using either an .alpha.-hydroxylated substrate of Formula
XXXV or the corresponding .beta.-hydroxylated substrate.
Scheme 7
[0393] Scheme 7 provides for the synthesis of epoxymexrenone and
other compounds of Formula I using a starting substrate comprising
.beta.-sitosterol, cholesterol, stigmasterol or other compound of
Formula XXXVII ##STR170## where -A-A-, R.sup.3, and --B--B-- are as
defined in Formula XIII; D-D is --CH.sub.2--CH.sub.2-- or
--CH.dbd.CH--; and each of R.sup.13, R.sup.14, R.sup.15 and
R.sup.16 is independently selected from among hydrogen or C.sub.1
to C.sub.4 alkyl. R.sup.3 is preferably hydrogen.
[0394] In the first step of the synthesis
11.alpha.-hydroxyandrostendione or other compound of Formula XXXVI
is prepared by bioconversion of the compound of Formula XXXVII. The
bioconversion process is carried out substantially in accordance
with the method described hereinabove for the
11.alpha.-hydroxylation of canrenone (or other substrate of Formula
XIII).
[0395] In the synthesis 11.alpha.-hydroxyandrostendione,
4-androstene-3,17-dione is initially prepared by bioconversion of
the compound of Formula XXXVII. This initial bioconversion may be
carried out in the manner described in U.S. Pat. No. 3,759,791,
which is expressly incorporated herein by reference. Thereafter,
4-androstene-3,17-dione is converted to
11.alpha.-hydroxyandrostenedione substantially in accordance with
the method described hereinabove for the 11.alpha.-hydroxylation of
canrenone (or other substrate of Formula XIII).
[0396] The remainder of the synthesis of Scheme 7 is identical to
Scheme 6. In a particularly preferred embodiment, the overall
process of Scheme 7 proceeds as follows: ##STR171##
[0397] It is hypothesized that epoxymexrenone and other compounds
corresponding to Formula I likewise can be prepared in accordance
with the general process set forth in Scheme 7 when the product of
the bioconversion of .beta.-sitosterol or other compounds of
Formula XXXVIII is 11.beta.-hydroxyandrostendione or other
compounds of Formula XXXV which have been 11.beta.-hydroxylated. In
other words, epoxymexrenone and other compounds corresponding to
Formula I can be prepared in accordance with the general process
set forth in Scheme 7 when the bioconversion of .beta.-sitosterol
or other compounds of Formula XXXVIII results in the preparation of
either an .alpha.-hydroxylated substrate of Formula XXXV or the
corresponding .beta.-hydroxylated substrate.
Scheme 8
[0398] A significant complication in the synthesis of
epoxymexrenone and related compounds is the need for
stereoselective introduction of an .alpha.-alkoxycarbonyl
substituent at the 7-carbon, without unwanted modifications at
other sites of the steroidal structure. In accordance with the
invention, it has been discovered that an effective synthesis path
for introduction of a 7.alpha.-alkoxycarbonyl substituent involves
the following steps: (i) initial cyanidation at the 7-carbon of the
steroid, (ii) hydrolysis of the 7-cyano steroid to form a mixture
of 7.alpha.-carboxylic acid and 7.beta.-carboxylic acid steroids,
(iii) formation of a 5,7-lactone steroid from the
7.alpha.-carboxylic acid steroid, and (iv) separation of the
7.beta.-carboxylic acid steroid from the 5,7-lactone steroid. A
base-mediated opening reaction of the 5,7-lactone steroid with an
alkylating reagent produces the desired 7.alpha.-alkoxycarbonyl
steroid.
[0399] Accordingly, the process of Scheme 8 is generally directed
to a process for the preparation of a
3-keto-7.alpha.-alkoxycarbonyl substituted .DELTA..sup.4,5-steroid
comprising reacting an alkylating reagent with a
3-keto-4,5-dihydro-5,7-lactone steroid substrate in the presence of
a base. The lactone substrate is substituted with keto at the
3-carbon, and further comprises the moiety: ##STR172## where C(5)
represents the 5-carbon and C(7) represents the 7-carbon of the
steroid structure of the substrate. Conversion of the 5,7-lactone
to the 7.alpha.-alkoxycarbonyl is preferably effected by reaction
with an alkyl halide in the presence of the base. The alkyl halide
reagent is preferably an iodide, most preferably methyl iodide.
[0400] Further in accordance with the invention, an advantageous
process has been discovered for the preparation of the
4,5-dihydro-5,7-lactone steroid compound described above. In this
process, a 3-keto-.DELTA..sup.4,5-7.alpha.-cyano substituted
steroid substrate is converted to the 7-carboxylic acid, and the
acid in turn reacts with the trialkyl orthoformate in an acidified
lower alcohol solvent to yield the 5,7-lactone. Reaction with
orthoformate esters also converts the 3-keto group to the 3-acyclic
or cyclic ketal 5,7-lactone (the lactone is understood to form
first). Preferably, the 3-ketal 5,7-lactone is a 3-dialkyl ketal
5,7-lactone. More preferably, the alkyl moiety of the alcohol
solvent is the same as the alkyl moiety of the orthoformate alkoxy
groups (and most preferably all are methyl) because: the alkoxy
moieties of the ketal can derive either from the orthoformate or
the alcohol; mixed ketals are not preferred; and 3-dimethoxy is
preferred. Where the ketal is an ethylene ketal, the alkyl moiety
of the alcohol solvent need not be the same as the alkyl moiety of
the orthoformate alkoxy groups. The 3-ketal-5,7-lactone is readily
hydrolyzed to the 3-keto-5,7-lactone, a crystalline compound which
can be easily purified. Since only the 7.alpha.-carboxylic acid
undergoes the lactonization reaction, complete stereospecificity is
realized. The 75-acid may then be removed from the reaction mixture
in the form of its salt, e.g., by treating the 70-acid with a mild
base such as sodium bicarbonate.
[0401] The 7-cyano substrate for the formation of the 5,7-lactone
can be prepared in a known manner. For example, a substrate
unsubstituted at the 7-carbon may be reacted with a slight excess
of cyanide ion, preferably about 1.05 to about 1.25 equivalents per
equivalent substrate in a weakly acidic solution comprising a
water/DMSO solvent mixture. Preferably, the reaction mixture
includes a carboxylic acid, e.g., about one equivalent acetic acid
per equivalent substrate. Both the 7.alpha.- and 7.beta.-CN isomers
are formed with the 7.alpha.-isomer as the major isomer. The
7.alpha.-cyano steroid may be recovered in a conventional manner.
Other methods known to the art are useful in this ancillary
preparation.
[0402] Generally in accordance with Scheme 8, the 5,7-lactone may
be formed from a 7-carboxy intermediate (which itself is prepared
by hydrolyzing a 7-cyano intermediate) that is substituted at the
17-position with either keto, R.sup.8 or R.sup.9, where R.sup.8 and
R.sup.9 are as described above, and having either an aliphatic,
olefin, epoxide or hydroxy substituted configuration at C-9 and
C-11, i.e., ##STR173## where -A-A-, --B--B-- and R.sup.3 are as
defined above, R.sup.80 and R.sup.90 are the same as R.sup.8 and
R.sup.9, or R.sup.80 and R.sup.90 together constitute keto, and
R.sup.18 is as described below regarding Scheme 9, and -E-E- is
selected from among ##STR174## The compound of Formula XLII is then
converted to the 7.alpha.-alkoxy-carbonyl: ##STR175## In each of
XL, XLI, XLII and XLVIII, R.sup.80 and R.sup.90 together preferably
comprise keto or ##STR176## where Y.sup.1, Y.sup.2, X and, C(17)
are as defined above, and most preferably R.sup.80 and R.sup.90
together comprise ##STR177## R.sup.1 is preferably H, R.sup.1 is
preferably methoxycarbonyl, and -A-A- and --B--B-- are each
preferably --CH.sub.2--CH.sub.2--. It will be understood that the
reactions can also be carried out with the 3-keto group protected
by converting it to and maintaining it in each ether or ketal form
throughout the reaction sequence. Alternative processes of Scheme 8
comprise use of various intermediates within the scope of Formulae
XLI and XLII as recited hereinabove.
[0403] Noting that the reagent for formation of the 5,7-lactone
from the 3-keto-.DELTA..sup.4,5-7-carboxylic acid per Scheme 8 is
the trialkyl orthoformate, the same reagent used in conversion of
the 11.alpha.-hydroxyandrostendione to the 3-enol
ether-3,5-diene-11.alpha.-hydroxy intermediate 101 of Scheme 6, it
is believed that the path of the Scheme 8 reaction is dependent on
substitution at C-7. Reaction with orthoformate in the presence of
H.sup.+ forms an intermediate carbonium ion having a carboxyl at
C-7 and its positive charge in equilibrium between C-3 and C-5.
Upon loss of the proton, the C-3 carbonium ion yields the compound
of Formula 101, while the C-5 carbonium ion yields the lactone.
With hydrogen at C-7, it is believed that 3,5-dien-3-alkoxy (enol
ether) is favored because of the double bond conjugation. With the
7.alpha.-CO.sub.2 substituent at C-7, the C-5 carbonium ion is
captured by the carboxy and the 5,7-lactone is formed. At this
point the 3-keto group is preferentially converted to the ketal,
thereby driving the reaction to completion.
[0404] Preferred embodiments of Scheme 8 are described in Schemes 9
and 10, infra.
Scheme 9
[0405] Scheme 9 begins with the same substrate as Scheme 4, i.e.,
the compound of Formula XX. This substrate is first oxidized to the
compound of Formula B: ##STR178## where -A-A-, R.sup.3, and
--B--B-- are as defined in Formula XIII. The oxidation reaction is
conducted in accordance with any of the reaction schemes described
above for conversion of the compound of Formula XXIV to the
intermediate of Formula XXIII in the synthesis of Scheme 4. Using
the methods described for Scheme 8, the compound of Formula B is
converted to the 7-cyano intermediate of Formula C: ##STR179##
where -A-A-, R.sup.3, and --B--B-- are as defined in Formula XIII.
Next, the compound of Formula C is converted to the 5,7-lactone of
Formula D: ##STR180## where -A-A-, R.sup.3, and --B--B-- are as
defined in Formula XIII and R.sup.17 is C.sub.1-C.sub.4 alkyl,
using the trialkyl orthoformate reagent utilized previously in
Scheme 6. The 5,7-lactone of Formula D is readily separated from
the unreacted 7-.beta.-COOH, e.g., by removal of the acid via a
bicarbonate wash, thereby establishing the desired C-7
stereochemistry and impeding epimerization in subsequent reactions
that are conducted under basic conditions. Esterification of the
lactone per reaction with alkyl halide, as described in Scheme 8,
yields the enester intermediate of Formula II.
[0406] Continuing the Scheme 9 synthesis, the compound of Formula D
is converted to a compound of Formula II. With the 3-keto group
protected by having been converted to the ketal, a 20-spiroxane
group of Formula XXXIII is selectively introduced at the
17-position in accordance with the reaction scheme described above
for Schemes 3 and 6, supra, thereby producing a compound of Formula
E ##STR181##
[0407] Because the 3-ketone is protected, hydrolysis conditions may
be selected which are optimal for attacking the 17-ketone without
concern for the formation of by-products through reaction at the
3-position. After hydrolysis of the 3-ketal compound of Formula E
to the 3-keto group structure of Formula F ##STR182## the latter
intermediate is reacted with alkyl iodide in the presence of base,
per the conversion of Scheme 8, to produce the intermediate enester
of Formula II. Finally, the latter intermediate is converted to
epoxymexrenone or other compound of Formula I, using any of the
methods described above for Scheme 1.
[0408] Scheme 9 benefits not only from the control of
stereochemistry afforded by the 5,7-lactone intermediate, but
enjoys the further advantage of allowing for a wider is range of
hydrolysis conditions without interference of the
17-spirolactone.
[0409] Like the reactions for other synthesis schemes of this
invention, the reactions of Scheme 9 may be used for conversion of
substrates other than those particularly described above. Thus, for
example, the conversion of 3-keto- or 3-ketal-7-cyano steroids to
3-keto- or 3-ketal-5,7-lactone, or the conversion of the 3-keto- or
3-ketal-5,7-lactone to 7.alpha.-alkoxycarbonyl, may be carried out
on compounds substituted at the 17-carbon by R.sup.8 and R.sup.9 as
defined above, or more particularly by a substituent of Formula:
##STR183## where X, Y.sup.1 and Y.sup.2 are as defined above and
C(17) indicates the 17-carbon. However, important advantages are
realized, especially in process economics, by use of the specific
reaction sequence using 17-keto substrates and following the
specific reaction scheme described above for introduction of
17-spirolactone and 7.alpha.-alkoxycarbonyl into a
3-keto-.DELTA..sup.9,11 steroid.
[0410] The lactones of Formula D, E, and F are novel compounds
which are useful in the preparation of epoxymexrenone and other
compounds of Formula I and IA in accordance with the synthesis of
Scheme 9. In these compounds -A-A- and --B--B-- are preferably
--CH.sub.2--CH.sub.2-- and R.sup.3 is hydrogen, lower alkyl or
lower alkoxy. Most preferably the compound of Formula D is where
R.sup.17 is methoxy.
[0411] In a particularly preferred embodiment, the overall process
of Scheme 9 proceeds as follows: ##STR184##
[0412] Scheme 10 is the same as Scheme 9 through the formation of
the 7-cyano intermediate of Formula C. In the next step of Scheme
10, 7-cyano steroid is reacted with trialkyl orthoformate in an
alkanol solvent, preferably trimethyl orthoformate in methanol, to
simultaneously protect the 3-keto and 17-keto groups, by converting
the former to the enol ether and the latter to the ketal.
Thereafter the 7-cyano group is reduced to 7-formyl, e.g., by
reaction with a dialkyl aluminum hydride, preferably diisobutyl
aluminum hydride, thereby producing a compound of Formula 203:
##STR185## where -A-A-, R.sup.3, and --B--B-- are as defined in
Formula XIII, and R.sup.11 is C.sub.1-C.sub.4 alkyl. Prior
protection of the keto groups, as described above, prevents their
reduction by the dialkyl aluminum hydride. The intermediate of
Formula 203 is next reacted with dilute aqueous acid to selectively
hydrolyze the 17-ketal, in the presence of excess alcohol
(R.sup.19OH), producing the intermediate of Formula 204: ##STR186##
where R.sup.19 is selected from among lower alkyl (preferably
C.sub.1 to C.sub.4), or the R.sup.19 groups at the 3-position
forming a cyclic O,O-oxyalkyleneoxy substituent at the 3-carbon.
The hemiacetal (204) is further protected by treatment with alkanol
(R.sup.19OH) in the presence of non-aqueous acid to produce the
intermediate of Formula 205: ##STR187## where -A-A-, --B--B--,
R.sup.3 and R.sup.19 are as defined above, and R.sup.20 is C.sub.1
to C.sub.4 alkyl. The 17-spirolactone moiety can then be introduced
in accordance with the reaction steps described above for Schemes 3
and 6, thus proceeding through the sequence outlined below:
##STR188## wherein -A-A-, --B--B--, R.sup.3, R.sup.19, and R.sup.20
are as defined above and R.sup.25 is C.sub.1 to C.sub.4 alkyl.
[0413] Thereafter the 3-position is deprotected by conventional
hydrolysis to reintroduce the 3-keto group and 5,7-hemiacetal,
producing the further intermediate corresponding to Formula 209:
##STR189## where -A-A-, --B--B-- and R.sup.3 are as defined above.
Next, a 9,11 epoxide moiety is introduced in accordance with any of
the methods described above for conversion of the compounds of
Formula II to the compounds of Formula I. Under the oxidizing
conditions of the epoxidation reaction, the hemiacetal partially
converts to the 5,7-lactone, thereby producing a further
intermediate corresponding to Formula 211 ##STR190## where -A-A-,
--B--B-- and R.sup.3 are as defined above. Any remaining
9,11-epoxy-5,7-hemiacetal intermediate reaction product of Formula
210: ##STR191## wherein -A-A-, --B--B-- and R.sup.3 are as defined,
is readily oxidized by conventional means to the compound of
Formula 211. Finally, the intermediate of Formula 211 is converted
to epoxymexrenone or other compound of Formula I using the method
described in Scheme 8 for the conversion of the 5,7-lactone to the
7.alpha.-alkoxycarbonyl compound. Thus, overall, Scheme 10 proceeds
as follows, it being understood that at least the following steps
may be carried out in situ without recovery of the is intermediate.
Overall, the synthesis of Scheme 10 proceeds as follows:
##STR192##
[0414] As in the case of Scheme 9, the reactions described above
for Scheme 10 offer important advantages, especially with regard to
process economics; but the novel reactions of Scheme 10 also have
more generic application to substrates other than those
particularly described. For example, introduction of the 7-formyl
group into a 3-enol ether steroid, protection of the resulting
7-formyl-.DELTA.-5,6-3,4-enol ether, hydrolysis to the
5,7-hemiacetal, and subsequent deprotection can be conducted on
steroids substituted at the 17-position by R.sup.8 and R.sup.9 as
defined above, or more particularly by a substituent of Formula:
##STR193## where X, Y.sup.1, Y.sup.2, and C(17) are as defined
above.
[0415] Alternative processes of Scheme 10 comprise use of the
various intermediates within the scope of Formulae A203 through
A210, respectively, hereinabove. Each of the intermediates of
Formulae A203 through A211 is a novel compound which is useful in
the preparation of epoxymexrenone and other compounds of Formula I
and IA in accordance with the synthesis of Scheme 10.
[0416] In a particularly preferred embodiment, the overall process
of Scheme 10 proceeds as follows: ##STR194##
[0417] From the several schemes that are illustrated above, it will
be understood that the reaction steps selected for use in the
processes of the invention provide substantial flexibility in the
manufacture of epoxymexrenone and related compounds. The key
features include, inter alia: (a) bioconversion of a substrate such
as canrenone, androstendione, or .beta.-sitosterol to an 11.alpha.-
or 9.alpha.-hydroxy derivative (with simultaneous conversion of
.beta.-sitosterol to a 17-keto structure; (b) introduction of the
9,11 double bond by dehydration of a compound containing either an
11.alpha.- or 9.alpha.-hydroxy group, followed by introduction of
the epoxy group by oxidation of the 9,11 double bond; (c)
attachment of a 7.alpha.-alkoxycarbonyl by formation of the
enamine, hydrolysis of the enamine to the diketone, and reaction of
the diketone with an alkali metal alkoxide; (d) formation of the
20-spiroxane ring at the 17 position; (e) formation of the
5,7-lactone, and esterification of the lactone to the
7-alkoxycarbonyl; (f) protection of the 3-ketone by conversion to
3-enol ether or 3-ketal during various of the conversions at other
positions (including formation of the 20-spiroxane ring at the
17-position. With few limitations, these four component process
elements (b) to (d) can be conducted in almost any sequence.
Process elements (e) and (f) offer comparable flexibility. They
provide a route to epoxymexrenone and other compounds of Formula I
which are much simplified as compared to the process of U.S. Pat.
No. 4,559,332. Moreover, they provide important benefits in
productivity and yield.
[0418] In the descriptions of the reaction schemes as set forth
above, recovery, isolation and purification of reaction products
can generally be carried out by methods well known to those skilled
in the art. Except where otherwise indicated, conditions, solvents,
and reagents are either conventional, not narrowly critical, or
both. However, certain of the specific procedures as particularly
described above provide advantages which contribute to the
favorable overall yield and/or productivity of the various process
steps and process schemes, and/or to high quality of the
intermediates and ultimate 9,11-epoxy steroid products.
[0419] The utility of 20-Spiroxane compounds produced in accordance
with the invention is described in Grob U.S. Pat. No. 4,559,332
which is expressly incorporated herein by reference.
[0420] 20-Spiroxane compounds produced in accordance with the
invention are distinguished by favorable biological properties and
are, therefore, valuable pharmaceutical active ingredients. For
example, they have a strong aldosterone-antagonistic action in that
they reduce and normalize unduly high sodium retention and
potassium excretion caused by aldosterone. They therefore have, as
potassium-saving diuretics, an important therapeutic application,
for example in the treatment of hypertension, cardiac insufficiency
or cirrhosis of the liver.
[0421] 20-Spiroxane derivatives having an aldosterone-antagonistic
action are known, cf., for example, Fieser and Fieser: Steroids;
page 708 (Reinhold Publ. Corp., New York, 1959) and British Patent
Specification No. 1,041,534; also known are analogously active
17.beta.-hydroxy-21-carboxylic acids and their salts, cf., for
example, U.S. Pat. No. 3,849,404. Compounds of this kind that have
hitherto been used in therapy, however, have a considerable
disadvantage in that they always possess a certain sexual-specific
activity which has troublesome consequences sooner or later in the
customary long-term therapy. Especially undesirable are the
troublesome effects that can be attributed to the anti-androgenic
activity of the known anti-aldosterone preparations.
[0422] The methods, processes and compositions of the invention,
and the conditions and reagents used therein, are further described
in the following examples.
EXAMPLE 1
[0423] Slants were prepared with a growth medium as set forth in
Table 1 TABLE-US-00004 TABLE 1 YPDA (medium for slants and plates)
yeast extract 20 g peptone 20 g glucose 20 g agar 20 g distilled
water, q.s. to 1000 ml pH as is 6.7 adjust at pH 5 with
H.sub.3PO.sub.4, 10% w/v Distribute for slants: 7.5 ml in 180
.times. 18 mm tubes for plates (10 cm of .phi.) 25 ml in 200
.times. 20 mm tubes sterilize at 120.degree. C. for 20 minutes pH
after sterilization: 5
To produce first generation cultures, a colony of Aspergillus
ochraceus was suspended in distilled water (2 ml) in a test tube;
and 0.15 ml aliquots of this suspension applied to each of the
slants that had been prepared as described above. The slants were
incubated for seven days at 25.degree. C., after which the
appearance of the surface culture was that of a white cottony
mycelium. The reverse was pigmented in orange in the lower part, in
yellow-orange in the upper part.
[0424] The first generation slant cultures were suspended in a
sterile solution (4 ml) containing Tween 80 nonionic surfactant (3%
by weight), and 0.15 ml aliquots of this suspension were used to
inoculate second generation slants that had been prepared with the
growth medium set forth in Table 2 TABLE-US-00005 TABLE 2 (for
second generation and routine slants) malt extract 20 g peptone 1 g
glucose 20 g agar 20 g distilled water q.s. to 1000 ml pH as is 5.3
distribute in tubes (180 .times. 18 mm) ml 7.5 sterilize at
120.degree. C. for 20 minutes
The second generation slants were incubated for 10 days at
25.degree. C., producing a heavy mass of golden-colored spores;
reverse pigmented in brown orange.
[0425] A protective medium was prepared having the composition set
forth in Table 3. TABLE-US-00006 TABLE 3 PROTECTIVE MEDIUM Skim
milk 10 g distilled water 100 ml In a 250 ml flask containing 100
ml of distilled water at 50.degree. C., add skim milk. Sterilize at
120.degree. C. for 15 minutes. Cool at 33.degree. C. and use before
the day is over
Cultures from five of the second generation slants were suspended
in the protective solution (15 ml) in a 100 ml flask. The
suspension was distributed in aliquots (0.5 ml each) among
100.times.10 mm tubes for lyophilization. These were pre-frozen at
-70.degree. to -80.degree. C. in an acetone/dry ice bath for 20
minutes, then transferred immediately to a drying room pre-cooled
to -40.degree. to -50.degree. C. The pre-frozen aliquots were
lyophilized at a residual pressure of 50 .mu.Hg and
.ltoreq.-30.degree. C. At the end of the lyophilization, two to
three granules of sterile silica gel were added to each tube with
moisture indicator and flame seal.
[0426] To obtain mother culture slants suitable for industrial
scale fermentation, a single aliquot of lyophilized culture, which
had been prepared in the manner described above, was suspended in
distilled water (1 ml) and 0.15 ml aliquots of the suspension were
used to inoculate slants that had been provided with a growth
medium having the composition set forth in Table 2. The mother
slants were incubated for seven days at 25.degree. C. At the end of
incubation, the culture developed on the slants was preserved at
4.degree. C.
[0427] To prepare a routine slant culture, the culture from a
mother slant was suspended in a sterile solution (4 ml) containing
Tween 80 (3% by weight) and the resulting suspension distributed in
0.15 ml aliquots among slants which had been coated with the growth
medium described in Table 2. The routine slant cultures may be used
to inoculate the primary seed flasks for laboratory or industrial
fermentations.
[0428] To prepare a primary seed flask culture, the culture from a
routine slant, which had been prepared as described above, was
removed and suspended in a solution (10 ml) containing Tween 80 (3%
by weight). A 0.1 aliquot of the resulting suspension was
introduced into a 500 ml baffled flask containing a growth medium
having the composition set forth in Table 4. TABLE-US-00007 TABLE 4
(for primary and transformation flask culture and round bottomed
flask) glucose 20 g peptone 20 g yeast autolysate 20 g distilled
water q.s to pH as is 5.2 adjust at pH 5.8 with NaOH 20% distribute
in 500 ml baffled flask 100 ml distribute in 2000 ml round bottomed
flasks (3 baffles) 500 ml sterilize 120.degree. C. .times. 20 min.
pH after sterilization about 5.7
The seed flask was incubated on a rotating shaker (200 rpm, 5 cm
displacement) for 24 hours at 28.degree. C., thereby producing a
culture in the form of pellet-like mycelia having diameters of 3 to
4 mm. On microscopic observation, the seed culture was found to be
a pure culture, with synnematic growth, with big hyphae and well
twisted. The pH of the suspension was 5.4 to 5.6. PMV was 5 to 8%
as determined by centrifugation (3000 rpm.times.5 min.).
[0429] A transformation flask culture was prepared by inoculating a
growth medium (100 ml) having the composition set forth Table 4 in
a second 500 ml shaker flask with biomass (1 ml) from the seed
culture flask. The resulting mixture was incubated on a rotating
shaker (200 rpm, 5 cm displacement) for 18 hours at 28.degree. C.
The culture was examined and found to comprise pellet like mycelia
with a 3-4 mm diameter. On microscopic examination, the culture was
determined to be a pure culture, with synnematic and filamentous
growth in which the apical cells were full of cytoplasm and the
olden cells were little vacuolated. The pH of the culture
suspension was 5 to 5.2 and the PMV was determined by
centrifugation to be between 10% and 15%. Accordingly, the culture
was deemed suitable for transformation of canrenone to
11.alpha.-hydroxycanrenone.
[0430] Canrenone (1 g) was micronized to about 5.mu. and suspended
in sterile water (20 ml). To this suspension were added: a 40%
(w/v) sterile glucose solution; a 16% (w/v) sterile solution of
autolyzed yeast; and a sterile antibiotic solution; all in the
proportions indicated for 0 hours reaction time in Table 5. The
antibiotic solution had been prepared by dissolving kanamicyn
sulfate (40 mg), tetracycline HCl (40 mg) and cefalexin (200 mg) in
water (100 ml). The steroid suspension, glucose solution, and
autolyzed yeast solution were added gradually to the culture
contained in the shaker flask. TABLE-US-00008 TABLE 5 Indicative
Additions of Steroid and Solutions (additives and antibiotics) in
the Course of Bioconversion of Canrenone in Shake Flask Steroid
yeast Reaction Suspension glucose autolised antibiotic time approx.
solution sol. solution hours ml mg. ml ml. ml 0 1 50 1 0.5 1 8 2
100 2 1 24 2 100 1 0.5 1 32 5 250 2 1 48 2 100 1 0.5 1 56 5 250 2 1
72 3 150 1 0.5 1 90
[0431] As reaction proceeded, the reaction mixture was periodically
analyzed to determine glucose content, and by thin layer
chromatography to determine conversion to
11.alpha.-hydroxycanrenone. Additional canrenone substrate and
nutrients were added to the fermentation reaction mixture during
the reaction at rates controlled to maintain the glucose content in
the range of about 0.1% by weight. The addition schedule for
steroid suspension, glucose solution, autolyzed yeast solution and
antibiotic solution is set forth in Table 5. The transformation
reaction continued for 96 hours at 25.degree. C. on a rotary shaker
(200 rpm and 5 cm displacement). The pH ranged between 4.5 and 6
during the fermentation. Whenever the PMV rose to or above 60%, a
10 ml portion of broth culture was withdrawn and replaced with 10
ml distilled water. The disappearance of canrenone and appearance
of 11.alpha.-hydroxycanrenone were monitored during the reaction by
sampling the broth at intervals of 4, 7, 23, 31, 47, 55, 71, 80,
and 96 hours after the start of the fermentation cycle, and
analyzing the sample by TLC. The progress of the reaction as
determined from these samples is set forth in Table 6
TABLE-US-00009 TABLE 6 Time Course of Bioconversion of Canrenone in
Shake Flask Transformation Ratio Time Canrenone Rf.
11.alpha.hydroxy Canrenone hours RF. = 0.81 RF. = 0.29 0 100 0.0 4
50 50 7 20 80 23 20 80 31 30 70 47 20 80 55 30 70 71 25 75 80 15 85
96 .about.10 .about.90
EXAMPLE 2
[0432] A primary seed flask culture was prepared in the manner
described in Example 1. A nutrient mixture was prepared having the
composition set forth in Table 7 TABLE-US-00010 TABLE 7 For
Transformation Culture in 10 l glass fermenter quantity g/l glucose
80 g 20 peptone 80 g 20 yeast autolyzed 80 g 20 antifoam SAG 471
0.5 g deionized water q.s. to 4 l sterilize the empty fermenter for
30 minutes at 130.degree. C. load it with 3 l of deionized water,
heat at 40.degree. C. add while stirring the components of the
medium stir for 15 minutes, bring to volume of 3.9 l pH as is 5.1
adjust of 5.8 with NaOH 20% w/v sterilize at 120.degree. C. .times.
20 minutes pH after sterilization 5.5-5.7
An initial charge of this nutrient mixture (4 L) was introduced
into a transformation fermenter of 10 L geometric volume. The
fermenter was of cylindrical configuration with a height to
diameter ratio of 2.58. It was provided with a 400 rpm turbine
agitator having two No. 2 disk wheels with 6 blades each. The
external diameter of the impellers was 80 mm, each of the blades
was 25 mm in radial dimension and 30 mm high, the upper wheel was
positioned 280 mm below the top of the vessel, the lower wheel was
365 mm below the top, and baffles for the vessel were 210 mm high
and extended radially inwardly 25 mm from the interior vertical
wall of the vessel.
[0433] Seed culture (40 ml) was mixed with the nutrient charge in
the fermenter, and a transformation culture established by
incubation for 22 hours at 28.degree. C., and an aeration rate of
0.5 l/l-min. at a pressure of 0.5 kg/cm.sup.2. At 22 hours, the PMV
of the culture was 20-25% and the pH 5 to 5.2.
[0434] A suspension was prepared comprising canrenone (80 g) in
sterile water (400 ml), and a 10 ml portion added to the mixture in
the transformation fermenter. At the same time a 40% (w/v) sterile
glucose solution, a 16% (w/v) sterile solution of autolyzed yeast,
and a sterile antibiotic solution were added in the proportions
indicated in Table 8 at 0 hours reaction time. The antibiotic
solution was prepared in the manner described in Example 1.
TABLE-US-00011 TABLE 8 Indicative Additions of Steroid and
Solutions (additives and antibiotics) in the Course of
Bioconversion of Canrenone in 10 l Glass Fermenter Steroid yeast
Reaction Suspension glucose autolyzed antibiotic time approx
solution solution solution hours ml gr ml ml ml 0 10 4 25 12.5 40 4
25 12.5 8 10 4 25 12.5 12 25 12.5 16 10 4 25 12.5 20 25 12.5 24 10
4 25 12.5 40 28 10 4 25 12.5 32 12.5 5 25 12.5 36 12.5 5 25 12.5 40
12.5 5 25 12.5 44 12.5 5 25 12.5 48 12.5 5 25 12.5 40 52 12.5 5 25
12.5 56 12.5 5 25 12.5 60 12.5 5 25 12.5 64 12.5 5 25 12.5 68 12.5
5 25 12.5 72 12.5 5 25 12.5 40 76 12.5 5 25 12.5 80 84 88
As reaction proceeded, the reaction mixture was periodically
analyzed to determine glucose content, and by thin layer
chromatography to determine conversion to
11.alpha.-hydroxycanrenone. Based on TLC analysis of reaction broth
samples as described hereinbelow, additional canrenone was added to
the reaction mixture as canrerone substrate was consumed. Glucose
levels were also monitored and, whenever glucose concentration
dropped to about 0.05% by weight or below, supplemental glucose
solution was added to bring the concentration up to about 0.25% by
weight. Nutrients and antibiotics were also added at discrete times
during the reaction cycle. The addition schedule for steroid
suspension, glucose solution, autolyzed yeast solution and
antibiotic solution is set forth in Table 8. The transformation
reaction continued for 90 hours at an aeration rate of 0.5 vol. air
per vol. liquid per minute (vvm) at a positive head pressure of 0.3
kg/cm.sup.2. The temperature was maintained at 28.degree. C. until
PVM reached 45%, then decreased to 26.degree. C. and maintained at
that temperature as PVM grew from 45% to 60%, and thereafter
controlled at 24.degree. C. The initial agitation rate was 400 rpm,
increasing to 700 rpm after 40 hours. The pH was maintained at
between 4.7 and 5.3 by additions of 2M orthophosphoric acid or 2M
NaOH, as indicated. Foaming was controlled by adding a few drops of
Antifoam SAG 471 as foam developed. The disappearance of canrenone
and appearance of 11.alpha.-hydroxycanrenone were monitored at 4
hour intervals during the reaction by TLC analysis of broth
samples. When most of the canrenone had disappeared from the broth,
additional increments were added.
[0435] After all canrenone additions had been made, the reaction
was terminated when TLC analysis showed that the concentration of
canrenone substrate relative to 11.alpha.-hydroxycanrenone product
had dropped to about 5%.
[0436] At the conclusion of the reaction cycle, the fermentation
broth was filtered through cheese cloth for separation of the
mycelium from the liquid broth. The mycelia fraction was
resuspended in ethyl acetate using about 65 volumes (5.2 liters)
per gram canrenone charged over the course of the reaction. The
suspension of mycelia in ethyl acetate was refluxed for one hour
under agitation, cooled to about 20.degree. C., and filtered on a
Buchner. The mycelia cake was washed sequentially with ethyl
acetate (5 vol. per g canrenone charge; 0.4 L) and deionized water
(500 ml) to displace the ethyl acetate extract from the cake. The
filter cake was discarded. The rich extract, solvent washing and
water washing were collected in a separator, then allowed to stand
for 2 hours for phase separation.
[0437] The aqueous phase was then discarded and the organic phase
concentrated under vacuum to a residual volume of 350 ml. The still
bottoms were cooled to 15.degree. C. and kept under agitation for
about one hour. The resulting suspension was filtered to remove the
crystalline product, and the filter cake was washed with ethyl
acetate (40 ml). After drying, the yield of
11.alpha.-hydroxycanrenone was determined to be 60 g.
EXAMPLE 3
[0438] A spore suspension was prepared from a routine slant in the
manner described in Example 1. In a 2000 ml baffled round bottomed
flask (3 baffles, each 50 mm.times.30 mm), an aliquot (0.5 ml) of
the spore suspension was introduced into a nutrient solution (500
ml) having the composition set forth in Table 4. The resulting
mixture was incubated in the flask for 24 hours at 25.degree. C. on
an alternating shaker (120 strokes per min.; displacement 5 cm),
thereby producing a culture which, on microscopic examination, was
observed to appear as a pure culture with hyphae well twisted. The
pH of the culture was between about 5.3 and 5.5, and the PMV (as
determined by centrifugation at 3000 rpm for 5 min.) was 8 to
10%.
[0439] Using the culture thus prepared, a seed culture was prepared
in a stainless steel fermenter of vertical cylindrical
configuration, having a geometric volume of 160 L and an aspect
ratio of 2.31 (height=985 mm; diameter=425 mm). The fermenter was
provided with a disk turbine type agitator having two wheels, each
wheel having six blades with an external diameter of 240 mm, each
blade having a radial dimension of 80 mm and a height of 50 mm. The
upper wheel was positioned at a depth of 780 mm from the top of the
fermenter, and the second at a depth of 995 mm. Vertical baffles
having a height of 890 mm extended radially inwardly 40 mm from the
interior vertical wall of the fermenter. The agitator was operated
at 170 rpm. A nutrient mixture (100 L) having the composition set
forth in Table 9 was introduced into the fermenter, followed by a
portion of preinoculum (1 L) prepared as described above and having
a pH of 5.7. TABLE-US-00012 TABLE 9 For Vegetative Culture in 160 L
Fermenter About 8 L are needed to Seed Productive fermenter
Quantity g/L glucose 2 kg 20 peptone 2 kg 20 yeast autolysed 2 kg
20 antifoam SAG 471 0.010 Kg traces deionized water q.s. to 100 L
sterilize the empty fermenter for 1 hour at 130.degree. C. load it
with 6 L of deionized water; heat at 40.degree. C. add while
stirring the components of the medium stir for 15 minutes, bring to
volume of 95 L sterilization at 121.degree. C. for 30 minutes post
sterilization pH is 5.7 add sterile deionized water to 100 L
[0440] The inoculated mixture was incubated for 22 hours at an
aeration rate of 0.5 L/L-min. at a head pressure of 0.5
kg/cm.sup.2. The temperature was controlled at 28.degree. C. until
PMV reached 25%, and then lowered to 25.degree. C. The pH was
controlled in the range of 5.1 to 5.3. Growth of mycelium volume is
shown in Table 10, along with pH and dissolved oxygen profiles of
the seed culture reaction. TABLE-US-00013 TABLE 10 Time Course for
Mycelial Growth in Seed Culture Fermentation packed mycelium volume
(pmv)% Fermentation (3000 rpms dissolved period h pH 5 min) oxygen
% 0 5.7 .+-. 0.1 100 4 5.7 .+-. 0.1 100 8 5.7 .+-. 0.1 12 .+-. 3 85
.+-. 5 12 5.7 .+-. 0.1 15 .+-. 3 72 .+-. 5 16 5.5 .+-. 0.1 25 .+-.
5 40 .+-. 5 20 5.4 .+-. 0.1 30 .+-. 5 35 .+-. 5 22 5.3 .+-. 0.1 33
.+-. 5 30 .+-. 5 24 5.2 .+-. 0.1 35 .+-. 5 25 .+-. 5
[0441] Using the seed culture thus produced, a transformation
fermentation run was carried out in a vertical cylindrical
stainless steel fermenter having a diameter of 1.02 m, a height of
1.5 m and a geometric volume of 1.4 m.sup.3. The fermenter was
provided with a turbine agitator having two impellers, one
positioned 867 cm below the top of the reactor and the other
positioned 1435 cm from the top. Each wheel was provided with six
blades, each 95 cm in radial dimension and 75 cm high. Vertical
baffles 1440 cm high extended radially inwardly 100 cm from the
interior vertical wall of the reactor. A nutrient mixture was
prepared having the composition set forth in Table 11
TABLE-US-00014 TABLE 11 For Bioconversion Culture in 1000 L
Fermenter Quantity g/L glucose 16 kg 23 peptone 16 kg 23 yeast
autolysed 16 kg 23 antifoam SAG 471 0.080 Kg traces deionized water
q.s. to 700 L sterilize the empty fermenter for 1 hour at
130.degree. C. load it with 600 L ofdeionized water; heat at
40.degree. C. add while stirring the components of the medium stir
for 15 minutes, bring to volume of 650 L sterilization at
121.degree. C. for 30 minutes post sterilization pH is 5.7 add
sterile deionized water to 700 L
An initial charge (700 L) of this nutrient mixture (pH=5.7) was
introduced into the fermenter, followed by the seed inoculum of
this example (7 L) prepared as described above.
[0442] The nutrient mixture containing inoculum was incubated for
24 hours at an aeration rate of 0.5 L/L-min at a head pressure of
0.5 kg/cm.sup.2. The temperature was controlled at 28.degree. C.,
and the agitation rate was 110 rpm. Growth of mycelium volume is
shown in Table 12, along with pH and dissolved oxygen profiles of
the seed culture reaction. TABLE-US-00015 TABLE 12 Time Course for
Mycelial Growth in Fermenter of the Transformation Culture packed
mycelium Fermentation volume (pmv) % dissolved period h pH (3000
rpm .times. 5 min) oxygen % 0 5.6 .+-. 0.2 100 4 5.5 .+-. 0.2 100 8
5.5 .+-. 0.2 12 .+-. 3 95 .+-. 5 12 15 .+-. 3 90 .+-. 5 16 5.4 .+-.
0.1 20 .+-. 5 75 .+-. 5 20 5.3 .+-. 0.1 25 .+-. 5 60 .+-. 5 22 5.2
.+-. 0.1 30 .+-. 5 40 .+-. 5
At the conclusion of the incubation, pelleting of the mycelium was
observed, but the pellets were generally small and relatively
loosely packed. Diffuse mycelium was suspended in the broth. Final
pH was 5.1 to 5.3.
[0443] To the transformation culture thus produced was added a
suspension of canrenone (1.250 kg; micronized to 5.mu.) in sterile
water (5 L). Sterile additive solution and antibiotic solution were
added in the proportions indicated at reaction time 0 in Table 14.
The composition of the additive solution is set forth in Table 13.
TABLE-US-00016 TABLE 13 ADDITIVE SOLUTION (for transformative
culture) quantity dextrose 40 Kg yeast autolysate 8 Kg antifoam SAG
471 0.010 Kg deionized water q.s. to 100 l sterilize a 150 l empty
fermenter for 1 hour at 130.degree. C. load it with 70 l of
deionized water; heat at 40.degree. C. add while stirring the
components of "additive solution" stir for 30 minutes, bring to
volume of 95 l pH as is 4.9 sterilize at 120.degree. C. .times. 20
minutes pH after sterilization about 5
[0444] Bioconversion was carried out for about 96 hours with
aeration at 0.5 L/L-min. at a head pressure of 0.5 kg/cm.sup.2 and
a pH of ranging between 4.7 and 5.3, adjusted as necessary by
additions of 7.5 M NaOH or 4 M H.sub.3PO.sub.4. The agitation rate
was initially 100 rpm, increased to 165 rpm at 40 hours and 250 rpm
at 64 hours. The initial temperature was 28.degree. C., lowered to
26.degree. C. when PMV reached 45%, and lowered to 24.degree. C.
when PMV rose to 60%. SAG 471 in fine drops was added as necessary
to control foaming. Glucose levels in the fermentation were
monitored at 4 hour intervals and, whenever the glucose
concentration fell below 1 gpl, an increment of sterile additive
solution (10 L) was added to the batch. Disappearance of canrenone
and appearance of 11.alpha.-hydroxycanrenone were also monitored
during the reaction by HPLC. When at least 90% of the initial
canrenone charge had been converted to 11.alpha.-hydroxycanrenone,
an increment of 1.250 kg canrenone was added. When 90% of the
canrenone in that increment was shown to have been converted,
another 1.250 kg increment was introduced. Using the same criterion
further increments (1.250 kg apiece) were added until the total
reactor charge (20 kg) had been introduced. After the entire
canrenone charge had been delivered to the reactor, reaction was
terminated when the concentration of unreacted canrenone was 5%
relative to the amount of 11.alpha.-hydroxycanrenone produced. The
schedule for addition of canrenone, sterile additive solution, and
antibiotic solution is as shown in Table 14. TABLE-US-00017 TABLE
14 Additions of the Steroid and Solutions (additives and
antibiotics) in the Course of Bioconversion of Canrenone in
Fermenter CANRENONE Sterile Reaction in suspension additive
antibiotic volume time Progressive solution solution liters hours
Kg Kg liters liters about 0 1.250 1.25 10 8 700 4 10 8 1.250 2.5 10
12 10 16 1.250 10 20 10 24 1.250 5 10 8 800 28 1.250 10 32 1.250 10
36 1.250 10 40 1.250 10 44 1.250 10 48 1.250 12.5 10 8 900 52 1.250
10 56 1.250 10 60 1.250 10 64 1.250 10 68 1.250 10 72 1.250 20 10 8
1050 76 0 80 84 88 92 Total
[0445] When bioconversion was complete, the mycelia were separated
from the broth by centrifugation in a basket centrifuge. The
filtrate was determined by HPLC to contain only 2% of the total
quantity of 11.alpha.-hydroxycanrenone in the harvest broth, and
was therefore eliminated. The mycelia were suspended in ethyl
acetate (1000 L) in an extraction tank of 2 m.sup.3 capacity. This
suspension was heated for one hour under agitation and is ethyl
acetate reflux conditions, then cooled and centrifuged in a basket
centrifuge. The mycelia cake was washed with ethyl acetate (200 L)
and thereafter discharged. The steroid rich solvent extract was
allowed to stand for one hour for separation of the water phase.
The water phase was extracted with a further amount of ethyl
acetate solvent (200 L) and then discarded. The combined solvent
phases were clarified by centrifugation and placed in a
concentrator (500 L geometric volume) and concentrated under vacuum
to a residual volume of 100 L. In carrying out the evaporation, the
initial charge to the concentrator of combined extract and wash
solutions was 100 L, and this volume was kept constant by continual
or periodic additions of combined solution as solvent was taken
off. After the evaporation step was complete, the still bottoms
were cooled to 20.degree. C. and stirred for two hours, then
filtered on a Buchner filter. The concentrator pot was washed with
ethyl acetate (20 L) and this wash solution was then used to wash
the cake on the filter. The product was dried under vacuum for 16
hours at 50.degree. C. Yield of 11.alpha.-hydroxycanrenone was 14
kg.
EXAMPLE 4
[0446] Lyophilized spores of Aspergillus ochraceus NRRL 405 were
suspended in a corn steep liquor growth medium (2 ml) having the
composition set forth in Table 15: TABLE-US-00018 TABLE 15 Corn
Steep Liquor Medium (Growth Medium for Primary Seed Cultivation)
Corn steep liquor 30 g Yeast extract 15 g Ammonium phosphate 3 g
Monobasic Glucose (charge after sterilization) 30 g distilled
water, q.s. to 1000 ml pH as is: 4.6, adjust to pH 6.5 with 20%
NaOH, distribute 50 ml to 250 ml Erlenmeyer flask sterilize
121.degree. C. for 20 minutes.
[0447] The resulting suspension was used in an inoculum for the
propagation of spores on agar plates. Ten agar plates were
prepared, each bearing a solid glucose/yeast extract/phosphate/agar
growth medium having the composition set forth in Table 16:
TABLE-US-00019 TABLE 16 GYPA (Glucose/Yeast Extract/Phosphate Agar
for Plates) Glucose (charge after sterilization) 10 g Yeast extract
2.5 g K.sub.2HPO.sub.4 3 g Agar 20 g distilled water, q.s. to 1000
ml adjust pH to 6.5 sterilize 121.degree. C. for 30 minutes
[0448] A 0.2 ml aliquot of the suspension was transferred onto the
surface of each plate. The plates were incubated at 25.degree. C.
for ten days, after which the spores from all the plates were
harvested into a sterile cryogenic protective medium having the
composition set forth in Table 17: TABLE-US-00020 TABLE 17
GYP/Glycerol (Glucose/Yeast Extract/ Phosphate/Glycerol medium for
stock vials) Glucose (charge after sterilization) 10 g Yeast
extract 2.5 g K.sub.2HPO.sub.4 3 g Glycerol 20 g Distilled water,
q.s. to 1000 mL Sterilize at 121.degree. C. for 30 minutes
The resulting suspension was divided among twenty vials, with one
ml being transferred to each vial. These vials constitute a master
cell bank that can be drawn on to produce working cell banks for
use in generation of inoculum for bioconversion of canrenone to
11.alpha.-hydroxycanrenone. The vials comprising the master cell
bank were stored in the vapor phase of a liquid nitrogen freezer at
-130.degree. C.
[0449] To begin preparation of a working cell bank, the spores from
a single master cell bank vial were resuspended in a sterile growth
medium (1 ml) having the composition set forth in Table 15. This
suspension was divided into ten 0.2 ml aliquots and each aliquot
used to inoculate an agar plate bearing a solid growth medium
having the composition set forth in Table 16. These plates were
incubated for ten days at 25.degree. C. By the third day of
incubation, the underside of the growth medium was brown-orange. At
the end of the incubation there was heavy production of golden
colored spores. The spores from each plate were harvested by the
procedure described hereinabove for the preparation of the master
cell bank. A total of one hundred vials was prepared, each
containing 1 ml of suspension. These vials constituted the working
cell bank. The working cell bank vials were also preserved by
storage in the vapor phase of a liquid nitrogen freezer at
-130.degree. C.
[0450] Growth medium (50 ml) having the composition set forth in
Table 15 was charged to a 250 ml Erlenmeyer flask. An aliquot (0.5
ml) of working cell suspension was introduced into the flask and
mixed with the growth medium. The inoculated mixture was incubated
for 24 hours at 25.degree. C. to produce a primary seed culture
having a percent packed mycelial volume of approximately 45%. Upon
visual inspection the culture was found to comprise pellet-like
mycelia of 1 to 2 mm diameter; and upon microscopic observation it
appeared as a pure culture.
[0451] Cultivation of a secondary seed culture was initiated by
introducing a growth medium having the composition set forth in
Table 15 into a 2.8 L Fernbach flask, and inoculating the medium
with a portion (10 ml) of the primary seed culture of this example,
the preparation of which was as described above. The inoculated
mixture was incubated at 25.degree. C. for 24 hours on a rotating
shaker (200 rpm, 5 cm displacement). At the end of the incubation,
the culture exhibited the same properties as described above for
the primary seed culture, and was suitable for use in a
transformation fermentation in which canrenone was bioconverted to
11.alpha.-hydroxycanrenone.
[0452] Transformation was conducted in a Braun E Biostat fermenter
configured as follows: TABLE-US-00021 Capacity: 15 liters with
round bottom Height: 53 cm Diameter: 20 cm H/D: 2.65 Impellers:
7.46 cm diameter, six paddles 2.2 .times. 1.4 cm each Impeller
spacing: 65.5, 14.5 and 25.5 cm from bottom of tank Baffles: four
1.9 .times. 48 cm Sparger: 10.1 cm diameter, 21 holes -1 mm
diameter Temperature control: provided by means of an external
vessel jacket
[0453] Canrenone at a concentration of 20 g/L was suspended in
deionized water (4 L) and a portion (2 L) of growth medium having
the composition set forth in Table 18 was added while the mixture
in the fermenter was stirred at 300 rpm. TABLE-US-00022 TABLE 18
(Growth medium for bioconversion culture in 10 L fermenter)
Quantity Amount/L glucose (charge after 160 g 20 g sterilization)
peptone 160 g 20 g yeast extract 160 g 20 g antifoam SAF471 4.0 ml
0.5 ml Canrenone 160 g 20 g deionized water q.s. to 7.5 L sterilize
121.degree. C. for 30 minutes
The resulting suspension was stirred for 15 minutes, after which
the volume was brought up to 7.5 L with additional deionized water.
At this point the pH of the suspension was adjusted from 5.2 to 6.5
by addition of 20% by weight NaOH solution, and the suspension was
then sterilized by heating at 121.degree. C. for 30 minutes in the
Braun E fermenter. The pH after sterilization was 6.3.+-.0.2, and
the final volume was 7.0 L. The sterilized suspension was
inoculated with a portion (0.5 L) of the secondary seed culture of
this example that has been prepared as described above, and the
volume brought up to 8.0 L by addition of 50% sterile glucose
solution. Fermentation was carried out at a temperature of
28.degree. C. until the PMV reached 50%, then lowered to 26.degree.
C., and further lowered to 24.degree. C. when PMV exceeded 50% in
order to maintain a consistent PMV below about 60%. Air was
introduced through the sparger at a rate of 0.5 vvm based on
initial liquid volume and the pressure in the fermenter was
maintained at 700 millibar gauge. Agitation began at 600 rpm and
was increased stepwise to 1000 rpm as needed to keep the dissolved
oxygen content above 30% by volume. Glucose concentration was
monitored. After the initial high glucose concentration fell below
1% due to consumption by the fermentation reaction, supplemental
glucose was provided via a 50% by weight sterile glucose solution
to maintain the concentration in the 0.05% to 1% range throughout
the remainder of the batch cycle. Prior to inoculation the pH was
6.3.+-.0.2. After the pH dropped to about 5.3 during the initial
fermentation period, it was maintained in the range of 5.5.+-.0.2
for the remainder of the cycle by addition of ammonium hydroxide.
Foam was controlled by adding a polyethylene glycol antifoam agent
sold under the trade designation SAG 471 by OSI Specialties,
Inc.
[0454] Growth of the culture took place primarily during the first
24 hours of the cycle, at which time the PMV was about 40%, the pH
was about 5.6 and the dissolved oxygen content was about 50% by
volume. Canrenone conversion began even as the culture was growing.
Concentrations of canrenone and 11.alpha.-hydroxycanrenone were
monitored during the bioconversion by analyzing daily samples.
Samples were extracted with hot ethyl acetate and the resulting
sample solution analyzed by TLC and HPLC. The bioconversion was
deemed complete when the residual canrenone concentration was about
10% of the initial concentration. The approximate conversion time
was 110 to 130 hours.
[0455] When bioconversion was complete, mycelial biomass was
separated from the broth by centrifugation. The supernatant was
extracted with an equal volume of ethyl acetate, and the aqueous
layer discarded. The mycelial fraction was resuspended in ethyl
acetate using approximately 65 volumes per g canrenone charged to
the fermentation reactor. The mycelial suspension was refluxed for
one hour under agitation, cooled to about 20.degree. C., and
filtered on a Buchner funnel. The mycelial filter cake was washed
twice with 5 volumes of ethyl acetate per g of canrenone charged to
the fermenter, and then washed with deionized water (1 L) to
displace the residual ethyl acetate. The aqueous extract, rich
solvent, solvent washing and water washing were combined. The
remaining exhausted mycelial cake was either discarded or extracted
again, depending on analysis for residual steroids therein. The
combined liquid phases were allowed to settle for two hours.
Thereafter, the aqueous phase was separated and discarded, and the
organic phase concentrated under vacuum until the residual volume
was approximately 500 ml. The still bottle was then cooled to about
15.degree. C. with slow agitation for about one hour. The
crystalline product was recovered by filtration, and washed with
chilled ethyl acetate (100 ml). Solvent was removed from the
crystals by evaporation, and the crystalline product dried under
vacuum at 50.degree. C.
EXAMPLE 5
[0456] Lyophilized spores of Aspergillus ochraceus ATCC 18500 were
suspended in a corn steep liquor growth medium (2 ml) as described
in Example 4. Ten agar plates were prepared, also in the manner of
Example 4. The plates were incubated and harvested as described in
Example 4 to provide a master cell bank. The vials comprising the
master cell bank were stored in the vapor phase of a liquid
nitrogen freezer at -130.degree. C.
[0457] From a vial of the master cell bank, a working cell bank was
prepared as described in Example 4, and stored in the nitrogen
freezer at -130.degree. C.
[0458] Growth medium (300 mL) having the composition set forth in
Table 19 was charged to a 2 L baffled flask. An aliquot (3 mL) of
working cell suspension was introduced into the flask. The
inoculated mixture was incubated for 20 to 24 hours at 28.degree.
C. on a rotating shaker (200 rpm, 5 cm displacement) to produce a
primary seed culture having a percent packed mycelial volume of
approximately 45%. Upon visual inspection the culture was found to
comprise pellet like mycelia of 1 to 2 mm diameter; and upon
microscopic observation it appeared as a pure culture.
TABLE-US-00023 TABLE 19 Growth medium for primary and secondary
seed cultivation Amount/L glucose (charge after 20 g sterilization)
peptone 20 g Yeast extract 20 g distilled water q.s. to 1000 mL
sterilize 121.degree. C. for 30 minutes
[0459] Cultivation of a secondary seed culture was initiated by
introducing 8 L growth medium having the composition set forth in
Table 19 into a 14 L glass fermenter. Inoculate the fermenter with
160 mL to 200 mL of the primary seed culture of this example. The
preparation of which was as described above.
[0460] The inoculated mixture was cultivated at 28.degree. C. for
18-20 hours, 200 rmp agitation, aeration rate was 0.5 vvm. At the
end of the propagation, the culture exhibited the same properties
as described above for the primary seed.
[0461] Transformation was conducted in a 60 L fermenter,
substantially in the manner described in Example 4, except that the
growth medium had the composition set forth in Table 20, and the
initial charge of secondary seed culture was 350 mL to 700 mL.
Agitation rate was initially 200 rpm, but increased to 500 rpm as
necessary to maintain dissolved oxygen above 10% by volume. The
approximate bioconversion time for 20 g/L canrenone was 80 to 160
hours. TABLE-US-00024 TABLE 20 Growth Medium for Bioconversion
Culture in 60 L Fermenter Quantity Amount/L glucose (charge after
17.5 g 0.5 g sterilization) peptone 17.5 g 0.5 g yeast extract 17.5
g 0.5 g Canrenone (charge as a 700 g 20 g 20% slurry in sterile
water) deionized water, q.s. to 35 L sterilize 121.degree. C. for
30 minutes
EXAMPLE 6
[0462] Using a spore suspension from the working cell bank produced
in accordance with the method described in Example 4, primary and
secondary seed cultures were prepared, also substantially in the
manner described in Example 4. Using secondary seed culture
produced in this manner, two bioconversion runs were made in
accordance with a modified process of the type illustrated in FIG.
1, and two runs were made with the process illustrated in FIG. 2.
The transformation growth medium, canrenone addition schedules,
harvest times, and degrees of conversion for these runs are set
forth in Table 21. Run R2A used a canrenone addition scheme based
on the same principle as Example 3, while run R2C modified the
Example 3 scheme by making only two additions of canrenone, one at
the beginning of the batch, and one after 24 hours. In runs R2B and
R2D, the entire canrenone charge was introduced at the beginning of
the batch and the process generally carried in the manner described
in Example 4, except that the canrenone charge was sterilized in a
separate vessel before it was charged to the fermenter and glucose
was added as the batch progressed. A Waring blender was used to
reduce chunks produced on sterilization. In runs R2A and R2B,
canrenone was introduced into the batch in methanol solution, in
which respect these runs further differed from the runs of Examples
3 and 4, respectively. TABLE-US-00025 TABLE 21 Descriptions of the
Initial Canrenone Bioconversion Processes Run Number R2A R2B R2C
R2D Medium (g/L) Corn steep liq. 30 the same as run 30 the same as
run Yeast extract 15 R2A 15 R2C NH.sub.4H.sub.2PO.sub.4 3 3 Glucose
15 30 OSA 0.5 ml 0.5 ml pH adjusted to 6.0 adjusted to with 2.5N
NaOH 6.5 with 2.5N NaOH Canrenone 10 g/80 ml MEOH 80 g/640 ml MEOH
Sterilized and Sterilized and added at 0, 18, added at 0 hr all
blended; added blended; added 24, 30, 36, 42, at once at: 0 hr: 25
g at: 0 hr: 200 g. 50, 56, 62 and 24 hr: 200 g 68 hr. Harvest time
143 hrs. 166 hrs. 125 hrs. 104 hrs. Bioconversion 45.9% 95.6% 98.1%
95.1%
In runs R2A and R2B, the methanol concentration accumulated to
about 6.0% in the fermentation beer, which was found to be
inhibitory to the growth of culture and bioconversion. However,
based on the results of these runs, it was concluded that methanol
or other water-miscible solvent could serve effectively at lower
concentrations to increase the canrenone charge and provide
canrenone as a fine particle precipitate providing a large
interfacial area for supply of canrenone to the subject to the
reaction.
[0463] Canrenone proved stable at sterilization temperature
(121.degree. C.) but aggregated into chunks. A Waring blender was
employed to crush the lumps into fine particles, which were
successfully converted to product.
EXAMPLE 7
[0464] Using a spore suspension from the working cell bank produced
in accordance with the method described in Example 4, primary and
secondary seed cultures were prepared, also substantially in the
manner described in Example 4. The description and results of
Example 7 are shown in Table 22. Using secondary seed culture
produced in this manner, one bioconversion (R3C) was carried out
substantially as described in Example 3, and three bioconversions
were carried out in accordance with the process generally described
in Example 5. In the latter three runs (R3A, R3B and R3D),
canrenone was sterilized in a portable tank, together with the
growth medium except for glucose. Glucose was aseptically fed from
another tank. The sterilized canrenone suspension was introduced
into the fermenter either before inoculation or during the early
stage of bioconversion. In run R3B, supplemental sterile canrenone
and growth medium was introduced at 46.5 hours. Lumps of canrenone
formed on sterilization were delumped through a Waring blender thus
producing a fine particulate suspension entering the fermenter. The
transformation growth media, canrenone addition schedules, nutrient
addition schedules, harvest times, and degrees of conversion for
these runs are set forth in Tables 22 and 23. TABLE-US-00026 TABLE
22 Descriptions of Process for Canrenone Bioconversion Run Number
R3A R3B R3C R3D Medium (g/L) Corn steep liq. 30 the same as run
Peptone: 20 the same as run Yeast extract 15 R3A Yeast Ext.: 20 R3A
NH.sub.4H.sub.2PO.sub.4 3 Glucose: 20 Glucose 15 OSA: 3 ml OSA 0.5
ml pH adjusted to 6.5 adjusted to 6.5 with 2.5N NaOH with 2.5N NaOH
Canrenone charge canrenone was the same as run Non-sterile The same
as run at sterilized and R3A canrenone: R3A blended. BI: 50 g BI:
50 g charged by the BI: 50 g 16.5 hrs: 110 g 16.5 hrs: 110 g
scheduled listed 16.5 hrs: 110 g 46.5 hrs: 80 g in Table 23
Feedings see Table 23 see Table 23 see Table 23 see Table 23
Harvest time 118.5 hrs. 118.5 hrs. 118.5 hrs. 73.5 hrs.
Bioconversion 93.7% 94.7% 60.0% 68.0%
[0465] TABLE-US-00027 TABLE 23 The Feeding Schedule for Canrenone,
Glucose and Growth Medium in the Development Experiment R3C
Antibiotics R3A R3B R3D Peptone 20 mg kanamycin Canrenone/
Canrenone/ Canrenone/ & Yeast 20 mg Growth Growth Growth
canrenone ext. tetracycline Medium Medium Medium 200 g/2 L Glucose
20 g 100 mg see Table see Table see Table Addition sterile 50% each
in cefalexin in 22 22 22 Time hr. DI g solution g IL water g 50 ml
g/L g/L g/L 0 -- -- -- -- 50 g/0.4 L 50 g/0.4 L 50 g/0.4 L 14.5 16
100 25 50 ml -- -- -- 16.5 -- -- -- -- 110 g/1.2 L 110 g/1.2 L 110
g/1.2 L 20.5 16 140 25 -- -- -- -- 28.5 16 140 25 -- -- -- -- 34.5
16 150 25 -- -- -- -- 40.5 16 150 25 50 ml -- -- -- 46.5 880 130 25
-- -- 80 g/0.8 L -- 52.5 160 120 25 -- -- -- -- 58.5 160 150 25 --
-- -- -- 64.5 160 180 25 50 ml -- -- -- 70.5 160 140 25 -- -- --
--
Due to filamentous growth, a highly viscous fermenter broth was
seen in all four of the runs of this Example. To overcome obstacles
which high viscosity created with respect to aeration, mixing, pH
control and temperature control, the aeration rate and agitation
speed were increased during these runs. Conversions proceeded
satisfactorily under the more severe conditions, but a dense cake
formed above the liquid broth surface. Some unreacted canrenone was
carried out of the broth by this cake.
EXAMPLE 8
[0466] The description and results of Example 8 are summarized in
Table 24. Four fermentation runs were made in which
11.alpha.-hydroxycanrenone was produced by bioconversion of
canrenone. In two of these runs (R4A and R4D), the bioconversion
was conducted in substantially the same manner as runs R3A and R3D
of Example 6. In run R4C, canrenone was converted to
11.alpha.-hydroxycanrenone generally in the manner described in
Example 3. In Run R4B, the process was carried out generally as
described in Example 4, i.e., with sterilization of canrenone and
growth medium in the fermenter just prior to inoculation, all
nitrogen and phosphorus nutrients were introduced at the start of
the batch, and a supplemental solution containing glucose only was
fed into the fermenter to maintain the glucose level as the batch
proceeded. In the latter process (run R4B), glucose concentration
was monitored every 6 hours and glucose solution added as indicated
to control glucose levels in the 0.5 to 1% range. The canrenone
addition schedules for these runs are set forth in Table 25.
TABLE-US-00028 TABLE 24 Descriptions of the Process Development
Experiment of Canrenone Bioconversions Run Number R4A R4B R4C R4D
Medium (g/L) Corn steep liq. 30 the same as run Peptone: 20 the
same as run Yeast extract 15 R4A Yeast ext.: 20 R4A NH4H2PO4 3
Glucose: 20 Glucose 15 OSA 3 ml OSA 0.5 ml pH adjusted to 6.5
adjusted to with 2.5N NaOH 6.5 with 2.5N NaOH Canrenone charge
Canrenone was 160 g canrenone Nonsterile Canrenone was at
sterilized and is sterilized canrenone: sterilized and blended. in
the charged by the blended. BI: 40 g fermenter schedule BI: 40 g
23.5 hrs: 120 g listed in 23.5 hrs: 120 g Table 25 Medium charge
see Table 25 see Table 25 see Table 25 see Table 25 Harvest time
122 hrs. 122 hrs. 122 hrs. 122 hrs. Bioconversion 95.6% 97.6% 95.4%
96.7%
[0467] TABLE-US-00029 TABLE 25 The Feeding Schedule of Canrenone,
Glucose and Growth Medium in the Development Experiment R4C
Antibiotics 20 mg kanamycin Peptone 20 mg tetracycline R4A R4B R4D
& Yeast 100 mg Growth Growth Growth Canrenone ext. 20 g
cefalexin in 50 ml Medium Medium Medium 200 g/2 L Glucose each in
(added in see see see Addition sterile 50% 1 L canrenone Table
Table Table Time hr. water g solution g water g slurry) 24 24 24 14
600 135 25 50 ml -- -- -- 20 -- 100 -- -- -- -- -- 23 -- -- -- --
120 g/ -- 120 g/ 1.2 L 1.2 L 26 -- 100 25 -- -- -- -- 32 -- 135 25
-- -- -- -- 38 500 120 25 50 ml -- -- -- 44 -- 100 25 -- -- -- --
50 -- 100 25 -- -- -- -- 56 -- 150 25 -- -- -- -- 62 500 150 25 50
ml -- -- -- 68 -- 200 25 -- -- -- -- 74 -- 300 25 -- -- -- -- 8--
-- 100 25 -- -- -- -- 86 -- 125 25 -- -- -- -- 92 -- 175 25 -- --
-- -- 98 -- 150 -- -- -- -- -- 104 -- 175 -- -- -- -- -- 110 -- 175
-- -- -- -- -- 116 -- 200 -- -- -- -- --
All fermenters were run under high agitation and aeration during
most of the fermentation cycle because the fermentation beer had
become highly viscous within a day or so after inoculation.
EXAMPLE 9
[0468] The transformation growth media, canrenone addition
schedules, harvest times, and degrees of conversion for the runs of
this Example are set forth in Table 26.
[0469] Four bioconversion runs were carried out substantially in
the manner described for run R4B of Example 8, except as described
below. In run R5B, the top turbine disk impeller used for agitation
in the other runs was replaced with a downward pumping marine is
impeller. The downward pumping action axially poured the broth into
the center of the fermenter and reduced cake formation. Methanol
(200 ml) was added immediately after inoculation in run R5D. Since
canrenone was sterilized in the fermenter, all nutrients except
glucose were added at the start of the batch, obviating the need
for chain feeding of sources of nitrogen, sources of phosphorus or
antibiotics. TABLE-US-00030 TABLE 26 Process Description of the
Process Development Experiment of 10 L Scale Bioconversions Run
Number R5A R5B R5C R5D Medium (g/L) Corn steep liq. 30 the same as
run Peptone: 20 the same as run Yeast Extract 15 R5A Yeast Ext.: 20
R5A NH.sub.4H.sub.2PO.sub.4 3 Glucose: 20 Glucose 15 OSA 3 ml OSA
0.5 ml pH adjusted to 6.5 adjusted to 6.5 with 2.5N NaOH with 2.5N
NaOH Canrenone charge 160 g canrenone 160 g canrenone 160 g
canrenone 160 g canrenone sterilized in sterilized in sterilized in
sterilized in the fermenter the fermenter the fermenter the
fermenter Medium feeding glucose feeding glucose feeding glucose
feeding glucose feeding Harvest time 119.5 hrs. 119.5 hrs. 106
119.5 hrs. Bioconversion 96% 94.1% 88.5% 92.4%
In order to maintain immersion of the solid phase growing above the
liquid surface, growth medium (2 L) was added to each fermenter 96
hours after the beginning of the batch. Mixing problems were not
entirely overcome by either addition of growth medium or use of a
downward pumping impeller (run R5B) but the results of the runs
demonstrated the feasibility and advantages of the process, and
indicated that satisfactory mixing could be provided according to
conventional practices.
EXAMPLE 10
[0470] Three bioconversion runs were carried out substantially in
the manner described in Example 9. The transformation growth media,
canrenone addition schedules, harvest times, and degrees of
conversion for is the runs of this Example are set forth in Table
27: TABLE-US-00031 TABLE 27 Process Description of the Experiment
10 L Scale Bioconversion Run Number R6A R6B R6C Medium (g/L) Corn
steep liq. 30 the same Peptone: 20 Yeast Extract 15 as run Yeast
Ext.: 20 NH.sub.4H.sub.2PO.sub.4 3 R6A Glucose: 20 Glucose 15 OSA
OSA 0.5 ml 0.5 ml pH adjusted to 6.5 adjusted to 6.5 with 2.5N NaOH
with 2.5N NaOH Canrenone charge 160 g canrenone 160 g 160 g
canrenone sterilized in the canrenone sterilized in fermenter
sterilized the fermenter in the fermenter Medium feeding glucose
feeding; glucose glucose 1.3 L medium and feeding; feeding; no 0.8
L sterile 0.5 L other addition water at 71 hrs. medium and 0.5 L
sterile water at 95 hrs Harvest time 120 hrs. 120 hrs. 120 hrs.
Bioconversion 95% 96% 90% Mass Balance 59% 54% 80%
Growth medium (1.3 L) and sterile water (0.8 L) were added after 71
hours in run R6A to submerge mycelial cake which had grown above
the surface of the liquid broth. For the same purpose, growth
medium (0.5 L) and sterile water (0.5 L) were added after 95 hours
in run R6B. Material balance data showed that a better mass balance
could be determined where cake buildup above the liquid surface was
minimized.
EXAMPLE 11
[0471] Fermentation runs were made to compare pre-sterilization of
canrenone with sterilization of canrenone and growth medium in the
transformation fermenter. In run R7A, the process was carried out
as illustrated in FIG. 2, under conditions comparable to those of
runs R2C, R2D, R3A, R3B, R3D, R4A, and R4D. Run R7B was as
illustrated in FIG. 3 under conditions comparable to those of
Examples 4, 9 and 10, and run R4B. The transformation growth media,
canrenone addition schedules, harvest times, and degrees of
conversion for the runs of this Example are set forth in Table 28:
TABLE-US-00032 TABLE 28 Process Description of the Experiment of 10
L Scale Bioconversions Run Number R7A R7B Medium (g/L) corn steep
liq. 30 the same as run Yeast extract 15 R7A
NH.sub.4H.sub.2PO.sub.4 3 Glucose 15 OSA 0.5 ml pH adjusted to 6.5
with 2.5N NaOH Canrenone charge 160 g canrenone 160 g canrenone was
sterilized & was sterilized blended outside in the fermenter
the fermenter Medium charge Glucose feeding; Glucose feeding;
canrenone was no other added with 1.6 L addition growth medium
Harvest time 118.5 hrs. 118.5 hrs. Bioconversion 93% 89%
A mass balance based on the final sample taken from run R7B was
89.5%, indicating that no significant substrate loss or degradation
in bioconversion. Mixing was determined to be adequate for both
runs.
[0472] Residual glucose concentration was above the desired 5-10 g
per liter control range during the initial 80 hours. Run
performance was apparently unaffected by a light cake that
accumulated in the head space of both the fermenters.
EXAMPLE 12
[0473] Extraction efficiency was determined in a series of 1 L
extraction runs as summarized in Table 29. In each of these runs,
steroids were extracted from the mycelium using ethyl acetate (1
L/L fermentation volume). Two sequential extractions were performed
in each run. Based on RP-HPLC, About 80% of the total steroid was
recovered in the first extraction; and recovery was increased to
95% by the second extraction. A third extraction would have
recovered another 3% of steroid. The remaining 2% is lost in the
supernatant aqueous phase. The extract was drawn to dryness using
vacuum but was not washed with any additional solvent. Chasing with
solvent would improve recovery from the initial extraction if
justified by process economics. TABLE-US-00033 TABLE 29 Recovery of
11.alpha.-Hydroxycanrenone at 1 Liter Extraction (% of Total) 1st
2nd 3rd Run Number Extract Extract Extract Supernatant R5A 79% 16%
2% 2% R5A 84% 12% 2% 2% R4A 72% 20% 4% 4% R4A 79% 14% 2% 5% R4B 76%
19% 4% 1% R4B 79% 16% 3% 2% R4B 82% 15% 2% 1% Average 79% 16% 3%
2%
Methyl isobutyl ketone (MIBK) and toluene were evaluated as
extraction/crystallization solvents for 11.alpha.-hydroxycanrenone
at the 1 L broth scale. Using the extraction protocol as described
hereinabove, both MIBK and toluene were comparable to ethyl acetate
in both extraction efficiency and crystallization performance.
EXAMPLE 13
[0474] As part of the evaluation of the processes of FIGS. 2 and 3,
particle size studies were conducted on the canrenone substrate
provided at the start of the fermentation cycle in each of these
processes. As described above, canrenone fed to the process of FIG.
1 was micronized before introduction into the fermenter. In this
process, the canrenone is not sterilized, growth of unwanted
microorganisms being controlled by addition of antibiotics. The
processes of FIGS. 2 and 3 sterilize the canrenone before the
reaction. In the process of FIG. 2, this is accomplished in a
blender before introduction of canrenone into the fermenter. In the
process of FIG. 3, a suspension of canrenone in growth medium is
sterilized in the fermenter at the start of the batch. As discussed
hereinabove, sterilization tends to cause agglomeration of
canrenone particles. Because of the limited solubility of canrenone
in the aqueous growth medium, the productivity of the process
depends on mass transfer from the solid phase, and thus may be
expected to depend on the interfacial area presented by the solid
particulate substrate which in turn depends on the particle size
distribution. These considerations initially served as deterrents
to the processes of FIGS. 2 and 3.
[0475] However, agitation in the blender of FIG. 2 and the
fermentation tank of FIG. 3, together with the action of the shear
pump used for transfer of the batch in FIG. 2, were found to
degrade the agglomerates to a particle size range reasonably
approximate that of the unsterilized and micronized canrenone fed
to the process of FIG. 1. This is illustrated by the particle size
distributions for the canrenone as available at the outset of the
reaction cycle in each of the three processes. See Table 30 and
FIGS. 4 and 5. TABLE-US-00034 TABLE 30 Particle Distributions of
Three Different Canrenone Samples mean Run #: % Sample 45-125.mu.
<180.mu. size .mu. Bioconversion Canrenone 75% 95% -- R3C:
shipment 93.1% (120 h) R4C: 96.3% (120 h) Blended 31.2% 77.2% 139.5
R3A: Sample 94.6% (120 h) R3B: 95.2% (120 h) Sterilized 24.7% 65.1%
157.4 R4B: Sample 97.6% (120 h) R5B: 93.8% (120 h)
From the data in Table 30, it will be noted that agitators and
shear pump were effective to reduce the average particle size of
the sterilized canrenone to the same order of magnitude as the
unsterilized substrate, but a significance size difference remained
in favor of the unsterilized substrate. Despite this difference,
reaction performance data showed that the pre-sterilization
processes were at least as productive as the process of FIG. 1.
Further advantages may be realized in the process of FIG. 2 by
certain steps for further reducing and controlling particle size,
e.g., wet milling of sterilized canrenone, and/or by pasteurizing
rather than sterilizing.
EXAMPLE 14
[0476] A seed culture was prepared in the manner described in
Example 5. At 20 hours, the mycelia in the inoculum fermenter was
pulpy with a 40% PMV. Its pH was 5.4 and 14.8 gpl glucose remained
unused.
[0477] A transformation growth medium (35 L) was prepared having
the composition shown in Table 20. In the preparation of feeding
medium, glucose and yeast extract were sterilized separately and
mixed as a single feed at an initial concentration of 30% by weight
glucose and 10% by weight yeast extract. pH of the feed was
adjusted to 5.7.
[0478] Using this medium, (Table 20), two bioconversion runs were
made for the conversion of canrenone to 11.alpha.-hydroxycanrenone.
Each of the runs was conducted in a 60 L fermenter provided with an
agitator comprising one Rushton turbine impeller and two Lightnin'
A315 impellers.
[0479] Initial charge of the growth medium to the fermenter was 35
L. Micronized and unsterilized canrenone was added to an initial
concentration of 0.5%. The medium in the fermenter was inoculated
with a seed culture prepared in the manner described in Example 5
at an initial inoculation ratio of 2.5%. Fermentation was carried
out at a temperature of 28.degree. C., an agitation rate of 200 to
500 rpm, an aeration rate of 0.5 vvm, and backpressure sufficient
to maintain a dissolved oxygen level of at least 20% by volume. The
transformation culture developed during the production run was in
the form of very small oval pellets (about 1-2 mm). Canrenone and
supplemental nutrients were chain fed to the fermenter generally in
the manner described in Example 1. Nutrient additions were made
every four hours at a ratio of 3.4 g glucose and 0.6 g yeast
extract per liter of broth in the fermenter.
[0480] Set forth in Table 31 are the aeration rate, agitation rate,
dissolved oxygen, PMV, and pH prevailing at stated intervals during
each of the runs of this Example, as well as the glucose additions
made during the batch. Table 32 shows the canrenone conversion
profile. Run R11A was terminated after 46 hours; Run R11B continued
for 96 hours. In the latter run, 93% conversion was reached at 81
hours; one more feed addition was made at 84 hours; and feeding
then terminated. Note that a significant change in viscosity
occurred between the time feeding was stopped and the end of the
run. TABLE-US-00035 TABLE 31 air PMV Time (lpm) rpm % DO Backpress
(%) pH Gluc cc (g/l) Fermentation R11A 0.1 20 200 93 0 2 6.17 5.8 7
20 200 85.1 0 5 6.03 5.5 12.4 20 300 50.2 0 5.43 21.8 20 400 25.5 0
38 6.98 0 29 20 500 17 0 35 5.22 30.2 20 500 18.8 10 5.01 31 20 500
79 10 4.81 1 35.7 20 500 100 10 45 5.57 0 46.2 20 500 23 6 45 5.8 1
Total glucose: 27.5 g/l Total yeast extract: 8.75 g/l Fermentation
R11B 0.1 20 200 92.9 0 2 5.98 5.4 7 20 200 82.3 0 5 5.9 5 12.4 20
300 49.5 0 5.48 21.8 20 400 18 0 40 7.12 0 29 20 500 36.8 0 35 5.1
3 35.7 20 500 94.5 10 4.74 0 46.2 20 500 14.5 6 45 5.32 2 55 20 500
16.7 10 5.31 0.5 58.6 20 500 19.4 15 5.32 1 61.9 20 500 13 15 40
5.36 2 71.7 20 500 13 15 42 5.37 0 81.1 20 500 22.9 15 5.42 2.5
85.6 20 500 22 15 45 5.48 1 97.5 20 500 108 15 45 6.47 0 117.7 20
500 15 7.38 0 Total glucose: 63 g/l Total yeast extract: 14.5
g/l
[0481] TABLE-US-00036 TABLE 32 Concentrations (g/l) Conversion Calc
OH-can Conv.rates (g/l/h) Sample Time OH-can Canren. Total (%)
(g/l) Calculated Measured Fermentation R11A: Canrenone conversion
R11A-0 0.10 0.00 5.41 5.41 R11A-7 7.00 0.18 4.89 5.07 3.58 0.18
0.03 0.03 R11A- 21.80 2.02 2.12 4.14 48.75 2.44 0.15 0.12 22 R11A-
29.00 3.67 4.14 7.81 47.03 4.48 0.28 0.23 29 R11A- 35.70 6.68 1.44
8.12 82.27 7.74 0.49 0.45 36 R11A- 46.20 7.09 0.41 7.51 94.48 8.59
0.08 0.04 46 Fermentation R11B: Canrenone conversion R11B-0 0.1
0.00 5.60 5.60 R11B-7 7.0 0.20 4.98 5.18 3.78 0.19 0.03 0.03 R11B-
21.8 2.51 2.46 4.97 50.49 2.52 0.16 0.16 22 R11B- 29.0 4.48 16.99
21.47 20.87 4.69 0.30 0.27 29 R11B- 35.7 8.18 10.35 18.53 44.16
9.70 0.75 0.55 36 R11B- 55.0 17.03 13.20 30.23 56.33 19.50 0.32
0.36 55 R11B- 58.6 20.80 11.73 32.53 63.95 21.97 0.69 1.05 59 R11B-
61.9 22.19 8.62 30.81 72.02 24.50 0.77 0.42 62 R11B- 71.7 26.62
3.61 30.23 88.06 29.46 0.51 0.45 72 R11B- 81.1 27.13 2.05 29.18
92.97 30.32 0.09 0.05 81 R11B- 85.6 26.87 2.02 28.88 93.02 30.11
-0.04 -0.06 86 R11B- 97.5 23.95 1.71 25.66 93.34 30.22 0.01 -0.25
97 R11B- 117.7 24.10 1.68 25.79 93.47 30.26 0.00 0.01 118
EXAMPLE 15
[0482] Various cultures were tested for effectiveness in the
bioconversion of canrenone to 11.alpha.-hydroxycanrenone according
to the methods generally described above.
[0483] A working cell bank of each of Aspergillus niger ATCC 11394,
Rhizopus arrhizus ATCC 11145 and Rhizopus stolonifer ATCC 6227b was
prepared in the manner described in Example 5. Growth medium (50
ml) having the composition set forth in Table 18 was inoculated
with a suspension of spores (1 ml) from the working cell bank and
placed in an incubator. A seed culture was prepared in the
incubator by fermentation at 26.degree. C. for about 20 hours. The
incubator was agitated at a rate of 200 rpm.
[0484] Aliquots (2 ml) of the seed culture of each microorganism
were used to inoculate transformation flasks containing the growth
medium (30 ml) of Table 18. Each culture was used for inoculation
of two flasks, a total of six. Canrenone (200 mg) was dissolved in
methanol (4 ml) at 36.degree. C., and a 0.5 ml aliquot of this
solution was introduced into each of the flasks. Bioconversion was
carried out generally under the conditions described in Example 5
with additions of 50% by weight glucose solution (1 ml) each day.
After the first 72 hours the following observations were made on
the development of mycelia in the respective transformation
fermentation flasks:
ATCC 11394--good even growth
ATCC 11145--good growth in first 48 hours, but mycelial clumped
into a ball; no apparent growth in last 24 hours;
ATCC 6227b--good growth; mycelial mass forming clumped ball.
[0485] Samples of the broth were taken to analyze for the extent of
bioconversion. After three days, the fermentation using ATCC 11394
provided conversion to 11.alpha.-hydroxycanrenone of 80 to 90%;
ATCC 11145 provided a conversion of 50%; and ATCC 6227b provided a
conversion of 80 to 90%.
EXAMPLE 16
[0486] Using the substantially the method described in Example 15,
the additional microorganisms were tested for effectiveness in the
conversion of canrenone to 11.alpha.-hydroxycanrenone. The
organisms tested and the results of the tests are set forth in
Table 33: TABLE-US-00037 TABLE 33 Cultures tested for Bioconversion
of canrenone to 11 alpha-hydroxy-canrenone approximate Culture
ATTC# media.sup.1 results conversion Rhizopus oryzae 1145 CSL + 50%
-- Rhizopus stolonifer 6227b CSL + 80-90% -- Aspergillus nidulans
11267 CSL + 50% 80% Aspergillus niger 11394 CSL + 80-90% --
Aspergillus ochraceus NRRL CSL + 90% 405 Aspergillus ochraceus
18500 CSL + 90% Bacillus subtilis 31028 P&CSL - 0% 0% Bacillus
subtilis 31028 CSL - 0% 0% Bacillus sp. 31029 P&CSL - 0% 0%
Bacillus sp. 31029 CSL - 0% * Bacillus megaterium 14945 P&CSL +
5% 80%* Bacillus megaterium 14945 CSL + 5% 10%* Trichothecium
roseum 12519 CSL + 80%* 90%* Trichothecium roseum 8685 CSL + 80%*
90%* Streptomyces fradiae 10745 CSL + <5% <10% Streptomyces
fradiae 10745 TSB - * * Streptomyces 13664 CSL - 0% * lavendulae
Streptomyces 13664 TSB - 0% 0% lavendulae Nocardiodes simplex 6946
BP - 0% 0% Nocardiodes simplex 13260 BP - * * Pseudomonas sp. 14696
BP - * * Pseudomonas sp. 14696 CSL + <5% <10% Pseudomonas sp.
14696 TSB - 0% * Pseudomonas sp. 13261 BP + * <10% Pseudomonas
cruciviae 13262 BP # <10% Pseudomonas putida 15175 BP - 0% 0% *
formation of other unidentified products .sup.1Media: CSL--corn
steep liquor; TSB--tryptic soy broth; P&CSL--peptone and acorn
steep liquor; BP--beef extract and peptone.
EXAMPLE 17
[0487] Various microorganisms were tested for effectiveness in the
conversion of canrenone to 9.alpha.-hydroxycanrenone. Fermentation
media for the runs of this Example were prepared as set forth in
Table 34: TABLE-US-00038 TABLE 34 Soybean Meal: dextrose 20 g
soybean meal 5 g NaCl 5 g yeast extract 5 g KH.sub.2PO.sub.4 5 g
water to 1 L pH 7.0 Peptone/yeast extract/glucose: glucose 40 g
bactopeptone 10 g yeast extract 5 g water to 1 L Mueller-Hinton:
beef infusion 300 g casamino acids 17.5 g starch 1.5 g water to 1
L
Fungi were grown in soybean meal medium and in peptone-yeast
extract glucose; atinomycetes and eubacteria were grown in soybean
meal (plus 0.9% by weight Na formate for biotransformations) and in
Mueller-Hinton broth.
[0488] Starter cultures were inoculated with frozen spore stocks
(20 ml soybean meal in 250 ml Erlenmayer flask). The flasks were
covered with a milk filter and bioshield. Starter cultures (24 or
48 hours old) were used to inoculate metabolism cultures (also 20
ml in 250 ml Erlenmeyer flask)--with a 10% to 15% crossing
volume--and the latter incubated for 24 to 48 hours before addition
of steroid substrate for the transformation reaction.
[0489] Canrenone was dissolved/suspended in methanol (20 mg/ml),
filter sterilized, and added to the cultures to a final
concentration of 0.1 mg/ml. All transformation fermentation flasks
were shaken at 250 rpm (2'' throw) in a controlled temperature room
at 26.degree. C. and 60% humidity.
[0490] Biotransformations were harvested at 5 and 48 hours, or at
24 hours, after addition of substrate. Harvesting began with the
addition of ethyl acetate (23 ml) or methylene chloride to the
fermentation flask. The flasks were then shaken for two minutes and
the contents of each flask poured into a 50 ml conical tube. To
separate the phases, tubes were centrifuged at 4000 rpm for 20
minutes in a room temperature unit. The organic layer from each
tube was transferred to a 20 ml borosilicate glass vial and
evaporated in a speed vac. Vials were capped and stored at
-20.degree. C.
[0491] To obtain material for structure determination,
biotransformations were scaled up to 500 ml by increasing the
number of shake flask fermentations to 25. At the time of harvest
(24 or 48 hours after addition of substrate), ethyl acetate was
added to each flask individually, and the flasks were capped and
put back on the shaker for 20 minutes. The contents of the flasks
were then poured into polypropylene bottles and centrifuged to
separate the phases, or into a separatory funnel in which phases
were allowed to separate by gravity. The organic phase was dried,
yielding crude extract of steroids contained in the reaction
mixture.
[0492] Reaction product was analyzed first by thin layer
chromatography on silica gel (250 .mu.m) fluorescence backed plates
(254 nm). Ethyl acetate (500 .mu.L was added to each vial
containing dried ethyl acetate extract from the reaction mixture.
Further analyses were conducted by high performance liquid
chromatography and mass spectrometry. TLC plates were developed in
a 95:5 v/v chloroform/methanol solvent mixture.
[0493] Further analysis was conducted by high performance liquid
chromatography and mass spectrometry. A waters HPLC with Millennium
software, photodiode array detector and autosampler was used.
Reversed phase HPLC used a waters NovaPak C-18 (4 .mu.m particle
size) RadialPak 4 mm cartridge. The 25 minute linear solvent
gradient began with the column initialized in water:acetonitrile
(75:25), and ended at water:acetonitrile (25:75). This was followed
by a three minute gradient to 100% acetonitrile and 4 minutes of
isocratic wash before column regeneration in initial
conditions.
[0494] For LC/MS, ammonium acetate was added to both the
acetonitrile and water phases at a concentration of 2 nM.
Chromatography was not significantly affected. Eluant from the
column was split 22:1, with the majority of the material directed
to the PDA detector. The remaining 4.5% of the material was
directed to the electrospray ionizing chamber of an Sciex API III
mass spectrometer. Mass spectrometry was accomplished in positive
mode. An analog data line from the PDA detector on the HPLC
transferred a single wave length chromatogram to the mass
spectrometer for coanalysis of the UV and MS data.
[0495] Mass spectrometric fragmentation patterns proved useful in
sorting from among the hydroxylated substrates. The two expected
hydroxylated canrenones, 11.alpha.-hydroxy- and 9.alpha.-hydroxy,
lost water at different frequencies in a consistent manner which
could be used as a diagnostic. Also, the 9.alpha.-hydroxycanrenone
formed an ammonium adduct more readily than did
11.alpha.-hydroxycanrenone. Set forth in Table 35 is a summary of
the TLC, HPLC/UV and LC/MS data for canrenone fermentations,
showing which of the tested microorganism were effective in the
bioconversion of canrenone to 9.alpha.-hydroxycanrenone. Of these,
the preferred microorganism was Corynespora cassiicola ATCC 16718.
TABLE-US-00039 TABLE 35 Summary of TLC, HPLC/UV, and LC/MS Data for
Canrenone Fermentations Evidence for 9.alpha.OH-canrenone HPLC-peak
MS: TLC spot at 9.alpha.OH- 357 (M + H), at 9.alpha.QH- canrenone
339 (-H.sub.2O) & Culture AD w/UV 375 (+NH.sub.4) Absidia
coerula ATCC n y y/n 6647 Absidia glauca ATCC n 22752 Actinomucor
elegans ATCC tr y tr 6476 Aspergillus flavipes tr ATCC 1030
Aspergillus fumigatus tr y n ATCC 26934 Aspergillus nidulans tr y y
ATCC 11267 Aspergillus niger ATCC n y y 16888 Aspergillus niger
ATCC n y n 26693 Aspergillus ochraceus n y n ATCC 18500 Bacterium
cyclo-oxydans n tr n (Searle) ATCC 12673 Beauveria bassiana ATCC tr
y y 7159 Beauveria bassiana ATCC y y y 13144 Botrvospnaeria obtusa
y tr tr IMI 038560 Calonectria decora ATCC n tr y 14767 Chaetomium
cochliodes tr tr y/n ATCC 10195 Comomonas testosteroni tr tr n
(Searle) ATCC 11996 Corvnespora cassiicola y y y ATCC 16718
Cunninghamella y y y blakesleana ATCC 8688a Cunninghamella y y y
echinulata ATCC 3655 Cunninghamella elegans y y y ATCC 9245
Curcularia clavata ATCC n y y/n 22921 Curvularia lunata ATCC y n n
12071 Cylindrocarpon tr n n radicicola (Searle) ATCC 11011
Epicoccum humucola ATCC y y y 12722 Epicoccum oryzae ATCC tr tr tr
12724 Fusarium oxysporum ATCC tr 7601 Fusarium oxysporum f. sp. n
cepae ATCC 11171 Gibberella fujikuroi tr y y ATCC 14842 Gliocladium
deliguescens y tr tr ATCC 10097 Gongronella butieri ATCC y y UV? y
22822 Hypomyces chrvsospermus y y y Tul. IMI 109891 Lipomyces
lipofer ATCC n 10792 Melanospora ornata ATCC tr n n 26180
Mortierella isabellinay y y n ATCC 42613 Mucor grisco-cyanus ATCC n
1207a Mucor mucedo ATCC 4605 tr y y Mycobacterium fortuitumn NRRL
B8119 Myrothecium verrucaria tr tr y ATCC 9095 Nocardia aurentia n
tr n (Searle) ATCC 12674 Nocardia cancicruria y y n ATCC 31548
Nocardia corallina ATCC n 19070 Paecilomyces carneus n y n ATCC
46579 Penicillium chrysogenum n ATCC 9480 Penicillium patulum ATCC
y y y/n 24550 Penicillium purpurogenum tr y y ATCC 46581 Pithomyces
atro- tr y tr olivaceus ATCC 6651 Pithomyces cynodontis n tr tr
ATCC 26150 Phvcomvces blakesleeanus y y y/n IMI 118496 Pvcnosporium
sp. ATCC y y y/n 12231 Rhizopogon sp. ATCC 36060 Rhizopus arrhizus
ATCC tr y n 11145 Rhizopus stolonifer ATCC n 6227b Rhodococcus egui
ATCC n tr n 14887 Rhodococcus egui ATCC tr tr n 21329 Rhodococcus
sp. ATCC n n n 19070 Rhodococcus rhodochrous n tr n ATCC 19150
Saccharopolyspora y y y erythaea ATCC 11635 Sepedonium
ampullosporum n n n IMI 203033 Sepedonium chrvsospermum n ATCC
13378 Septomyxa affinis ATCC n y UV? y/n 6737 Stachylidium bicolor
y y y/n ATCC 12672 Streptomyces n californicus ATCC 15436
Streptomyces n cinereocrocatus ATCC 3443 Streptomyces coelicolor n
ATCC 10147 Streptomyces flocculus ATCC 25453 Streptomyces fradiae n
ATCC 10745 Streptomyces griseus n subsp. griseus ATCC 13968
Streptomyces griseus n ATCC 11984 Streptomyces hydrogenans n ATCC
19631 Streptomyces y y y hvgroscopicus ATCC 27438 Streptomyces
lavendulae n Panlab 105 Streptomyces n paucisporogenes ATCC 25489
Streptomyces n tr tr purpurascens ATCC 25489 Streptomyces
roseochromogenes ATCC 13400 Streptomyces spectabilis n ATCC 27465
Stysanus microsporus ATCC 2833 Syncephalastrum n racemosum ATCC
18192 Thamnidium elegans ATCC 18191 Thamnostylum piriforme y tr y
ATCC 8992 Thielavia terricolan n ATCC 13807 Trichoderma viride ATCC
n 26802 Trichothecium roseum tr y y/n ATCC 12543 Verticillium
theobromae y tr tr ATCC 12474
EXAMPLE 18
[0496] Various cultures were tested for effectiveness in the
bioconversion of androstendione to 11.alpha.-hydroxyandrostendione
according to the methods generally described above.
[0497] A working cell bank of each of Aspergillus ochraceus NRRL
405 (ATCC 18500); Aspergillus niger ATCC 11394; Aspergillus
nidulans ATCC 11267; Rhizopus oryzae ATCC 11145; Rhizopus
stolonifer ATCC 6227b; Trichothecium roseum ATCC 12519 and ATCC
8685 was prepared essentially in the manner described in Example 4.
Growth medium (50 ml) having the composition set forth in Table 18
was inoculated with a suspension of spores (1 ml) from the working
cell bank and placed in an incubator. A seed culture was prepared
in the incubator by fermentation at 26.degree. C. for about 20
hours. The incubator was agitated at a rate of 200 rpm.
[0498] Aliquots (2 ml) of the seed culture of each microorganism
were used to inoculate transformation flasks containing the growth
medium (30 ml) of Table 15. Each culture was used for inoculation
of two flasks, a total of 16. Androstendione (300 mg) was dissolved
in methanol (6 ml) at 36.degree. C., and a 0.5 ml aliquot of this
solution was introduced into each of the flasks. Bioconversion was
carried out generally under the conditions described in Example 6
for 48 hours. After 48 hours samples of the broth were pooled and
extracted with ethyl acetate as in Example 17. The ethyl acetate
was concentrated by evaporation, and samples were analyzed by thin
layer chromatography to determine whether a product having a
chromatographic mobility similar to that of
11.alpha.-hydroxy-androstendione standard (Sigma Chemical Co., St.
Louis) was present. The results are shown in Table 36. Positive
results are indicated as "+". TABLE-US-00040 TABLE 36 Bioconversion
of androstendione to 11 alpha- hydroxy-androstendione TLC Culture
ATTC# media results Rhizopus oryzae 11145 CSL + Rhizopus stolonifer
6227b CSL + Aspergillus nidulans 11267 CSL + Aspergillus niger
11394 CSL + Aspergillus ochraceus NRRL 405 CSL + Aspergillus
ochraceus 18500 CSL + Trichothecium roseum 12519 CSL +
Trichothecium roseum 8685 CSL +
[0499] The data in Table 36 demonstrate that each of listed
cultures was capable of producing a compound from androstendione
having the same Rf value as that of the
11.alpha.-hydroxyandrostendione standard.
[0500] Aspergillus ochraceus NRRL 405 (ATCC 18500) was retested by
the same procedure described above, and the culture products were
isolated and purified by normal phase silica gel column
chromatography using methanol as the solvent. Fractions were
analyzed by thin layer chromatography. TLC plates were Whatman K6F
silica gel 60 .ANG., 10.times.20 size, 250.mu. thickness. The
solvent mixture was chloroform:methanol, 95:5, v/v. The
crystallized product and 11.alpha.-hydroxyandrostendione standard
were both analyzed by LC-MS and NMR spectroscopy. Both compounds
yielded similar profiles and molecular weights.
EXAMPLE 19A
[0501] Various microorganisms were tested for effectiveness in the
bioconversion of androstendione to 11.beta.-hydroxyandrostendione
essentially by the methods described above in Examples 17 and
18.
[0502] Cultures of each of Aspergillus fumigatus ATCC 26934,
Aspergillus niger ATCC 16888 and ATCC 26693, Epicoccum oryzae ATCC
7156, Curvularia lunata ATCC 12017, Cunninghamella blakesleeana
ATCC 8688a, and Pithomyces atro-olivaceus IFO 6651 were grown
essentially in the manner described in Example 17. Growth and
fermentation media (30 ml) had the composition shown in Table
34.
[0503] The 11.beta.-hydroxylation of androstendione by the
above-listed microorganisms was analyzed using essentially the same
methods of product identification described in Examples 17 and 18.
The results are set forth in Table 19A-1. TABLE-US-00041 TABLE
19A-1 11.beta.-Hydroxylation of Androstendione by Various
Microorganisms Organism TLC LC/MS Aspergillus fumigatus + + ATCC
26934 Aspergillus niger + + ATCC 16888 and ATCC 26693 Epicoccum
oryzae + + ATCC 7156 Curvularia lunata + + ATCC 12017
Cunninghamella blakesleeana + + ATCC 8688a Pithomyces
atro-olivaceus + + IFO 6651
[0504] In Table 19A-1, a "+" indicates a positive result, i.e., an
R.sub.f as expected in thin layer chromatography and an
approximately correct molecular weight upon LC/MS.
[0505] These results demonstrate that the listed is micro-organisms
are capable of carrying out the 11.beta.-hydroxylation of
androstendione.
EXAMPLE 19B
[0506] Various microorganisms were tested for effectiveness in the
conversion of mexrenone to 11.beta.-hydroxymexrenone. Fermentation
media for this example were prepared as described in Table 34.
[0507] The fermentation conditions and analytical methods were the
same as those in Example 17. TLC plates and the solvent system were
as described in Example 18. The rationale for chromatographic
analysis is as follows: 11.alpha.-hydroxymexrenone and
11.alpha.-hydroxycanrenone have the same chromatographic mobility.
11.alpha.-hydroxycanrenone and 9.alpha.-hydroxycanrenone exhibit
the same mobility pattern as 11.alpha.-hydroxyandrostendione and
11.beta.-hydroxyandrostendione. Therefore,
11.beta.-hydroxymexrenone should have the same mobility as
9.alpha.-hydroxycanrenone. Therefore, compounds extracted from the
growth media were run against 9.alpha.-hydroxycanrenone as a
standard. The results are shown in Table 36. TABLE-US-00042 TABLE
37 Summary of TLC Data for 11.beta.-hydroxymexrenone Formation from
Mexrenone Spot Microorganism Medium.sup.1 Character.sup.2 Absidia
coerula ATCC 6647 M, S strong Aspergillus niger ATCC S, P faint (S)
16888 ? (P) Beauveria bassiana ATCC P strong 7159 Beauveria
bassiana ATCC S, P ?, ? 13144 Botryosphaeria obtusa IMI faint
038560 Cunninghamella blakesleeana ATCC 8688a S, P strong
echinulata ATCC 3655 S, P strong elegans ATCC 9245 S, P strong
Curvularia lunata ATCC S strong 12017 Gongronella butleri ATCC S, P
strong 22822 Penicillium patulum ATCC S, P strong 24550 Penicillium
purpurogenum S, P strong ATCC 46581 Pithomyces atro-olivaceus S, P
faint IFO 6651 Rhodococcus equi ATCC M faint 14887
Saccharopolyspora erythaea M, SF faint ATCC 11635 Streptomyces
hygroscopicus M, SF strong ATCC 27438 Streptomyces purpurascens M,
SF faint ATCC 25489 Thamnidium elegans ATCC S, P faint 18191
Thamnostylum piriforme S, P faint ATCC 8992 Trichothecium roseum
ATCC P, S faint (P) 12543 ? (S) .sup.1M = Mueller-Hinton P = PYG
(peptone/yeast extract/glucose) S = soybean meal SF = soybean meal
plus formate .sup.2? = questionable difference from no substrate
control
[0508] These data suggest that the majority of the organisms listed
in this table produce a product similar or identical to
11.beta.-hydroxymexrenone from mexrenone.
EXAMPLE 19C
[0509] Various microorganisms were tested for effectiveness in the
conversion of mexrenone to 11.alpha.-hydroxymexrenone,
.DELTA..sup.1,2-mexrenone, 60-hydroxymexrenone,
12.beta.-hydroxymexrenone, and 9.alpha.-hydroxymexrenone. Mexrenone
can be prepared in the manner set forth in Weier, U.S. Pat. No.
3,787,396 and R. M. Weier et al., J. Med. Chem., Vol. 18, pp.
817-821 (1975), which are incorporated herein by reference.
Fermentation media were prepared as described in Example 17, except
that mexrenone was included. The fermentation conditions were
essentially the same as those in Example 17; analytical methods
were also the same as those in Examples 17 and 18. TLC plates and
the solvent system were as described in Examples 17 and 18.
[0510] The microorganisms tested and results obtained therewith are
shown in Table 19C-1. TABLE-US-00043 TABLE 19C-1 Production of
11.alpha.-hydroxymexrenone from Mexrenone by Various Microorganisms
Organism TLC HPLC m/z 457:399 Beauveria bassiana + + 5:1 ATCC 7159
Beauveria bassiana + + 10:1 ATCC 13144 Mortierella isabella + + 1:1
ATCC 42613 Cunninghamella blakesleeana + + 1:1 ATCC 8688a
Cunninghamella echinulata + + 1:2 ATCC 3655 Cunninghamella elegans
+ + 1:1 ATCC 9245 Absidia coerula + + 1:1 ATCC 6647 Aspergillus
niger + + 4:1 ATCC 16888 Gongronella butieri + + 3:1 ATCC 22822
Pithomyces atro-olivaceus + + 3:1 ATCC 6651 Streptomyces
hygroscopicus + + 3:1 ATCC 27438
[0511] In Table 19C-1, a "+" indicates a positive result, i.e., an
R.sub.f as expected in thin layer chromatography and a retention
time as expected in HPLC m/z 417:399 indicates the peak height
ratio of the 417 molecule (hydroxymexrenone) and the 399 molecule
(mexrenone). The standard has a 10:1 ratio of peak height for m/z
417 to m/z 399.
[0512] The product obtained from Beauveria bassiana ATCC 13144 was
isolated from the incubation mixture and analyzed by NMR, and the
structural profile thereby confirmed to be
11.alpha.-hydroxymexrenone. By analogy, the products obtained from
the other microorganisms listed in Table 19C-1 were also presumed
to be 11.alpha.-hydroxymexrenone. TABLE-US-00044 TABLE 19C-2
Production of .DELTA..sup.1,2-Mexrenone from Mexrenone by Various
Microorganims Organism m/z 399 HPLC TLC Rhodococcus egui + + + ATCC
148875 Bacterium cyclo-oxydans + + + ATCC 12673 Comomonas
testosteroni + + + ATCC 11996 Nocardia aurentia + + + ATCC 12674
Rhodococcus egui + + + ATCC 21329
[0513] In Table 19C-2, a "+" indicates a positive result, e.g., an
R.sub.f as expected in thin layer chromatography, a retention time
as expected in HPLC, etc.
[0514] The product obtained from Bacterium cyclo-oxydans ATCC 12673
was isolated from the incubation mixture and analyzed by NMR, and
the structural profile thereby confirmed to be
.DELTA..sup.1,2-mexrenone. By analogy, the products obtained from
the other microorganisms listed in Table 19C-2 were also presumed
to be .DELTA..sup.1,2-mexrenone.
Production of 6.beta.- and 12.beta.-hydroxymexrenone
[0515] Mortierella isabella ATCC 42613 was grown as in Example 17
in the presence of mexrenone. The fermentation products were
isolated and purified by flash chromatography. The purified
products were analyzed by LC/MS as in Examples 17 and 18, and
proton NMR and carbon-13 NMR. The data indicated that the products
included 6.beta.- and 12.beta.-hydroxymexrenone. TABLE-US-00045
TABLE 19C-3 Production of 9.alpha.-Hydroxymexrenone from Mexrenone
by Various Microorganisms Organism m/z 417 HPLC TLC Streptomyces
hygroscopicus + + + ATCC 27438 Gongronella butleri + + + ATCC 22822
Cunninghamella blakesleeana + + + ATCC 8688a Cunninghamella
echinulata + + + ATCC 3655 Cunninghamella elegans + + + ATCC 9245
Mortierella isabellina + + + ATCC 42613 Absidia coerula + + + ATCC
6647 Beauveria bassiana + + + ATCC 7159 Beauveria bassiana + + +
ATCC 13144 Aspergillus niger + + + ATCC 16888
[0516] The microorganisms listed in Table 19C-3 were grown under
the same conditions as in Example 17, in the presence of mexrenone.
The fermentation products were analyzed by TLC and LC/MS as in
Examples 17 and 18. A "+" indicates a positive result, e.g., an
R.sub.f as expected in thin layer chromatography, a retention time
as expected in HPLC, etc. The data suggest that the products
include 9.alpha.-hydroxymexrenone.
EXAMPLE 19D
[0517] Various microorganisms were tested for effectiveness in the
conversion of canrenone to .DELTA..sup.9,11-canrenone. The
fermentation media and growth conditions were essentially the same
as in Example 17, except that canrenone was included in the medium.
The analytical methods were as described in Examples 17 and 18. The
microorganisms and results are shown in Table 19D-1, below.
TABLE-US-00046 TABLE 19D-1 Production of .DELTA..sup.9,11-Canrenone
from Canrenone by Various Microorganisms Organism m/z 339 HPLC TLC
Bacterium cyclo-oxydans + + + ATCC 12673 Comomonas testosteroni + +
+ ATCC 11996 Cylindrocarpon radicicola + + + ATCC 11011
Paecilomyces carneus + + + ATCC 46579 Septomyxa affinis + + + ATCC
6737 Rhodococcus spp. + + + ATCC 19070
[0518] The fermentation products were analyzed by TLC and LC/MS as
in Examples 17 and 18. A "+" indicates a positive result, e.g., an
R.sub.f as expected in thin layer chromatography, a retention time
as expected in HPLC, etc.
[0519] The product obtained from Comomonas testosteroni ATCC 11996
was isolated from the growth medium and analyzed by UV
spectroscopy. The spectroscopic profile confirmed the presence of
.DELTA..sup.9,11-canrenone. By analogy, the products obtained from
the other microorganisms listed in Table 19D-1 were also presumed
to be .DELTA..sup.9,11-canrenone.
EXAMPLE 20A
[0520] Scheme 1: Step 1: Method A: Preparation of
5'R(5'.alpha.),7'.beta.-20'-Aminohexadecahydro-11'.beta.-hydroxy-10'.alph-
a.,13'.alpha.-dimethyl-3',5-dioxospiro[furan-2(3H),17'.alpha.(5'H)-[7,4]me-
theno[4H[cyclopenta[a]phenanthrene]-5'-carbonitrile.
[0521] Into a 50 gallon glass-line reactor was charged 61.2 L (57.8
kg) of DMF followed by 23.5 Kg of 11-hydroxycanrenone 1 with
stirring. To the mixture was added 7.1 kg of lithium chloride. The
mixture was stirred for 20 minutes and 16.9 kg of acetone
cyanohydrin was charged followed by 5.1 kg of triethylamine. The
mixture was heated to 85.degree. C. and maintained at this
temperature for 13-18 hours. After the reaction 353 L of water was
added followed by 5.6 kg of sodium bicarbonate. The mixture was
cooled to 0.degree. C., transferred to a 200 gallon glass-lined
reactor and quenched with 130 kg of 6.7% sodium-hypochlorite
solution slowly. The product was filtered and washed with
3.times.40 L portions of water to give 21.4 kg of the product
enamine. ##STR195##
[0522] H.sup.1 NMR (DMSO-d.sub.6): 7.6 (2H, bd), 4.53 (1H, d,
J=5.9), 3.71 (1H, m), 3.0-1.3 (17H, m), 1.20 (5H, m), 0.86 (3H, s),
0.51 (1H, t, J=10).
EXAMPLE 20B
Preparation of
7.alpha.-cyano-11.alpha.,17-dihydroxy-3-oxo-17.alpha.-pregn-4-ene-21-carb-
oxylic acid, .gamma.-lactone
[0523] ##STR196##
[0524] 50.0 g of 11-hydroxycanrenone and 150.0 mL of
dimethylacetamide were added to a clean, dry three-necked flask
equipped with a mechanical stirrer, condenser, thermocouple and
heating mantle. 16.0 mL of a sulfuric acid solution (prepared by
mixing 50.0 mL of sulfuric acid (98.7% Baker grade) with 50.0 mL of
water) was added to this mixture. A sodium cyanide solution
comprising 15.6 g of sodium cyanide and 27.0 mL of water was then
added.
[0525] The resulting mixture was heated at 80.degree. C. for 7
hours, the degree of conversion being periodically checked by TLC
or HPLC. After approximately 7 hours, HPLC of the mixture indicated
the presence of the 7-cyano compound. The mixture was then stirred
overnight and allowed to cool to room temperature (about 22.degree.
C.). 200 mL of water was added to the mixture followed by 200 mL of
methylene chloride and the resulting two phase mixture stirred and
the phases were then allowed to separate. The aqueous layer was a
gel. 100 mL of sodium bicarbonate solution was added to the aqueous
layer in an unsuccessful attempt to break up the gel. The aqueous
layer was then discarded.
[0526] The separated methylene chloride layer was washed with 100
mL of water and the resulting two phase mixture stirred. The phases
were then allowed to separate and the separated methylene chloride
layer was filtered through 200 g of silica gel (Aldrich 200-400
mesh, 60 .ANG.). The filtrate was concentrated to dryness under
reduced pressure at 45.degree. C. using a water aspirator to
provide about 53.9 g of a crude solid product. The crude solid
product then was dissolved in 50 mL of methylene chloride and
treated with 40 mL of 4N hydrochloric acid in a separatory funnel
and the two phase mixture allowed to separate. The methylene
chloride layer was washed with 50 mL of water. The combined aqueous
layers were extracted with 50 mL of methylene chloride chloride.
The combined methylene chloride layers were then dried over sodium
sulfate to provide 45 g of a solid which was a mixture of
11.alpha.-hydroxycanrenone and the product,
7.alpha.-cyano-11.alpha.,17-dihydroxy-3-oxo-17.alpha.-pregn-4-ene-21-carb-
oxylic acid, .gamma.-lactone.
[0527] A sample of the product was analyzed by HPLC (column: 25
cm.times.4.6 mm, 5.mu. Altima C.sub.18LL); solvent gradient:
solvent A=water/trifluoroacetic acid=99.9/0.1, solvent
B=acetonitrile/trifluoroacetic acid=99.9/0.1, flow rate=1.00
mL/minute, gradient=65:30 (v/v) (A:B--initial), 35:65 (v/v)
(A:B--after 20 minutes), 10:90 (v/v) (A:B--after 25 minutes); diode
array detector) which revealed a .lamda..sub.max of 238 nm.
[0528] The reaction mixture was analyzed by HPLC-NMR using the
following conditions: HPLC--column: Zorbax RX-C8 (25 cm.times.4.6
mm, 5.mu.) using a solvent gradient from 75% D.sub.2O, 25%
acetonitrile to 25% D.sub.2O, 75% acetonitrile over 25 minutes with
a flow of 1 mL/minute; .sup.1H NMR (obtained using WET solvent
suppression): 5.84 (s, 1H), 4.01 (m, 1H), 3.2 (m, 1H), 2.9-1.4 (m,
integral not meaningful due to solvent suppression of
acetonitrile), 0.93-0.86 (s, overlapping 3H, and t, 2H).
EXAMPLE 20C
Preparation of
5.beta.,7.alpha.-dicyano-17-hydroxy-3-oxo-17.alpha.-pregnane-21-carboxyli-
c acid, .gamma.-lactone
[0529] ##STR197##
[0530] 102 g (0.3 mol) of
17-hydroxy-3-oxo-17.alpha.-pregna-4,6-diene-21-carboxylic acid,
.gamma.-lactone (canrenone) was slurried with 46.8 g (0.72 mol) of
potassium cyanide, 78.6 mL (1.356 mols) of acetic acid, and 600 mL
of methanol in a three liter, three neck, round bottom flask. 64.8
mL (0.78 mol) of pyrrolidine was added to the mixture and the
combined slurry heated to reflux (64.degree. C.) and maintained for
about 1.5 hours. The temperature of the slurry was then lowered to
25.degree. C. to 30.degree. C. over a ten minute period with a
cooling bath. 120 mL of a concentrated hydrochloric acid was slowly
added during the cooldown as a tan colored solid precipitated.
[0531] The mixture was stirred at 25.degree. C. to 30.degree. C.
for 1.5 hours, then an additional 500 mL of water added in 30
minutes. The mixture was cooled to 5.degree. C. with an ice bath
and the pH adjusted from 3 to 5.5 (monitored using pH strips) with
the addition of 100 mL of aqueous 9.5M sodium hydroxide (0.95 mol).
Excess cyanide was destroyed with the addition of household bleach.
25 mL (0.020 mol) was added to achieve a negative starch iodide
test. The cold mixture (10.degree. C.) was filtered and the solid
washed with water until the rinse exhibited a neutral pH (pH
strips). The solid was dried at 60.degree. C. to a constant weight
of 111.4 g.
[0532] The isolated solid melted at 244.degree. C. to 246.degree.
C. on a Fisher Johns block. A methanol solution containing the
solid exhibited no absorption throughout the UV region of 210 to
240 nm. IR (CHCl.sub.3)cm.sup.-1 2222 (cyanide), 1775 (lactone),
1732 (3-keto). .sup.1H NMR (pyridine d.sub.5) ppm 0.94 (s, 3H),
1.23 (s, 3H).
EXAMPLE 21A
Scheme 1: Step 2: Preparation of
4'S(4'.alpha.),7'.alpha.-Hexadecahydro-11'.alpha.-hydroxy-10'.beta.,13'.b-
eta.-dimethyl-3',5,20'-trioxospiro(furan-2(3H),17'.beta.-[4,7]methano[17H]-
cyclopenta[a]phenanthrene]-5'.beta.(2'H)-carbonitrile
[0533] Into a 200 gallon glass-lined reactor was charged 50 kg of
enamine 2, approximately 445 L of 0.8 N dilute hydrochloric acid
and 75 L of methanol. The mixture was heated to 8.degree. C. for 5
hours, cooled to 0.degree. C. for 2 hours. The solid product was
filtered to give 36.5 kg of dry product diketone. ##STR198##
[0534] H.sup.1 NMR (DMSO-d.sub.6): 4.53 (1H, d, J=6), 3.74 (2H, m),
2.73 (1H, dd, J=14, 7) 2.65-2.14 (8H, m), 2.05 (1H, t, J=11),
1.98-1.71 (4H, m), 1.64 (1H, m), 1.55 (1H, dd, J=13, 5), 1.45-1.20
(7H, m), 0.86 (3H, s).
EXAMPLE 21B
Scheme 1: Steps 1 and 2: In Situ Preparation of
4'S(4'.alpha.),7.alpha.-Hexadecahydro-11.alpha.-hydroxy-10'.beta.,13'.bet-
a.-dimethyl-3',5,20'-trioxospiro[furan-2(3H),17'.beta.-[4,7]methano-[17H]c-
yclopenta[a]phenanthrene]-5'.beta.(2'H)-carbonitrile from
11.alpha.-hydroxycanrenone
[0535] Into a reactor fitted with a cooling condenser, mechanical
stirrer, heating mantle and controller, and funnel was charged 100
g (280.54 mmol) of 11-hydroxycanrenone prepared as in the manner of
Example 1 followed by 300 mL of dimethylacetamide (Aldrich). The
mixture was stirred until the 11-hydroxycanrenone dissolved. To
this mixture was added 31.5 mL of 50% sulfuric acid (Fisher) which
caused the temperature of the resulting mixture to rise about
10.degree. C. to 15.degree. C. A sodium cyanide solution prepared
by dissolving 31.18 g (617.20 mmol) (Aldrich) of sodium cyanide in
54 mL of deionized water was then added to the
11.alpha.-hydroxycanrenone mixture over a 2 to 3 minute period. The
temperature of the resulting mixture rose about 20.degree. C. to
25.degree. C. after addition of the sodium cyanide solution.
[0536] The mixture was heated to 80.degree. C. and maintained at
this temperature for 2-3 hours. Once HPLC analysis indicated the
reaction for the conversion of the 11.alpha.-hydroxycanrenone to
the enamine was substantially complete (greater than 98%
conversion), the heat source was removed. Without isolation of the
enamine contained in the mixture, an additional 148 mL of 50%
sulfuric acid was added to the mixture over a 3-5 minute period.
Over a 10 minute period 497 mL of deionized water was then added to
the mixture.
[0537] The mixture was heated to 102.degree. C. and maintained at
that temperature until approximately 500 g of distillate had been
removed from the mixture. During the reaction/distillation, 500 mL
of deionized water was added to the mixture in four separate 125 mL
portions. Each portion was added to the mixture after an equivalent
amount of distillate (approximately 125 mL) had been removed. The
reaction continued for over 2 hours. When HPLC analysis indicated
that the reaction hydrolyzing the enamine to the diketone was
substantially completed (greater than 98% conversion), the mixture
was cooled to about 80.degree. C. over a 20 minute period.
[0538] The mixture was filtered through a glass funnel. The reactor
was rinsed with 1.2 L of deionized water to remove residual
product. The solid on the filter was washed three times using
approximately equal portions (about 0.4 L) of the rinse water. A 1
L solution of methanol and deionized water (1:1 v/v) was prepared
in the reactor and the filtrate was washed with 500 mL of this
solution. The filtrate was then washed a second time with the
remaining 500 mL of the methanol/water solution. Vacuum was applied
to the funnel to dry the filtrate sufficiently for transfer. The
filtrate was transferred to a drying oven where it was dried under
vacuum for 16 hours to yield 84 g of dry product diketone,
4'S(4'.alpha.),7'.alpha.-Hexadecahydro-11'.alpha.-hydroxy-10'.beta.,13'.b-
eta.-dimethyl-3',5,20'-trioxospiro[furan-2(3H),17'.beta.-[4,7]methano-[17H-
]cyclopenta[a]phenanthrene]-5'.beta.(2'H)-carbonitrile. HPLC assay
indicated 94% of the desired diketone.
EXAMPLE 22
Scheme 1: Step 3A: Method A: Preparation of Methyl Hydrogen
11.alpha.,17.alpha.-Dihydroxy-3-oxopregn-4-ene-7.alpha.,21-dicarboxylate,
.gamma.-Lactone
[0539] A 4-neck 5-L bottom flask was equipped with mechanical
stirrer, pressure equalizing addition funnel with nitrogen inlet
tube, thermometer and condenser with bubbler. The bubbler was
connected via tygon tubing to two 2-L traps, the first of which was
empty and placed to prevent back-suction of the material in the
second trap (1 L of concentrated sodium hypochlorite solution) into
the reaction vessel. The diketone 3 (79.50 g; [weight not corrected
for purity, which was 85% ]) was added to the flask in 3 L
methanol. A 25% methanolic sodium methoxide solution (64.83 g) was
placed in the funnel and added dropwise, with stirring under
nitrogen, over a 10 minute period. After the addition was complete,
the orangish yellow reaction mixture was heated to reflux for 20
hours. After this period, 167 mL of 4 N HCl was added (Caution: HCN
evolution at this point!) dropwise through the addition funnel to
the still refluxing reaction mixture. The reaction mixture
lightened in color to a pale golden orange. The condenser was then
replaced with a take-off head and 1.5 L of methanol was removed by
distillation while 1.5 L of water was simultaneously added to the
flask through the funnel, in concert with the distillation rate.
The reaction mixture was cooled to ambient temperature and
extracted twice with 2.25 L aliquots of methylene chloride. The
combined extracts were washed successively with 750 mL aliquots of
cold saturated NaCl solution, 1N NaOH and again with saturated
NaCl. The organic layer was dried over sodium sulfate overnight,
filtered and reduced in volume to -250 mL in vacuo. Toluene (300
mL) was added and the remaining methylene chloride was stripped
under reduced pressure, during which time the product began to form
on the walls of the flask as a white solid. The contents of the
flask were cooled overnight and the solid was removed by
filtration. It was washed with 250 mL toluene and twice with 250 mL
aliquots of ether and dried on a vacuum funnel to give 58.49 g of
white solid was 97.3% pure by HPLC. On concentrating the mother
liquor, an additional 6.76 g of 77.1% pure product was obtained.
The total yield, adjusted for purity, was 78%.
[0540] H.sup.1 NMR (CDCl.sub.3): 5.70 (1H, s), 4.08 (1H, s), 3.67
(3H, s), 2.9-1.6 (19H, m), 1.5-1.2 (5H, m), 1.03 (3H.s).
EXAMPLE 23
Scheme 1: Step 3B: Conversion of Methyl Hydrogen
11.alpha.,17.alpha.-Dihydroxy-3-oxopregn-4-ene-7.alpha.,21-dicarboxylate,
.gamma.-Lactone to Methyl Hydrogen
17.alpha.-Hydroxy-11.alpha.-(methylsulfonyl)oxy-3-oxopregn-4-ene-7.alpha.-
,21-dicarboxylate, .gamma.-Lactone
[0541] A 5-L four neck flask was equipped as in the above example,
except that no trapping system was installed beyond the bubbler. A
quantity of 138.70 g of the hydroxyester was added to the flask,
followed by 1425 mL methylene chloride, with stirring under
nitrogen. The reaction mixture was cooled to -5.degree. C. using a
salt/ice bath. Methanesulfonyl chloride (51.15 g, 0.447 mole) was
added rapidly, followed by the slow dropwise addition of
triethylamine (54.37 g) in 225 mL methylene chloride. Addition,
which required -30 minutes, was adjusted so that the temperature of
the reaction-never rose about 5.degree. C. Stirring was continued
for 1 hour post-addition, and the reaction contents were
transferred to a 12-L separatory funnel, to which was added 2100 mL
methylene chloride. The solution was washed successively with 700
mL aliquots each of cold 1N HCl, 1N NaOH, and saturated aqueous
NaCl solution. The aqueous washes were combined and back-extracted
with 3500 mL methylene chloride. All of the organic washes were
combined in a 9-L jug, to which was added 500 g neutral alumina,
activity grade II, and 500 g anhydrous sodium sulfate. The contents
of the jug were mixed well for 30 minutes and filtered. The
filtrate was taken to dryness in vacuo to give a gummy yellow foam.
This was dissolved in 350 mL methylene chloride and 1800 mL ether
was added dropwise with stirring. The rate of addition was adjusted
so that about one-half of the ether was added over 30 minutes.
After about 750 mL had been added, the product began to separate as
a crystalline solid. The remaining ether was added in 10 minutes.
The solid was removed by filtration, and the filter cake was washed
with 2 L of ether and dried in a vacuum oven at 50.degree. C.
overnight, to give 144.61 g (88%) nearly white solid, m.p.
149.degree.-150.degree. C. Material prepared in this fashion is
typically 98-99% pure by HPLC (area %). In one run, material having
a melting point of 1530-153.5.degree. C. was obtained, with a
purity, as determined by HPLC area, of 99.5%.
[0542] H.sup.1 NMR (CDCl.sub.3): 5.76 (1H, s), 5.18 (1H, dt), 3.68
(3H, s), 3.06 (3H, s), 2.85 (1H, m), 2.75-1.6 (19H, m), 1.43 (3H,
s), 1.07 (3H, s).
EXAMPLE 24
Scheme 1: Step 3C: Method A: Preparation of 7-Methyl Hydrogen
17.alpha.-Hydroxy-3-oxopregna-4,9(11)-diene-7.alpha.,21-dicarboxylate,
.gamma.-Lactone
[0543] A 1-L four neck flask was equipped as in the second example.
Formic acid (250 mL) and acetic anhydride (62 mL) were added to the
flask with stirring under nitrogen. Potassium formate (6.17 g) was
added and the reaction mixture was heated with an oil bath to an
internal temperature of 40.degree. C. (this was later repeated at
70.degree. C. with better results) for 16 hours. After 16 hours,
the mesylate was added and the internal temperature was increased
to 100.degree. C. Heating and stirring were continued for 2 hours,
after which the solvent was removed in vacuo on a rotavap. The
residue was stirred with 500 mL ice water for fifteen minutes, then
extracted twice with 500 mL aliquots of ethyl acetate. The organic
phases were combined and washed successively with cold 250 mL
aliquots of saturated sodium chloride solution (two times), 1 N
sodium hydroxide solution, and again with saturated sodium
chloride. The organic phase was then dried over sodium sulfate,
filtered and taken to dryness in vacuo to give a yellowish white
foam, which pulverized to a glass when touched with a spatula. The
powder that formed, 14.65 g analyzed (by HPLC area %) as a mixture
of 82.1% 7-Methyl Hydrogen
17.alpha.-Hydroxy-3-oxopregna-4,9(11)-diene-7.alpha.,21-dicarboxylate,
.gamma.-Lactone; 7.4% 7-Methyl Hydrogen
17.alpha.-Hydroxy-3-oxopregna-4,11-diene-7.alpha.,21-dicarboxylate,
.gamma.-Lactone; and 5.7%
9.alpha.,17-dihydroxy-3-oxo-17.alpha.-pregn-4-ene-7.alpha.,21-dicarboxyli-
c acid, bis(.gamma.-lactone).
[0544] H.sup.1 NMR (CDCl.sub.3): 5.74 (1H, 5), 5.67 (1H, m), 3.61
(3H, s), 3.00 (1H, m), 2.84 (1H, ddd, J=2, 6.15), 2.65-2.42 (6H,
m), 2.3-2.12 (5H, m), 2.05-1.72 (4H, m), 1.55-1.45 (2H, m), 1.42
(3H, s), 0.97 (3H, s).
EXAMPLE 25
Scheme 1: Step 3C: Method B: Preparation of Methyl Hydrogen
17.alpha.-Hydroxy-3-oxopregna-4,9(11)-diene-7.alpha.,21-dicarboxylate,
.gamma.-Lactone
[0545] A 5-L four neck flask was equipped as in the above example
and 228.26 g acetic acid and 41.37 g sodium acetate were added with
stirring under nitrogen. Using an oil bath, the mixture was heated
to an internal temperature of 100.degree. C. The mesylate (123.65
g) was added, and heating was continued for thirty minutes. At the
end of this period, heating was stopped and 200 mL of ice water was
added. The temperature dropped to 40.degree. C. and stirring was
continued for 1 hour, after which the reaction mixture was poured
slowly into 1.5 L of cold water in a 5-L stirred flask. The product
separated as a gummy oil. The oil was dissolved in 1 L ethyl
acetate and washed with 1 L each cold saturated sodium chloride
solution, 1 N sodium hydroxide, and finally saturated sodium
chloride again. The organic phase was dried over sodium sulfate and
filtered. The filtrate was taken to dryness in vacuo to give a foam
which collapsed to a gummy oil. This was triturated with ether for
some time and eventually solidified. The solid was filtered and
washed with more ether to afford 79.59 g of a yellow white solid.
This consisted of 70.4% of the desired .DELTA..sup.9,11 enester 6,
12.3% of the .DELTA..sup.11,12 enester 8, 10.8% of the
7-.alpha.,9-.alpha.-lactone 9 and 5.7% unreacted 5.
EXAMPLE 26
Scheme 1: Step 3D: Method A: Synthesis of Methyl Hydrogen
9,11.alpha.-Epoxy-17.alpha.-hydroxy-3-oxopregn-4-ene-7.alpha.,21-dicarbox-
ylate, .gamma.-Lactone
[0546] A 4-neck jacketed 500 mL reactor was equipped with
mechanical stirrer, condenser/bubbler, thermometer and addition
funnel with nitrogen inlet-tube. The reactor was charged with 8.32
g of the crude enester in 83 mL methylene chloride, with stirring
under nitrogen. To this was added 4.02 g dibasic potassium
phosphate, followed by 12 mL of trichloroacetonitrile. External
cooling water was run through the reactor jacket and the reaction
mixture was cooled to 8.degree. C. To the addition funnel 36 mL of
30% hydrogen peroxide was added over a 10 minute period. The
initially pale yellow colored reaction mixture turned almost
colorless after the addition was complete. The reaction mixture
remained at 9.+-.1.degree. C. throughout the addition and on
continued stirring overnight (23 hours total). Methylene chloride
(150 mL) was added to the reaction mixture and the entire contents
were added to -250 mL ice water. This was extracted three times
with 150 mL aliquots of methylene chloride. The combined methylene
chloride extracts were washed with 400 mL cold 3% sodium sulfite
solution to decompose any residual peroxide. This was followed by a
330 mL cold 1 N sodium hydroxide wash, a 400 mL cold 1 N
hydrochloric acid wash, and finally a wash with 400 mL brine. The
organic phase was dried over magnesium sulfate, filtered, and the
filter cake was washed with 80 mL methylene chloride. Solvent was
removed in vacuo to give 9.10 g crude product as a pale yellow
solid. This was recrystallized from -25 mL 2-butanone to give 5.52
g nearly white crystals. A final recrystallization from acetone
(-50 mL gave 3.16 g long, acicular crystals, mp 241-243.degree.
C.
[0547] H.sup.1 NMR (CDCl.sub.3): 5.92 (1H, s), 3.67 (3H, s), 3.13
(1H, d, J=5), 2.89 (1H, m), 2.81-2.69 (15H, m), 1.72 (1H, dd,
J=5.15), 1.52-1.22 (5H, m), 1.04 (3H, s).
EXAMPLE 27
[0548] Scheme 1: Step 3: Option 1: From
4'S(4'.alpha.),7'.alpha.-Hexadecahydro-11'.alpha.-hydroxy-10'.beta.,13'.b-
eta.-dimethyl-3',5,20'-trioxospiro[furan-2(3H),17'.beta.-[4,7]methano[17H]-
cyclopenta[a]phenanthrene]-5'.beta.(2'H)-carbonitrile to Methyl
Hydrogen
9,11.alpha.-Epoxy-17.alpha.-hydroxy-3-oxopregn-4-ene-7.alpha.,21-dicarbox-
ylate, .gamma.-Lactone
[0549] Diketone (20 g) was charged into a clean and dried reactor
followed by the addition of 820 ml of MeOH and 17.6 ml of 25%
NaOMe/MeOH solution. The reaction mixture was heated to reflux
condition (.about.67.degree. C.) for 16-20 hours. The product was
quenched with 40 mL of 4N HCl. The solvent was removed at
atmospheric pressure by distillation. 100 mL of toluene was added
and the residual methanol was removed by azeotrope distillation
with toluene. After concentration, the crude hydroxyester 4 was
dissolved in 206 mL of methylene chloride and cooled to 0.degree.
C. Methanesulfonyl chloride (5 mL) was added followed by a slow
addition of 10.8 ml of triethylamine. The product was stirred for
45 minutes. The solvent was removed by vacuum distillation to give
the crude mesylate 5.
[0550] In a separate dried reactor was added 5.93 g of potassium
formate, 240 mL of formic acid and followed by 118 mL of acetic
anhydride. The mixture was heated to 70.degree. C. for 4 hours.
[0551] The formic acid mixture was added to the concentrated
mesylate solution 5 prepared above. The mixture was heated to
95-105.degree. C. for 2 hours. The product mixture was cooled to
50.degree. C. and the volatile components were removed by vacuum
distillations at 50.degree. C. The product was partitioned between
275 ml of ethyl acetate and 275 ml of water. The aqueous layer was
back extracted with 137 ml of ethyl acetate, washed with 240 ml of
cold 1N sodium hydroxide solution and then 120 ml of saturated
NaCl. After phase separation, the organic layer was concentrated to
under vacuum distillation to give crude enester.
[0552] The product was dissolved in 180 mL of methylene chloride
and cooled to 0 to 15.degree. C. 8.68 g of dipotassium hydrogen
phosphate was added followed by 2.9 mL of trichloroacetonitrile. A
78 mL solution of 30% hydrogen peroxide was added to the mixture
over a 3 minute period. The reaction mixture was stirred at
0-15.degree. C. for 6-24 hours. After the reaction, the two phase
mixture was separated. The organic layer was washed with 126 mL of
3% sodium sulfite solution, 126 mL of 0.5 N sodium hydroxide
solution, 126 mL of 1 N hydrochloric acid and 126 mL of 10% brine.
The product was dried over anhydrous magnesium sulfate or filtered
over Celite and the solvent methylene chloride was removed by
distillation at atmospheric pressure. The product was crystallized
from methylethyl ketone twice to give 7.2 g of epoxymexrenone.
##STR199##
EXAMPLE 28
Scheme 1: Step 3: Option 2: Conversion of
1'S(4'.alpha.),7'.alpha.-Hexadecahydro-11'.alpha.-hydroxy-10'.beta.,13'.b-
eta.-dimethyl-3',5,20'-trioxospiro[furan-2(3H),17'.beta.-[4,7]methano[17H]-
cyclopenta[a]phenanthrene]-5'.beta.(2'H)-carbonitrile to Methyl
Hydrogen
9,11.alpha.-Epoxy-17.alpha.-hydroxy-3-oxopregn-4-ene-7.alpha.,21-dicarbox-
ylate, .gamma.-Lactone without intermediate isolation
[0553] A 4-neck 5-L round bottom flask was equipped with mechanical
stirrer, addition funnel with nitrogen inlet tube, thermometer and
condenser with bubbler attached to a sodium hypochlorite scrubber.
The diketone (83.20 g) was added to the flask in 3.05 L methanol.
The addition funnel was charged with 67.85 g of a 25% (w:w) is
solution of sodium methoxide in methanol. With stirring under
nitrogen, the methoxide was added dropwise to the flask over a 15
minute period. A dark orange/yellow slurry developed. The reaction
mixture was heated to reflux for 20 hours and 175 mL-4 N
hydrochloric acid was added dropwise while refluxing continued.
(Caution, HCN evolution during this operation!) The reflux
condenser was replaced with a takeoff head and 1.6 L of methanol
was removed by distillation while 1.6 L of aqueous 10% sodium
chloride solution was added dropwise through the funnel, at a rate
to match the distillation rate. The reaction mixture was cooled to
ambient temperature and extracted twice with 2.25 L of aliquots of
methylene chloride. The combined extracts were washed with cold 750
mL aliquots of 1 N sodium hydroxide and saturated sodium chloride
solution. The organic layer was dried by azeotropic distillation of
the methanol at one atmosphere, to a final volume of 1 L (0.5% of
the total was removed for analysis).
[0554] The concentrated organic solution (hydroxyester) was added
back to the original reaction flask equipped as before, but without
the HCN trap. The flask was cooled to 0.degree. C. and 30.7 g
methanesulfonyl chloride was added with stirring under nitrogen.
The addition funnel was charged with 32.65 g triethylamine, which
was added dropwise over a 15 minute period, keeping the temperature
at 5.degree. C. Stirring was continued for 2 hours, while the
reaction mixture warmed to ambient. A column consisting of 250 g
Dowex 50 W.times.8-100 acid ion exchange resin was prepared and was
washed before using with 250 mL water, 250 mL methanol and 500 mL
methylene chloride. The reaction mixture was run down this column
and collected. A fresh column was prepared and the above process
was repeated. A third 250 g column, consisting of Dowex
1.times.8-200 basic ion exchange resin was prepared and pretreated
as in the acid resin treatment described above. The reaction
mixture was run down this column and collected. A fourth column of
the basic resin was prepared and the reaction mixture again was run
down the column and collected. Each column pass was followed by two
250 mL methylene chloride washes down the column, and each pass
required -10 minutes. The solvent washes were combined with the
reaction mixture and the volume was reduced in vacuo to -500 mL and
2% of this was removed for qc. The remainder was further reduced to
a final volume of 150 mL (crude mesylate solution).
[0555] To the original 5-L reaction set-up was added 960 mL formic
acid, 472 mL acetic anhydride and 23.70 g potassium formate. This
mixture was heated with stirring under nitrogen to 70.degree. C.
for 16 hours. The temperature was then increased to 100.degree. C.
and the crude mesylate solution was added over a thirty minute
period via the addition funnel. The temperature dropped to
85.degree. C. as methylene chloride was distilling out of the
reaction mixture. After all of it had been removed, the temperature
climbed back to 100.degree. C., and was held there for 2.5 hours.
The reaction mixture was cooled to 40.degree. C. and the formic
acid was removed under pressure until the minimum stir volume had
been reached (.about.150 mL). The residue was cooled to ambient and
375 mL methylene chloride was added. The diluted residue was washed
with cold 1 L portions of saturated sodium chloride solution, 1 N
sodium carbonate, and again with sodium chloride solution. The
organic phase was dried over magnesium sulfate (150 g), and
filtered to give a dark reddish brown solution (crude enester
solution).
[0556] A 4-neck jacketed 1 L reactor was equipped with mechanical
stirrer, condenser/bubbler, thermometer and addition funnel with
nitrogen inlet tube. The reactor was charged with the crude enester
solution (estimated 60 g) in 600 mL methylene chloride, with
stirring under nitrogen. To this was added 24.0 g dibasic potassium
phosphate, followed by 87 mL trichloroacetonitrile. External
cooling water was run through the reactor jacket and the reaction
mixture was cooled to 10.degree. C. To the addition funnel 147 mL
30% hydrogen peroxide was added mixture over a 30 minute period.
The initially dark reddish brown colored reaction mixture turned a
pale yellow after the addition was complete. The reaction mixture
remained at 10.+-.1.degree. C. throughout the addition and on
continued stirring overnight (23 hours total). The phases were
separated and the aqueous portion was extracted twice with 120 mL
portions of methylene chloride. The combined organic phases were
then washed with 210 mL 3% sodium sulfite solution was added. This
was repeated a second time, after which both the organic and
aqueous parts were negative for peroxide by starch/iodide test
paper. The organic phase was successively washed with 210 mL
aliquots of cold 1 N sodium hydroxide, 1 N hydrochloric acid, and
finally two washes with brine. The organic phase was dried
azeotropically to a volume of -100 mL, fresh solvent was added (250
mL and distilled azeotropically to the same. 100 mL and the
remaining solvent was removed in vacuo to give 57.05 g crude
product as a gummy yellow foam. A portion (51.01 g) was further
dried to a constant weight of 44.3 g and quantitatively analyzed by
HPLC. It assayed at 27.1% epoxymexrenone.
EXAMPLE 29
Formation of 3-ethoxy-11.alpha.-hydroxy-androsta-3,5-diene-17-one
from 11.alpha.-hydroxyandrostendione
[0557] 11.alpha.-Hydroxyandrostendione (429.5 g) and toluene
sulfonic acid hydrate (7.1) were charged to a reaction flask under
nitrogen. Ethanol (2.58 L) was added to the reactor, and the
resulting solution cooled to SOC. Triethyl orthoformate (334.5 g)
was added to the solution over a 15 minute period at 0.degree. to
15.degree. C. After the triethyl orthoformate addition was complete
the reaction mixture was warmed to 40.degree. C. and reacted at
that temperature for 2 hours, after which the temperature was
increased to reflux and reaction continued under reflux for an
additional 3 hours. The reaction mixture was cooled under vacuum
and the solvent removed under vacuum to yield
3-ethoxy-11.alpha.-hydroxy-androsta-3,5-diene-17-one.
EXAMPLE 30
Formation of Enamine from 11.alpha.-hydroxycanrenone
Scheme 1: Step 1: Method B: Preparation of
5'R(5'.alpha.),7'.beta.-20'-Aminohexadecahydro-11'.beta.-hydroxy-10'a,13'-
.alpha.-dimethyl-3',5-dioxospiro[furan-2(3H),
17'.beta.(5'H)-[7,4]metheno[4H[cyclopenta[a]phenanthrene]-5'-carbonitrile
[0558] ##STR200##
[0559] Sodium cyanide (1.72 g) was placed in 25 mL 3-neck flask
fitted with a mechanical stirrer. Water (2.1 mL) was added and the
mixture was stirred with heating until the solids dissolved.
Dimethylformamide (15 mL) was added followed by
11.alpha.-hydroxycanrenone (5.0 g). A mixture of water (0.4 mL) and
sulfuric acid (1.49 g) was added to mixture. The mixture was heated
to 85.degree. C. for 2.5 hours at which time HPLC analysis showed
complete conversion to product. The reaction mixture was cooled to
room temperature. Sulfuric acid (0.83 g) was added and the mixture
stirred for one half hour. The reaction mixture was added to 60 mL
water cooled in an ice bath. The flask was washed with 3 mL DMF and
5 mL water. The slurry was stirred for 40 min. and filtered. The
filter cake was washed twice with 40 mL water and dried in a vacuum
oven at 60.degree. C. overnight to yield the 11.alpha.-hydroxy
enamine, i.e.,
5'R(5'.alpha.),7'.beta.-20'-aminohexadecahydro-11'.beta.-hydroxy-10'.alph-
a.,13'.alpha.-dimethyl-3',5-dioxospiro[furan-2(3H),17'.alpha.(5'H)-[7,4]me-
theno[4H]cyclopenta[a]phenanthrene]-5'-carbonitrile (4.9 g)
EXAMPLE 31
One-Rot Conversion of 11.alpha.-hydroxycanrenone to Diketone
[0560] ##STR201##
[0561] Sodium cyanide (1.03 g) was added to a 50 mL 3-neck flask
fitted with a mechanical stirrer. Water (1.26 mL) was added and the
flask was heated slightly to dissolve the solid. Dimethylacetamide
[or dimethylormamide] (9 mL) was added followed by
11.alpha.-hydroxycanrenone (3.0 g). A mixture of sulfuric acid
(0.47 mL) and water (0.25 mL) was added to the reaction flask while
stirring. The mixture was heated to 95.degree. C. for 2 hours. HPLC
analysis indicated that the reaction was complete. Sulfuric acid
(0.27 mL) was added and the mixture stirred for 30 min. Additional
water (25 mL) and sulfuric acid (0.90 mL) were introduced and the
reaction mixture stirred for 16 hours. The mixture was then cooled
in an ice bath to 5-10.degree. C. The solid was isolated by
filtering through a sintered glass filter followed by washing twice
with water (20 mL). The solid diketone, i.e.,
4'S(4'.alpha.),7'.alpha.-Hexadecahydro-11'.alpha.-hydroxy-10'.beta.,13'.b-
eta.-dimethyl-3',5,20'-trioxospiro[furan-2(3H),17'.beta.-[4,7]methano[17H]-
cyclo-penta[a]phenanthrene]-5'.beta.(2'H)-carbonitrile was dried in
a vacuum oven to yield 3.0 g of a solid.
EXAMPLE 32A-1
Scheme 1: Step 3A: Method B: Preparation of Methyl Hydrogen
11.alpha.,17.alpha.-Dihydroxy-3-oxopregn-4-ene-7.alpha.,21-dicarboxylate,
.gamma.-Lactone
[0562] A suspension of 5.0 g of the diketone produced in the manner
described in Example 31 in methanol (100 mL) was heated to reflux
and a 25% solution of potassium methoxide in methanol (5.8 mL) was
added over 1 min. The mixture became homogeneous. After 15 min., a
precipitate was present. The mixture was heated at reflux and again
became homogeneous after about 4 hours. Heating at reflux was
continued for a total of 23.5 hours and 4.0 N HCl (10 mL) was
added. A total of 60 mL of a solution of hydrogen cyanide in
methanol was removed by distillation. Water (57 mL) was added to
the distillation residue over 15 min. The temperature of the
solution was raised to 81.5.degree. C. during water addition and an
additional 4 mL of hydrogen cyanide/methanol solution was removed
by distillation. After water addition was complete, the mixture
became cloudy and the heat source was removed. The mixture was
stirred for 3.5 hours and product slowly crystallized. The
suspension was filtered and the collected solid was washed with
water, dried in a stream of air on the funnel, and dried at
92.degree. (26 in. Hg) for 16 hours to give 2.98 g of an off-white
solid. The solid was 91.4% of the hydroxyester, i.e., methyl
hydrogen
11.alpha.,17.alpha.-dihydroxy-3-oxopregn-4-ene-7.alpha.,21-dicarboxylate,
.gamma.-lactone by weight. The yield was 56.1%.
EXAMPLE 32A-2
Preparation of Methyl Hydrogen
11.alpha.,17.alpha.-Dihydroxy-3-oxopregn-4-ene-7.alpha.,21-dicarboxylate,
.gamma.-Lactone
[0563] The diketone (40 g) produced in the manner described in
Example 31 is charged to a clean, dry, jacketed 1 L reactor
equipped with a bottom drain, condenser, RTD probe and fraction
collecting receiver. Methanol (800 mL) is then charged to the
reactor and the mixture stirred. The resulting slurry is heated to
about 60.degree. C. to 65.degree. C. and a 25% solution of
potassium methoxide (27.8 mL) is added. The mixture becomes
homogeneous.
[0564] The mixture is heated at reflux. After about 1.5 hours at
reflux an additional 16.7 mL of 25% potassium methoxide solution is
added to the mixture while maintaining reflux. The mixture is
maintained at reflux for an additional 6 hours. Conversion of
diketone to hydroxyester is analyzed by HPLC. Once HPLC indicates
the ratio of diketone to hydroxyester is less than about 10%, a
charge of 77 mL of 4M HCl (a comparable amount of 1.5 M to 3 M
sulfuric acid could be substituted for the hydrochloric acid) is
added to the mixture over a period of about 15 minutes as refluxing
is continued.
[0565] The mixture is then distilled and about 520 mL of
methanol/HCN distillate is collected and discarded. The
concentrated mixture is cooled to about 65.degree. C. About 520 mL
of water is added to the mixture over a period of about 90 minutes
and the temperature is maintained at about 65.degree. C. during the
addition. The mixture is gradually cooled to about 15.degree. C.
over a period of about four hours, and then is stirred and
maintained at about 15.degree. C. for an additional two hours. The
mixture is filtered and the filtered product is washed twice with
about 200 mL of water each time. The filtered product is dried in
vacuo (90.degree. C., 25 mm Hg). Approximately 25 to 27 g of an
off-white solid comprising principally methyl hydrogen
11.alpha.,17.alpha.-dihydroxy-3-oxopregn-4-ene-7.alpha.,21-dicarboxylate,
.gamma.-lactone is obtained.
EXAMPLE 32B-1
Preparation of 7-methyl hydrogen
50-cyano-11.alpha.,17-dihydroxy-3-oxo-17.alpha.-pregnane-7.alpha.,21-dica-
rboxylate, .gamma.-lactone
[0566] ##STR202## A reaction flask was charged with 4.1 g of the
diketone produced in the manner described in Example 31, 75 mL of
methanol and 1 mL of 1N methanolic sodium hydroxide. The suspension
was stirred at room temperature. A homogeneous solution was
obtained within minutes and a precipitate observed after about 20
minutes. Stirring was continued for 70 minutes at room temperature.
At the end of this time the solid was filtered and washed with
methanol. The solid was dried in a steam cabinet resulting in 3.6 g
of 7-methyl hydrogen
50-cyano-11.alpha.,17-dihydroxy-3-oxo-17.alpha.-pregnane-7.alpha.,21-dica-
rboxylate, .gamma.-lactone.
[0567] .sup.1H NMR (CDCl.sub.3) ppm 0.95 (s, 3H), 1.4 (s, 3H), 3.03
(d, 1H, J15), 3.69 (s, 3H), 4.1 (m, 1H).
[0568] .sup.13C NMR (CDCl.sub.3) ppm 14.6, 19.8, 22.6, 29.0, 31.0,
33.9, 35.17, 35.20, 36.3, 37.7, 38.0, 38.9, 40.8, 42.8, 43.1, 45.3,
45.7, 47.5, 52.0, 68.0, 95.0, 121.6, 174.5, 176.4, 207.0.
EXAMPLE 32B-2
Preparation of 7-methyl hydrogen
5.beta.-cyano-9,11.alpha.-epoxy-17-hydroxy-3-oxo-17.alpha.-pregnane-7.alp-
ha.,21-dicarboxylate, .gamma.-lactone
[0569] ##STR203##
[0570] To 2.0 g (4.88 mmol) of the 9,11-epoxydiketone of Formula 21
suspended in 30 mL of anhydrous methanol was added 0.34 mL (2.4
mmol) of triethylamine. The suspension was heated to reflux and
after 4.5 hours no starting material remained as judged by HPLC
(Zorbax SB-C8 150.times.4.6 mm, 2 ml/min., linear gradient 35:65
A:B to 45:55 A:B over 15 min, A=acetonitrile/methanol 1:1,
B=water/0.1% trifluoroacetic acid, detection at 210 nm). The
mixture was allowed to cool and maintained at about 25 degrees for
about 16 hours. The resulting suspension was filtered to give 1.3 g
of 7-methyl hydrogen
5.beta.-cyano-9,11.alpha.-epoxy-17-hydroxy-3-oxo-17.alpha.-pregnane-7.alp-
ha.,21-dicarboxylate, .gamma.-lactone as a white solid. The
filtrate was concentrated to dryness on a rotary evaporator and the
residue was triturated with 3-5 mL of methanol. Filtration gave an
additional 260 mg of 7-methyl hydrogen
50-cyano-9,11.alpha.-epoxy-17-hydroxy-3-oxo-17.alpha.-pregnane-7.alpha.,2-
1-dicarboxylate, .gamma.-lactone. The yield was 74.3%.
[0571] 1H-nmr (400 MHz, deuterochloroform) d 1.00 (s, 3H), 1.45 (m,
1H), 1.50 (s, 3H), 1.65 (m, 2H), 2.10 (m, 2H), 2.15-2.65 (m, 8H),
2.80 (m, 1H), 2.96 (m, 1H), 3.12 (d, J=13, 1H), 3.35, (d, J=7, 1H),
3.67 (s, 3H).
EXAMPLE 32C
Preparation of
5.beta.-cyano-11-.alpha.,17-dihydroxy-3-oxo-17.alpha.-pregnane-7.alpha.,2-
1-dicarboxylic acid, .gamma.-lactone
[0572] ##STR204##
[0573] A reaction flask was charged with 6.8 g of the diketone
(prepared in the manner described in Example 31), 68 mL of
acetonitrile, 6.0 g of sodium acetate and 60 mL of water. The
mixture was warmed and stirred at reflux. After about 1.5 hours the
mixture was almost homogeneous. At the end of three hours 100 mL of
water was added as 50 mL of acetonitrile was distilled. The mixture
was cooled and the precipitated solid (1.7 g) was removed via
filtration. The filtrate (pH=5.5) was treated with hydrochloric
acid to reduce the pH to about 4.5 and a solid precipitated. The
solid was isolated, washed with water and dried to give 4.5 g of
5.beta.-cyano-11-.alpha.,17-dihydroxy-3-oxo-17.alpha.-pregnane-7.alpha.,2-
1-dicarboxylic acid, .gamma.-lactone.
[0574] .sup.1H NMR (DMSO) ppm 0.8 (s, 3H), 1.28 (s, 3H), 3.82 (m,
1H).
[0575] .sup.13C NMR (DMSO) ppm 14.5, 19.5, 22.0, 28.6, 30.2, 33.0,
34.1, 34.4, 36.0, 37.5, 37.7, 38.5, 42.4, 42.6, 45.08, 45.14, 47.6,
94.6, 122.3, 176.08, 176.24, 207.5.
EXAMPLE 33
Scheme 1: Step 3A: Method C: Preparation of Methyl Hydrogen
11.alpha.,17.alpha.-Dihydroxy-3-oxopregn-4-ene-7.alpha.,21-dicarboxylate,
.gamma.-Lactone
[0576] Diketone (30 g) prepared in the manner described in Example
31 was charged into a cleaned and dried 3-neck reaction flask
equipped with a thermometer, a Dean Stark trap and a mechanical
stirrer. Methanol (24 mL) was charged to the reactor at room
temperature (22.degree. C.) and the resulting slurry stirred for 5
min. A 25% by weight solution of sodium methoxide in methanol (52.8
mL) was charged to the reactor and the mixture stirred for 10 min.
at room temperature during which the reaction mixture turned to a
light brown clear solution and a slight exotherm was observed
(2-3.degree. C.). The addition rate was controlled to prevent the
pot temperature from exceeding 30.degree. C. The mixture was
thereafter heated to reflux conditions (about 67.degree. C.) and
continued under reflux for 16 hrs. A sample was then taken and
analyzed by HPLC for conversion. The reaction was continued under
reflux until the residual diketone was not greater than 3% of the
diketone charge. During reflux 4 N HCl (120 mL) was charged to the
reaction pot resulting in the generation of HCN which was quenched
in a scrubber.
[0577] After conclusion of the reaction, 90-95% of the methanol
solvent was distilled out of the reaction mixture at atmospheric
pressure. Head temperature during distillation varied from
67-75.degree. C. and the distillate which contained HCN was treated
with caustic and bleach before disposal. After removal of methanol
the reaction mixture was cooled to room temperature, solid product
beginning to precipitate as the mixture cooled in the 40-45.degree.
C. range. An aqueous solution containing optionally 5% by weight
sodium bicarbonate (1200 mL) at 25.degree. C. was charged to the
cooled slurry and the resultant mixture then cooled to 0.degree. C.
in about 1 hr. Sodium bicarbonate treatment was effective to
eliminate residual unreacted diketone from the reaction mixture.
The slurry was stirred at 0.degree. C. for 2 hrs. to complete the
precipitation and crystallization after which the solid product,
Methyl Hydrogen
11.alpha.,17.alpha.-Dihydroxy-3-oxopregn-4-ene-7.alpha.,21-dicar-
boxylate, .gamma.-Lactone, was recovered by filtration and the
filter cake washed with water (100 mL). The product was dried at
80-90.degree. C. under 26'' mercury vacuum to constant weight.
Water content after drying was less than 0.25% by weight. Adjusted
molar yield was around 77-80% by weight.
EXAMPLE 34
Scheme 1: Step 3A: Method D: Preparation of Methyl Hydrogen
11.alpha.,17.alpha.-Dihydroxy-3-oxopregn-4-ene-7.alpha.,21-dicarboxylate,
.gamma.-Lactone
[0578] Diketone as prepared in accordance with Example 31 (1 eq.)
was reacted with sodium methoxide (4.8 eqs.) in a methanol solvent
in the presence of zinc iodide (1 eq.). Work up of the reaction
product can be either in accordance with the extractive process
described herein, or by a non-extractive process in which methylene
chloride extractions, brine and caustic washes, and sodium sulfate
drying steps are eliminated. Also in the non-extractive process,
toluene was replaced with 5% by weight sodium bicarbonate solution.
Methyl Hydrogen
11.alpha.,17.alpha.-Dihydroxy-3-oxopregn-4-ene-7.alpha.,21-dicarboxylate,
.gamma.-Lactone was isolated as the product.
EXAMPLE 35
Scheme 1: Step 3C: Method C: Preparation of Methyl Hydrogen
17.alpha.-Hydroxy-3-oxopregna-4,9(11)-diene-7.alpha.,21-dicarboxylate,
.gamma.-Lactone
[0579] The hydroxyester prepared as by Example 34 (1.97 g) was
combined with tetrahydrofuran (20 mL) and the resulting mixture
cooled to -70.degree. C. Sulfuryl chloride (0.8 mL) was added and
the mixture was stirred for 30 min., after which imidazole (1.3 g)
was added. The reaction mixture was warmed to room temperature and
stirred for an additional 2 hrs. The mixture was then diluted with
methylene chloride and extracted with water. The organic layer was
concentrated to yield crude product Methyl Hydrogen
17.alpha.-Hydroxy-3-oxopregna-4,9(11)-diene-7.alpha.,21-dicarboxylate,
.gamma.-Lactone (1.97 g). A small sample of the crude product was
analyzed by HPLC. The analysis showed that the ratio of
9,11-olefin: 11,12-olefin: 7,9-lactone was 75.5:7.2:17.3. When
carried out at 0.degree. C. but otherwise as described above, the
reaction yielded a product in which the 9,11-olefin: 11,12-olefin:
7,9-lactone distribution was 77.6:6.7:15.7. This procedure combines
into one step the introduction of a leaving group and elimination
thereof for the introduction of the 9,11-olefin structure of the
enester, i.e., reaction with sulfuryl chloride causes the
11.alpha.-hydroxy group of the hydroxy ester of Formula V to be
replaced by halide and this is followed by dehydrohalogenation to
the .DELTA..sup.9,11 structure. Thus formation of the enester is
effected without the use of a strong acid (such as formic) or a
drying agent such as acetic anhydride. Also eliminated is the
refluxing step of the alternative process which generates carbon
monoxide.
EXAMPLE 36A
Scheme 1: Step 3C: Method D: Preparation of Methyl Hydrogen
17.alpha.-Hydroxy-3-oxopregna-4,9(11)-diene-7.alpha.,21-dicarboxylate,
.gamma.-Lactone
[0580] Hydroxyester (20 g) prepared as by Example 34, and methylene
chloride (400 mL) were added to a clean dry three-neck round bottom
flask fitted with a mechanical stirrer, addition funnel and
thermocouple. The resulting mixture was stirred at ambient
temperature until complete solution was obtained. The solution was
cooled to 5.degree. C. using an ice bath. Methanesulfonyl chloride
(5 mL) was added to the solution of CH.sub.2Cl.sub.2 containing the
hydroxyester, rapidly followed by the slow dropwise addition of
triethylamine (10.8 mL). The addition rate was adjusted so that the
temperature of the reaction did not exceed 5.degree. C. The
reaction was very exothermic; therefore cooling was necessary. The
reaction mixture was stirred at about 5.degree. C. for 1 h. When
the reaction was complete (HPLC and TLC analysis), the mixture was
concentrated at about 0.degree. C. under 26 in Hg vacuum until it
became a thick slurry. The resulting slurry was diluted with
CH.sub.2Cl.sub.2 (160 mL), and the mixture was concentrated at
about 0.degree. C. under 26 in Hg vacuum to obtain a concentrate.
The purity of the concentrate (mesylate product of Formula IV
wherein R.sup.3.dbd.H and -A-A- and --B--B-- are both --CH,
--CH.sub.2--, i.e., methyl hydrogen
11.alpha.,17.alpha.-dihydroxy-3-oxooregn-4-ene-7.alpha.,21-dicarboxylate,
.gamma.-lactone to methyl hydrogen
17.alpha.-hydroxy-11.alpha.-(methylsulfonyl)oxy-3-oxopregn-4-ene-7.alpha.-
,21-dicarboxylate, .gamma.-lactone was found to be 82% (HPLC area
%). This material was used for the next reaction without
isolation.
[0581] Potassium formate (4.7 g), formic acid (16 mL) and acetic
anhydride (8 mL, 0.084 mol) were added to a clean dry reactor
equipped with mechanical stirrer, condenser, thermocouple and
heating mantle. The resulting solution was heated to 70.degree. C.
and stirred for about 4-8 hours. The addition of acetic anhydride
is exothermic and generated gas (CO), so that the rate of addition
had to be adjusted to control both temperature and gas generation
(pressure). The reaction time to prepare the active eliminating
reagent was dependent on the amount of water present in the
reaction (formic acid and potassium formate contained about 3-5%
water each). The elimination reaction is sensitive to the amount of
water present; if there is >0.1% water (KF), the level of the
7,9-lactone impurity may be increased. This by product is difficult
to remove from the final product. When the KF showed <0.1%
water, the active eliminating agent was transferred to the
concentrate of mesylate (0.070 mol) prepared in the previous step.
The resulting solution was heated to 95.degree. C. and the volatile
material was distilled off and collected in a Dean Stark trap. When
volatile material evolution ceased, the Dean Stark trap was
replaced with the condenser and the reaction mixture was heated for
additional 1 h at 95.degree. C. Upon completion (TLC and HPLC
analysis; <0.1% starting material) the content was cooled to
50.degree. C. and vacuum distillation was started (26 in
Hg/50.degree. C.). The mixture was concentrated to a thick slurry
and then cooled to ambient temperature. The resulting slurry was
diluted with ethyl acetate (137 mL) and the solution was stirred
for 15 min. and diluted with water (137 mL). The layers were
separated, and the aqueous lower layer was re-extracted with ethyl
acetate (70 mL). The combined ethyl acetate solution was washed
once with brine solution (120 mL) and twice with ice cold 1N NaOH
solution (120 mL each). The pH of aqueous was measured, and the
organic layer rewashed if the pH of the spent wash liquor was
<8. When the pH of the spent wash was observed to be >8, the
ethyl acetate layer was washed once with brine solution (120 mL)
and concentrated to dryness by rotary evaporation using a
50.degree. C. water bath. The resulting enester, solid product
i.e., methyl hydrogen
17.alpha.-hydroxy-3-oxopregna-4,9(11)-diene-7.alpha.,21-dicarboxylate,
.gamma.-lactone weighed 92 g (77% mol yield).
EXAMPLE 36B
Preparation of Methyl Hydrogen
17.alpha.-Hydroxy-3-oxopregna-4,9(11)-diene-7.alpha.,21-dicarboxylate,
.gamma.-Lactone
[0582] To a clean dry 250 mL three-neck round bottom flask fitted
with a mechanical stirrer, addition funnel and thermocouple was
added 25 g (53.12 mmol) of the hydroxyester Methyl Hydrogen
11.alpha.,17.alpha.-Dihydroxy-3-oxopregn-4-ene-7.alpha.,21-dicarboxylate,
.gamma.-Lactone prepared as by Example 34, followed by 150 mL of
methylene chloride (Burdick & Johnson). The resulting mixture
was stirred at ambient temperature until a light slurry was
obtained. The solution was cooled to -5.degree. C. using an ice
bath. Methanesulfonyl chloride (7.92 g, 69.06 mmol) (Aldrich) was
added to the solution of methylene chloride containing the
hydroxyester, rapidly followed by the slow dropwise addition of
triethylamine (7.53 g) (Aldrich). The addition rate was adjusted so
that the temperature of the reaction did not exceed 0.degree. C.
The reaction was very exothermic; therefore cooling was necessary.
Addition time was 35 minutes. The reaction mixture was stirred at
about 0.degree. C. for an additional 45 minutes. When the reaction
was complete (less than 1% hydroxyester remaining indicated by HPLC
and TLC analysis), the mixture was concentrated by stripping
approximately 110 mL to 125 mL of the methylene chloride solvent at
atmospheric pressure. The reactor temperature reached
approximately. 40.degree. C. to 45.degree. C. during stripping.
Where the reaction is not complete after the additional 45 minutes,
an additional 0.1 equivalent of methanesulfonyl chloride and an
additional 0.1 equivalent triethylamine can be charged to the
reactor and the reaction checked for completion. The resulting
mixture contained the crude product methyl hydrogen
17.alpha.-hydroxy-11.alpha.-(methylsulfonyl)oxy-3-oxopregn-4-ene-7.alpha.-
,21-dicarboxylate, .gamma.-lactone. This material was used for the
next reaction without isolation.
[0583] Anhydrous sodium acetate (8.7 g) (Mallinkrodt), glacial
acetic acid (42.5 mL) (Fisher) and acetic anhydride (0.5 mL)
(Fisher) were added to a second 250 mL clean dry reactor equipped
with mechanical stirrer, condenser, thermocouple and heating
mantle. The resulting solution was heated to 90.degree. C. and
stirred for about 30 minutes. The addition of acetic anhydride is
exothermic, so that the rate of addition had to be adjusted to
control both temperature and pressure. Acetic anhydride was added
to reduce the water content of the solution to an acceptable level
(less than about 0.1%). When the KF showed <0.1% water, the
acetic acid solution was transferred to the concentrate of mesylate
prepared as discussed in the first paragraph of this example. The
temperature of the resulting mixture was about 55.degree. C. to
60.degree. C. after the transfer. By using acetic acid and sodium
acetate instead of formic acid and potassium formate as in Example
36A, gas generation was reduced.
[0584] The mixture was heated to 135.degree. C. and maintained at
that temperature for about 60 to 90 minutes until volatile material
evolution ceased. The volatile material distilled from the mixture
was collected in a Dean Stark trap. Upon completion (TLC and HPLC
analysis; <0.1% starting material) the heat source was removed.
When the temperature of the mixture reached 80.degree. C., 150 mL
of water was slowly added to the mixture over 60 to 90 minutes. At
the end of the water addition, the mixture had cooled to a
temperature of about 35.degree. C. to 45.degree. C. and a slurry
had begun to form. The mixture was further-cooled to 15.degree. C.
and maintained at that temperature for about 30 to 60 minutes.
[0585] The mixture was filtered through a glass funnel. The
filtrate was rinsed with 100 mL of water. The filtrate was then
washed a second time with an additional 100 mL of water. The
resulting filtrate was dried at 70.degree. C. in vacuo to yield
25.0 g of dry product enester, methyl hydrogen
17.alpha.-hydroxy-3-oxopregna-4,9(11)-diene-7.alpha.,21-dicarbox-
ylate, .gamma.-lactone. HPLC assay indicated 70% of the desired
9,11-olefin, 15% of the 11,12-olefin, and 5% of the
7,0-lactone.
[0586] This method had the beneficial result (relative to similar
processes) of (i) reducing solvent volumes, (ii) reducing the
number of separate operational steps needed to produce the enester
from the hydroxyester, (iii) reducing the washes needed, (iv)
replacing extraction with water precipitation in isolating the
final product, and (v) eliminating safety concerns previously
associated with mixed anhydride formation and gas generation when
formic acid is used instead of acetic acid.
EXAMPLE 37A
Scheme 1: Step 3C: Method E: Preparation of Methyl Hydrogen
17.alpha.-Hydroxy-3-oxopregna-4,9(11)-diene-7.alpha.,21-dicarboxylate,
.gamma.-Lactone
[0587] Hydroxyester (100 g; 0.22 mol) prepared as by Example 34 was
charged to a 2 L 3-neck round bottom flask equipped with mechanical
stirrer, addition funnel, and thermocouple. A circulating cooling
bath was used with automatic temperature control. The flask was
dried prior to reaction because of the sensitivity of
methanesulfonyl chloride to water.
[0588] Methylene chloride (1 L) was charged to the flask and the
hydroxyester dissolved therein under agitation. The solution was
cooled to 0.degree. C. and methane sulfonyl chloride (25 mL; 0.32
mol) was charged to the flask via the addition funnel.
Triethylamine (50 mL; 0.59 mol) was charged to the reactor via the
addition funnel and the funnel was rinsed with additional methylene
chloride (34 mL). Addition of triethylamine was highly exothermic.
Addition time was around 1 min. under agitation and cooling. The
charge mixture was cooled to 0.degree. C. and held at that
temperature under agitation for an additional 45 min. during which
the head space of the reaction flask was flushed with nitrogen. A
sample of the reaction mixture was then analyzed by thin layer
chromatography and high performance liquid chromatography to check
for reaction completion. The mixture was thereafter stirred at
0.degree. C. for an additional 30 min. and checked again for
reaction completion. Analysis showed the reaction to be
substantially complete at this point; the solvent methylene
chloride was stripped at 0.degree. C. under 26'' mercury vacuum.
Gas chromatography analysis of the distillate indicated the
presence of both methane sulfonyl chloride and triethylamine.
Methylene chloride (800 mL) was thereafter charged to the reactor
and the resulting mixture was stirred for 5 min. at a temperature
in the range of 0-15.degree. C. The solvent was again stripped at
0-5.degree. C. under 26'' mercury vacuum yielding the mesylate of
Formula IV wherein R.sup.3 is H, -A-A- and --B--B-- are
--CH.sub.2--CH.sub.2-- and R.sup.1 is methoxy carbonyl. The purity
of the product was about 90-95 area %.
[0589] To prepare an elimination reagent, potassium formate (23.5
g; 0.28 mol), formic acid (80 mL) and acetic anhydride (40 mL) were
mixed in a separate dried reactor. Formic acid and acetic anhydride
were pumped into the reactor and the temperature was maintained not
greater than 40.degree. C. during addition of acetic anhydride. The
elimination reagent mixture was heated to 70.degree. C. to scavenge
water from the reaction system. This reaction was continued until
the water content was lower than 0.3% by weight as measured by Karl
Fisher analysis. The elimination reagent solution was then
transferred to the reactor containing the concentrated crude
mesylate solution prepared as described above. The resulting
mixture was heated to a maximum temperature of 95.degree. C. and
volatile distillate collected until no further distillate was
generated. Distillation ceased at about 90.degree. C. After
distillation was complete, the reaction mixture was stirred at
95.degree. C. for an additional 2 hrs. and completion of the
reaction was checked for thin layer chromatography. When the
reaction was complete, the reactor was cooled to 50.degree. C. and
the formic acid and solvent removed from the reaction mixture under
26'' mercury vacuum at 50.degree. C. The concentrate was cooled to
room temperature and thereafter ethyl acetate (688 mL) was
introduced and the mixture of ethyl acetate and concentrate stirred
for 15 min. At this point, a 12% brine solution (688 mL) was
introduced to assist in removing water soluble impurities from the
organic phase. The phases were then allowed to settle for 20 min.
The aqueous layer was transferred to another vessel to which an
additional amount of ethyl acetate (350 mL) was charged. This back
extraction of the aqueous layer was carried out for 30 min. after
which the phases were allowed to settle and the ethyl acetate
layers combined. To the combined ethyl acetate layers, saturated
sodium chloride solution (600 mL) was charged and stirring carried
out for 30 min. The phases were then allowed to settle. The aqueous
layer was removed. An additional sodium chloride (600 mL) wash was
carried out. The organic phase was separated from the second
spent-wash liquor. The organic phase was then washed with 1 N
sodium hydroxide (600 mL) under stirring for 30 min. The phases
were settled for 30 min. to remove the aqueous layer. The pH of the
aqueous layer was checked and it found to be >7. A further wash
was carried out with saturated sodium chloride (600 mL) for 15 min.
The organic phase was finally concentrated under 26'' mercury
vacuum at 50.degree. C. and the product recovered by filtration.
The final product was a foamy brown solid when dried. Further
drying at 45.degree. C. under reduced pressure for 24 hrs. yielded
95.4 g of the enester product Methyl Hydrogen
17.alpha.-Hydroxy-3-oxopregna-4,9(11)-diene-7.alpha.,21-dicarboxylate,
.gamma.-Lactone which assayed at 68.8%. The molar yield was 74.4%
corrected for both the starting hydroxy ester and the final
enester.
EXAMPLE 37B
Preparation of 7-methyl hydrogen
17-methyl-3-oxo-18-norpregna-4,9(11),13-triene-7.alpha.,21-dicarboxylate
[0590] ##STR205##
[0591] A reaction flask was charged with 5.5 g of the mesylate
prepared in the manner of Example 23, 55 mL of 94.3% formic acid
and 1.38 g of potassium formate. The mixture was heated and stirred
at reflux (104.degree. C.) for two hours. At the end of the two
hour period the formic acid was distilled under reduced pressure.
The residue was dissolved in ethyl acetate and washed with 10%
potassium carbonate (50 mL). The recovered aqueous portion was
yellow in color. The ethyl acetate was washed with 5% sodium
hydroxide (50 mL). The aqueous portions were combined and acidified
with dilute hydrochloric acid and the insoluble material extracted
with ethyl acetate. The ethyl acetate was evaporated to dryness
under reduced pressure to give 1.0 g of a residue, 7-methyl
hydrogen
17-methyl-3-oxo-18-norpregna-4,9(11),13-triene-7.alpha.,21-dicar-
boxylate.
[0592] .sup.1H NMR (CDCl.sub.3) ppm 1.5 (s, 3H), 1.4 (s, 3H), 3.53
(s, 3H), 5.72 (m, 1H).
[0593] .sup.13C NMR (CDCl.sub.3) ppm 25.1 and 25.4 (18 CH.sub.3 and
19 CH.sub.3), 40.9 (10 C), 48.5 (17 C), 51.4 (OCH.sub.3), 118.4 (11
CH), 125.4 (4 CH), 132.4 (9 C), 138.5 and 139.7 (13 C and 14 C),
168.2 (5 C), 172.4 (7 CO), 179.6 (22 CO), 198.9 (3 CO).
EXAMPLE 37C
Preparation of 7-methyl hydrogen
5.beta.-cyano-17-hydroxy-3-oxo-17.alpha.-pregn-11-ene-7.alpha.,21-dicarbo-
xylate, .gamma.-lactone
[0594] ##STR206##
[0595] A reaction flask was charged with 5.5 g of the mesylate
prepared in the manner of Example 23, 55 mL of 94.3% formic acid
and 1.38 g of potassium formate. The mixture was heated and stirred
at reflux (104.degree. C.) for two hours. At the end of the two
hour period the formic acid was distilled under reduced pressure.
The residue was dissolved in ethyl acetate and washed with 10%
potassium carbonate (50 mL). The recovered aqueous portion was
yellow in color. The ethyl acetate was washed with 5% sodium
hydroxide (50 mL). The ethyl acetate was, evaporated to dryness
under reduced pressure to give a 3.7 g residue. A 3.4 g portion of
the residue was chromatographed on 267 g of Merck silica gel
(40-63.mu.). The product was recovered with an elution scheme of
ethyl acetate and toluene 37:63 (v/v). After drying this product,
0.0698 g of a residue, 7-methyl hydrogen
5.beta.-cyano-17-hydroxy-3-oxo-17.alpha.-pregn-11-ene-7.alpha.,21-dicarbo-
xylate, .gamma.-lactone, was obtained.
[0596] .sup.1H NMR (CDCl.sub.3) ppm 1.03 (s, 3H), 1.22 (s, 3H),
3.70 (s, 3H), 5.60 (d, 1H, J10), 5.98 (d, 1H, J10).
[0597] MIR cm.sup.-1 2229 (CN), 1768 (lactone), 1710 (ester).
EXAMPLE 37D
Isolation of
9.alpha.,17-dihydroxy-3-oxo-17.alpha.-pregn-4-ene-7.alpha.,21-dicarboxyli-
c acid, bis(.gamma.-lactone)
[0598] ##STR207##
[0599]
9.alpha.,17-dihydroxy-3-oxo-17.alpha.-pregn-4-ene-7.alpha.,21-dica-
rboxylic acid, bis(.gamma.-lactone) is a byproduct of the
11-mesylate elimination. A pure sample was isolated from the
reaction mixture of Example 37A via preparative liquid
chromatography followed by reverse phase preparative HPLC. Thus, a
73 g residue was chromatographed over 2.41 kg of Merck silica gel
(40-63.mu.) with a gradient elution scheme of ethyl acetate and
toluene (20:80, 30:70, 40:60, 60:40, v/v). An enriched mixture
(10.5 g) of the enamine and the 7,9-lactone was obtained in the
60:40 fractions. The progress of the purification was observed via
TLC on EMF plates with a 60:40 (v/v) ethyl acetate, toluene eluent
and visualization-via sulfuric acid, SWUV. A portion (10.4 g) of
the mixture was further purified via reverse phase HPLC on Kromasil
C8 (7.mu.) and a 30:70 (v/v) milliQ water and acetonitrile mobile
phase. The 7,9-lactone (2.27 g) was isolated as crystals from the
mobile phase.
[0600] MIR cm.sup.-1 1762 (7,9-lactone and 17-lactone), 1677, 1622
(3-keto-.DELTA..sup.4,5).
[0601] .sup.1H NMR (CDCl.sub.3) ppm 1.00 (s, 3H), 1.4 (s, 3H), 2.05
(d, 1H), 2.78 (d, 1H), 5.87 (s, 1H).
[0602] .sup.13C NMR (CDCl.sub.3) ppm 13.2, 19.0, 22.2, 23.2, 26.8,
28.8, 29.5, 30.8, 33.1, 34.4, 35.1, 42.5, 43.6, 43.9, 45.0, 45.3,
89.9, 94.7, 129.1, 161.5, 176.0, 176.4, 196.9.
[0603] Theory C, 71.85 and H, 7.34; Found C, 71.68 and H, 7.30.
EXAMPLE 37E
Isolation of 7-methyl hydrogen
5-cyano-17-hydroxy-3-oxo-17.alpha.-pregn-11-ene-7.alpha.,21-dicarboxylate-
, .gamma.-lactone
[0604] ##STR208##
[0605] The compound 7-methyl hydrogen
5-cyano-17-hydroxy-3-oxo-17.alpha.-pregn-11-ene-7.alpha.,21-dicarboxylate-
, .gamma.-lactone was isolated after multiple preparative liquid
chromatography on the reaction mixture obtained after elimination
of the 11-mesyloxy group (Example 24). It was part of a cluster of
less polar impurities as viewed via TLC on EMF plates with a 30:70
(v/v) ethyl acetate and methylene chloride eluting system and
visualized via sulfuric acid, SWUV. Generally, these less polar
impurities were separated from the crude enamine via preparative
liquid chromatography. Specifically, 9.6 g of the crude enamine
solution was chromatographed over 534 g of Merck silica gel
(40-63.mu.) using an ethyl-acetate and toluene gradient elution
scheme (20:80, 30:70, 40:60, 60:40, v/v). The less polar impurities
were concentrated in the 30:70 fractions. A 12.5 g pool of the less
polar impurities was collected in this fashion. This material was
further chromatographed over 550 g of Merck silica gel (40-63.mu.)
using an ethyl acetate and methylene chloride gradient elution
scheme (5:95, 10:90, 20:80, 30:70, v/v). An enriched portion of the
20:80 fractions yielded 1.2 g of residue. Additional chromatography
of the 1.2 g residue over 53 g of Merck silica gel (40-63.mu.)
using an acetone and methylene chloride gradient (3:97, 6:94,
10:90, 15:85, v/v) yielded 0.27 g of 7-methyl hydrogen
5-cyano-17-hydroxy-3-oxo-17.alpha.-pregn-11-ene-7.alpha.,21-dicarboxylate-
, .gamma.-lactone from an enriched portion of the 10:90
fractions.
[0606] MS M+425, calculated for C.sub.25H.sub.31NO.sub.5
(425.52).
[0607] MIR 2222 cm.sup.-1 (nitrile), 1767 cm.sup.-1 (lactone), 1727
cm.sup.-1 (ester and 3-ketone).
[0608] .sup.1H NMR (CDCl.sub.3) ppm 0.92 (s, 3H), 1.47 (s, 3H),
2.95 (m, 1H), 3.65 (s, 3H), 5.90 (m, 1H).
[0609] .sup.13C NMR (CDCl.sub.3) ppm 14.0 (18 CH.sub.3), 23.5 (15
CH.sub.2), 27.0 (19 CH.sub.3), 37.8, 38.5 and 40.9 (7, 8 and 14
CH), 52.0 (OCH.sub.3), 95.0 (17 C), 121.5 (23 CN), 123.5 (11 CH),
135.3 (9 C), 174.2 and 176.2 (22 and 24 CO), 206 (3CO).
EXAMPLE 37F
Preparation of 7-methyl hydrogen
17-hydroxy-3-oxo-11.alpha.-(2,2,2-trifluoro-1-oxoethoxy)-17.alpha.-pregn--
4-ene-7.alpha.,21-dicarboxylate
[0610] ##STR209##
[0611] Hydroxyester (2.0 g, 4.8 mmols) prepared in the manner of
Example 34 was added to 40 mL of methylene chloride in a clean, dry
3-neck, round bottom flask equipped with a mechanical stirrer.
Triethyl amine (0.61 g, 6.10 mmols) and trifluoracetic anhydride
(1.47 g, 7.0 mmols) were then added to the solution. This mixture
was stirred at ambient temperature overnight.
[0612] The mixture then was diluted with an additional 40 mL of
methylene chloride. The mixture then was washed successively with
40 mL of water, 40 mL of 1N HCl, and 40 mL of 1N NaOH solution. The
resulting solution was then dried over magnesium sulfate and
concentrated to dryness to afford 3.2 g of a light brown solid,
7-methyl hydrogen
17-hydroxy-3-oxo-11.alpha.-(2,2,2-trifluoro-1-oxoethoxy)-17.alpha.-pregn--
4-ene-7.alpha.,21-dicarboxylate.
[0613] The residue was further analyzed and purified by
chromatography. HPLC conditions: column--Waters Symmetry C18 (150
mm.times.4.6 mm i.d., 5 micron particle size); column
temperature--ambient; mobile phase--acetonitrile/water, 30/70 by
volume; flow rate--1.0 mL/minute; injection volume--20 microliters;
sample concentration--1.0 mg/mL; detection--UV at 210 nm;
pressure--1500 psi; and run time--45 minutes. TLC conditions:
adsorbent--Merck Silica Gel 60 F.sub.254; solvent system--ethyl
acetate/toluene, 65/35 by volume; visualization
technique--shortwave; and application amount--100 micrograms.
EXAMPLE 37G
Preparation of 7-methyl hydrogen
11.alpha.-(acetyloxy)-17-hydroxy-3-oxo-17.alpha.-pregn-4-ene-7,21-dicarbo-
xylate, .gamma.-lactone
[0614] ##STR210##
[0615] Hydroxyester (2.86 g, 6.87 mmole) prepared in the manner of
Example 34 was added to 15 mL of methylene chloride in a clean, dry
3-neck, round bottom flask equipped with a mechanical stirrer.
Triethyl amine (1.39 g, 13.7 mmol), dimethylaminopyridine (0.08 g,
0.6 mmol) and acetic anhydride (1.05 g, 10.3 mmol) were then added
to the solution. This mixture was stirred at ambient temperature
overnight.
[0616] The mixture then was diluted with 150 mL of ethyl acetate
and 25 mL of water. This ethyl acetate solution then was washed 25
mL of citric acid solution. The solution was then dried over
magnesium sulfate and concentrated to dryness to afford 3.33 g of a
light brown solid, 7-methyl hydrogen
11.alpha.-(acetyloxy)-17-hydroxy-3-oxo-17.alpha.-pregn-4-ene-7,2-
1-dicarboxylate, .gamma.-lactone.
[0617] The residue was further analyzed and purified by
chromatography. HPLC conditions: column--waters Symmetry C18 (150
mm.times.4.6 mm i.d., 5 micron particle size); column
temperature--ambient; mobile phase--acetonitrile/water, 30/70 by
volume; flow rate--1.0 mL/minute; injection volume--20 microliters;
sample concentration--1.0 mg/mL; detection--UV at 210 nm;
pressure--1500 psi; and run time--45 minutes. TLC conditions:
adsorbent--Merck Silica Gel 60 F.sub.254; solvent system--methylene
chloride/methanol, 95/5 by volume; visualization
technique--shortwave; and application amount--100 micrograms.
EXAMPLE 37H
Scheme 1: Step 3C: Method F: Preparation of 7-methyl hydrogen
17-hydroxy-3-oxo-17.alpha.-pregna-4,9(11)-diene-7,21-dicarboxylate,
.gamma.-lactone
[0618] ##STR211##
[0619] Potassium formate (1.5 g, 0.018 mol), formic acid 60 mL, 1.6
mol) and acetic anhydride (29.5 mL, 0.31 mol) were added to a
clean, dry 250 mL reactor equipped with a mechanical stirrer,
condenser, thermocouple and heating mantle. The solution was then
was stirred at 70.degree. C. for 4 hours and cooled to ambient
temperature to provide an elimination reagent useful for converting
the mesylate of Formula IV to the product of this example.
[0620] The preformed TFA/TFA anhydride elimination reagent was
added to 70.0 g (0.142 mol) of the mesylate prepared in the manner
of Example 23. The resulting mixture was heated to 95.degree. C. to
105.degree. C. for 2.5 hrs., the degree of conversion being
periodically checked by TLC or HPLC. The resulting residue was
cooled to 50.degree. C., diluted with ice water (1.4 L) and stirred
for 1 hour. The mixture was left standing overnight at ambient
temperature. The layers were separated and aqueous phase was
re-extrated with ethyl acetate (75 mL). The ethyl acetate solution
was then successively washed with a water/brine mixture (70 mL),
another water/brine mixture (60 mL), 1N sodium hydroxide (60 mL),
and a third water/brine mixture (60 mL). Brine strength was 12% by
weight. The ethyl acetate solution was then dried over sodium
sulfate, filtered and concentrated to dryness by rotary evaporator
to afford a 4.5 g mixture of both the desired product and an
unknown impurity. The ratio of the impurity/product by HPLC area
was about 50/15 respectively. The major product from this reaction
was the impurity which was identified as 7-methyl hydrogen
17-hydroxy-3-oxo-17.alpha.-pregna-4,9(11)-diene-7,21-dicarboxylate,
.gamma.-lactone.
[0621] The mixture was purified by column chromatography to afford
1.9 g of analytically pure 7-methyl hydrogen
17-hydroxy-3-oxo-17.alpha.-pregna-4,9(11)-diene-7,21-dicarboxylate,
.gamma.-lactone.
[0622] The residue was further analyzed and purified by
chromatography. HPLC conditions: column--Waters Symmetry C18 (150
mm.times.4.6 mm i.d., 5 micron particle size); column
temperature--ambient; mobile phase--acetonitrile/water, 30/70 by
volume; flow rate--1.0 mL/minute; injection volume--20 microliters;
sample concentration--1.0 mg/mL; detection--UV at 210 nm;
pressure--1500 psi; and run time--45 minutes. TLC conditions:
adsorbent--Merck Silica Gel 60 F.sub.254; solvent
system--chloroform/methyl t-butyl ether/isopropanol, 70/28/2 by
volume; visualization technique--50% by volume aqueous
H.sub.2SO.sub.4/LWUV and 50% by volume
H.sub.2SO.sub.4/phosphomolybdic acid; and application amount--100
micrograms.
EXAMPLE 37I
Preparation of 7-methyl hydrogen
17-hydroxy-3,11-dioxo-17.alpha.-pregn-4-ene-7.alpha.,21-dicarboxylate,
.gamma.-lactone
[0623] ##STR212##
[0624] A Jones Reagent was prepared by dissolving 6.7 g of chromic
anhydride (Crop) in 6 mL of concentrated sulfuric acid and
carefully diluting that mixture with distilled water to 50 mL. One
mL of this reagent is sufficient to oxidize 2 mmol of a secondary
alcohol to a ketone.
[0625] Hydroxyester (10.0 g, 24.0 mmole) prepared in the manner of
Example 34 was dissolved/suspended in 1200 mL of acetone. To this
mixture was added 8.992 mL of the Jones Reagent and the combined
mixture was stirred for 10 minutes. An aliquot of the reaction
mixture, after being treated with water and extracted with
methylene chloride, was analyzed by HPLC (column: Beckman
Ultrasphere ODS C18, 4.6 mm.times.250 mm, 5 micron; solvent
gradient: acetonitrile/water=1/99 to 100/0 in 20 minutes at a flow
rate of 1.5 mL/minute; detector: UV210 nm). The reaction was
complete as evidenced by the lack of any significant amount of the
starting material in the reaction mixture. The HPLC retention time
for the starting material (hydroxester) is 13.37 minute and for the
product ketone was 14.56 min.
[0626] The reaction was worked up by adding 200 mL of water and 300
mL of methylene chloride. The organic layer was separated from the
aqueous layer and washed again with 200 mL of water. The organic
layer was separated from the aqueous layer and dried over magnesium
sulfate. The solvent was evaporated to provide 9.52 g of the
off-white solid (95.6% crude yield) 7-methyl hydrogen
17-hydroxy-3,11-dioxo-17.alpha.-pren-4-ene-7.alpha.,21-dicarboxylate,
.gamma.-lactone
[0627] The structure assignment was based on the mass spectrum (m/e
414), HNMR (DMF-d7) and CNMR (DMF-d7). In HNMR, the characteristic
peak of the 11-H (4.51 ppm, doublet, j=5.8 Hz) found in the
hydroxester was absent.
[0628] In CNMR, a peak appeared at 208.97 ppm which is expected for
the 11-keto carbon.
[0629] CNMR (400 MHz, DMF-d7) 208.97 (11-keto), 197.70 (3-keto),
176.00 (22-lactone), 173.34 (7-COOMe), 167.21 (C5), 125.33 (C4),
93.63 (C17), and other peaks in the region of 15 to 57 ppm.
EXAMPLE 37J
Preparation of dimethyl
11.alpha.,17-dihydroxy-3-oxo-17.alpha.-pregn-4-ene-7.alpha.,21-dicarboxyl-
ate, .gamma.-lactone
[0630] ##STR213##
[0631] A solution of 3.5 g (8.4 mmols) of the hydroxyester prepared
in the manner of Example 34 in 42 mL of methanol was mixed with 4
mL of methanolic 4N potassium hydroxide (8 mmols). The slurry was
stirred at room temperature overnight and heated to reflux for one
hour. The methanol was evaporated under vacuum and the residue
mixed with 50 mL of ethyl acetate. The ethyl acetate was evaporated
under vacuum and the residue digested with 50 mL of ethyl acetate.
The dried solid was combined with 50 mL of acetone and 2 mL of
methyl iodide (32.1 mmols). The mixture was stirred at room
temperature for 18 hours. During this time most of the solids
dissolved. The mixture was filtered and the filtrate evaporated to
dryness under vacuum. The residue was digested with ethyl acetate,
the solids then removed via filtration and the solvent removed
under vacuum distillation. The residue was determined to be a 78:22
(v/v) mixture of dimethyl
11.alpha.,17-dihydroxy-3-oxo-17.alpha.-pregn-4-ene-7.alpha.,21-dicarboxyl-
ate, .gamma.-lactone and the starting material hydroxyester via
H.sup.1 NMR. This mixture was adequate for use as an HPLC marker
without further purification.
[0632] .sup.1H NMR (CDCl.sub.3) indicated the following features:
ppm 0.93 (s, 3H), 1.37 (s, 3H), 3.64 (s, 3H), 3.69 (s, 3H).
EXAMPLE 37K
Preparation of
11.alpha.,17-dihydroxy-3-oxo-17.alpha.-pregn-4-ene-7.alpha.,21-dicarboxyl-
ate, .gamma.-lactone
[0633] ##STR214##
[0634] To 11.86 g (28.5 mmol) of the hydroxyester prepared in the
manner of Example 34 was added 50 mL of methanol and 20 mL of 2.5 M
NaOH. The suspension was heated to reflux. After 25 minutes, a
portion of the starting ester remained unreacted as judged by HPLC
(Zorbax SB-C8 150.times.4.6 mm, 2 ml/min., linear gradient 35:65
A:B to 45:55 A:B over 15 min, A=acetonitrile/methanol 1:1,
B=water/0.1% trifluoroacetic acid, detection at 210 nm) and 10 mL
of 10 M NaOH was added. After 1.5 hours, a trace of ester remained
unreacted as judged by HPLC. The reaction mixture was allowed to
stand at about 25 degrees for 64 hours.
[0635] The mixture was diluted with 100 mL of water and then made
strongly acidic with 20 mL of concentrated HCl. The resulting gummy
precipitate was stirred until the precipitate became a suspension.
The solid was isolated by filtration, resuspended in methanol and
filtered to give 3.75 g of a brown solid. The material was
dissolved in 8 mL of hot DMF and the mixture was diluted with 40 mL
of methanol. The acid crystallized and was isolated by filtration
to give 1.7 g of a fluffy white solid,
11.alpha.,17-dihydroxy-3-oxo-17.alpha.-pregn-4-ene-7.alpha.,21-dicarboxyl-
ate, .gamma.-lactone.
[0636] 1H-nmr (400 MHz, deuterodimethyl sulfoxide) d 0.80 (s, 3H),
1.25 (s, 3H), 1.2-2.7 (m, 20H), 3.8 (brs, 1H), 4.45 (m, 1H), 5.50
(s, 1H). The carboxyl proton was not observed due to the presence
of an HOD peak at 3.4 ppm.
EXAMPLE 38
Scheme 1: Step 3C: Method G: Preparation of 7-methyl Hydrogen
17-hydroxy-3-oxo-17.alpha.-pregna-4,9(11)-diene-7,21-dicarboxylate,
.gamma.-lactone
[0637] The procedure of Example 37A was repeated except that the
multiple washing steps were avoided by treating the reaction
solution with an ion exchange resin, basic alumina or basic silica.
Conditions for treatment with basic alumina or basic silica are set
forth in Table 38. Each of these treatments was found effective for
removal of impurities without the multiple washes of Example 44 to
produce 7-methyl hydrogen
17-hydroxy-3-oxo-17.alpha.-pregna-4,9(11)-diene-7,21-dicarboxylate,
.gamma.-lactone. TABLE-US-00047 TABLE 38 Factor Set point Purpose
of Experiment Key results Basic 2 g/125 g Treating the reaction
mixture The yield alumina product with basic alumina to remove was
93% Et.sub.3N.cndot.HCl salt and to eliminate the 1N NaOH and 1N
HCl washes Basic 2 g/125 g Treating the reaction mixture The yield
silica product with basic silica which is was 95% cheaper to remove
Et.sub.3N.cndot.HCl salt and eliminate 1N NaOH and 1N HCl
washes
EXAMPLE 39
Scheme 1: Step 3C: Method H: Preparation of 7-methyl hydrogen
17-hydroxy-3-oxo-17.alpha.-pregna-4,9(11)-diene-7,21-dicarboxylate,
.gamma.-lactone
[0638] Potassium acetate (4 g) and trifluoroacetic acid (42.5 mL)
were mixed in a 100 mL reactor. trifluoroacetic anhydride (9.5 mL)
was added to the mixture at a rate controlled to maintain
temperature during addition below 30.degree. C. The solution was
then heated to 30.degree. C. for 30 min. to provide an elimination
reagent useful for converting the mesylate of Formula IV to the
enester of Formula II.
[0639] The preformed TFA/TFA anhydride elimination reagent was
added to a solution of the mesylate of Formula IV previously
prepared as in Example 37A. The resulting mixture was heated at
40.degree. C. for 4% hrs., the degree of conversion being
periodically checked by TLC or HPLC. When the reaction was
complete, the mixture was transferred to a 1-neck flask and
concentrated to dryness under reduced pressure at room temperature
(22.degree. C.). Ethyl acetate (137 mL) was added to the mixture to
obtain complete dissolution of solid phase material after which a
water/brine mixture (137 mL) was added and the resulting two phase
mixture stirred for 10 min. The phases were then allowed to
separate for 20 min. Brine strength was 24% by weight. The aqueous
phase was contacted with an additional amount of ethyl acetate (68
mL) and the two phase mixture thus prepared was stirred for 10 min.
after which it was allowed to stand for 15 min. for phase
separation. The ethyl acetate layers from the two extractions were
combined and washed with 24% by weight brine (120 mL), another
aliquot of 24% by weight brine (60 mL), 1 N sodium hydroxide
solution (150 mL) and another portion of brine (60 mL). After each
aqueous phase addition, the mixture was stirred for 10 min. and
allowed to stand for 15 min. for separation. The resulting solution
was concentrated to dryness under reduced pressure at 45.degree. C.
using a water aspirator. The solid product (8.09 g) was analyzed by
HPLC and found to include 83.4 area % of the enester 7-methyl
hydrogen
17-hydroxy-3-oxo-17.alpha.-pregna-4,9(11)-diene-7,21-dicarboxylate,
.gamma.-lactone; 2.45 area % of the 11,12-olefin; 1.5% of the
7,9-lactone; and 1.1% of unreacted mesylate.
EXAMPLE 40
Scheme 1: Step 3C: Method I: Preparation of 7-methyl hydrogen
17-hydroxy-3-oxo-17.alpha.-pregna-4,9(11)-diene-7,21-dicarboxylate,
.gamma.-lactone
[0640] The mesylate having the structure prepared per Example 23
(1.0 g), isopropenyl acetate (10 g) and p-toluenesulfonic acid (5
mg) were placed in a 50 ml flask and heated to 90.degree. C. with
stirring. After 5 hours the mixture was cooled to 25.degree. C. and
concentrated in vacuo at 10 mm of Hg. The residue was dissolved in
CH.sub.2Cl.sub.2 (20 ml) and washed with 5% aqueous NaHCO.sub.3.
The CH.sub.2Cl.sub.2 layer was concentrated in vacuo to give 1.47 g
of a tan oil. This material was recrystallized from
CH.sub.2Cl.sub.2/Et.sub.2O to give 0.50 g of enol acetate of
Formula IV(Z).
[0641] This material was added to a mixture of sodium acetate (0.12
g) and acetic acid (2.0 ml) that had been previously heated to
100.degree. C. with stirring. After 60 minutes the mixture was
cooled to 25.degree. C. and diluted with CH.sub.2Cl.sub.2 (20 ml).
The solution was washed with water (20 ml) and dried over
MgSO.sub.4. The drying agent was removed by filtration and the
filtrate was concentrated in vacuo to give 0.4 g of the desired
9,11-olefin, 7-methyl hydrogen
17-hydroxy-3-oxo-17.alpha.-pregna-4,9(11)-diene-7,21-dicarboxylate,
.gamma.-lactone. The crude product contained less than 2% of the
7,9-lactone impurity.
EXAMPLE 41
Thermal Elimination of Mesylate in DMSO
Scheme 1: Step 3C: Method J: Preparation of 7-methyl hydrogen
17-hydroxy-3-oxo-17.alpha.-pregna-4,9(11)-diene-7,21-dicarboxylate,
.gamma.-lactone
[0642] ##STR215##
[0643] A mixture of 2 g of mesylate and 5 ml of DMSO in a flask was
heated at 80.degree. C. for 22.4 hours. HPLC analysis of the
reaction mixture indicated no starting material was detected. To
the reaction was added water (10 ml) and the precipitate was
extracted with methylene chloride three times. The combined
methylene chloride layers were washed with water, dried over
magnesium sulfate, and concentrated to give the enester 7-methyl
hydrogen
17-hydroxy-3-oxo-17.alpha.-pregna-4,9(11)-diene-7,21-dicarboxylate,
.gamma.-lactone.
EXAMPLE 42
Scheme 1: Step 3D: Method B: Synthesis of Methyl Hydrogen
9,11.alpha.-Epoxy-17.alpha.-hydroxy-3-oxopregn-4-ene-7.alpha.,21-dicarbox-
ylate, .gamma.-Lactone
[0644] In a 50 mL pear-shaped flask under stirring the enester of
Formula IIA (1.07 g assaying 74.4% enester), trichloroacetamide
(0.32 g), dipotassium hydrogen phosphate (0.70 g) as solid were
mixed with methylene chloride (15.0 mL). Hydrogen peroxide (30% by
weight; 5.0 mL) was added via a pipet over a 1 min. period. The
resulting mixture was stirred for 6 hrs. at room temperature at
which point HPLC analysis showed that the ratio of epoxymexrenone
to enester in the reaction mixture was approximately 1:1.
Additional trichloroacetamide (0.32 g) was added to the reaction
mixture and reaction continued under agitation for 8 more hours
after which time the remaining proportion of enester was shown to
have been reduced to 10%. Additional trichloroacetamide (0.08 g)
was added and the reaction mixture was allowed to stand overnight
at which point only 5% of unreacted enester remained relative to
epoxymexrenone in the mixture.
EXAMPLE 43
Scheme 1: Step 3D: Method C: Synthesis of Methyl Hydrogen
9,11.alpha.-Epoxy-17.alpha.-hydroxy-3-oxopregn-4-ene-7.alpha.,21-dicarbox-
ylate, .gamma.-Lactone
[0645] Enester of Formula IIA (5.4 g, assaying 74.4% enester) was
added to a 100 mL reactor. Trichloroacetamide (4.9 g) and
dipotassium hydrogen phosphate (3.5 g) both in solid form were
added to the enester followed by methylene chloride (50 mL). The
mixture was cooled to 15.degree. C. and a 30% hydrogen peroxide (25
g) was added over a ten min. period. The reaction mixture was
allowed to come to 20.degree. C. and stirred at that temperature
for 6 hrs., at which point conversion was checked by HPLC.
Remaining enester was determined to be less than 1% by weight.
[0646] The reaction mixture was added to water (100 mL), the phases
were allowed to separate, and the methylene chloride layer was
removed. Sodium hydroxide (0.5 N; 50 mL) was added to the methylene
chloride layer. After 20 min. the phases were allowed to separate
HCl (0.5 N; 50 mL) was added to the methylene chloride layer after
which the phases were allowed to separate and the organic phase was
washed with saturated brine (50 mL). The methylene chloride layer
was dried over anhydrous magnesium sulfate and the solvent removed.
A white solid (5.7 g) was obtained. The aqueous sodium hydroxide
layer was acidified and extracted and the extract worked up to
yield an additional 0.2 g of product. Yield of epoxymexrenone was
90.2%.
EXAMPLE 44
Scheme 1: Step 3D: Method D: Synthesis of Methyl Hydrogen
9,11.alpha.-Epoxy-17.alpha.-hydroxy-3-oxopregn-4-ene-7.alpha.,21-dicarbox-
ylate, .gamma.-Lactone
[0647] Enester of Formula IIA was converted to epoxymexrenone in
the manner described in Example 43 with the following differences:
the initial charge comprised of enester (5.4 g assaying 74.4%
enester), trichloroacetamide (3.3 g), and dipotassium hydrogen
phosphate (3.5 g). Hydrogen peroxide solution (12.5 mL) was added.
The reaction was conducted overnight at 20.degree. C. after which
HPLC showed a 90% conversion of enester to epoxymexrenone.
Additional trichloroacetamide (3.3 g) and 30% hydrogen peroxide
(5.0 mL) was added and the reaction carried out for an additional 6
hrs. at which point the residual enester was only 2% based on the
enester charge. After work up as described in Example 43, 5.71 g of
epoxymexrenone resulted.
EXAMPLE 45
Scheme 1: Step 3D: Method E: Synthesis of Methyl Hydrogen
9,11.alpha.-Epoxy-17.alpha.-hydroxy-3-oxopregn-4-ene-7.alpha.,21-dicarbox-
ylate, .gamma.-Lactone
[0648] The enester of Formula IIA was converted to epoxymexrenone
in the manner generally described in Example 43. In the reaction of
this Example, enester charge was 5.4 g (assaying 74.4% enester),
the trichloroacetamide charge was 4.9 g, hydrogen peroxide charge
was 25 g, dipotassium hydrogen phosphate charge was 3.5 g. The
reaction was run at 20.degree. C. for 18 hrs. The residual enester
was less than 2%. After work up, 5.71 g of epoxymexrenone
resulted.
EXAMPLE 46
Scheme 1: Step 3D: Method F: Synthesis of Methyl Hydrogen
9,11.alpha.-Epoxy-17.alpha.-hydroxy-3-oxopregn-4-ene-7.alpha.,21-dicarbox-
ylate, .gamma.Lactone
[0649] Enester of Formula IIA was converted to epoxymexrenone in
the manner described in Example 43 except that the reaction
temperature in this Example was 28.degree. C. The materials charged
in the reactor included enester (2.7 g), trichloroacetamide (2.5
g), dipotassium hydrogen phosphate (1.7 g), hydrogen peroxide (17.0
g) and methylene chloride (50 mL). After 4 hrs. reaction, unreacted
enester was only 2% based on the enester charge. After work up as
described in Example 43, 3.0 g of epoxymexrenone was obtained.
EXAMPLE 47-1
Scheme 1: Step 3D: Method G: Synthesis of Methyl Hydrogen
9,11.alpha.-Epoxy-17.alpha.-hydroxy-3-oxopregn-4-ene-7.alpha.,21-dicarbox-
ylate, .gamma.-Lactone
[0650] Enester of Formula IIA (40.0 g, assaying 68.4% enester) was
charged into a 1000 mL, jacketed reactor and dissolved in 175 mL of
methylene chloride. The solution was stirred as trichloroacetamide
(22.3 g) and dipotassium hydrogen phosphate (6.0 g) were added as
solids. The mixture was stirred at 400 RPM and the temperature
adjusted to 27.degree. C. with a constant temperature bath to
control the liquid circulating through the reactor jacket. Hydrogen
peroxide (72.8 mL at 30% assay) was added over a 3 to 5 minute
period. Following the hydrogen peroxide addition, the mixture was
stirred at 400 RPM and 27.degree. C. HPLC assay indicated that the
reaction was 99% complete within 5 hours. At the end of six hours,
72.8 mL of water was added. The aqueous hydrogen peroxide was
separated and back extracted one time with 50 mL of methylene
chloride. The combined methylene chloride was washed with 6% sodium
sulfite (62.3 mL) to destroy any contained peroxide. The methylene
chloride removal was started with atmospheric distillation and
concluded under vacuum. A yellowish residue (48.7 g, 55.4% assay)
was obtained. This correlated with a 94.8% assay adjusted molar
yield.
[0651] A portion (47.8 g) of the residue was combined with 498 mL
of ethanol 3A (95% ethanol denatured with 5% methanol). The mixture
was heated to reflux and 249 mL of distillate removed at
atmospheric pressure. The mixture was cooled to 25.degree. C. and
filtered. An ethanol 3A rinse (53 mL) was used to assist the
transfer. The dried solid weighed 27.6 g (87.0% assay) which
correlated with a 91% recovery. A portion of the solid (27.0 g) was
dissolved in 292 mL of methyl ethyl ketone at reflux. The hot
solution was filtered through a pad of solka floc (powdered
cellulose) with another 48.6 mL of methyl ethyl ketone used to
assist the transfer. A 146 mL portion of the methyl ethyl ketone
was removed via atmospheric distillation. The solution was cooled
to 50.degree. C. and stirred for one hour as the product
crystallized. After one hour the mixture was cooled to 25.degree.
C. Stirring was continued for one hour and the solid filtered with
48.6 mL of methyl ethyl ketone used as a rinse. The solid was dried
to a constant weight of 20.5 g which represented an 87.2%
recrystallization recovery. The reaction yield and ethanol, methyl
ethyl ketone recoveries combined for a 75% overall yield.
[0652] The methyl ethyl ketone mother liquor was suitable for
recycling with an incoming methylene chloride solution from a
subsequent reaction. The combined methylene chloride and methyl
ethyl ketone mixture was evaporated to dryness with atmospheric and
vacuum distillation. The residue was combined with 19 volumes of
ethanol 3A based on epoxymexrenone content. One half of the solvent
was removed under atmospheric distillation. After cooling to
25.degree. C. the solid was filtered and dried. The dry solid was
dissolved in 12 volumes of methyl ethyl ketone at reflux. The hot
solution was filtered through a solka floc pad with 2 volumes of
methyl ethyl ketone added as a rinse. The filtrate was concentrated
with the atmospheric distillation of 6 volumes of methyl ethyl
ketone. The solution was cooled to 5.degree. C. and stirred for one
hour as the product crystallized. After one hour the mixture was
cooled to 25.degree. C. Stirring was continued for one hour and the
solid filtered with 2 volumes of methyl ethyl ketone used as a
rinse. The solid was dried to a constant weight. The incorporation
of the methyl ethyl ketone mother liquor raised the overall yield
to 80-85%.
[0653] This method appears particularly suited for scaleup since it
maximizes throughput and minimizes washing volumes and waste.
EXAMPLE 47A
Scheme 1: Step 3D: Method H: Synthesis of Methyl Hydrogen
9,11.alpha.-Epoxy-17.alpha.-hydroxy-3-oxopregn-4-ene-7.alpha.,21-dicarbox-
ylate, .gamma.-Lactone
[0654] Enester of Formula IIA (17 g, assaying 72% enester) was
dissolved in methylene chloride (150 mL) after which
trichloroacetamide (14.9 g) was added under slow agitation. The
temperature of the mixture was adjusted to 25.degree. C. and a
solution of dipotassium hydrogen phosphate (10.6 g) in water (10.6
mL) was stirred into the enester substrate solution under 400 rpm
agitation. Hydrogen peroxide (30% by weight solution; 69.4 mL) was
added to the substrate/phosphate/trichloroacetamide mixture over a
3-5 min. period. No exotherm or oxygen evolution was observed. The
reaction mixture thus prepared was stirred at 400 rpm and
25.degree. C. for 18.5 hrs. No oxygen evolution was observed
throughout the course of the reaction, but analysis of the hydrogen
peroxide consumption indicated that some oxygen was formed during
the reaction. The reaction mixture was diluted with water (69.4 mL)
and the mixture stirred at about 250 rpm for 15 min. No temperature
control was necessary for this operation and it was conducted
essentially at room temperature (any temperature in the range of
5-25.degree. C. being acceptable). The aqueous and organic layers
were allowed to separate and the lower methylene chloride layer was
removed.
[0655] The aqueous layer was back extracted with methylene chloride
(69.4 mL) for 15 min. under agitation of 250 rpm. The layers were
allowed to separate and the lower methylene chloride layer was
removed. The aqueous layer (177 g; pH=7) was submitted for hydrogen
peroxide determination. The result (12.2%) indicated that 0.0434
mol of hydrogen peroxide were consumed in the reaction of 0.0307
mol of olefin. The excess hydrogen peroxide consumption was a
measure of oxygen generation in the reaction. Back extraction with
a small amount of methylene chloride volume was sufficient to
insure no loss of epoxymexrenone in the aqueous layer. This result
was confirmed with the application of a second large methylene
chloride extraction in which only trichloroacetamide was
recovered.
[0656] The combined methylene chloride solutions from the above
described extractions were combined and washed with 3% by weight
sodium sulfite solution (122 mL) for at least 15 min. at about 250
rpm. A negative starch iodide test (KI paper; no color observed; in
a positive test a purple coloration indicates the presence of
peroxide) was observed at the end of the stir period.
[0657] The aqueous and organic layers were allowed to separate and
the lower methylene chloride layer removed. The aqueous layer
(pH=6) was discarded. Note that addition of sodium sulfite solution
can cause a slight exotherm so that such addition should be carried
out under temperature control.
[0658] The methylene chloride phase was washed with 0.5 N sodium
hydroxide (61 mL) for 45 min. at about 250 rpm and a temperature in
the range of 15-25.degree. C. (pH 12-13). Impurities derived from
trichloroacetamide were removed in this process. Acidification of
the alkaline aqueous fraction followed by extraction of the
methylene chloride confirmed that very little epoxymexrenone was
lost in this operation.
[0659] The methylene chloride phase was washed once with 0.1 N
hydrochloric acid (61 mL) for 15 min. under 250 rpm agitation at a
temperature in the range 15-25.degree. C. The layers were then
allowed to separate and the lower methylene chloride layer removed
and washed again with 10% by weight aqueous sodium chloride (61 mL)
for 15 min at 250 rpm at a temperature in the range of
15-25.degree. C. Again the layers were allowed to separate and the
organic layer removed. The organic layer was filtered through a pad
of Solkafloc and then evaporated to dryness under reduced pressure.
Drying was completed with a water bath temperature of 65.degree. C.
An off-white solid (17.95 g) was obtained and submitted for HPLC
assay. Epoxymexrenone assay was 66.05%. An adjusted molar yield for
the reaction was 93.1%.
[0660] The product was dissolved in hot methyl ethyl ketone (189
mL) and the resulting solution was distilled at atmospheric
pressure until 95 mL of the ketone solvent had been removed. The
temperature was lowered to 50.degree. C. as the product
crystallized. Stirring was continued at 50.degree. C. for 1 hr. The
temperature was then lowered to 20-25.degree. C. and stirring
continued for another 2 hrs. The solid was filtered and rinsed with
MEK (24 mL) and the solid dried to a constant weight of 9.98 g,
which by HPLC assay contain 93.63% epoxymexrenone. This product was
re-dissolved in hot MEK (106 mL) and the hot solution filtered
through a 10 micron line filter under pressure. Another 18 mL of
MEK was applied as a rinse and the filtered MEK solution distilled
at atmospheric pressure until 53 mL of solvent had been removed.
The temperature was lowered to 50.degree. C. as the product
crystallized; and stirring was continued at 50.degree. C. for 1 hr.
The temperature was then lowered to 20-25.degree. C. and held at
that temperature while stirring was continued for another 2 hrs.
The solid product was filtered and rinsed with MEK (18 mL). The
solid product was dried to a constant weight of 8.32 g which
contained 99.6% epoxymexrenone per quantitative HPLC assay. The
final loss on drying was less than 1.0%. Overall yield of
epoxymexrenone in accordance with the reaction and work up of this
Example is 65.8%. This overall yield reflected a reaction yield of
93%, an initial crystallization recovery of 78.9%, and a
recrystallization recovery of 89.5%.
EXAMPLE 47B
Preparation of 7-methyl hydrogen
11.alpha.,12.alpha.-epoxy-17-hydroxy-3-oxo-17.alpha.-pregn-4-ene-7.alpha.-
,21-dicarboxylate, .gamma.-lactone
[0661] ##STR216##
[0662] The .DELTA..sup.11,12 olefin of the enester is a byproduct
of the 11-mesylate elimination. A pure sample was isolated from a
reaction mixture prepared in the manner of Example 37A via
repetitive preparative liquid chromatography. Thus, a 73 g residue
(prepared as described in Example 37A) was chromatographed-over
2.41 kg of Merck silica gel (40-63.mu.) with an ethyl acetate,
toluene gradient elution scheme (20:80, 30:70, 40:60, 60:40, v/v).
Enriched .DELTA..sup.11,12 olefin portions were combined from
selected 30:70 fractions. TLC on EMF plates using ethyl
acetate/toluene 60:40 (v/v) with sulfuric acid SWUV visualization
served as a guide for choosing the appropriate fractions. The 7.9 g
of crude .DELTA..sup.11,12 olefin (80 area % via HPLC) obtained
after the removal of solvent was chromatographed over 531 g of
Merck silica gel (40-63.mu.) with an ethyl acetate/methylene
chloride gradient elution scheme (10:90, 20:80, 35:65, v/v). Pure
7-methyl hydrogen
17-hydroxy-3-oxo-17.alpha.-pregna-4,11-diene-7.alpha.,21-dicarboxylate,
.gamma.-lactone (3.72 g) was obtained from selected 20:80
fractions. The selection of fractions was based on TLC evaluation
as in the previous situation.
[0663] MIR cm-.sup.1 1767 (lactone) 1727 (ester), 1668 and 1616
(3-Keto-.DELTA..sup.4,5).
[0664] .sup.1H NMR (CDCl.sub.3) ppm 1.05 (s, 3H), 1.15 (s, 3H),
3.66 (s, 3H), 5.58 (dd, 1H), 5.80 (s, 1H), 5.88 (dd, 1H).
[0665] .sup.13C NMR (CDCl.sub.3) ppm 17.41, 18.58, 21.73, 28.61,
32.28, 33.63, 34.91, 35.64, 35.90, 38.79, 42.07, 44.12, 48.99,
49.18, 51.52, 93.81, 126.43, 126.69, 133.76, 166.24, 172.91,
176.64, 198.56.
[0666] A solution of 1.6 g (3.9 mmols) of 7-methyl hydrogen
17-hydroxy-3-oxo-17.alpha.-pregna-4,11-diene-7.alpha.,21-dicarboxylate,
.gamma.-lactone in 16 mL of methylene chloride was mixed with 2.2
mL of trichloroacetonitrile (22.4 mmols) and 0.75 g of dipotassium
phosphate (4.3 mmols). The mixture was stirred and combined with
6.7 mL of 30% hydrogen peroxide (66 mmols). Stirring was continued
at 25.degree. C. for 45 hours. At the end of this time 28 mL of
methylene chloride and 39 mL of water were added. The organic
portion was isolated and washed in succession with a) 74 mL of 3%
sodium sulfite, b) 62 mL of 1 N sodium hydroxide, c) 74 mL of 1 N
hydrochloric acid, and d) 31 mL of 10% brine. The organic portion
was separated again, dried over magnesium sulfate, and evaporated
to dryness under vacuum. The 1.25 g residue was chromatographed
over 138.2 g of Merck silica gel (40-63.mu.) using a methyl-t-butyl
ether and toluene gradient system (40:60, 60:40, 75:25, v/v).
Appropriate portions of the 60:40 and 75:25 fractions were combined
after TLC evaluation to give 0.66 g of pure 7-methyl hydrogen
11.alpha.,12.alpha.-epoxy-17-hydroxy-3-oxo-17-pregn-4-ene-7.alpha.,21-dic-
arboxylate, .gamma.-lactone. The TLC system utilized EMF plates and
a 75:25 (v/v) methyl-t-butyl ether and toluene elution scheme with
sulfuric acid and SWUV for visualization.
[0667] .sup.1H NMR (CDCl.sub.3) ppm 1.09 (s, 3H), 1.30 (s, 3H),
3.05 (AB.sup.11,12 2H for), 3.67 (s, 3H), 5.80 (s, 1).
[0668] .sup.13C NMR (CDCl.sub.3) ppm 14.2, 18.0, 21.2, 28.8, 31.9,
33.5, 34.6, 34.7, 35.1, 35.5, 37.4, 38.3, 41.8, 46.0, 47.2, 50.4,
51.7, 56.7, 94.0, 126.7, 165.2, 172.5, 176.7, 198.1.
[0669] Theory: C, 69.54 and H, 7.30; Found: C, 69.29 and H,
7.17.
EXAMPLE 47C
Isolation of 7-methyl hydrogen
4.alpha.,5.alpha.:9.alpha.,11.alpha.-diepoxy-17-hydroxy-3-oxo-17.alpha.-p-
regnane-7.alpha.,21-dicarboxylate, .gamma.-lactone
[0670] ##STR217##
[0671] Crude epoxymexrenone (157 g) prepared from 200 g of the
enester in the manner of Example 26 was subjected to chromatography
over 4.4 kg of Merck silica gel (40-63.mu.). An 88.1 g portion was
recovered with an acetonitrile and toluene 10:90 (v/v) elution
scheme. The isolated solid was dissolved in 880 mL of hot methyl
ethyl ketone and filtered through a pad of solka floc. Another 88
mL of methyl ethyl ketone was applied as a rinse. The filtrate was
concentrated via the removal of 643 mL of solvent and the mixture
cooled to room temperature. The solids were filtered and rinsed
with methyl ethyl ketone. After drying, 60.2 g of epoxymexrenone
assaying 96.8% via HPLC was obtained. The filtrate was concentrated
to dryness under reduced pressure. The 9.3 g residue was
recrystallized from 99 mL of methyl ethyl ketone to yield 2.4 g of
dry solid. A 400 mg portion of the solid was subjected to reverse
phase preparative HPLC on a YMC ODS AQ column. Pure 7-methyl
hydrogen
4.alpha.,5.alpha.:9.alpha.,11.alpha.-diepoxy-17-hydroxy-3-oxo-17.alpha.-p-
regnane-7.alpha.,21-dicarboxylate, .gamma.-lactone (103 mg) was
isolated with an elution scheme of acetonitrile (24%), methanol
(4%) and water (72%).
[0672] .sup.1H NMR (CDCl.sub.3) ppm 0.98 (s, 3H), 1.32 (s, 3H),
2.89 (m, 1H), 3.07 (s, d, 2H), 3.73 (s, 3H).
[0673] MS, M+430, calculated for C.sub.24H.sub.30O.sub.7
(430.50).
EXAMPLE 47D
Isolation of 7-methyl hydrogen
17-hydroxy-3,12-dioxo-17.alpha.-pregna-4,9(11)-diene-7.alpha.,21-dicarbox-
ylate, .gamma.-lactone
[0674] ##STR218##
[0675] A methyl ethyl ketone mother liquor obtained in the manner
of Example 26 was evaporated to dryness under reduced pressure. A
4.4 g portion of the residue was subjected to chromatography on
58.4 g of BTR Zorbax LP (40.mu.). Elution with a gradient of methyl
ethyl ketone and methylene chloride (25:75 to 50:50, v/v) yielded
1.38 g of material. A 1.3 g portion of this material was further
purified via reverse phase preparative HPLC using acetonitrile
(30%), methanol (5%) and water (65%) as the mobile phase and a YMC
ODS AQ column (10.mu.). The product was obtained from the enriched
fractions via methylene chloride extraction. The methylene chloride
was evaporated to dryness and the 175 mg residue repurified via
reverse phase preparative HPLC using acetonitrile (24%), methanol
(4%) and water (72%) as the mobile phase and a YMC ODS AQ column.
Methylene chloride extraction of enriched fractions yielded 30.6 mg
of pure 7-methyl hydrogen
17-hydroxy-3,12-dioxo-17.alpha.-pregna-4,9(11)-diene-7.alpha.,21-dicarbox-
ylate, .gamma.-lactone.
[0676] .sup.1H NMR (CDCl.sub.3) ppm 1.17 (s, 3H), 1.49 (s, 3H),
3.13 (m, 1H), 3.62 (s, 3H), 5.77 (s, 1H), 5.96 (s, 1H).
[0677] .sup.13C NMR (CDCl.sub.3) ppm 13.1, 21.0, 28.0, 29.4, 33.1,
33.4, 33.9, 35.5, 36.7, 40.3, 41.5, 43.0, 43.4, 52.0, 55.0, 91.0,
123.7, 126.7, 163.2, 167.9, 171.8, 176.8, 197.4, 201.0.
EXAMPLE 47E
Preparation of
9,11.alpha.-epoxy-17-hydroxy-3-oxo-17.alpha.-pregn-4-ene-7.alpha.,21-dica-
rboxylic acid, dihydrate, dipotassium salt
[0678] ##STR219##
[0679] A suspension containing 2.0 g (4.8 mmol) of epoxymexrenone
prepared in the manner of Example 43, 10 mL of water, 3 mL of
dioxane and 9.3 mL of 1.04 N aqueous potassium hydroxide (9.7 mmol)
was prepared. The mixture was stirred at 25.degree. C. for 3 hours.
A yellow, homogenous solution was formed during the first two
hours. The temperature was raised to 70.degree. C. and stirring
continued for an additional 3 hours. The solvent was removed via
vacuum distillation and the residue purified via reverse phase
chromatography over 90 g of C18 silica gel using water as the
eluent. The desired fractions were combined after review via TLC on
EMF plate, using methylene chloride, methanol (7:3) as the eluent
and SWUV for visualization. The combined fractions were
concentrated to dryness under vacuum and the residue subjected to
reverse phase purification was repeated as previously described.
The desired fractions were concentrated to dryness under reduced
pressure and the residue dissolved in ethanol. Ethyl acetate was
added to the cloud point, then heptane added to complete the
precipitation. 0.55 g of the product,
9,11.alpha.-epoxy-17-hydroxy-3-oxo-17.alpha.-pregn-4-ene-7.alpha.,21-dica-
rboxylic acid, dihydrate, dipotassium salt, was isolated as a
yellow solid. The carbon analysis correlated with a hydrated
structure C.sub.23H.sub.28O.sub.7K.sub.2.1.75H.sub.2O: Theory C,
52.50 vs 55.85 for anhydrous form; Found C, 52.49. After TLC on EMF
plates with methylene chloride, methanol, water (6:3:0.5, v/v) as
the eluent and visualization via SWUV, an R.sub.f of 0.29 was
observed.
EXAMPLE 47F
Preparation of
9,11.alpha.-epoxy-17-hydroxy-3-oxo-17.alpha.-pregn-4-ene-7.alpha.,21-dica-
rboxylic acid, disodium salt
[0680] ##STR220##
[0681] About 5 mg (0.01 mmol) of epoxymexrenone prepared in the
manner of Example 43 was suspended in about 200 .mu.L of methanol
in a 4 mL vial and diluted with about 200 .mu.L of 2.5 N NaOH. The
resulting mixture was yellow and homogeneous. The mixture was then
heated in an oil bath at 70.degree. C. After 10 minutes a 1 .mu.m
sample from the mixture was analyzed by HPLC (Zorbax SB-C8
150.times.4.6 nn, 2 mL/minute, gradient=35:65 (v/v) A:B,
A=acetonitrile/methanol (1:1), B=water/0.1% trifluoroacetic acid,
detection at 210 nm) showed two materials at 4.86 and 2.93 minute
retention times consistent with the hydroxyacid (open lactone) and
the open lactone 7-carboxylic acid, respectively. After 30 minutes
a second (0.05 mL) sample was removed and acidified with 0.05 mL of
3 N HCl followed by neutralization with about 0.5 mL of sodium
bicarbonate. HPLC analysis as above showed the expected ring-closed
steroids with retention times of 6.59 and 10.71 minutes. The ratio
of 7-methyl hydrogen
9,11.alpha.-epoxy-17-hydroxy-3-oxo-17.alpha.-pregn-4-ene-7.alpha.,21-dica-
rboxylate, .gamma.-lactone (10.71 minutes) to the corresponding
7-carboxylic acid was 7:89.
[0682] Selective hydrolysis of the lactone was possible under mild
conditions. A second 4 mL vial was prepared as above but was not
heated. The mixture was sonicated for 5 minutes. A 0.05 mL sample
was diluted in 0.5 mL of a 1:1 (v/v) mixture of
methanol/acetonitrile and was analyzed by HPLC without prior
acidification. The resulting open lactone carboxylic acid 7-ester
had a retention time of 4.85 minutes as observed above and was
uncontaminated by the 7-carboxylic acid.
EXAMPLE 47G
Isolation of 7-methyl hydrogen
9.alpha.,11.beta.,17-trihydroxy-3-oxo-17.alpha.-pregn-4-ene-7.alpha.,21-d-
icarboxylate, .gamma.-lactone
[0683] ##STR221## and
7-methyl hydrogen
12.alpha.,17-dihydroxy-3-oxo-17.alpha.-pregna-4,9(11)-diene-7.alpha.,21-d-
icarboxylate, .gamma.-lactone
[0684] ##STR222##
[0685] 7-methyl hydrogen
9.alpha.,11.alpha.,17-trihydroxy-3-oxo-17.alpha.-pregn-4-ene-7.alpha.,21--
dicarboxylate, .gamma.-lactone and 7-methyl hydrogen
12.alpha.,17-dihydroxy-3-oxo-17.alpha.-pregna-4,9(11)-diene-7.alpha.,21-d-
icarboxylate, T-lactone were isolated after preparative liquid
chromatography of the 2-butanone mother liquor recovered from the
epoxidation of the enester as described in example 26
(trichloroacetonitrile protocol). Thus, the first crystallization
was carried out using 2-butanone as indicated. The
recrystallization, however, utilized 2-butanone (10 vols per g) in
place of acetone. A 2.8 g residue was obtained in this manner and
was purified via reverse phase preparative HPLC. Cromasil C8
(10.mu.) was used as the stationary phase with a mobile phase
composed of milliQ water and acetonitrile in a ratio of 70:30
(v/v). Crystallization was observed in one of the enriched
fractions. The solid (46.7 mg) was isolated and identified as
7-methyl hydrogen 9.alpha.,
11.beta.,17-trihydroxy-3-oxo-17.alpha.-pregn-4-ene-7.alpha.,21-dicarboxyl-
ate, .gamma.-lactone. The mother liquor was evaporated to dryness
under reduced pressure and the residue (123 mg) identified as
7-methyl hydrogen
12.alpha.,17-dihydroxy-3-oxo-17.alpha.-pregna-4,9(11)-diene-7.alpha.,21-d-
icarboxylate, .gamma.-lactone.
7-methyl hydrogen
9.alpha.,11.beta.,17-trihydroxy-3-oxo-17.alpha.-pregn-4-ene-7.alpha.,21-d-
icarboxylate, .gamma.-lactone
[0686] MS M+432, calculated for C.sub.24H.sub.32O.sub.5
(432.51).
[0687] .sup.1H NMR (CDCl.sub.3) ppm 1.23 (s, 3H), 1.54 (s, 3H),
3.00 (m, 1H), 3.14 (m, 1H), 3.74 (s, 3H), 5.14 (s, 1H, slowly
exchangeable), 5.79 (s, 1H).
[0688] .sup.13C NMR (CDCl.sub.3) ppm 16.8, 22.7, 24.8, 29.0, 29.3,
32.1, 34.1, 34.7, 35.2, 35.7, 36.8, 40.7, 43.0, 45.0, 45.9, 52.9,
72.8, 77.4, 95.9, 127.4, 163.7, 176.7, 177.3, 199.4.
7-methyl hydrogen
12.alpha.,17-dihydroxy-3-oxo-17.alpha.-pregna-4,9(11)-diene-7.alpha.,21-d-
icarboxylate, .gamma.-lactone
[0689] MS M+ 441, calculated for C.sub.24H.sub.30O.sub.6
(414.50).
[0690] .sup.1H NMR (CDCl.sub.3) ppm 0.87 (s, 1H), 1.40 (s, 1H),
3.05 (m, 1H), 3.63 (s, 3H), 3.99 (m, 1H), 5.72 (s, 1H), 5.96 (m,
1H).
[0691] .sup.13C NMR (CDCl.sub.3) ppm 14.8, 24.0, 26.1, 29.7, 33.6,
33.8, 34.0, 36.3, 37.0, 37.4, 40.7, 40.9, 43.8, 48.1, 51.9, 69.1,
95.5, 122.7, 126.3, 145.9, 164.5, 173.2, 177.6, 198.2.
EXAMPLE 47H
Preparation of 7-methyl hydrogen
9,11.alpha.-epoxy-3-ethoxy-17-hydroxy-17.alpha.-pregn-4-ene-7.alpha.,21-d-
icarboxylate, .gamma.-lactone
[0692] ##STR223## and
7-methyl hydrogen
6.beta.,17-dihydroxy-9.alpha.,11.beta.-epoxy-3-oxo-17.alpha.-pregn-4-ene--
7.alpha.,21-dicarboxylate, .gamma.-lactone
[0693] ##STR224##
[0694] 7-methyl hydrogen
9,11.alpha.-epoxy-3-ethoxy-17-hydroxy-17.alpha.-pregn-4-ene-7.alpha.,21-d-
icarboxylate, .gamma.-lactone was prepared according to the method
of R. M. Weier and L. M. Hofmann (J. Med Chem 1977, 1304) which is
incorporated by reference. 148 g (357 mmols) of 7-methyl hydrogen
9,11.alpha.-epoxy-17-hydroxy-3-oxo-17.alpha.-pregn-4-ene-7.alpha.,21-dica-
rboxylate, .gamma.-lactone prepared in the manner of Example 43
were combined with 311 mL of absolute ethanol and 155 mL (932
mmols) of triethylorthoformate. The slurry was stirred at room
temperature and 10.4 g (54.7 mmols) of toluene sulfonic acid
(monohydrate) added as a catalyst. Stirring was continued for 30
minutes and the reaction quenched with the addition of 41.4 g (505
mmols) of powdered sodium acetate and 20.7 mL (256 mmols) of
pyridine. Solids (70.2 g) were removed by filtration and the
filtrate concentrated to dryness under vacuum. The residue was
digested with 300 mL of ethyl acetate and 9.8 g of solids were
removed via filtration.
[0695] The filtrate was concentrated to dryness and the residue
digested with 100 mL of methanol containing 2 mL of pyridine. 29.7
g of solids were removed via filtration. Additional precipitation
was observed in the filtrate. Therefore, the filtrate was
refiltered to remove an additional 21.9 of solids. The filtrate was
concentrated to dryness and the residue digested with 50 mL of
methanol containing 1 mL of pyridine. 33.8 g of solids were
isolated via filtration. Qualitative HPLC indicated that this last
portion of solids was sufficiently pure (90 area percent of
7-methyl hydrogen
9,11.alpha.-epoxy-3-ethoxy-17-hydroxy-17.alpha.-pregn-4-ene-7.al-
pha.,21-dicarboxylate, .gamma.-lactone) for use in the next step of
the reaction.
[0696] 7-methyl hydrogen
9,11.alpha.-epoxy-3-ethoxy-17-hydroxy-17.alpha.-pregn-4-ene-7.alpha.,21-d-
icarboxylate, .gamma.-lactone: .sup.1H NMR (CDCl.sub.3) ppm 1.02
(s, 3H), 1.27 (s, 3H), 1.30 (t, 3H), 3.12 (m, 1H), 3.28 (m, 1H),
3.66 (s, 3H), 3.78 (m, 2H), 5.20 (s, 1H), 5.29 (d, 1H).
[0697] An 8 g portion of the enol ether (7-methyl hydrogen
9,11.alpha.-epoxy-3-ethoxy-17-hydroxy-17.alpha.-pregn-4-ene-7.alpha.,21-d-
icarboxylate, .gamma.-lactone) (18 mmols) prepared in the prior
step was dissolved in 120 mL of 1,4-dioxane. The solution was
combined with a mixture of 6.8 g of 53% m-chloroperbenzoic acid
(20.9 mmols), 18.5 mL of 1.0 N sodium hydroxide (18.5 mmols) and 46
mL of dioxane/water (9:1). The temperature was maintained at
-3.degree. C. and the mixture stirred for two hours. The
temperature was raised to 25.degree. C. and stirring continued for
another twenty hours. The mixture was combined with 400 mL of cold
water (10.degree. C.) and 23.5 mL of 1.0 N sodium hydroxide (23.5
mmols). The mixture was extracted four times with 100 mL portions
of methylene chloride each time. The combined methylene chloride
portions were dried over magnesium sulfate and the supernatant
solvent removed under vacuum distillation. The 13.9 g residue was
triturated with 50 mL of ethyl ether to give 2.9 g of a white
solid. A 2.4 g portion of the solid was chromatographed over 100 g
of Merck silica gel (60.mu.). After an initial washing with 1 L of
1:1 ethyl acetate/heptane, the product was eluted with a 7:3 ratio
of ethyl acetate/heptane. Enriched fractions were combined on the
basis of TLC evaluation (EMF plates; ethyl acetate/heptane 7:3
(v/v) eluent; SWUV visualization). Thus, 0.85 g of enriched
material was obtained and recrystallized from 10 mL of isopropanol
to give 0.7 g of 7-methyl hydrogen
6.beta.,17-dihydroxy-9,11.alpha.-epoxy-3-oxo-17.alpha.-pregn-4-ene-7.alph-
a.,21-dicarboxylate, .gamma.-lactone. The more contaminated
fractions were combined and 0.87 g of crude 7-methyl hydrogen
6.beta.,17-dihydroxy-9,11.alpha.-epoxy-3-oxo-17.alpha.-pregn-4-ene-7.alph-
a.,21-dicarboxylate, .gamma.-lactone obtained. This material was
chromatographed over 67.8 g of Merck silica gel (40-63.mu.). An
additional 0.69 g of product was recovered with toluene containing
0.5 to 2.5% methanol.
7-methyl hydrogen
6.beta.,17-dihydroxy-9,11.alpha.-epoxy-3-oxo-17.alpha.-pregn-4-ene-7.alph-
a.,21-dicarboxylate, .gamma.-lactone
[0698] Theory: C, 66.96 and H, 7.02: Found: C, 66.68 and H,
7.16.
[0699] .sup.1H NMR (CDCl.sub.3) ppm 1.06 (s, 3H), 1.36 (dm, 1H),
1.63 (s, 3H), 2.92 (m, 1H), 3.02 (dd, 1H), 3.12 (d, 1H), 3.64 (s,
3H), 4.61 (d, 1H), 5.96 (s, 1H).
[0700] .sup.13C NMR (CDCl.sub.3) ppm 16.17, 21.32, 21.79, 24.36,
27.99, 28.94, 30.86, 31.09, 32.75, 33.19, 34.92, 36.77, 39.16,
43.98, 47.74, 51.56, 51.66, 65.36, 72.23, 94.79, 165.10, 171.36,
176.41, 199.59.
EXAMPLE 47I
Preparation of 7-methyl hydrogen
9,11.alpha.-epoxy-17-hydroxy-3-oxo-17.alpha.-pregn-4-ene-7.beta.,21-dicar-
boxylate, .gamma.-lactone
[0701] ##STR225##
[0702] To 2 g (4.8 mmol) of 7-methyl hydrogen
9,11.alpha.-epoxy-17-hydroxy-3-oxo-17.alpha.-pregn-4-ene-7.alpha.,21-dica-
rboxylate, .gamma.-lactone prepared in the manner of Example 43 was
added 3.3 mL (14.4 mmol) of 25% sodium methoxide in methanol. The
resulting yellow suspension was heated to 5.degree. C. The solid
did not dissolve. To the mixture was added 3.3 ml of methanol
(Aldrich anhydrous). The mixture was heated to reflux conditions
(65.degree. C.) and became homogeneous. After 30 minutes a solid
precipitate prevented stirring.
[0703] About 25 ml of anhydrous methanol was added and the mixture
was transferred to a 100 mL flask. The mixture was heated at reflux
conditions for 16 hours at which time the mixture was dark and
homogeneous. The mixture was cooled to 25.degree. C. and 70 ml of
3N HCl was added (exothermic). Several grams of ice were added to
cool the mixture and the solution was extracted with two successive
25 mL portions of methylene chloride. The dark solution was dried
over sodium sulfate and filtered through a 2.5 cm pad of silica gel
(E. Merck, 70-230 mesh 60 .ANG. pore size). The silica was eluted
with 100 mL of methylene chloride. The eluted methylene chloride
was then concentrated in vacuo to give 1 g of a brown foam which
crystallized upon addition of ethyl acetate. The silica pad was
eluted a second time with 100 ml of 10% ethyl acetate/methylene
chloride and the eluted solution was concentrated to give 650 mg of
a brown foam.
[0704] Thin layer chromatography (E. Merck 60 F-254 silica gel 0.25
mm, toluene/ethyl acetate (1:1, v/v)) revealed the presence of
7-methyl hydrogen
9,11.alpha.-epoxy-17-hydroxy-3-oxo-17.alpha.-pregn-4-ene-7.alpha-
.,21-dicarboxylate, .gamma.-lactone and 7-methyl hydrogen
9,11.alpha.-epoxy-17-hydroxy-3-oxo-17.alpha.-pregn-4-ene-70,21-dicarboxyl-
ate, .gamma.-lactone in both samples, although very little of the
7.alpha.-carboxy epimer was present in the first sample. The first
sample was triturated with hot ethyl acetate (77.degree. C.) and
allowed to cool to 25.degree. C. The mixture then was filtered to
give 400 mg of an off-white solid, mp 254-258.degree. C. H,
.sup.13C and .sup.13C-APT were consistent with the assigned
structure. A small amount of ethyl acetate remained in the sample
but no starting material was evident by HPLC (Zorbax SB-C8
150.times.4.6 nn, 2 mL/minute, isocratic 40:60 (v/v) A:B,
A=acetonitrile/methanol (1:1), B=water/0.1% trifluoroacetic acid,
detection at 210 nm) (HPLC showed 98.6 area percent), and by TLC
(toluene-ethyl acetate 1:1, v/v).
[0705] FAB-MS confirmed a molecular weight of 414 with M.sub.+H at
415.2.
[0706] .sup.1H NMR (400 MHz, deuterochloroform) .delta. 0.95 (s,
3H), 1.50 (s, 3H), 1.45 (m, 3H), 1.55-2.7 (m, 15H), 2.85 (t, J=13,
1H), 3.25 (d, J=6, 1H), 3.65 (s, 3H), 5.78 (s, 1H).
EXAMPLE 47J
Preparation of
9,11.alpha.-epoxy-17-hydroxy-3-oxo-17.alpha.-pregn-4-ene-7.alpha.,21-dica-
rboxylic acid, .gamma.-lactone
[0707] ##STR226##
[0708] To 774 mg (1.82 mml) of 7-methyl hydrogen
9,11.alpha.-epoxy-17-hydroxy-3-oxo-17.alpha.-pregn-4-ene-7.alpha.,21-dica-
rboxylate, .gamma.-lactone prepared in the manner of Example 43 and
suspended in 3 ml of acetonitrile was added 3 mL (7.5 mmol, 2.0
equivalents) of 2.5 M sodium hydroxide. The mixture became yellow
and after 10 minutes was homogeneous.
[0709] To monitor the progress of the reaction, aliquots (0.1 mL)
of the mixture were quenched in 0.01 mL of 3M sulfuric acid and
extracted in a 4 mL glass vial with ethyl acetate (0.2 mL). The
phases were separated by removal of the lower aqueous phase with a
pipette. The organic phase was stripped and the residue analyzed by
HPLC using the method described in Example 47H. After 50 minutes at
25.degree. C. there was little change in the composition of the
mixture.
[0710] The mixture was heated to reflux conditions (about
90.degree. C.) for 50 minutes. HPLC analysis of the mixture showed
6 area percent of the starting material remained. The mixture was
stirred at 25.degree. C. for 65 hours. Acidification, extraction
and HPLC analysis of an aliquot as described above confirmed that
no starting material remained.
[0711] The mixture was made strongly acidic by addition of about 4
mL of 3M sulfuric acid and was extracted with two portions (about
10 mL) of methylene chloride. The organic phases were combined and
dried over sodium sulfate. Concentration on a rotary evaporator
yielded 780 g of a solid. The solid was recrystallized from
dimethyl formamide/methanol to give 503 mg (67%) of a tan
crystalline solid. The sample melted with gas evolution near
260.degree. C. when heated rapidly. The sample slowly darkened but
remained solid when slowly heated to 285.degree. C.
[0712] .sup.1H NMR (dimethylsulfoxide d-6, 400 MHz) .delta. 0.85
(s, 3H), 1.4 (s, 3H), 1.3-2.9 (m, 19H), 3.15 (m, 1H), 5.55 (s, 1H),
11.8 (br, 1H).
EXAMPLE 47K
Scheme 1: Step 3D: Method I: Synthesis of Methyl Hydrogen
9,11.alpha.-Epoxy-17.alpha.-hydroxy-3-oxopregn-4-ene-7.alpha.,21-dicarbox-
ylate, .gamma.-Lactone
[0713] A 0.2 M solution of the enester of Formula IIA in methylene
chloride was combined with 2 equivalents of dipotassium phosphate
dissolved in an equal weight of water (50% w/w aqueous solution), 3
equivalents of chlorodifluoroacetamide and 22 equivalents of
hydrogen peroxide (added as a 30% aqueous solution). The mixture
was stirred at 25.degree. C. for 23 hours. The reaction was diluted
with an amount of water equal to the hydrogen peroxide charge and
the methylene chloride separated. The methylene chloride portion
was washed one time with a 3% sodium sulfite solution (volume equal
to 1.75 times the hydrogen peroxide charge). The methylene chloride
portion was separated and dried over sodium sulfate. The solution
was concentrated under atmospheric distillation until a head
temperature of 70.degree. C. was achieved. The residue was
evaluated via HPLC, .sub.1H and .sup.13C NMR (CDCl.sub.3). The
yield of epoxymexrenone was determined to be 54.2 area % by
HPLC.
EXAMPLE 47L
Scheme 1: Step 3D: Method J: Synthesis of Methyl Hydrogen
9,11.alpha.-Epoxy-17.alpha.-hydroxy-3-oxopregn-4-ene-7.alpha.,21-dicarbox-
ylate, .gamma.-Lactone
[0714] The procedure of Example 47K was repeated using
heptafluorobutyramide (CF.sub.3CF.sub.2CF.sub.2CONH.sub.2) instead
of chlorodifluoroacetamide. The yield of epoxymexrenone was
determined to be 58.4 area % by HPLC.
EXAMPLE 48
Epoxidation of Enester of Formula IIA Using Toluene
Scheme 1: Step 3D: Method K: Synthesis of Methyl Hydrogen
9,11.alpha.-Epoxy-17.alpha.-hydroxy-3-oxopregn-4-ene-7.alpha.,21-dicarbox-
ylate, .gamma.-Lactone
[0715] The enester of Formula IIA was converted to epoxymexrenone
in the method generally described in Example 46 except that toluene
was used as the solvent. The materials charged to the reactor
included enester (2.7 g) trichloroacetamide (2.5 g), dipotassium
hydrogen phosphate (1.7 g), hydrogen peroxide (17.0 g) and toluene
(50 ml). The reaction was allowed to exotherm to 28.degree. C. and
was complete in 4 hours. The resulting three phase mixture was
cooled to 15.degree. C., filtered, washed with water and dried in
vacuo to yield 2.5 g of product.
EXAMPLE 49
Scheme 4: Method A: Epoxidation of 9,11-Dienone
[0716] A compound designated XVIIA (compound XVII wherein -A-A- and
--B--B-- are both --CH.sub.2--CH.sub.2--) (40.67 g) was dissolved
in methylene chloride (250 mL) in a one liter 3 necked flask and
cooled by ice salt mixture externally. Dipotassium phosphate (22.5
g), and trichloroacetonitrile (83.5 g) were added and mixture
cooled to 2.degree. C. after which 30% Hydrogen peroxide (200 g)
was slowly added over a period of 1 hour. The reaction mixture was
stirred at 12.degree. for 8 hours and 14 hours at room temperature.
A drop of the organic layer was taken and checked for any starting
enone and was found to be <0.5%. Water (400 mL) was added,
stirred for 15 min. and layers separated. The organic layer was
washed successively with 200 mL of potassium iodide (10%), 200 mL
of sodium thiosulfate (10%) and 100 mL of saturated sodium
bicarbonate solution separating layers each time. The organic layer
was dried over anhydrous magnesium sulfate and concentrated to
yield crude epoxide (41 g). The product crystallized from ethyl
acetate:methylene chloride to give 14.9 g of pure material.
EXAMPLE 50
Scheme 4: Method B: Epoxidation of Compound XVIIA Using
m-chloroperbenzoic acid
[0717] Compound XVIIA (18.0 g) was dissolved in 250 mL of methylene
chloride and cooled to 10.degree. C. Under stirring solid
m-chloroperbenzoic acid, (50-60% pure, 21.86 g) was added during 15
min. No rise in temperature was observed. The reaction mixture was
stirred for 3 hours and checked for the presence of the dienone.
The reaction mixture was treated successively with sodium sulfite
solution (10%), sodium hydroxide solution (0.5N), hydrochloric acid
solution (5%) and finally with 50 mL of saturated brine solution.
After drying with anhydrous magnesium sulfate and evaporation,
17.64 g of the epoxide resulted and was used directly in the next
step. The product was found to contain Baeyer-Villiger oxidation
product that had to be removed by trituration from ethyl acetate
followed by crystallization from methylene chloride. On a 500 g
scale, the precipitated m-chlorobenzoic acid was filtered followed
by the usual work up.
EXAMPLE 51
Scheme 4: Method C: Epoxidation of Compound XVIIA Using
Trichloroacetamide
[0718] Compound XVIIA (2 g) was dissolved in 25 mL of methylene
chloride. Trichloroacetamide (2 g), dipotassium phosphate (2 g)
were added. Under stirring at room temperature 30% hydrogen
peroxide (10 mL) was added and stirring continued for 18 hours to
yield the epoxide (1.63 g). Baeyer-Villiger product was not
formed.
EXAMPLE 52
[0719] Potassium hydroxide (56.39 g; 1005.03 mmol; 3.00 eq.) was
charged to a 2000 mL flask and slurried with dimethylsulfoxide
(750.0 mL) at ambient temperature. A trienone corresponding to
Formula XX (wherein R.sup.3 is H and -A-A- and --B--B-- are each
--CH.sub.2--CH.sub.2--) (100.00 g; 335.01 mmol; 1.00 eq.) was
charged to the flask together with THF (956.0 mL).
Trimethylsulfonium methylsulfate (126.14 g; 670.02 mmol; 2.00 eq.)
was charged to the flask and the resulting mixture heated at
reflux, 80 to 85.degree. C. for 1 hr. Conversion to the
17-spirooxymethylene was checked by HPLC. THF approximately 1 L was
stripped from the reaction mixture under vacuum after which water
(460 mL) was charged over a 30 min. period while the reaction
mixture was cooled to 15.degree. C. The resulting mixture was
filtered and the solid oxirane product washed twice with 200 mL
aliquots of water. The product was observed to be highly
crystalline and filtration was readily carried out. The product was
thereafter dried under vacuum at 40.degree. C. 104.6 g of the
3-methyl enol ether .DELTA.-5,6,9,11,-17-oxirane steroid product
was isolated.
EXAMPLE 53
[0720] Sodium ethoxide (41.94 g; 616.25 mmol; 1.90 eq.) was charged
to a dry 500 mL reactor under a nitrogen blanket. Ethanol (270.9
mL) was charged to the reactor and the sodium methoxide slurried in
the ethanol. Diethyl malonate (103.90 g; 648.68 mmol; 2.00 eq.) was
charged to the slurry after which the oxirane steroid prepared in
the manner described in Example 52 (104.60 g; 324.34 mmol; 1.00
eq.) was added and the resulting mixture heated to reflux, i.e., 80
to 85.degree. C. Heating was continued for 4 hrs. after which
completion of the reaction was checked by HPLC. Water (337.86 mL)
was charged to the reaction mixture over a 30 min. period while the
mixture was being cooled to 15.degree. C. Stirring was continued
for 30 min. and then the reaction slurry filtered producing a
filter cake comprising a fine amorphous powder. The filter cake was
washed twice with water (200 mL each) and thereafter dried at
ambient temperature under vacuum. 133.8 g of the 3-methyl
enolether-.DELTA.5,6,9,11,-17-spirolactone-21-ethoxycarbonyl
intermediate was isolated.
EXAMPLE 54
[0721] The 3-methyl
enolether-.DELTA.5,6,9,11,-17-spirolactone-21-ethoxycarbonyl
intermediate (Formula XVIII where R.sup.3 is H and -A-A- and
--B--B-- are each --CH.sub.2--CH.sub.2--; 133.80 g; 313.68 mmol;
1.00 eq., as produced in Example 53, was charged to the reactor
together with sodium chloride (27.50 g; 470.52 mmol; 1.50 eq.)
dimethyl formamide (709 mL) and water (5 mL) were charged to a 2000
mL reactor under agitation. The resulting mixture was heated to
reflux, 138 to 142.degree. C. for 3 hrs. after which the reaction
mixture was checked for completion of the reaction by HPLC. Water
was thereafter added to the mixture over a 30 min. period while the
mixture was being cooled to 15.degree. C. Agitation was continued
for 30 min. after which the reaction slurry was filtered recovering
amorphous solid reaction product as a filter cake. The filter cake
was washed twice (200 mL aliquots of water) after which it was
dried. The product 3-methylenolether-17-spirolactone was dried
yielding 91.6 g (82.3% yield; 96 area % assay).
EXAMPLE 55
[0722] The enol ether produced in accordance with Example 54 (91.60
g; 258.36 mmol; 1.00 eq.) ethanol (250 mL) acetic acid (250 mL) and
water (250 mL) were charged to a 2000 mL reactor and the resulting
slurry heated to reflux for 2 hrs. Water (600 mL) was charged over
a 30 min. period while the reaction mixture was being cooled to
15.degree. C. The reaction slurry was thereafter filtered and the
filter cake washed twice with water (200 mL aliquots). The filter
cake was then dried; 84.4 g of product 3-keto
.DELTA.4,5,9,11,-17-spirolactone was isolated (compound of Formula
XVII where R.sup.3 is H and -A-A- and --B--B-- are
--CH.sub.2--CH.sub.2--; 95.9% yield).
EXAMPLE 56
[0723] Compound XVIIA (1 kg; 2.81 moles) was charged together with
carbon tetrachloride (3.2 L) to a 22 L 4-neck flask.
N-bromo-succinamide (538 g) was added to the mixture followed by
acetonitrile (3.2 L). The resulting mixture was heated to reflux
and maintained at the 68.degree. C. reflux temperature for
approximately 3 hrs. producing a clear orange solution. After 5
hrs. of heating, the solution turned dark. After 6 hrs. the heat
was removed and the reaction mixture was sampled. The solvent was
stripped under vacuum and ethyl acetate (6 L) added to the residue
in the bottom of the still. The resultant mixture was stirred after
which a 5% sodium bicarbonate solution (4 L) was added and the
mixture stirred for 15 min. after which the phases were allowed to
settle. The aqueous layer was removed and saturated brine solution
(4 L) introduced into the mixture which was then stirred for 15
min. The phases were again separated and the organic layer stripped
under vacuum producing a thick slurry. Dimethylformamide (4 L) was
then added and stripping continued to a pot temperature of
55.degree. C. The still bottoms were allowed to stand overnight and
DABCO (330 g) and lithium bromide (243 g) added. The mixture was
then heated to 70.degree. C. After one and one-half hrs. heating, a
liquid chromatography sample was taken and after 3.50 hrs. heating,
additional DABCO (40 g) was added. After 4.5 hrs. heating, water (4
L) was introduced and the resulting mixture was cooled to
15.degree. C. The slurry was filtered and the cake washed with
water (3 L) and dried on the filter overnight. The wet cake (978 g)
was charged back into the 22 L flask and dimethylformamide (7 L)
added. The mixture thus produced was heated to 105.degree. C. at
which point the cake had been entirely taken up into solution. The
heat was removed and the mixture in the flask was stirred and
cooled. Ice water was applied to the reactor jacket and the mixture
within the reactor cooled to 14.degree. C. and held for two hours.
The resulting slurry was filtered and washed twice with 2.5 L
aliquots of water. The filter cake was dried under vacuum
overnight. A light brown solid product 510 g was obtained.
EXAMPLE 57
[0724] To a 2 L 4-neck flask were charged: 9,11-epoxy canrenone as
produced in Example 56 (100.00 g; 282.1 mmol; 1.00 eq.),
dimethylformamide (650.0 mL), lithium chloride (30.00 g; 707.7
mmol; 2.51 eq.), and acetone cyanohydrin (72.04 g; 77.3 mL; 846.4
mmol; 3.00 eq.). The resulting suspension was mechanically stirred
and treated with tetramethyl guanidine (45.49 g; 49.6 mL; 395.0
mmol; 1.40 eq.). The system was then filtered with a water cooled
condenser and a dry ice condenser (filled with dry ice in acetone)
to prevent escape of HCN. The vent line from the dry ice condenser
passed into a scrubber filled with a large excess of chlorine
bleach. The mixture was heated to 80.degree. C.
[0725] After 18 hrs., a dark reddish-brown solution was obtained
which was cooled to room temperature with stirring. During the
cooling process, nitrogen was sparged into the solution to remove
residual HCN with the vent line being passed into bleach in the
scrubber. After two hrs. the solution was treated with acetic acid
(72 g) and stirred for 30 min. The crude mixture was then poured
into ice water (2 L) with stirring. The stirred suspension was
further treated with 10% aqueous HCl (400 mL) and stirred for 1 hr.
Then the mixture was filtered to give a dark brick-red solid (73
g). The filtrate was placed in a 4 L separatory funnel and
extracted with methylene chloride (3.times.800 mL); and the organic
layers were combined and back extracted with water (2.times.2 L).
The methylene chloride solution was concentrated in vacuo to give
61 g of a dark red oil.
[0726] After the aqueous wash fractions were allowed to sit
overnight, a considerable precipitate developed. This precipitate
was collected by filtration and determined to be pure product
enamine (14.8 .mu.g).
[0727] After drying the original red solid (73 g) was analyzed by
HPLC and it was determined that the major component was the
9,11-epoxyenamine. HPLC further showed that enamine was the major
component of the red oil obtained from methylene chloride workup.
Calculated molar yield of enamine was 46%.
EXAMPLE 58
[0728] 9,11-epoxyenamine (4.600 g; 0.011261 mol; 1.00 eq.) as
prepared in accordance with Example 57 was introduced into a 1000
mL round bottom flask. Methanol (300 mL) and 0.5% by weight aqueous
HCl (192 mL) were added to the mixture which was thereafter
refluxed for 17 hrs. Methanol was thereafter removed under vacuum
reducing the amount of material in the still pot to 50 mL and
causing a white precipitate to be formed. Water (100 mL) was added
to the slurry which was thereafter filtered producing a white solid
cake which was washed three times with water. Yield of solid
9,11-epoxydiketone product was 3.747 g (81.3%).
EXAMPLE 59
[0729] The epoxydiketone prepared in accordance with Example 58
(200 mg; 0.49 mmol) was suspended in methanol (3 mL) and
1,8-diazabicyclo[5.4.0]undec-7-ene(DBU) added to the mixture. Upon
heating under reflux for 24 hrs. the mixture became homogeneous. It
was then concentrated to dryness at 30.degree. C. on a rotary
evaporator and the residue partitioned between methylene chloride
and 3.0 N HCl. Concentration of the organic phase yielded a yellow
solid (193 mg) which was determined to be 22% by weight epoxy
mexrenone. The yield was 20%. ##STR227##
EXAMPLE 60
[0730] To 100 mg of diketone (prepared in accordance with Example
58) suspended in 1.5 mL of methanol was added 10 microliters (0.18
eq) of a 25% (w/w) solution of sodium methoxide in methanol. The
solution was heated to reflux. After 30 min. no diketone remained
and the 5-cyanoester was present. To the mixture was added 46
microliters of 25% (w/w) sodium methanol solution in methanol. The
mixture was heated at reflux for 23 hours at which time the major
product was epoxymexrenone as judged by HPLC. ##STR228##
EXAMPLE 61
[0731] To 2 g of diketone (prepared in accordance with Example 58)
suspended in 30 ml of dry methanol was added 0.34 mL of
triethylamine. The suspension was heated at reflux for 4.5 hours.
The mixture was stirred at 25.degree. C. for 16 hours. The
resulting suspension was filtered to give 1.3 g of the 5-cyanoester
as a white solid.
[0732] To 6.6 g of the diketone suspended in 80 mL of methanol was
added 2.8 mL of triethylamine. The mixture was heated at reflux for
4 hours and was stirred at 25 rpm for 88 hours during which time
the product crystallized from solution. Filtration followed by a
methanol wash gave 5.8 g of the cyanoester as a white powder. The
material was recrystallized from chloroform/methanol to give 3.1 g
of crystalline material which was homogeneous by HPLC.
[0733] In view of the above, it will be seen that the several
objects of the invention are achieved and other advantageous
results attained.
[0734] As various changes could be made in the above compositions
and processes without departing from the scope of the invention, it
is intended that all matter contained in the above description and
shown in the accompanying drawings shall be interpreted as
illustrative and not in a limiting sense.
The Novel Compounds
[0735] The present invention is further directed to additional
polycyclic organic moieties useful as chromatographic markers in
the preparation of steroid compounds having favorable biological
activity such as spironolactone or epoxymexrenone.
[0736] In brief, it has been discovered that certain compounds
comprising a substituted or unsubstituted steroid nucleus and a
substituted or unsubstituted carbocyclic ring fused to the 13,17
position of the steroid nucleus can used as internal or
chromatographic markers in the preparation of steroids such as
spironolactone and epoxymexrenone. In particular, the compound
methyl
2,3,3a,4,6,7,9,10,11,11a,12,13-dodecahydro-3a.beta.,11a.beta.-dimethyl-1,-
9-dioxo-1H-pentaleno[1,6a-a]phenanthrene-6.alpha.-carboxylate:
##STR229## is useful as a chromatographic marker. One of the novel
features of this compound and the related compounds of this
invention is the fused carbocyclic ring attached to the D ring of
the steroid nucleus. Spironolactone and epoxymexrenone lack this
feature and instead possess a 20-spirolactone ring.
[0737] As used herein, the steroid nucleus of the compound
corresponds to the following structure: ##STR230## This structure
reflects the conventional numbering and ring designation for
steroid compounds. The steroid nucleus may be saturated,
unsaturated, substituted or unsubstituted. Preferably, it contains
at least one to four unsaturated bonds. More preferably, the A, C
and D rings each contain at least one unsaturated bond. The steroid
nucleus also may be substituted as more specifically discussed
below. Preferably, the nucleus is substituted with at least a C7
ester group.
[0738] As used herein, the carbocyclic ring fused to the steroid
nucleus corresponds to a four, five or six carbon cyclic skeleton.
It may be saturated, unsaturated, substituted or unsubstituted.
Preferably, it is saturated or has one double bond, and it is
substituted with a hydroxy or keto group. In addition, the
carbocyclic ring preferably has the .alpha.-orientation relative to
the steroid nucleus.
[0739] In a preferred embodiment, the carbocyclic ring comprises a
five carbon cyclic skeleton and has the .alpha.-orientation, and
the compound is selected from the group consisting of those
compounds corresponding to the following formulae: ##STR231##
wherein
[0740] -A-A- represents the group
--CR.sup.102R.sup.102a--CR.sup.103R.sup.103a-- or
--CR.sup.102.dbd.CR.sup.103--, wherein --CR.sup.102R.sup.102a and
--CR.sup.102.dbd. correspond to the C2 carbon, and
--CR.sup.103R.sup.103a-- and .dbd.CR.sup.103-- correspond to the C3
carbon;
[0741] -D-D- represents the group --CHR.sup.104--CH-- or
--CR.sup.104.dbd.C--;
[0742] -E-E- represents the group --CH.sub.2--CR.sup.110-- or
--CH.dbd.C--;
[0743] -A-E- represents the group
--CR.sup.102R.sup.102a--CH.sub.2-- or --CR.sup.102.dbd.CH, wherein
--CR.sup.102R.sup.102a-- and --CR.sup.102-- correspond to the C2
carbon and --CH.sub.2-- and .dbd.CH-- correspond to the C1
carbon;
[0744] -G-G- represents the group
--CR.sup.106R.sup.106a--CHR.sup.107 or
--CR.sup.106.dbd.CR.sup.107--, wherein --CR.sup.106R.sup.106a-- and
--CR.sup.106.dbd. correspond to the C6 carbon and --CHR.sup.107--
and .dbd.CR.sup.107-- correspond to the C7 carbon;
[0745] -J-J- represents the group --CR.sup.108--CR.sup.109-- or
--C.dbd.C--, wherein --CR.sup.108-- corresponds to the C8 carbon
and --CR.sup.109-- corresponds to the C9 carbon;
[0746] -L-L- represents the group
--CR.sup.111R.sup.111a--CH.sub.2-- or --CR.sup.111.dbd.CH--,
wherein --CR.sup.111R.sup.111a-- and --CR.sup.111.dbd. correspond
to the C11 carbon and --CHR.sup.2--, and .dbd.CH-- correspond to
the C.sub.1-2 carbon;
[0747] -J-L- represents the group
--CR.sup.109--CR.sup.111R.sup.111a-- or --C.dbd.CR.sup.111--,
wherein --CR.sup.109-- and --C.dbd. correspond to the C9 carbon,
and --CR.sup.111R.sup.111a-- and --CR.sup.111-- correspond to the
C11 carbon;
[0748] -M-M- represents the group --CR.sup.114--CH.sub.2-- or
--C.dbd.CH--, wherein --CR.sup.114-- and --C.dbd. correspond to the
C14 carbon, and --CH.sub.2-- and --C.dbd.H-- correspond to the
C.sub.1-5 carbon;
[0749] -J-M- represents the group --CR.sup.108--CR.sup.114-- or
--C.dbd.C--, wherein --CR.sup.108-- corresponds to the C8 carbon
and --CR.sup.114-- corresponds to the C14 carbon;
[0750] -Q-Q- represents the group
--CR.sup.120R.sup.120a--CR.sup.119R.sup.119a-- or
--CR.sup.120.dbd.CR.sup.119--, wherein --CR.sup.120R.sup.120a-- and
--CR.sup.120.dbd.CR.sup.119-- correspond to the C20 carbon, and
--CR.sup.119R.sup.119a-- and .dbd.CR.sup.119-- correspond to the
C19 carbon;
[0751] -Q-T- represents the group
--CR.sup.119R.sup.119a--CHR.sup.118-- or
--CR.sup.119.dbd.CR.sup.118--, wherein --CR.sup.119R.sup.119a-- and
--CR.sup.119.dbd. correspond to the C19 carbon, and --CHR.sup.118--
and .dbd.CR.sup.118-- correspond to the C18 carbon;
[0752] R.sup.102 is hydrogen, alkyl, alkenyl or alkynyl;
[0753] R.sup.102a is hydrogen; or represents a bond between the C2
carbon atom and the C3 carbon atom when -A-A- represents the group
--CR.sup.102.dbd.CR.sup.103-- and -A-E- represents the group
--CR.sup.102R.sup.102a--CH.sub.2--; or represents a bond between
the C1 carbon atom and the C2 carbon atom when -A-E- represents the
group --CR.sup.102.dbd.CH-- and -A-A- represents the group
--CR.sup.102R.sup.102a--CR.sup.103R.sup.103a--;
[0754] R.sup.103 is hydrogen, hydroxy, protected hydroxy,
--R.sup.130O--, R.sup.130C(O)O--, R.sup.130OC(O)O--, or together
with R.sup.103a forms an Oxo; provided that -A-A- is
--CR.sup.102R.sup.102a--CR.sup.103R.sup.103a-- when R.sup.103
together with R.sup.103a form an oxo;
[0755] R.sup.103a is hydrogen or together with R.sup.103 forms an
oxo; provided that -A-A- is
--CR.sup.102R.sup.102a--CR.sup.103R.sup.103a-- when R.sup.103
together with R.sup.103 form an oxo;
[0756] R.sup.104 is hydrogen, alkyl, alkenyl or alkynyl;
[0757] R.sup.106 is hydrogen, hydroxy or protected hydroxy, or
together with R.sup.106a forms an oxo, or together with R.sup.106a
and the carbon atom to which they are attached form a cyclopropyl,
cyclobutyl or cyclopentyl ring; provided that -G-G- is
--CR.sup.106R.sup.106a--CR.sup.107R.sup.107a-- when R.sup.106
together with R.sup.106a form an oxo;
[0758] R.sup.106a is hydrogen, hydroxy or protected hydroxy, or
together with R.sup.106a forms an oxo, or together with R.sup.106
and the carbon atom to which they are attached form a cyclopropyl,
cyclobutyl or cyclopentyl ring, or R.sup.106a together with
R.sup.107 and the carbon atoms to which they are attached form a
cyclopropyl, cyclobutyl or cyclopentyl ring;
[0759] R.sup.107 is hydrogen; hydroxycarbonyl; lower alkyl,
alkenyl, alkynyl, aryl, heteroaryl or aralkyl; haloalkyl;
hydroxyalkyl; alkoxyalkyl; lower alkanoyl, alkenoyl, alkynoyl,
aryloyl, heteroaryloyl or aralkanoyl; lower alkoxycarbonyl,
alkenoxycarbonyl, alkynoxycarbonyl, aryloxycarbonyl,
heteroaryloxycarbonyl or aralkoxycarbonyl; lower alkanoylthio,
alkenoylthio, alkynoylthio, aryloylthio, heteraroylthio or
aralkanoylthio; lower alkylthio, alkenylthio, alkynylthio,
arylthio, heteroarylthio or aralkylthio; carbamyl;
alkoxycarbonylamino; or cyano; or
[0760] R.sup.107 together with R.sup.106a and the carbon atoms to
which they are attached form a cyclopropyl, cyclobutyl or
cyclopentyl ring; or
[0761] R.sup.107 and R.sup.114 together with the C7, C8 and C14
carbon atoms form a .gamma.-lactone;
[0762] R.sup.108 is hydrogen, hydroxy, protected hydroxy, alkyl,
alkenyl, alkynyl, R.sup.140O--, R.sup.140C(O)O--, or
R.sup.140OC(O)O--; or represents a bond between the C8 carbon atom
and the C9 carbon atom when -J-J- represents the group --C.dbd.C--
and -J-M- represents the group --CR.sup.108--CR.sup.114--; or
represents a bond between the C8 carbon atom and the C.sub.1-4
carbon atom when -J-M- represents the group --C.dbd.C-- and -J-J-
represents the group --CR.sup.108--CR.sup.114--;
[0763] R.sup.109 is hydrogen, hydroxy, protected hydroxy, alkyl,
alkenyl, alkynyl, R.sup.150O--, R.sup.150C(O)O--, or
R.sup.150OC(O)O--; or represents a bond between the C9 carbon atom
and the C11 carbon atom when -J-L- represents the group
--C.dbd.CR.sup.111-- and -J-J- represents the group
--CR.sup.108--CR.sup.109--; or represents a bond between the C9
carbon atom and the C8 carbon atom when -J-J- represents the group
--C.dbd.C-- and -J-L- represents the group
--CR.sup.109--CR.sup.111R.sup.111a--;
[0764] R.sup.110 is hydrogen or methyl;
[0765] R.sup.111 is hydrogen, hydroxy or protected hydroxy, or
together with R.sup.111a form an oxo, provided that -J-L-represents
the group --CR.sup.109--CR.sup.111R.sup.111a-- and -L-L- represents
the group --CR.sup.111R.sup.111a--CH.sub.2-- when R.sup.111
together with R.sup.111a form an oxo;
[0766] R.sup.111a is hydrogen, or together with R.sup.111 form an
oxo, provided that -J-L- represents the group
--CR.sup.109--CR.sup.111R.sup.111a-- and -L-L- represents the group
--CR.sup.111R.sup.111a--CH.sub.2-- when R.sup.111a together with
R.sup.111 form an oxo; or R.sup.111a represents a bond between the
C11 carbon atom and the C9 carbon atom when -J-L- represents the
group --C.dbd.CR.sup.111-- and -L-L- represents the group
--CR.sup.111R.sup.111a--CH.sub.2--; or R.sup.111a represents a bond
between the C11 carbon atom and the C12 carbon atom when -L-L-
represents the group --CR.sup.111.dbd.CH-- and -J-L- represents the
group --CR.sup.109R.sup.109a--CR.sup.111R.sup.111a--;
[0767] R.sup.114 is hydrogen, hydroxy, protected hydroxy, alkyl,
alkenyl, alkynyl, R.sup.160O--, R.sup.160C(O)O--, or
R.sup.160OC(O)O--; or R.sup.114 and R.sup.107 together with the C7,
C8 and C14 carbons form a .gamma.-lactone; or R.sup.114 represents
a bond between the C14 carbon atom and the C8 carbon atom when
-J-M- represents the group --C.dbd.C-- and -M-M- represents the
group --CR.sup.114--CH.sub.2--; or R.sup.114 represents a bond
between the C14 carbon atom and the C15 carbon atom when -M-M-
represents the group --C.dbd.CH-- and -J-M- represents the group
--CR.sup.108--CR.sup.114--;
[0768] R.sup.118 is hydrogen, alkyl, alkenyl, alkynyl, aryl,
heteroaryl, alkylthio, alkenylthio or cyano;
[0769] R.sup.118a is hydrogen, or represents a bond between the C18
carbon atom and the C19 carbon atom when -Q-Q- represents the group
--CR.sup.118.dbd.CR.sup.119-- and -Q-Q- represents the group
--CR.sup.119R.sup.119a--CR.sup.120R.sup.120a--;
[0770] R.sup.119 is hydrogen, alkyl or alkenyl;
[0771] R.sup.119a is hydrogen, or represents a bond between the C19
carbon atom and the C20 carbon atom when -Q-Q- represents the group
--CR.sup.150.dbd.CR.sup.119-- and -Q-T- represents the group
--CR.sup.119R.sup.119a--CR.sup.118CR.sup.118a--; or represents a
bond between the C19 carbon atom and the C18 carbon atom when -Q-T-
represents --CR.sup.119.dbd.CR.sup.118-- and -Q-Q- represents the
group --CR.sup.119R.sup.119a--CR.sup.120R.sup.120a;
[0772] R.sup.120 is hydrogen, hydroxy, protected hydroxy, or
together with R.sup.120a forms an oxo; provided that -Q-Q-
represents the group --CR.sup.119R.sup.119a--CR.sup.120R.sup.120a
when R.sup.120 together with R.sup.120a form an oxo;
[0773] R.sup.120a is hydrogen or together with R.sup.120 form an
oxo; provided that -Q-Q- represents the group
--CR.sup.119R.sup.119a--CR.sup.120R.sup.120a a when R.sup.120a
together with R.sup.120 forms an oxo; and
[0774] R.sup.130, R.sup.140, R.sup.150 and R.sup.160 are
independently alkyl, alkenyl, alkynyl, aryl or heteroaryl.
[0775] More preferably, the compound corresponds to the compound of
Formula C-3 wherein R.sup.107 is hydrogen, hydroxycarbonyl, lower
alkyl, lower alkanoyl, lower alkoxycarbonyl, lower alkanoylthio,
lower alkylthio, carbamyl, or R.sup.107 together with R.sup.106a
and the carbon atoms to which they are attached form a cyclopropyl
ring; R.sup.106 is hydrogen, hydroxy, or together with R.sup.106a
and the carbon atom to which they are attached form a cyclopropyl
ring; R.sup.106a is hydrogen, hydroxy, or together with R.sup.106
and the carbon atom to which they are attached form a cyclopropyl
ring; or R.sup.106a together with R.sup.107 and the carbon atoms to
which they are attached form a cyclopropyl ring; and R.sup.120 is
keto.
[0776] The following definitions apply to the discussion relating
to the 13,17-fused ring compounds disclosed in the present
specification:
[0777] The term "lower alkyl" means an alkyl radical having from 1
to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl,
isobutyl, sec.-butyl and tert.-butyl, pentyl and hexyl. The radical
may be straight, branched chain or cyclic and substituted
(particularly with aryl), unsubstituted or heterosubstituted.
[0778] The term "lower alkanoyl" means a radical preferably derived
from a straight-chain alkyl having from 1 to 7 carbon atoms and
attached to the parent molecular moiety via a carbonyl group.
Especially preferred are formyl and acetyl.
[0779] The term "lower alkoxycarbonyl" means a radical preferably
derived from a straight-chain alkyl having from 1 to 7 carbon atoms
and attached to an oxygen atom, said oxygen atom being attached to
the parent molecular moiety via a carbonyl group. Especially
preferred are methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl
and n-hexyloxycarbonyl.
[0780] The term "lower alkenyl" means an alkenyl radical having
from 2 to 6 carbon atoms, such as ethenyl, propenyl, isopropenyl,
butenyl, isobutenyl, sec.-butenyl and tert.-butenyl, pentenyl and
hexenyl. The radical may be straight or branched chain and
substituted, unsubstituted or heterosubstituted. The terms "lower
alkenoyl" and "lower alkenoxycarbonyl" are defined in the same
manner as "lower alkanoyl" and "lower alkoxycarbonyl",
respectively, except that they are derived from straight chain
alkenyl instead of straight chain alkyl. Preferably; the oxygen
attached to the alkenyl radical of any alkenoxycarbonyl group is
separated from any unsaturated carbon by at least one methylene
group.
[0781] The term "lower alkynyl" means an alkynyl radical having
from 2 to 6 carbon atoms, such as ethynyl, propynyl, isopropynyl,
butynyl, isobutynyl, sec.-butynyl and tert.-butynyl, pentynyl and
hexynyl. The radical may be straight or branched chain and
substituted, unsubstituted or heterosubstituted. The terms "lower
alkynoyl" and "lower alkynoxycarbonyl" are defined in the same
manner as "lower alkanoyl" and "lower alkoxycarbonyl",
respectively, except that they are derived from straight chain
alkynyl instead of straight chain alkyl. Preferably, the oxygen
attached to the alkynyl radical of any alkynoxycarbonyl group is
separated from any unsaturated carbon by at least one methylene
group.
[0782] An "aryl" moiety preferably contains, either alone or with
various substituents, from 5 to 15 atoms and includes phenyl.
[0783] The term "lower alkylthio" means a radical preferably
derived from a straight-chain alkyl having from 1 to 7 carbon atoms
and attached to the parent molecular moiety via a sulfur atom.
Especially preferred is methylthio.
[0784] The term "lower alkanoylthio" means a radical preferably
derived from a straight-chain alkyl having from 1 to 7 carbon atoms
and attached to a carbonyl group, said carbonyl group being
attached to the parent molecular moiety via a sulfur atom.
Especially preferred is acetylthio.
[0785] The terms "lower alkenylthio" and "lower alkenoylthio" are
defined in the same manner as "lower alkylthio" and "lower
alkanoylthio", respectively, except is that they are derived from
straight chain alkenyl instead of straight chain alkyl. Preferably,
the sulfur atom of any alkenylthio group is separated from any
unsaturated carbon by at least one methylene group.
[0786] The terms "lower alkynylthio" and "lower alkynoylthio" are
defined in the same manner as "lower alkylthio" and "lower
alkanoylthio", respectively, except that they are derived from
straight chain alkynyl instead of straight chain alkyl. Preferably,
the sulfur atom of any alkynylthio group is separated from any
unsaturated carbon by at least one methylene group.
[0787] The term "carbamyl" means an --NH.sub.2 radical attached to
the parent molecular moiety via a carbonyl group. The carbamyl
group may be mono-substituted or di-substituted and the
substituents may include alkyl, alkenyl, alkynyl and aryl
radicals.
[0788] The groups defined above may be unsubstituted or
additionally substituted. Such additional substituents can include
alkyl, alkenyl, alkynyl, aryl, heteroaryl, carboxy such as alkoxy,
carboxyalkyl, acyl, acyloxy, halo such as chloro or fluoro,
haloalkoxy, nitro, amino, amido, and keto. The groups defined
above, as well as the additional substituents, also may contain
oxygen, sulfur, phosphorus and/or nitrogen.
[0789] As used herein, "Me" means methyl; "Et" means ethyl; and
"Ac" means acetyl.
[0790] In a still more preferred embodiment, the compound is
selected from the group consisting of compounds having the
following formulae: ##STR232## ##STR233## ##STR234## wherein
R.sup.107, R.sup.106, R.sup.106 and R.sup.120 are as defined above.
Preferably, R.sup.107 is hydrogen, hydroxycarbonyl, lower alkyl,
lower alkanoyl, lower alkoxycarbonyl, lower alkanoylthio, lower
alkylthio, carbamyl, or together with R.sup.106a and the carbon
atoms to which they are attached form a cyclopropyl ring; R.sup.106
is hydrogen, hydroxy, or together with R.sup.106a and the carbon
atom to which they are attached form a cyclopropyl ring; R.sup.106a
is hydrogen, hydroxy, or together with R.sup.106 and the carbon
atom to which they are attached form a cyclopropyl ring; or
together with R.sup.107 and the carbon atoms to which they are
attached form a cyclopropyl ring; and R.sup.120 is keto. Still more
preferably, the compound further corresponds to the compound of
Formula C-5.
[0791] In an even more preferred embodiment, the compound of
Formula C-3 is selected from the group consisting of the following
compounds: ##STR235## ##STR236## ##STR237## ##STR238## ##STR239##
##STR240## ##STR241## ##STR242## ##STR243## ##STR244## ##STR245##
##STR246##
[0792] In the most preferred embodiment, the compound is methyl
2,2,3a,4,6,7,9,10,11,11a,12,13-dodecahydro-3.alpha..beta.,11.alpha..beta.-
-dimethyl-1,9-dioxo-1H-pentaleno[1,6a-a]phenantrene-6.alpha.-carboxylate:
##STR247## This compound of Formula C-1 is a particularly desirable
chromotographic marker in the preparation of epoxymexrenone.
[0793] The novel compounds discussed herein may also be in the form
of their salts.
Preparation of the Novel Compounds
[0794] In general, the novel compounds described immediately above
may be obtained by reacting a steroid having a 20-spiroxane ring
and the steroid nucleus previously described above with a
trihalogenated alkanoic acid. Advantageously, the reagent for the
reaction further includes an alkali metal salt of the alkanoic acid
utilized. It is particularly preferred that the reagent for the
reaction comprise trifluoroacetic acid and an alkali metal salt of
that acid such as potassium trifluoroacetate. In addition, a drying
agent such as trifluoroacetic anhydride preferably is employed in
the reaction to reduce free water present in the acid.
[0795] The steroid compounds used as starting materials preferably
have the following structural formula: ##STR248## wherein -A-A-,
-D-D-, -E-E-, -A-E-, -G-G-, -J-J-, -L-L-, -J-L-, -M-M-, -J-M-,
-Q-Q-, and -Q-T- are as defined above. Such starting materials can
be prepared and/or isolated by processes analogous to those
disclosed in Scheme 1 of the epoxymexrenone synthesis process
previously discussed herein. Alternatively, the starting materials
are commercially available.
[0796] The initial concentration of the steroid compound of formula
C-3 is preferably at least about 0.1% by weight of the total
reaction mixture, more preferably about 2% to about 20% by weight,
and even more preferably about 5% to about 15% by weight. An excess
of the trihaloalkanoic acid is preferably present. Where
trifluoroacetic anhydride is used, it should be present in a
proportion of at least about 3% by weight of the total reaction
mixture, more preferably between about 5% to about 25% by weight,
most preferably between about 10% and about 15% by weight.
[0797] In addition, the reaction temperature should exceed room
temperature (22.degree. C.). Preferably, the reaction temperature
is between about 40.degree. C. and 100.degree. C., more preferably
between about 50.degree. C. and 80.degree. C., still more
preferably between about 60.degree. C. and 70.degree. C., and most
preferably between about 60.degree. C. and 65.degree. C. Increasing
the reaction temperature to about 70.degree. C. or higher increases
the amount of the by-product C14 lactone produced in the reaction.
The reaction time preferably should be at least about 30 minutes,
more preferably between about 30 minutes and about 6 hours, still
more preferably between about 45 minutes and about 4 hours, and
most preferably between about one hours to two hours. In a
preferred embodiment the reaction time is between about one hour to
two hours and the reaction temperature is maintained at about
60.degree. C.
[0798] Scheme S-1 below illustrates a particularly preferred
embodiment of the process: ##STR249##
[0799] Fused ring steroids having different substituents at various
positions throughout the steroid can be prepared as set forth in
the reaction schemes below. Those skilled in the art are aware of
additional procedures and methods not specifically disclosed herein
for introducing various substituents at different positions of the
steroid. The starting material may be either a fused ring steroid
or a 20-spiroxane ring steroid. To simplify the description where
the starting material is a fused ring steroid, the following
reaction schemes employ specific steroids or groups of steroids as
illustrative starting materials. It should be understood, however,
that other fused ring steroid derivatives or analogs may be
produced in the same series of reactions by using a different fused
ring steroid as the starting material. Similarly, to simplify the
description where the starting material is a 20-spiroxane ring
steroid, certain specific 20-spiroxane ring steroids are used as
the starting materials. It should be understood, however, that
other 20-spiroxane ring steroid derivatives or analogs may be
produced in the same series of reactions by using a different
20-spiroxane ring steroid as the starting material.
[0800] Steroids having a C7 carboxylic acid substituent may be
prepared by saponification of a steroid having a C7 alkoxycarbonyl
substituent such as the compound of Formula C-1. The saponification
reaction may be carried out by treatment of the starting steroid
with a basic reagent such as sodium or potassium hydroxide in a
suitable solvent such as methanol, ethanol, isopropanol or the like
at temperatures up to the boiling point of the solvent in the
presence or absence of water. As illustrated in Scheme S-2,
saponification of the compound of Formula C-1 yields the carboxylic
acid of Formula C-101.
[0801] Steroids having C7 carboxylic ester substituents other than
methanoate can be prepared using carboxylic acids such as C-101 as
the starting material. Treatment of such carboxylic acids with an
alkylating agent such as an alkyl halide in the presence of a base
(such as sodium bicarbonate, sodium carbonate, potassium
bicarbonate, or triethylamine) in a solvent such as
dimethylformamide yields the desired esters. Examples of suitable
alkylating agents are ethyl iodide, ethyl bromide, isopropyl
iodide, hexyl iodide, benzyl bromide, allyl iodide, and the
like.
[0802] Carboxylic acid C-101 is also a suitable starting material
for the synthesis of carbamyls. Treatment of the acid with a
chloroformate such as isobutyl chloroformate or ethyl chloroformate
in the presence of a base yields a mixed anhydride. Treatment of
the mixed anhydride with an amine (such as dimethylamine,
methylamine or benzylamine) yields the carbamyl wherein R.sub.1 and
R.sub.2 are the substituents on the various amines. ##STR250##
[0803] Several modifications at the C7 position are made using
unsaturated ketones such as the compound of Formula C-105 (shown
below in Scheme S-3) as the starting material. Sulfides are
synthesized by the addition of suitable thiols under basic
conditions. Examples of suitable thiols are methyl mercaptan, ethyl
mercaptan and the like. Suitable bases include piperidine,
triethylamine and the like.
[0804] Treatment of unsaturated ketones (such as the compound of
Formula C-105) with thioalkanoic acids such as thioacetic acid
provides C7 thioacyl compounds such as acetylthio.
[0805] A fused C6,C7 cyclopropyl substituent may be added by
treatment of unsaturated ketones (such as the compound of Formula
C-105) with dimethylsulfoxonium methylide, which is generated by
treatment of trimethylsulfoxonium halide with a suitable base (such
as sodium hydride) in a suitable solvent.
[0806] These various synthesis schemes are illustrated in Scheme
S-3 below: ##STR251##
[0807] Steroids bearing a C6 spirocyclopropyl ring are synthesized
according to the procedures described in Scheme S-4 below. Enones
such as the compound of Formula C-111 are first protected as a C3
enol ether by treatment with an ortho ester such as triethyl
orthoformate or trimethyl orthoformate in the presence of an acid
such as p-toluenesulfonic acid. The resultant enol ether is treated
with Vilsmeier reagent generated in situ by addition of phosphorous
oxychloride to dimethylformamide to provide a formyl compound such
as the compound of Formula C-112. Reduction of the formyl group is
effected using a hydride reducing agent, such as lithium
tri-tert-butoxyaluminum hydride, in a solvent such as
tetrahydrofuran. This produces an intermediate alcohol, which upon
treatment with acid, eliminates water to provide a 6-methylene
compound such as the compound of Formula C-113. Suitable acids
include hydrochloric acid in an aqueous medium. Treatment of the
6-methylene compound with diazomethane provides an intermediate
pyrazoline, which decomposes upon heating to give a product
spirocyclopropyl compound such as the compound of Formula C-114.
The protected enol ether (such as the compound of Formula C-111) is
a versatile intermediate and treatment with a hydride reducing
agent such as sodium borohydride, followed by acid hydrolysis,
provides hydroxy compounds such as the compounds of Formulae C-115
and C-116.
[0808] These various synthesis steps are illustrated in Scheme S-4
below: ##STR252##
[0809] Steroids having a C6 hydroxy substituent and a C7 ester
substituent may be synthesized according to the procedures
illustrated below in Scheme S-5. An ester (such as the compound of
Formula C-1) is protected at the C3 carbonyl by formation of the
3,5-dienol ether (such as the compound of Formula C-117) using an
ortho ester such as triethyl orthoformate or trimethyl orthoformate
in the presence of an acid. A suitable acid is p-toluene sulfonic
acid. Treatment of the enol ether with an oxidizing agent such as
meta-chloroperoxybenzoic acid results in the formation of a hydroxy
compound such as the compound of Formula C-118. ##STR253##
[0810] Scheme S-6 illustrates the introduction of a double bond at
C1-C2 position of the steroid. This is carried out by treatment of
the desired steroid (such as the compounds of Formulae C-1, C-108
and C-114), with a suitable oxidizing agent, such as
dichlorodicyano-benzoquinone in a suitable solvent (such as
dioxane) at temperatures ranging up to the boiling point. C1-C2
unsaturated compounds such as the compounds of Formulae C-127,
C-128 and C-129 can be prepared in accordance with this procedure.
##STR254##
[0811] Scheme S-7 illustrates the introduction of a double bond
into the fused ring. A steroid (such as the compound of Formula
C-114) is treated with an ortho ester (such as triethyl
orthoformate or trimethyl orthoformate) in the presence of an acid
catalyst (such as p-toluensulfonic acid) to give the enol ether
wherein the C3 carbonyl is protected. In the case of the compound
of Formula C-114, because the C6 position is fully substituted, the
enol ether formed is the C2-C3 enol ether (such as the compound of
Formula C-131). Treatment of the enol ether with a strong base such
as lithium diisopropylamide at low temperature (-78 C to -30 C),
followed by treatment with a selenating agent, such as phenyl
selenenyl chloride, gives the seleno derivative such as the
compound of Formula C-132. Oxidation of the seleno derivative with
an oxidizing agent such as hydrogen peroxide at, for example, room
temperature in the presence of a base such as pyridine in a solvent
such as methylene chloride, causes the elimination of the seleno
group and introduction of the double bond. Hydrolysis of the enol
ether gives a ketone such as the compound of Formula C-134.
##STR255##
[0812] Scheme S-8 illustrates the synthesis of double bond isomers
of the compound of Formula C-1. Treatment of various spiroxane
compounds, such as those shown in Scheme S-8, with potassium
acetate, trifluoracetic anhydride and trifluoroacetic acid under
conditions similar to those for the synthesis of the compound of
Formula C-1 give the compounds of Formulae C-121 and C-123.
##STR256##
[0813] Scheme S-9 illustrates an alternative method for the
synthesis of double bond isomers in this family of steroids.
Starting with a preformed enone (such as the compound of Formula
C-24 whose synthesis is described above) already bearing the fused
ring, the C6-C7 fused cyclopropane is introduced using the
chemistry described above in Scheme S-3 for the synthesis of
compounds such as those of Formulae C-108 and C-109. ##STR257##
wherein X is halogen.
[0814] Fused ring steroids having an aromatic A ring can be
prepared by treating steroids such as those described in P.
Compain, et al., Tetrahedron, 52(31), 10405-10416 (1996) (which is
incorporated by reference herein) with trifluoroacetic acid,
potassium acetate and trifluoroacetic anhydride in substantially
the same manner as discussed above with respect to Scheme S-1.
[0815] Those novel fused ring steroids possessing an aromatic A
ring and a 3-hydroxy substituent are expected to undergo all
chemical reactions which are typical of phenols. Scheme S-10
illustrates the synthesis of a 3-phenolic ether from a such a fused
ring steroid. In particular, treatment of these phenolic compounds
with a base and an alkyl halide or alkyl sulfonate is expected to
produce the corresponding phenolic ester. See, for example, Feuer
and Hooz, In The Chemistry of the Ether Linkage, Patai (Ed.),
Interscience: New York, pp. 446-450, 460-468 (1967); and Olson, W.
T., J. Am. Chem. Soc., 69, 2451 (1947); which are incorporated
herein by reference. ##STR258##
[0816] Similarly, Scheme S-11 illustrates the synthesis of a
3-phenolic ester from a fused ring steroid possessing an aromatic A
ring and a 3-hydroxy substituent. In particular, treatment of these
phenolic compounds with a carboxylic acid anhydride or with a
carboxylic acid halide is expected to provide the corresponding
phenolic ester. See, for example, March, J., Advanced Organic
Chemistry, Wiley: New York, pp. 346-347 (1985), which is
incorporated herein by reference. ##STR259##
[0817] Scheme S-12 illustrates the synthesis of a 3-phenolic
carbonate from a fused ring steroid possessing an aromatic A ring
and a 3-hydroxy substituent. In particular, treatment of these
phenolic compounds with an alkyl haloformate is expected to provide
the corresponding phenolic carbonate. See, for example, March, J.,
Advanced Organic Chemistry, Wiley: New York, pp. 346-347 (1985),
which is incorporated herein by reference. ##STR260##
[0818] Scheme S-13 illustrates the synthesis of ortho allyl
substituted phenyl derivatives from a fused ring steroid possessing
an aromatic A ring and a 3-hydroxy substituent. In particular,
treatment of these compounds with a base and an allyl halide is
expected to provide the corresponding allyl phenyl ether. The allyl
phenyl ether should give a mixture of ortho allyl substituted
phenyl derivatives upon thermal rearrangement. See, for example,
Shine, H., J. Aromatic Rearrangements; Reaction Mechanisms in
Organic Chemistry, Monograph 6, American Elsevier: New York, pp.
89-120 (1967), which is incorporated herein by reference.
##STR261##
[0819] Scheme S-14 illustrates the synthesis of ortho dialkylated
substituted phenyl derivatives from a fused ring steroid possessing
an aromatic A ring and a 3-hydroxy substituent. In particular,
treatment of these compounds with an alcohol and an acid is
expected to provide the corresponding ortho dialkylated phenyl
derivatives. See, for example, Calcott, W. S., J. Am. Chem. Soc.,
61, 1010 (1939), which is incorporated herein by reference.
##STR262##
[0820] Scheme S-15 illustrates the allylic oxidation of a fused
ring steroid possessing an aromatic A ring and a 3-hydroxy
substituent. In particular, these compounds may be oxidized at an
allylic position by reaction with selenium dioxide and t-butyl
hydroperoxide to form the corresponding alcohol. Dehydration of
this alcohol affords the corresponding olefin. See, for example,
Schmuff, N. R., J. Org. Chem., 48, 1404 (1983), which is
incorporated herein by reference. ##STR263##
[0821] Scheme S-16 illustrates the protection of the 3-carbonyl
group and the reduction of the 20-carbonyl group of the novel fused
ring steroids of the present invention. In particular, these
compounds may be reacted with trialkylorthoformate and an acid to
form the 3-enol ether. The 3-enol ether can be reacted with sodium
borohydride to reduce the C-20 carbonyl group to the corresponding
C-20 alcohol. Treatment of the C-20 alcohol with an acid and water
deprotects the 3-enol ether to form the 3-keto derivative.
##STR264##
[0822] Scheme S-17 illustrates the hydrogenation of olefinic bonds
in the novel fused ring steroids of the present invention. In
particular, hydrogenation is expected to proceed step-wise. The
C-6,C-7 double bond is first saturated followed by saturation of
the C-8,C-14 double bond. ##STR265##
[0823] Scheme S-18 illustrates the rearrangement of protected
11.alpha.-hydroxy fused ring steroids of the present invention. In
particular, the 11.alpha.-hydroxy group of the compound is first
protected with a suitable protecting group such as
2-methoxyethoxymethyl ether (MEM ether). Treatment of the protected
11.alpha.-hydroxy steroid with an alkali metal salt and a
trihaloalkaoic acid in the presence of an acid anhydride is
expected to lead to the rearrangement of the lactone moiety in
these molecules as illustrated below. Removal of the MEM ether with
zinc bromide will provide the rearranged alcohols shown below.
##STR266## ##STR267##
[0824] Scheme S-19 illustrates the protection of the 3-carbonyl
group and the alkylation of the 19-position of the novel fused ring
steroids of the present invention. In particular, the compound is
first converted to the 3-alkyl enol ether as illustrated in Scheme
S-16. Treatment of the 3-alkyl enol ether with lithium
diisopropylamide (LDA) followed by treatment with an alkyl halide
leads to the formation of the 19-alkyl derivative. Hydrolysis of
the 3-alkyl enol ether protecting group gives the 3-keto
derivative. ##STR268##
[0825] Scheme S-20 illustrates the conversion of estrone methyl
ether to the corresponding spirolactone. Rearrangement of the
lactone to the corresponding fused ring steroid should occur upon
treatment of the lactone with a trihalogenated alkanoic acid,
preferably in the presence of an alkali metal salt of the alkanoic
acid utilized, under the reaction conditions previously disclosed.
See, for example, Otsubo, K., Tetrahedron Letters, 27(47), 5763
(1986). ##STR269##
[0826] The novel compounds described herein additionally may be
subjected to bioconversion processes similar to those disclosed
previously to yield yet other novel fused ring steroids, such as
steroids having an 9.alpha., 9.beta., 11.alpha. or 11.beta.-hydroxy
substituent as well as other hydroxylated fused ring steroids. If
desired, such hydroxylated steroids then can be oxidized via
elimination of the hydroxy substituent to introduce an olefinic
double bond such as a .DELTA..sup.9,11 olefinic double bond.
[0827] In view of the above, it will be seen that the several
objects of the invention are achieved and other advantageous
results attained.
[0828] As various changes could be made in the above compositions
and processes without departing from the scope of the invention, it
is intended that all matter contained in the above description
shall be interpreted as illustrative and not in a limiting
sense.
[0829] The following non-limiting examples serve to illustrate
various aspects of the present invention:
EXAMPLE X-1A
Preparation of methyl
2,3,3a,4,6,7,9,10,11,11a,12,13-dodecahydro-3a.beta.,11a.beta.-dimethyl-1,-
9-dioxo-1H-pentaleno[1,6a-a]phenanthrene-6.alpha.-carboxylate
(Compound C-1)
[0830] ##STR270##
[0831] Potassium acetate (6.7 g, 7.1 mmol; Sigma-Aldrich 5128LG)
was added to a clean, dry reactor equipped with a mechanical
stirrer, condenser, thermocouple and heating mantle.
Trifluoroacetic acid (25.0 mL, 8.1 mol; Sigma-Aldrich 7125MG) and
trifluoroacetic anhydride (4.5 mL, 31.0 mmol; Sigma-Aldrich
11828PN) were successively added to the reactor. The solution was
then maintained at a temperature between 25.degree. to 30.degree.
C. for 30 minutes.
[0832] The preformed TFA/TFA anhydride reagent was added to 5.0 g
(9.6 mmol) of 7-methyl hydrogen
17-hydroxy-11.alpha.-(methylsulfonyl)oxy-3-oxo-17.alpha.-pregna-4-ene-7.a-
lpha.,21-dicarboxylate, .gamma.-lactone: ##STR271## which was
prepared in the manner described in Example 36. The resulting
mixture was heated at 60.degree. C. for 60 minutes, the degree of
conversion being periodically checked by TLC and/or HPLC. When the
reaction was complete (approximately 60 minutes), the mixture was
transferred to 1-neck flask and concentrated under reduced pressure
at 50.degree. C. until it became a thick slurry.
[0833] The resulting slurry was diluted with 150 mL of ethyl
acetate and 80 mL of a water/brine mixture. The phases were then
allowed to separate and the aqueous layer was reextracted with 80
mL of ethyl acetate. Brine strength was 12% by weight. The combined
ethyl acetate solution was washed once with 12% by weight brine (80
mL), then once with 1N NaOH solution (80 mL), and finally with 12%
by weight brine (80 mL). The mixture was allowed to stand for
separation and the separated ethyl acetate layer was concentrated
to dryness under reduced pressure at 45.degree. C. using a water
aspirator to provide about 3.8 g of a crude solid product. HPLC
analysis of the crude product revealed that the product contained
about 40 area % of compound C-1.
[0834] The solid product then was subjected to chromatographic
purification. The chromatographic purification produced 210 mg of
methyl
2,3,3a,4,6,7,9,10,11,11a,12,13-dodecahydro-3a.beta.,11a.beta.-dimethyl-1,-
9-dioxo-1H-pentaleno[1,6a-a]phenantrene-6.alpha.-carboxylate
(Compound C-1).
[0835] The mass spectrometry data indicated a molecular weight of
380 and a formula of C.sub.24H.sub.28O.sub.4 from high resolution
data. The EI mass spectrum had an M+ peak at m/z 380. The APCI mass
spectrum had peaks at m/z 381 (MH)+ and m/z 398 (MNH.sub.4)+.
Carbon and hydrogen analyses were consistent with the proposed
molecular formula.
[0836] The IR spectrum had two peaks in the carbonyl absorption
region: 1722 cm-.sup.1 and 1667 cm-.sup.1. The 1722 cm-.sup.1 peak
was assigned to two carbonyls, since the .sup.13C NMR spectrum had
signals at .delta. 217.7, due to a saturated ketone, and at .delta.
172.7, due to carbomethoxy carbonyl. Absence of the 1773 cm-.sup.1
peak in the IR spectrum indicated loss of the lactone moiety.
[0837] The .sup.13C APT and HETCOR NMR data indicated the presence
of the following types of carbons: 3 carbonyls (.delta. 217.7,
198.4, 172.2); 4 fully substituted olefinic carbons (.delta. 166.3,
141.8, 139.3, 121.8); 2 methine olefinic carbons (.delta. 124.9,
122.0); 3 quaternary aliphatic carbons (.delta. 61.1, 50.7, 39.7);
1 methine aliphatic carbon (.delta. 43.3); 8 methylene carbons
(.delta. 46.0, 37.5, 34.1, 33.3, 32.9, 31.9, 23.7, 22.2); and 3
methyl carbons (.delta. 51.9, 23.6, 23.1).
EXAMPLE X-1B
Preparation of
(7.alpha.,13R,17.beta.)-3',4',5',17-tetrahydro-14-hydroxy-17-methyl-3,5'--
dioxo-.gamma.-lactone,
cyclopenta[13,17]-18-norandrosta-4,9(11)-diene-7-carboxylic acid
(Compound C-201)
[0838] ##STR272##
[0839] Potassium acetate (8.9 g, 90 mmol), trifluoroacetic acid
(150 mL, 1.480 g/mL) and trifluoroacetic anhydride (33 mL, 1.487
g/mL) were added to the 250 mL round bottom reactor equipped with
mechanical stirrer, condenser, and heating mantel. The resulting
solution was stirred between about 25.degree. C. to 30.degree. C.
for about 10 minutes.
[0840] The preformed TFA/TFA anhydride reagent was added to 15 g
(30.0 mmol) of 7-methyl hydrogen
17-hydroxy-11.alpha.-(methylsulfonyl)oxy-3-oxo-17.alpha.-pregna-4-ene-7.a-
lpha.,21-dicarboxylate, .gamma.-lactone: ##STR273## which was
prepared in the manner described in Example 36. The resulting
mixture was heated between about 60 to 70.degree. C. for about 1 to
1.5 hours. This mixture was concentrated under reduced pressure at
50.degree. C. to give a thick slurry. The slurry was dissolved in
100 mL ethyl acetate and was washed 2 times with about 20%
water/brine (80 mL each time), 1 time with a 1N sodium hydroxide
(80 mL) solution, followed by 1 time with about 20% water/brine (80
mL). The crude product was dried over magnesium sulfate filtered
and concentrated to give about 18 g of crude wet material.
[0841] This material was purified by column chromatography twice to
afford about 3 g of pure
(7.alpha.,13R,17.beta.)-3',4',5',17-tetrahydro-14-hydroxy-17-methyl-3,5'--
dioxo-.gamma.-lactone,
cyclopenta[13,17]-18-norandrosta-4,9(11)-diene-7-carboxylic acid
(Compound C-201).
EXAMPLE X-1C
Preparation of
[13S,17.beta.]-3',4'-dihydro-3-hydroxy-9,17-dimethylcyclopenta[13,17]gona-
-1,3,5(10)-triene-5'[2'H]-one (Compound C-202)
[0842] ##STR274##
[0843] Potassium acetate (6 g, 61.1 mmol), trifluoroacetic acid
(150 mL, 1.480 g/mL) and trifluoroacetic anhydride (26 mL, 1.487
g/mL) were added to a 250 mL round bottom reactor equipped with
mechanical stirrer, condenser, and heating mantel. The resulting
solution was stirred between about 25.degree. C. to 30.degree. C.
for about 10 minutes.
[0844] The preformed TFA/TFA anhydride reagent was added to 15 g
(43.7 mmol) of 17-hydroxy-3-oxo-17.alpha.-pregn-4-ene-21-carboxylic
acid, .gamma.-lactone (also known as aldona; G.D. Searle &
Co.): ##STR275## The resulting mixture was heated between
60.degree. C. to 70.degree. C. for about 1 to 1.5 hours. The
reaction mixture was concentrated under reduced pressure at
50.degree. C. to give thick slurry. The slurry was dissolved in 100
ml ethyl acetate and was washed 2 times with about 20% water/brine
solution (80 mL each time), 1 time with 1N sodium hydroxide
solution (80 mL), followed by 1 time with about 20% water/brine
solution (80 mL). The crude product was dried over magnesium
sulfate, filtered and concentrated to dryness under reduced
pressure at 50.degree. C. to give about 20 g of crude wet
material.
[0845] This material was purified by column chromatography twice to
afford about 125 g of pure
[13S,17.beta.]-3',4'-dihydro-3-hydroxy-9,17-dimethylcyclopenta[13,17]gona-
-1,3,5(10)-triene-5'[2'H]-one (Compound C-202).
EXAMPLE X-1D
Preparation of
[13S,17.beta.]-3',4'-dihydro-3-hydroxy-9,17-dimethylcyclopenta[13,17]gona-
-1,3,5(10),6-tetraene-5'[2'H]-one (Compound C-203)
[0846] ##STR276##
[0847] Potassium acetate (6 g, 61.1 mmol), trifluoroacetic acid
(150 mL, 1.480 g/mL) and trifluoroacetic anhydride (26 mL, 1.487
g/mL) was added to a 250 mL round bottom reactor equipped with
mechanical stirrer, condenser, and heating mantel. The resulting
solution was stirred between about 25.degree. C. to 30.degree. C.
for about 10 minutes.
[0848] The preformed TFA/TFA anhydride reagent was added to 15 g
(45.9 mmol) of was added to
17-hydroxy-3-oxo-17.alpha.-pregn-4,9(11)-diene-21-carboxylic acid,
.gamma.-lactone (also known as .DELTA.-9,11-aldona): ##STR277##
which was prepared from 3-methoxy-3,5,9(11)-androstatriene-17-one
(Upjohn). The resulting mixture was heated between about 60.degree.
C. to 70.degree. C. for about 1 to 1.5 hours. The reaction mixture
was concentrated under reduced pressure at 50.degree. C. to give
thick slurry. The slurry was dissolved in 100 ml ethyl acetate and
was washed 2 times with about 20% water/brine solution (80 mL each
time), 1 time with 1N sodium hydroxide solution (80 mL) followed by
1 time with about 20% water/brine solution (80 mL). The crude
product was dried over magnesium sulfate, filtered and concentrated
to dryness under reduced pressure at 50.degree. C. to give about 18
g of crude wet material.
[0849] This material was purified by column chromatography twice to
afford about 340 g of pure
[13S,17.beta.]-3',4'-dihydro-3-hydroxy-9,17-dimethylcyclopenta[13,17]gona-
-1,3,5(10),6-tetraene-5'[2'H]-one (Compound C-203).
EXAMPLE X-1E
Preparation of
[13S,17.beta.]-3',4'-dihydro-17-methyl-cyclopenta[13,17]-18-norandrosta-4-
,6,8(14)-triene-3,5'[2'H]-dione (Compound C-204)
[0850] ##STR278##
[0851] Potassium acetate (8 g, 81.5 mmol), trifluoroacetic acid
(150 mL, 1.480 g/mL) and trifluoroacetic anhydride (33 mL, 1.487
g/mL) were added to a 250 mL round bottom reactor equipped with
mechanical stirrer, condenser, and heating mantel. The resulting
solution was stirred between about 25.degree. C. to 30.degree. C.
for about 10 minutes.
[0852] The preformed TFA/TFA anhydride reagent was added to 15 g
(44.0 mmol) of
17-hydroxy-3-oxo-17.alpha.-pregn-4,6-diene-21-carboxylic acid,
.gamma.-lactone (also known as canrenone; G.D. Searle & Co.):
##STR279## The resulting mixture was heated between about 60 to
70.degree. C. for about 1 to 1.5 hours. The reaction mixture was
concentrated under reduced pressure at 50.degree. C. to give thick
slurry. The slurry was dissolved in 100 ml ethyl acetate and was
washed 2 times with about 20% water/brine solution (80 mL each
time), 1 time with 1N sodium hydroxide solution (80 mL), followed
by 1 time with about 20% water/brine solution (80 mL). The crude
product was dried over magnesium sulfate, filtered and concentrated
to dryness under reduced pressure at 50.degree. C. to give about 18
g of crude wet material.
[0853] This material was purified by column chromatography twice to
afford about 2.2 g of pure
[13S,17.beta.]-3',4'-dihydro-17-methyl-cyclopenta[13,17]-18-norandrosta-4-
,6,8(14)-triene-3,5'[2'H]-dione (Compound C-204).
EXAMPLE X-2
Preparation of
[0854] ##STR280##
[0855] To a stirred, cold (0.degree. C.) solution of
11.alpha.-hydroxycanrenone (3.6 g, 10 mmol) and triethylamine (1.2
g, 12 mmol) in methylene chloride (20 mL) is added methanesulfonyl
chloride (1.1 g, 10 mmol). The mixture is stirred in the cold for 3
hours and allowed to warm to room temperature. Stirring is
continued until thin layer chromatography indicates the reaction is
complete. The mixture then is diluted with ethyl acetate and
extracted with water, aqueous 5% sodium bicarbonate solution and
water and dried over sodium sulfate. The drying agent is filtered
and the filtrate concentrated in vacuo to give the following crude
mesylate C-136 which is suitable for use in the next step:
##STR281##
[0856] A solution of mesylate C-136 (4.3 g, 10 mmol) is reacted
with trifluoracetic acid (25 mL), trifluoracetic anhydride (4.5 mL)
and potassium acetate (6.7 g, 7.1 mmol) according to the procedure
described for the synthesis of Compound C-1 in Example X-1. The
crude product is isolated according to the same procedure as for
Compound C-1 in Example X-1 and is purified by chromatography on
silica gel using mixtures of ethyl acetate and toluene or ethyl
acetate and hexane as eluents. The product thus obtained is further
purified by recrystallization from alcohol, alcohol and water, or
ethyl acetate and hexane to yield to tetraene C-105.
EXAMPLE X-3
Preparation of
[0857] ##STR282## is synthesized and isolated (according to the
procedure described in Example X-2 for the synthesis of mesylate
C-136) using
11.alpha.,17-dihydroxy-3-oxo-17-oxo-pregn-4-ene-21-carboxylic acid,
.gamma.-lactone (3.6 g, 10 mmol), triethylamine (1.2 g, 12 mol) and
methanesulfonyl chloride (1.1 g, 10 mmol) in methylene chloride (20
mL). The mesylate C-138 thus isolated is suitable for use in the
following step.
[0858] A solution of mesylate C-138 (4.4 g, 10 mmol) is reacted
with trifluoracetic acid (25 mL), trifluoracetic anhydride (4.5 mL)
and potassium acetate (6.7 g, 7.1 mmol) according to the procedure
described for the synthesis of Compound C-1 in Example X-1. The
crude product C-110 is isolated according to the same procedure as
for Compound C-1 in Example X-1 and is purified by chromatography
on silica gel using mixtures of ethyl acetate and toluene or ethyl
acetate and hexane as eluents. The product thus obtained is further
purified by recrystallization from alcohol, alcohol and water, or
ethyl acetate and hexane.
EXAMPLE X-4
Preparation of
3',4',5',17-tetrahydro-17.beta.-methyl-3,5'-dioxocyclopenta[13R,17]-18-no-
randrosta-4,8,14-triene-7.alpha.-carboxylic acid (Compound
C-101)
[0859] ##STR283## A solution of Compound C-1 (3.8 g, 10 mmol) and
aqueous 1N sodium hydroxide solution (35 mL) in ethanol (60 mL) is
refluxed for 8 hours. The reaction is cooled to room temperature,
concentrated on the rotary evaporator in vacuo and the residual
aqueous layer is extracted three times with ethyl acetate. The
aqueous layer is then acidified with 1N hydrochloric acid solution
and extracted three times with ethyl acetate. The combined organic
layers are washed with water and dried over sodium sulfate. The
drying agent is filtered and the filtrate is concentrated on the
rotary evaporator. The residual crude carboxylic-acid C-101 is
crystallized by treatment with ethyl acetate and is recrystallized
from ethyl acetate and hexane or methanol or ethanol and water.
EXAMPLE X-5
Preparation of 1-methylethyl
3',4',5',17-tetrahydro-17.beta.-methyl-3,5'-dioxocyclopenta[13R,17]-18-no-
randrosta-4,8,14-triene-7.alpha.-carboxylate (Compound C-102)
[0860] ##STR284##
[0861] A mixture of sodium bicarbonate (3.5 g) and a solution of
carboxylic acid C-101 (3.7 g, 10 mmol) and isopropyl iodide (3 mL)
in dimethylformamide (35 mL) is stirred at room temperature
overnight. The reaction is poured onto water and the aqueous
solution is extracted three times with ethyl acetate. The combined
organic layers are washed with water and dried over sodium sulfate.
The drying agent is filtered and the filtrate concentrated in
vacuo. The residual crude isopropyl ester C-102 is crystallized by
treatment with ethyl acetate or alcohol and is purified by
chromatography on silica gel and recrystallization from ethyl
acetate and hexane or alcohol or alcohol and water.
EXAMPLE X-6
Preparation of ethyl
3',4',5',17-tetrahydro-17.beta.-methyl-3,5'-dioxocyclopenta[13R,17]-18-no-
randrosta-4,8,14-triene-7.alpha.-carboxylate
[0862] ##STR285##
[0863] A mixture of sodium bicarbonate (3.5 g) and a solution of
carboxylic acid C-101 (3.7 g, 10 mmol) and ethyl iodide (3 mL) in
dimethylformamide (35 mL) is stirred at room temperature overnight.
The reaction is poured onto water and the aqueous solution is
extracted three times with ethyl acetate. The combined organic
layers are washed with water and dried over sodium sulfate. The
drying agent is filtered and the filtrate concentrated in vacuo.
The residual crude ethyl ester C-103 is crystallized by treatment
with ethyl acetate or alcohol and is purified by chromatography on
silica gel and recrystallization from ethyl acetate and hexane or
alcohol or alcohol and water.
EXAMPLE X-7
Preparation of hexyl
3',4',5',17-tetrahydro-17.beta.-methyl-3,5'-dioxocyclopenta[13R,17]-18-no-
randrosta-4,8,14-triene-7.alpha.-carboxylate (Compound C-104)
[0864] ##STR286##
[0865] A mixture of sodium bicarbonate (3.5 g) and a solution of
carboxylic acid C-101 (3.7 g, 10 mmol) and n-hexyl iodide (3 mL) in
dimethylformamide (35 mL) is stirred at room temperature overnight.
The reaction is poured onto water and the aqueous solution is
extracted three times with ethyl acetate. The combined organic
layers are washed with water and dried over sodium sulfate. The
drying agent is filtered and the filtrate concentrated in vacuo.
The residual crude n-hexyl ester C-104 is crystallized by treatment
with ethyl acetate or alcohol and is purified by chromatography on
silica gel and recrystallization from ethyl acetate and hexane or
alcohol or alcohol and water.
EXAMPLE X-8
Preparation of
3',4'-dihydro-17-methyl-7.alpha.(methylthio)-cyclopenta[13,17]-18-norandr-
osta-4,8,14-triene-3,5'(2'H)-dione (Compound C-106)
[0866] ##STR287##
[0867] A solution of tetraene C-105 (3.2 g, 10 mmol) in methanol
(40 mL) and piperidine (4 mL) is cooled to 5.degree. C. Gaseous
methyl mercaptan is passed through until a weight gain of 7 g is
observed. The pressure container is sealed and held at room
temperature for 20 hours. The solution is poured onto ice water and
the precipitate is filtered, washed with water and air dried. The
methylthio product C-106 is purified by recrystallization from
methanol or ethyl acetate and hexane. See, e.g., the procedure set
forth in A. Karim and E. A. Brown, Steroids, 20, 41 (1972), which
is incorporated herein by reference.
EXAMPLE X-9
Preparation of
7.alpha.-(acetylthio)-3,4'-dihydro-17-methyl-cyclopenta[13,17]-18-norandr-
osta-4,8,14-triene-3,5'(2'H)-dione (Compound C-107)
[0868] ##STR288##
[0869] A solution of tetraene C-105 (3.2 g, 10 mmol) in thioacetic
acid (10 mL) is heated t 85-95 C for 1 hour. Excess thioacetic acid
is removed in vacuo and the resultant crude 7.alpha.-thioacetate
C-107 is purified by recrystallization from a suitable solvent such
as methanol or ethyl acetate or ethyl acetate and hexane. See e.g.,
the procedure set forth in U.S. Pat. No. 3,013,012, J. A. Cella and
R. C. Tweit, Dec. 12, 1961 which is incorporated herein by
reference.
EXAMPLE X-10
Preparation of
1,2,4bR(4bR*),5,5aS*,7,7aR*,8,9,11,12bS*-dodecahydro-7a,12b-dimethyl-10aR-
*-cyclopropal[1]pentaleno[1,6a-a]phenanthrene-3,10-dione
[0870] ##STR289##
1,2,4bS(4bR*),5,5aS*,8,9,11,12,12bR*-dodecahydro-7a,12b-dimethyl-10aS*-cyc-
lopropal[1]pentaleno[1,6a-a]phenanthrene-3,10-dione (Compound
C-109)
[0871] ##STR290##
[0872] To a solution of trimethylsulfoxonium iodide (1 g, 4.6 mmol)
in dry dimethylsulfoxide (20 mL) is added sodium hydride (220 mg of
50% dispersion in mineral oil, 4.6 mmol). The mixture is stirred at
room temperature under nitrogen until the evolution of hydrogen
ceases. A solution of tetraene C-105 (1.12 g, 3.5 mmol) in
dimethylsulfoxide (4 mL) is then added and stirring is continued
for 4 hours under a nitrogen atmosphere. The reaction mixture is
diluted with water and the resultant precipitate is filtered and
air dried. The product is a mixture of the 6.beta.,7.beta.
(Compound C-108) and 6.alpha.,7.alpha. (Compound C-109) isomers.
Separation of these isomers is effected by chromatography on silica
gel and the individual isomers are further purified by
recrystallization from solvents such as ethyl acetate and hexane,
alcohol, or alcohol and water.
EXAMPLE X-11
Preparation of
[0873] ##STR291##
[0874] To a suspension of enone C-110 (3.2 g, 10 mmol) in ethyl
orthoformate (10 mL) and anhydrous ethanol (10 mL) is added
p-toluenesulfonic acid monohydrade (0.05 g). The reaction is
stirred for 30 minutes at room temperature and is quenched by the
addition of a few drops of pyridine. After stirring another 5
minutes at 0.degree. C., the resultant precipitate is filtered,
washed with a small amount of methanol, and recrystallized from
alcohol or ethyl acetate and hexane containing traces of pyridine
to provide pure enol ether C-111. Alternatively, the reaction may
be worked up after addition of pyridine by removing all solvents in
vacuo and crystallizing the residue by addition solvents such as
ether, ethyl acetate or hexane. The crude C-111 is recrystallized
as above. See, e.g., the procedure set forth in R. M. Weier and L.
M. Hofmann, J. Med. Chem., 20, 1304-1308 (1977) which is
incorporated herein by reference.
EXAMPLE X-12
Preparation of
[0875] ##STR292## Procedure of R. M. Weier and L. M. Hofmann, J.
Med. Chem., 20 1304-1308 (1977) See, e.g., which is incorporated
herein by reference.
[0876] Vilsmeyer reagent is prepared by adding phosphorus
oxychloride (4.59 g, 30 mmol) to dimethylformamide (30 mL) at
).degree. C. After 5 minutes, a solution of enol ether C-111 (3.5
g, 10 mmol) in dimethylformamide (5 mL) is added and the reaction
is stirred at 0.degree. C. for 2 hours and at room temperature
overnight. The reaction is poured onto aqueous sodium acetate
solution and stirred for 2 hours. The precipitate is filtered and
dried to give crude aldehyde C-112. Purification is effected by
recrystallization from solvents such as alcohol, alcohol and water
or ethyl acetate and hexane. Alternatively, the crude aldehyde
C-112 is isolated from the aqueous sodium acetate solution by
extraction with a solvent such as ethyl acetate. After drying over
sodium sulfate and removing the solvent, the residue is purified by
recrystallization or by chromatography over silica gel followed by
recrystallization.
EXAMPLE X-13
Preparation of
[0877] ##STR293##
[0878] To a stirred cold solution of lithium
tri-tert-butoxyaluminum hydride (3.05 g, 12 mmol) in
tetrahydrofuran is added aldehyde C-112 (3.78 g, 10 mmol). The
reaction is stirred at room temperature for 5 hours and quenched by
adding water and acetic acid, care being taken to keep the mixture
slightly basic. The mixture is concentrated in vacuo and the
resultant solid slurried in ethyl acetate. The solid is filtered
and the filtrate concentrated in in vacuo. The residue is dissolved
in a minimum volume of a solution of acetone and water (3:1) and
added to acidified aqueous acetone (pH 1.5-2.0). The acid reaction
mixture is stirred at room temperature for 1 hour and concentrated
in vacuo. The resultant crude dienone C-113 is purified by
recrystallization from solvents such as alcohol, alcohol and water
or ethyl acetate and hexane. Alternatively, the crude dienone C-113
is purified by chromatography on silica gel and then
recrystallized. See, e.g., the procedure set forth in R. M. Weier
and L. M. Hofmann, J. Med. Chem., 20, 1304-1308 (1977) which is
incorporated herein by reference.
EXAMPLE X-14
Preparation of
2',3',3'a.alpha.,4',6',10',11',11'a.alpha.,12',13'-decahydro-3'aR,3'a,11'-
a-dimethyl-13'aR*-spiro[cyclopropane-1,7'(9'H)-[1H]pentaleno[1,6a-a]phenan-
threne]-1'9'-dione
[0879] ##STR294##
[0880] To a solution of dienone C-113 (1.17 g, 0.05 mmol) in
tetrahydrofuran (30 mL) is added a solution of diazomethane (0.2 g,
0.07 mmol) in ether (7 mL). The resultant reaction solution is
stored at room temperature for several days. Acetic acid is then
added to destroy excess diazomethane and the reaction is
concentrated in vacuo. The residue, which is the intermediate
pyrazoline, is crystallized under a solvent such as acetone,
hexane, ethyl acetate or ethanol and is then recrystallized. This
material is converted to the spirocyclopropane C-114. Thus, the
solid C-114 is heated at 190.degree. C. in vacuo and the resulting
solid is recrystallized from alcohol, alcohol and water, acetone
and water or ethyl acetate and hexane. Alternatively, a solution of
the pyrazoline in acetone is treated with boron trifluoride
etherate at room temperature for about 1 hour. Water is added and
the resultant precipitate is filtered and air dried. Purification
is effected by recrystallization. See, e.g., the procedure set
forth in F. B. Colton and R. T. Nicholson, U.S. Pat. No. 3,499,891,
March 10, (1970) which is incorporated herein by reference.
EXAMPLE X-15
Preparation of
[0881] ##STR295##
[0882] To a solution of enol ether C-111 (3.2 g, 10 mmol) in
methanol (20 mL) is added sodium borohydride (38 mg). The reaction
is stirred at room temperature for 3 hours and is treated with 1N
hydrochloric acid for 30 minutes. The reaction is further diluted
with water and the precipitate filtered, washed with water and
dried. The product, consisting of a mixture of two epimeric
alcohols (Compound C-115 and Compound C-116), is chromatographed on
silica gel to effect separation. The purified alcohols C-115 and
C-116 are each recrystallized from solvents such alcohol, alcohol
and water, ethyl acetate and hexane and acetone and hexane.
Alternatively, the diluted reaction mixture is extracted with ethyl
acetate, the combined organic layers dried over sodium sulfate and
the crude product thus isolated is chromatographed as above to
provide the separated alcohols C-115 and C-116.
EXAMPLE X-16
Preparation of
[0883] ##STR296##
[0884] Compound C-1 (3.8 g, 10 mmol) is converted to enol ether
C-117 using ethyl orthoformate (3.2 g, 10 mmol), ethanol (10 mL)
and p-toluenesulfonic acid monohydrate (0.05 g) according to the
procedure described for the synthesis of Compound C-111 set forth
in Example X-11.
EXAMPLE X-17
Preparation of methyl
3',4',5',17-tetrahydro-6.alpha.-hydroxy-17.beta.-methyl-3,5'-dioxocyclope-
nta[13R,17]-18-norandrosta-4,8,14-triene-7.alpha.-carboxylate
(Compound C-118)
[0885] ##STR297##
[0886] A solution of 57% m-chloroperoxybenzoic acid (3.64 g) in 10%
aqueous dioxane (20 mL) is half-neutralized with 1N sodium
hydroxide solution. This solution is cooled to 0.degree. C. and
added in portions to a stirred, cold (0.degree. C.) solution of
enol ether C-117 (4.1 g, 10 mmol) in 10% aqueous dioxane (20 mL).
The reaction is stirred at room temperature overnight, poured onto
ice water and extracted with methylene chloride or ethyl acetate.
The combined organic layers are dried over sodium sulfate.
Evaporation of the solvent provides the crude hydroxy ester C-118,
which is purified by chromatography on silica gel. See, e.g., the
procedure set forth in R. M. Weier and L. M. Hofmann, J. Med.
Chem., 20, 1304-1308 (1977) which is incorporated herein by
reference.
EXAMPLE X-18
Preparation of
[0887] ##STR298##
[0888] Lithium dimethyl cuprate is prepared by adding methyllithium
(19 mL of a 1.6 M solution in ether, 30 mmol) to a stirred
suspension of cuprous iodide (2.86 g, 15 mmol) in ether (30 mL) at
0.degree. C. under an inert atmosphere of argon. After stirring in
the cold for 15 minutes a solution of tetraene C-105 (1.6 g, 15
mmol) in tetrahydrofuran (25 mL) is added dropwise over 25 minutes.
The reaction is continued for an additional 30 minutes and poured
onto saturated ammonium chloride solution with vigorous stirring.
The aqueous mixture is extracted with ethyl acetate or methylene
chloride. The combined organic layers are washed with aqueous
ammonium chloride solution, water and dried over sodium sulfate.
The solvent is removed in vacuo and the residue is dissolved in
ethyl acetate or methylene chloride and treated with
p-toluenesulfonic acid (100 mg) on the steam bath for 30 minutes to
1 hour. The organic solution is washed with water and dried over
sodium sulfate. The solvent is removed in vacuo to give the crude
enone C-119. Purification is effected by recrystallization from
alcohol, alcohol and water or ethyl acetate and hexane.
Alternatively, the crude enone C-119 is chromatographed on silica
gel and the product then recrystallized. See, e.g., the procedure
set forth in J. K. Grunwell et al., Steroids, 27, 759-771 (1976),
which is incorporated herein by reference.
EXAMPLE X-19
Preparation of
[0889] ##STR299##
[0890] Ester C-120 (4.00 g, 10 mmol): ##STR300## (prepared in
accordance with the procedure set forth in R. M. Weier and L. M.
Hofmann, J. Med. Chem., 18, 817 (1975), which is incorporated
herein by reference) is reacted with trifluoroacetic acid (25 mL),
trifluoroacetic anhydride (4.5 mL) and potassium acetate (6.7 g,
7.1 mmol) according to the procedure described for the synthesis of
Compound C-1 in Example X-1. The crude product C-121 is isolated
according to the same procedure as for Compound C-1 in Example X-1
and is purified by chromatography on silica gel using mixtures of
ethyl acetate and toluene or ethyl acetate and hexane as eluents
and the product thus obtained is further purified by
recrystallization from alcohol, alcohol and water, or ethyl acetate
and hexane.
EXAMPLE X-20
Preparation of
[0891] ##STR301## Enone C-122 (3.68 g, 10 mmol): ##STR302##
(prepared in accordance with the procedure set forth in F. B.
Colton and R. T. Nicholson, U.S. Pat. No. 3,499,891, Mar. 10, 1970,
which is incorporated herein by reference) is reacted with
potassium acetate (6.7 g, 7.1 mmol), trifluoracetic acid (25 mL)
and trifluoroacetic acid anhydride (4.5 mL) according to the
procedure described for the synthesis of Compound C-1 in Example
X-1. The crude product is isolated as described in the above
reference and is purified by chromatography on silica gel using
mixtures of ethyl acetate and hexane or ethyl acetate and toluene
as eluents. The purified C-123 is further purified by
recrystallization from alcohol, alcohol and water, or ethyl acetate
and hexane.
EXAMPLE X-21
Preparation of
[0892] ##STR303##
[0893] Compounds C-125 and C-126 are synthesized according to the
procedure used above for the synthesis of Compounds C-108 and C-1.
Thus, to a solution of trimethylsulfoxonium iodide (1 g, 4.6 mmol)
in dry dimethylsulfoxide (20 mL) is added sodium hydride (220 mg of
50% dispersion in mineral oil, 4.6 mmol). The mixture is stirred at
room temperature under nitrogen until the evolution of hydrogen
ceases.
[0894] A solution of trienone C-124 (1.14 g, 3.5 mmol): ##STR304##
in dimethylsulfoxide (4 mL) is then added and stirring is continued
for 4 hours under a nitrogen atmosphere. Trienone C-124 is prepared
in accordance with the procedure set forth in Example X-2 for the
preparation of Tetraene C-105 by using canrenone as the starting
material instead of 11.alpha.-hydroxy canrenone. The reaction
mixture is diluted with water and the resultant precipitate is
filtered and air dried. The product is a mixture of the
6.beta.,7.beta. (Compound C-125) and 6.alpha.,7.alpha. (Compound
C-126) isomers. Separation of these isomers is effected by
chromatography on silica gel and the individual isomers are further
purified by recrystallization from solvents such as ethyl acetate
and hexane, alcohol or alcohol and water.
EXAMPLE X-22
Preparation of methyl
3'4',5',17-tetrahydro-17.beta.-methyl-3,5'-dioxocyclopenta[13R,17]-18-nor-
androsta-1,4,8,14-tetraene-7.alpha.-carboxylate (Compound
C-127)
[0895] ##STR305##
[0896] Dienone C-127 is synthesized according to the procedure
described in R. M. Weier and L. M. Hofmann, J. Med. Chem. 18, 817
(1975) which is incorporated by reference herein. Thus, a solution
of Compound C-1 (3.8 g, 10 mmol) and dichlorodicyanobenzoquinone
(2.72 g, 12 mmol) in dioxane (80 mL) is refluxed with stirring for
24 hours. The reaction mixture is concentrated in vacuo, the
residue digested with methylene chloride, filtered and the filtrate
washed with a 2% sodium sulfite, 5% sodium hydroxide and saturated
sodium chloride solution and dried over sodium sulfate. The drying
agent is filtered and the filtrate is concentrated in vacuo. The
crude product dienone C-127 is purified by chromatography on silica
gel using mixtures of ethyl acetate and toluene as eluents and the
product 27 thus isolated is further purified by recrystallization
from alcohol.
EXAMPLE X-25
Preparation of
2',3',3'a.alpha.,4',6'11'a.alpha.,12',13'-octahydro-3'aR,3'a,11'a-dimethy-
l-13'aR*-spiro[cyclopropane-1,7'(9'H)-[1H]pentaleno,[1,6a-a]phenanthrene]1-
'9'-dione
[0897] ##STR306##
[0898] Using the procedure and workup described above in Example
X-22 for the synthesis of dienone C-127, enone C-114 (3.48 g, 10
mmol) is converted to dienone C-128 using
dichlorodicyanobenzoquione (2.72 g, 12 mmol) in dioxane (80
mL).
EXAMPLE X-24
Preparation of
4bR(4bR*),5,5aS*,7,7aR*,8,9,11,12,12bS*-decahydro-7A,12b-dimethyl-10R*-cy-
clopropa(1)pentaleno[1,6a-a]phenanthrene-3,10-dione
[0899] ##STR307## Using the procedure and workup described above in
Example X-22 for the synthesis of dienone C-127, enone C-108 (3.34
g, 10 mmol) is converted to dienone C-129 using
dichlorodicyanobenzoquione (2.72 g, 12 mmol) in dioxane (80
mL).
EXAMPLE X-25
Preparation of
[0900] ##STR308##
[0901] To a cold (0.degree. C.), stirred solution of carboxylic
acid C-101 (3.66 g, 10 mmol) and N-methylmorpholine (1.01 g, 10
mmol) in tetrahydrofuran (35 mL) is added isobutyl chloroformate
(1.36 g, 10 mmol). The reaction is stirred at 0.degree. C. for 20
minutes, filtered and the filtrate is concentrated in vacuo. The
residue is the mixed anhydride and is suitable for use in the next
step.
[0902] Gaseous dimethylamine is bubbled through a cold (0.degree.
C.) solution of mixed anhydride (4.6 g, 10 mmol) in tetrahydrofuran
(40 mL) in a pressure vessel. After 15 minutes, the pressure vessel
is sealed and the reaction is allowed to stand at room temperature
for 24 hour. The reaction is warmed to 40 C for 30 minutes and
cooled back to 0.degree. C. After warming to room temperature, the
vessel is vented to the atmosphere and excess dimethylamine is
permitted to evaporate. The reaction is concentrated in vacuo, the
residue dissolved in ethyl acetate and extracted with 1N sodium
hydroxide solution and water. After drying over sodium sulfate, the
organic layer is stripped and the residue purified by
chromatography on silica gel using mixtures of ethyl acetate and
toluene as the eluents. The amide C-130 thus isolated is further
purified by recrystallization from alcohol, alcohol and water or
ethyl acetate and hexane.
EXAMPLE X-26
Preparation of
[0903] ##STR309##
[0904] To a suspension of enone C-114 (3.5 g, 10 mmol) in ethyl
orthoformate (10 mL) and anhydrous ethanol (10 mL) is added
p-toluenesulfonic acid monohydrade (0.05 g). The reaction is
stirred for 30 minutes at room temperature and is quenched by the
addition of a few drops of pyridine. After stirring another 5
minutes at 0.degree. C., the resultant precipitate is filtered,
washed with a small amount of methanol, and recrystallized from
alcohol or ethyl acetate and hexane containing traces of pyridine
to provide pure enol ether C-131. Alternatively, the reaction may
be worked up after addition of pyridine by removing all solvents in
vacuo and crystallizing the residue by addition solvents such as
hexane, ether, ethyl acetate or hexane. The crude enol ether C-131
is recrystallized as above.
EXAMPLE X-27
Preparation of
[0905] ##STR310##
[0906] To a cold (-78.degree. C.) solution of lithium
bis(trimethylsilyl)amide (10 mL of a 1.0 M solution in
tetrahydrofuran) a solution of enol ether C-131 (3.8 g, 10 mmol) in
tetrahydrofuran (20 mL) is added dropwise over 20 minutes The
reaction is stirred at -78.degree. C. for 10 minutes and a solution
of phenylselenenyl chloride (1.9 g, 10 mmol) in tetrahydrofuran (5
mL) is added. The reaction is stirred 5 minutes and quenched by the
addition of 1% sodium bisulfate solution. The reaction is further
diluted with water and extracted with ethyl acetate. The combined
organic layers are washed with 5% aqueous sodium bicarbonate
solution, and water, and dried over sodium sulfate. The drying
agent is filtered and the filtrate concentrated in vacuo. The
residue is purified by chromatography on silica gel using mixtures
of ethyl acetate and toluene or ethyl acetate and hexane as eluents
to give pure selenide C-132.
EXAMPLE X-28
Preparation of
[0907] ##STR311##
[0908] To a cold (0.degree. C.) solution of selenide C-132 (g, 10
mmol) and pyridine (1.61 mL, 20 mmol) in methylene chloride (40 mL)
is gradually added a solution of hydrogen peroxide (3.1 g of 30%
solution, 27 mmol) in water (3 mL). The temperature is maintained
at less than 30-35.degree. C. After any exothermicity subsides, the
ice bath is removed and the reaction is stirred vigorously at room
temperature for 15 minutes. The reaction is diluted with methylene
chloride and washed with 5% sodium bicarbonate solution and water
and dried over sodium sulfate. The drying agent is filtered and the
filtrate is concentrated in vacuo. The residue is purified by
chromatography on silica gel using mixtures of ethyl acetate and
toluene or ethyl acetate and hexane as eluents to give pure
unsaturated ketone C-133 which is further purified by
recrystallization from ethyl acetate and hexane or alcohol.
EXAMPLE X-29
Preparation of
[0909] ##STR312## A solution of ketone C-133 (2 g) in acetone (15
mL) is treated with 1N hydrochloric acid (4 mL) for 1 hour at room
temperature. The reaction is concentrated in vacuo. The residue is
diluted with water and extracted with ethyl acetate. The combined
organic layers are washed with 5% sodium bicarbonate solution and
water and dried over sodium sulfate. The drying agent is filtered
and the filtrate concentrated in vacuo to give crude ketone C-134.
The crude product is purified by chromatography on silica gel using
mixtures of ethyl acetate and toluene as eluents to give pure
ketone C-134 which is further purified by recrystallization from
ethyl acetate and hexane or alcohol.
* * * * *