Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02553378 1996-12-11
PROCESSES FOR PREPARATION OF 7 ALPHA-CARBOXYL 9,11-EPOXY
STEROIDS AND INTERMEDIATES USEFUL THEREIN AND A GENERAL
PROCESS FOR THE EPOXIDATION OF OLIFINIC DOUBLE BONDS
' Backcrround of the Invention -
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 9,11x-epoxy-17a-
hydroxy-3-oxopregn-4-ene-7a,21-dicarboxylate, y-lactone
(eplerenone; epoxymexrenone).
Methods for the preparation of 20-spiroxane
series compounds are described in U.S. patent 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:
Y2
Y~
~..vC C H2~ 2 C=X
i ..~
A/A CH3 N\0 8 B
IA
in which
-A-A- represents the group -CH2-CHZ- or -CH=CH-,
R' represents an a-oriented lower alkoxycarbonyl
or hydroxycarbonyl radical.
-B-B- represents the group -CH2-CH2- or an a- or
~i-oriented group
R~ R~
\CH CH
-CH-CHZ-CH- III
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R6 and R' being hydrogen
X represents two hydrogen atoms or oxo,
Y1 and YZ together represent the oxygen bridge _
-0- , or
Y1 represents hydroxy, and
YZ represents hydroxy, lower alkoxy or, if X
represents H2, also lower alkanoyloxy,
and salts of such compounds in which X
represents oxo and Y~ represents hydroxy, that is to say
of corresponding 17~-hydroxy-21-carboxylic acids.
U.S. patent 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
related steroids.
Summary of the Invention
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.
Accordingly, the present invention is directed
to a series of synthesis schemes for epoxymexrenone;
intermediates useful in the manufacture of eplerenone;
and syntheses for such novel intermediates.
The novel synthesis schemes are described in
detail in the Description of Preferred Embodiments.
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Among the novel intermediates of this invention are those
described immediately below.
A compound of Formula IV corresponds to the
structure:
R9
ZV
wherein:
-A-A- represents the group -CHR4-CFiRs- or -
CR'=CRS-
R3, R4 and RS are independently selected from
the group consisting of hydrogen, halo,
hydroxy, lower alkyl, lower alkoxy,
hydroxyalkyl, alkoxyalkyl, hydroxy carbonyl,
cyano, aryloxy,
R1 represents an alpha-oriented lower
alkoxycarbonyl or hydroxycarbonyl radical,
R2 is an 11a- leaving group the abstraction of
which is effective for generating a double bond
between the 9- and 11- carbon atoms;
-B-B- represents the group -CHR6-CHR'- or an
alpha- or beta- oriented group:
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R° R~
\CH CH
I I
-cH-CH2-CH- III
where R6 and R' are independently selected from
the group consisting of hydrogen, halo, lower
alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,
hydroxycarbonyl, alkyl, alkoxycarbonyl,
acyloxyalkyl, cyano, aryloxy, and
Re and R9 are independently selected from the
group consisting of hydrogen, halo, lower
alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,
hydroxycarbonyl, alkyl, alkoxycarbonyl,
acyloxyalkyl, cyano, aryloxy, or Rg and R9
together comprise a carbocyclic or heterocyclic
ring structure, or RB or R9 together with R6 or
R' comprise a carbocyclic or heterocyclic ring
structure fused to the pentacyclic D ring.
A compound of Formula IVA corresponds to
Formula IV wherein Re and R9 together with the ring carbon
to which they are attached form the structure:
Y2
Y ~~
~C\1 7) W ii( CHp) p - C - X
xxxlv
where X, Y1, Y2 and C(17) are as defined above.
A compound of Formula IVB corresponds to
Formula IVA wherein RB and R9 together form the structure
of Formula XXXIII:
0
XXXIII
Compounds of Formulae IVC, IVD and IVE,
respectively, correspond to any of Formula IV, IVA, or
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IVB wherein each of -A-A- and -B-B- is -CH2-CH2-, R3 is
hydrogen, and R1 is alkoxycarbonyl, preferably
methoxycarbonyl. Compounds within the scope of Formula
IV may be prepared by reacting a lower alkylsulfonylating
5 or acylating reagent, or a halide generating agent, with
a corresponding compound within the scope of Formula V.
A compound of Formula V corresponds to the
structure:
R3 R8
R9
H 0,~~~
B
a i
a
~~~,,R ~
v
wherein -A-A-, -B-B-, Rl, R3, RB and R9 are as defined in
Formula IV.
A compound of Formula VA corresponds to Formula
V wherein R8 and R9 with the ring carbon to which they are
attached together form the structure:
Y2
Y ~~
~~~~ ~) my cH2) 2 - c - x
XXXIV
where X, Y1, Y~ and C ( 17 ) are as defined above .
A compound of Formula VB corresponds to F'oz-mula
VA wherein R8 and R9 together form the structure of
Formula XXXIII:
0
0
2 0 ,~~~~~ XXXI I I
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 -CH2-CH2-, R3 is
hydrogen, and R1 is alkoxycarbonyl, preferably
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methoxycarbonyl.~ Compounds within the scope of Formula V
may be prepared by reacting an alkali metal alkoxide with
a corresponding compound of Formula VI.
A compound of Formula VI corresponds to the
structure:
R3
oe
R9
,
VI
wherein -A-A-, -B-B-, R3, RB and R9 are as defined in
Formula IV.
A compound of Formula VIA corresponds to
Formula VI wherein RB and R9 together with the ring carbon
to which they are attached form the structure:
Y~
Y'~C( 1 7) mU( CH2) 2 . C ~ X
i ~ XXXIV
where X, Y1, YZ and C ( 17 ) are as def fined above .
A compound of Formula VIB corresponds to
Formula VIA wherein RB and R9 together form the structure
of Formula XXXIII:
0
XXXIII
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 -CHZ-CH2-, and R'
is hydrogen. Compounds of Formula VI, VIA, VIB and VIC
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are prepared by hydrolyzing a compound corresponding to
Formula VII, VIIA, VIIB or VIIC, respectively.
A compound of Formula VII corresponds to the
structure:
R3 R8
I ~ _R9
B
B
a , ~ ~..: ,
C
I
NHZ VII
wherein -A-A-, -B-B-, R', RB and R9 are as defined in
Formula IV.
A compound of Formula VIIA corresponds to
Formula VII wherein Re and R9 together with the ring
carbon to which they are attached form the structure:
YZ
Y~
~C( 1 7) 11111( Chl2) 2 - C ~ X
XXXIV
where X, Y1, YZ and C ( 17 ) are as def fined above .
A compound of Formula VIIB corresponds to
Formula VIIA wherein Re and R9 together form the structure
of Formula XXXIII:
0
0
XXXIII
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 -CH2-CH2-, and R3
is hydrogen. A compound within the scope of Formula VII
may be prepared by cyanidation of a compound within the
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scope of Formula VIII.
A compound of Formula VIII corresponds to the
structure:
R3 R8
I \ R9
8
8
VIII
wherein -A-A-, -B-B-, R', RB and R9 are as defined in
Formula IV.
A compound of Formula VIIIA corresponds to
Formula VIII wherein RB and R9 together with the ring
carbon to which they are attached form the structure:
Y2
Y'~C( 1 7) VIII( CH2) Z _ C - X
~ ~ XXXIV
where X, Y1, Y2 and C ( 17 ) are as def fined above .
A compound of Formula VIIIB corresponds to
Formula VIIIA wherein RB and R9 together form the
structure of Formula XXXIII:
0
0
'I I I I I XXX I I I
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 -CH2-CHz-, and
R' is hydrogen. Compounds within the scope of Formula
VIII are prepared by oxidizing a substrate comprising a
compound of Formula XIII as described hereinbelow by
fermentation effective for introducing an 11-hydroxy
group into the substrate in a-orientation.
A compound of Formula XIV corresponds to the
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structure:
R3 Re
\ H3 C ~ R9
CH3~ ~ ~B
AiA B
I .CN
o AXIV
wherein -A-A-, -B-B-, R', Re and R9 are as defined in
Formula IV.
A compound of Formula XIVA corresponds to
Formula XIV wherein RB and R9 together with the ring
carbon to which they are attached form the structure:
Y2
Y'
~cp ~~ my cH2~ 2 - c - X XXXIV
where X, Y1, Y~ and C(17) are as defined above.
A compound of Formula XIV corresponds to
Formula XIVA wherein RB and R9 together with the ring
carbon to which they are attached form the structure of
Formula XXXIII:
0
0
XXXIII
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 -CH2-CH2-, and R3
is hydrogen. Compounds within the scope of Formula XIV
can be prepared by hydrolysis of a corresponding compound
within the scope of Formula XV.
A compound of Formula XV corresponds to the
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1~
structure:
Ra Rs
~HB~ v R9
CH~~ ~ ~B
AiA B
I _CN
,
C
NH2 AXV
wherein -A-A-, -B-B-, R3, R8 and R9 are as defined in
Formula IV.
A compound of Formula XVA corresponds to
Formula XV wherein R8 and R9 together with the ring carbon
to which they are attached form the structure:
Y2
t
Y ~cp ~~ my cHz~ 2 - c - X XXXIV
where X, Yl, Y2 and C ( 17 ) are as defined above .
A compound of Formula XVB corresponds to
Formula XVA wherein R8 and R9 together with the ring
carbon to which they are attached form the structure of
Formula XXXIII:
0
,mn XXXIII
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 -CH2-CH2-, and R3
is hydrogen. Compounds within the scope of Formula XV
can be prepared by cyanidation of a corresponding
compound within the scope of Formula XVI.
A compound of Formula XXI corresponds to the
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structure:
H3C
8
R R9
CH ~~'~p B
A 3 ~~~ i
Ai 8
I _CN
f!
p XXI
wherein -A-A- , -B-B- , R3 , RB and R9 are as def fined in
Formula IV.
A compound of Formula XXIA corresponds to
Formula XXI wherein RB and R9 together with the ring
carbon to which they are attached form the structure:
Y$
Y ~C( 1 7) iiiii( CHp) 2 - C - X
XXXIV
where X, Y1, Yz and C(17) are as defined above.
A compound of Formula XXIB corresponds to
Formula XXIA wherein Re and R9 together form the structure
of Formula XXXIII:
0
,mn XXXIII
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 -CHZ-CH2-, and R3
is hydrogen. Compounds within the scope of Formula XXI
may be prepared by hydrolyzing a corresponding compound
within the scope of Formula XXII.
A compound of Formula XXII corresponds to the
structure:
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H3C
R3 RB
R9
i,~~
B
~0
~i v.
C
NHz
XXII
wherein -A-A-, -B-B-, R3, RB and R9 are as defined in
Formula IV.
A compound of Formula XXIIA corresponds to
Formula XXII wherein RB and R9 together with the ring
carbon to which they are attached form the structure:
Y2
t
Y ~C( 1 7) iiiii( CH2) 2 - C - X
v XXXIV
where X, Y1, Yz and C(17) are as defined above.
A compound of Formula XXIIB corresponds to
Formula XXIIA wherein RB and R9 together form the
structure of Formula XXXIII:
0
0
XXXIII
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 -CH2-CHZ-, and
R3 is hydrogen. Compounds within the scope of Formula
XXII may be prepared by cyanidation of a compound within
the scope of Formula XXIII.
A compound of Formula XXIII corresponds to the
structure:
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H3C
R3 R8
\ R9
B
a
XXIII
wherein -A-A-, -B-B-, R3, RB and R9 are as defined in
Formula IV.
A compound of Formula XXIIIA corresponds to
Formula XXIII wherein R8 and R9 together with the ring
carbon to which they are attached form the structure:
Y2
Y ~C( 1 7) i~iii( CH2) 2 - C - X
XXXIV
where X, Y1, YZ and C(17) are as defined above.
A compound of Formula XXIIIB corresponds to
Formula XXIIIA wherein RB and R9 together form the
structure of Formula XXXIII:
0
XXXIII
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 -CH2-
CH2-, and R3 is hydrogen. Compounds within the scope of
Formula XXIII can be prepared by oxidation of a compound
of Formula XXIV, as described hereinbelow.
A compound of Formula 104 corresponds to the
structure:
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0
R3
0
8
B
R" o~~~ 104
wherein -A-A-, -B-B-, and R' are as defined in Formula IV,
and R11 is C1 to C, alkyl.
A compound of Formula 104A corresponds to
Formula 104 wherein each of -A-A- and -B-B- is -CH2-CH2-,
and R3 is hydrogen. Compounds within the scope of Formula
104 may be prepared by thermal decomposition of a
compound of Formula 103.
A compound of Formula 103 corresponds to the
structure:
0
Ra O C02R~2
H 0,~~~ _ _
B
A~q
R' ~ O
103
wherein -A-A-, -B-B-, R3 and R11 are as defined in Formula
104 , and R1z is C1-C4 lower alkyl .
A compound of Formula 103A corresponds to
Formula 103 wherein each of -A-A- and -B-B- is -CH2-CH2-,
and R3 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.
A compound of Formula 102 corresponds to the
structure:
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R" 10 2
wherein -A-A-, -B-B-, R3 and Rll are as defined in Formula
104.
A compound of Formula 102A corresponds to
5 Formula 102 wherein each of -A-A- and -H-B- is -CH2-CH2-,
and R' 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.
10 A compound of Formula 101 corresponds to the
structure:
R'
n
0
101
wherein -A-A-, -B-B-, R' and R11 are as defined in Formula
104.
15 A compound of Formula 101A corresponds to
Formula 101 wherein each of -A-A- and -B-B- is -CHz-CHZ-,
and R3 is hydrogen. Compounds within the scope of Formula
101 may be prepared by reaction of 11a-hydroxyandrostene-
3,17-dione or other compound of Formula XXXVI with a
trialkyl orthoformate in the presence of an acid.
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. Use of the compounds of
this invention are useful as intermediates for
CA 02553378 1996-12-11
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epoxymexrenone and other steroids.
Other objects and features will be in part
apparent and in part pointed out hereinafter.
Brief Description of the Drawings
Fig. 1 is a schematic flow sheet of a process
for the bioconversion of canrenone or a canrenane
derivative to the corresponding 11a-hydroxy compound;
Fig. 2 is a schematic flow sheet of a preferred
process for the bioconversion of 11-a-hydroxylation of
canrenone and canrenone derivatives;
Fig. 3 is a schematic flow sheet of a
particularly preferred process for the bioconversion of
11-a-hydroxylation of canrenone and canrenone
derivatives;
Fig. 4 shows the particle size distribution for
canrenone as prepared in accordance with the process of
Fig. 2; and
'Fig. 5 shows the particle size distribution for
canrenone as sterilized in the transformation fermenter
in accordance with the process of Fig. 3.
Corresponding reference characters indicate
corresponding parts throughout the drawings.
Description of the Preferred Embodiments
In accordance with the present invention,
various novel process schemes have been devised for the
preparation of epoxymexrenone and other compounds
corresponding Formula I:
R3 n8
Rs
I
CA 02553378 1996-12-11
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wherein:
-A-A- represents the group -CHR4-CHRS- or -
CRS=CRS-
R3, R' and RS are independently selected from
the group consisting of hydrogen, halo,
hydroxy, lower alkyl, lower alkoxy,
hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl,
cyano, aryloxy,
R1 represents an alpha-oriented lower
alkoxycarbonyl or hydroxyalkyl radical,
-B-B- represents the group -CHR6-CHR'- or an
alpha- or beta- oriented group:
Re R~
\ i
CH CH
-CH-CN2-CH- III
where R6 and R' are independently selected from
the group consisting of hydrogen, halo, lower
alkoxy, aryl, hydroxyalkyl, alkoxyalkyl,
hydroxycarbonyl, alkyl, alkoxycarbonyl,
acyloxyalkyl, cyano, aryloxy, and
R8 and R9 are independently selected from the
group consisting of hydrogen, halo, lower
alkoxy, aryl, hydroxyalkyl, alkoxyalkyl,
hydroxycarbonyl, alkyl, alkoxycarbonyl,
acyloxyalkyl, cyano, aryloxy, or R8 and R9
together comprise a carbocyclic or heterocyclic
ring structure, or R8 or R9 together with R6 or
R7 comprise a carbocyclic or heterocyclic ring
structure fused to the pentacyclic D ring.
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.
A lower alkoxycarbonyl radical is preferably
one derived from an alkyl radical having from 1 to 4
CA 02553378 1996-12-11
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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 C1-C4 alkyl
radicals, especially from a primary C1-C4 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
20 preferred are fozmyl and acetyl.
A methylene bridge in the 15,16-position is
preferably ~i-oriented.
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. patent
4,559,332, i.e., those corresponding to Formula IA:
Y2
Y1
~..W~ CH2~ 2 C=X
C H ~ '~ B
AiA H
'/~/~~~~~R ~ IA
where:
-A-A- represents the group -CH2-CH2- or -CH=CH-,
-B-B- represents the group -CHZ-CH2- or an alpha-
or beta- oriented group of Formula IIIA:
- C H- C Hz- C H- I I IA
R1 represents an alpha-oriented lower
alkoxycarbonyl or hydroxycarbonyl radical,
X represents two hydrogen atoms, oxo or =S
Y' and YZ together represent the oxygen bridge -
O-, or
Y1 represents hydroxy, and
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Yz represents hydroxy, lower alkoxy or, if X
represents HZ, also lower alkanoyloxy,
Preferably, 20-spiroxane compounds produced by
the novel methods of the invention are those of Formula I
in which Y1 and Yz together represent the oxygen bridge.-
O-.
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 Y1 together with Y2
represents the oxygen bridge -O-.
As already mentioned, 17~i-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).
Also included amongst preferred compounds are
alkali metal salts, especially potassium salts, of
compounds of the formula IA in which R1 represents
alkoxycarbonyl, with X representing oxo and each of Y1 and
CA 02553378 1996-12-11
YZ representing hydroxy.
Especially preferred compounds of the formula I
and IA are, for example, the following:
9a,11a-epoxy-7a-methoxycarbonyl-20-spirox-4-
5 ene-3,21-dione,
9a,11a-epoxy-7a-ethoxycarbonyl-20-spirox-4-ene-
3,21-dione,
9a,11a-epoxy-7a-isopropoxycarbonyl-20-spirox-4-
ene-3,21-,dione,
10 and the 1,2-dehydro analogue of each of the
compounds,
9a,11a-epoxy-6a,7a-methylene-20-spirox-4-ene-
3,21-dione,
9a,11a-epoxy-6~,7~-methylene-20-spirox-4-ene-
15 3,21-dione,
9oc,11a-epoxy-6~i, 7~;15~i,16(3-bismethylene-20-
spirox-4-ene-3,21-dione,
and the 1,2-dehydro analogue of each of these
compounds, -
20 9a,11a-epoxy-7a-methoxycarbonyl-17(3-hydroxy-3-
oxo-pregn-4-ene-21-carboxylic acid,
9a,12a-epoxy-7a-ethoxycarbonyl-17~-hydroxy-3-
oxo-pregn-4-ene-21-carboxylic acid,
9a,11a-epoxy-7a-isopropoxycarbonyl-17~-hydroxy-
3-oxo-pregn-4-ene-21-carboxylic acid,
9a,11a-epoxy-17~-hydroxy-6a,7a-methylene-3-oxo-
pregn-4-ene-21-carboxylic acid,
9a,11a-epoxy-17~i-hydroxy-6(3,7-methylene-3-oxo-
pregn-4-ene-21-carboxylic acid,
9a,11a-epoxy-17(3-hydroxy-6~i, 7~3;15~,16~-
bismethylene-3-oxo-pregn-4-ene-21-carboxylic acid, 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.
9a,11a-epoxy-15(3,16~i-methylene-3, 21-dioxo-20-
CA 02553378 1996-12-11
21
spirox-4-ene-7a-carboxylic acid methyl ester, ethyl ester
and isopropyl ester,
9a,11a-epoxy-1565~i,16~i-methylene-3,21-dioxo-20-
spiroxa-1,4-dime-7a-carboxylic acid methyl ester,
ethyl ester and isopropyl ester,
and also 9a,11a-epoxy-3-oxo-20-spirox-4-ene-7a-
carboxylic acid methyl ester, ethyl ester and isopropyl
ester,
9a,11a-epoxy-6~3,6~i-methylene-20-spirox-4-en-3-
one,
9a, 11a-epoxy-6(3, 7~i;15~i, 16~i-bismethylene-20-
spirox-4-en-3-one,
and also 9a,11a-epoxy,l7(3-hydroxy-17a(3-
hydroxy-propyl)-3-oxo-androst-4-ene-7a-carboxylic acid
methyl ester, ethyl ester and isopropyl ester,
9a,11a-epoxy,l7(3-hydroxy-17a-(3-hydroxypropyl)-
6a,7a-methylene-androst-4-en-3-one,
9a,11a-epoxy-17(3-hydroxy-17a-(3-hydroxypropyl)-
6~3,7p-methylene-androst-4-en-3-one,
9a,11a-epoxy-17~i-hydroxy-17a-(3-hydroxypropyl)-
6~i, 7~3; 15~i, 16~i-bismethylene-androst-4-en-3-one,
including 17a-(3-acetoxypropyl) and 17a-(3-
fromyloxypropyl) analogues of the mentioned androstane
compounds,
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.
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 Y1 together with Yz
represents -O-, from 20-spiroxane (for example a compound
of the formula IA in which X represents oxo and Y1
together ~:ith Y2 represents -0- is derived from 20-
spiroxan-21-one); for those in which each of Y1 and Yz
CA 02553378 1996-12-11
22
represents hydroxy and X represents oxo, from 17~-
hydroxy-17a-pregnene-21-carboxylic acid; and for those in
which each of Y1 and Yz represents hydroxy and X
represents two hydrogen atoms, from 17~i-hydroxy-l7oc-(3-
hydroxypropyl)-androstane. Since the cyclic and open-
chain forms, that is to say lactones and 173-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.
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)
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
R9
XIII
wherein
-A-A- represents the group -CHR4-CHRS- or -
CA 02553378 1996-12-11
23
CR4=CRS-
R3, R4 and R5 are independently selected from
the group consisting of hydrogen, halo,
hydroxy, lower alkyl, lower alkoxy,
hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl,
cyano, aryloxy,
- -B-B- represents the group -CHR6-CIiR'- or an
alpha- or beta- oriented group:
R~ R'
\CH CN
-CH-CHZ-CH- III
where R6 and R' are independently selected from
the group consisting of hydrogen, halo, lower
alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,
hydroxycarbonyl, alkyl, alkoxycarbonyl,
acyloxyalkyl, cyano, aryloxy, and
Re and R9 are independently selected from the
group consisting of hydrogen, halo; lower
alkoxy, aryl, hydroxyalkyl, alkoxyalkyl,
hydroxycarbonyl, alkyl, alkoxycarbonyl,
acyloxyalkyl, cyano, aryloxy or R8 and R9
together comprise a carbocyclic or heterocyclic
ring structure, or R8 and R9 together with R6 or
R' comprise a carbocyclic or heterocyclic ring
structure fused to the pentacyclic D ring.
Using a bioconversion process of the type
illustrated in Figs. 1 and 2, an 11-hydroxy
group of oc-orientation is introduced in the
compound of Formula XIII, thereby producing a
compound of Formula VIII:
CA 02553378 1996-12-11
24
R3 R8
R9
H 0,~~~
B
A /
Ai
o~ / / VIII
where -A-A-, -B-B-, R3, RB and R9 are as defined above.
Preferably, the compound of Formula XIII has the
structure
Y2
1 I
H3C Y \\\\~ CH2~ 2 C-X
CH3 B
A /
Ai ~B
/ / /
~ XIIIA
and the lla-hydroxy product has the structure
Y2
1
H C Y -C-X
H 0 3 v~~ C C H 2' 2
ii~~
CH3 B
A /
Ai
/ /
VIIIA
in each of which
-A-A- represents the group -CHZ-CH2- or -CH=CH-,
-B-B- represents the group -CHz-CHZ- or an
alpha- or beta- oriented group:
-cH- cHZ-cH- IIIA
X represents two hydrogen atoms, oxo or =S,
Y1 and YZ together represent the oxygen bridge -
O- , or
CA 02553378 1996-12-11
Y1 represents hydroxy, and
Y2 represents hydroxy, lower alkoxy or, if X
represents H~, also lower alkanoyloxy,
and salts of compounds in which X represents oxo and Y2
5 represents hydroxy-, and the compound of Formula VIII
produced in the reaction corresponds to Formula VIIIA
Y2
I
HC', H3C ~~~~C CF12) z C-X
ii
CH3 B
~A 9
A
/ /
o VIIIA
wherein -A-A-, -B-B-, Y1, Y2, and X are as defined in
Formula XIIIA. More preferably, RB and R9 together form
10 the 20-spiroxane structure:
0
0
XXXIII
-A-A- and -B-B- are each -CH2-CHZ-, and R3 is hydrogen.
Among the preferred organisms that can be used
in this hydroxylation step are Aspergillus ochraceus NRRL
15 405, Aspergillus ochraceus ATCC 18500, Aspergillus niQer
ATCC 16888 and ATCC 26693, Asperctillus nidulans ATCC
11267, Rhizopus oryzae ATCC 11145, Rhizot~us stolonifer
ATCC 6227b, Streptomyces fradiae ATCC 10745, Bacillus
megaterium ATCC 14945, Pseudomonas cruciviae ATCC 13262,
20 and Trichothecium roseum ATCC 12543. Other preferred
organisms include Fusarium oxysporum f.sp.cepae ATCC
11171 and Rhizopus arrhizus ATCC 11145.
Other organisms that have exhibited activity
for this reaction include Absidia coerula ATCC 6647,
25 Absidia glauca ATCC 22752, Actinomucor elegans ATCC 6476,
Asperctillus flavipes ATCC 1030, Aspergillus fumictatus
CA 02553378 1996-12-11
26
ATCC 26934, Beauveria bassiana ATCC 7159 and ATCC 13144,
Botryosphaeria obtusa IMI 038560, Calonectria decora
ATCC 14767, Chaetomium cochliodes ATCC 10195, Corvnespora
cassiicola ATCC 16718, Cunninqhamella blakesleeana ATCC '
8688a, Cunnincthamella echinulata ATCC 3655,
Cunninahamella eleaans ATCC 9245, Curvularia clavata
ATCC 22921, Curvularia lunata ACTT 12071, Cylindrocarnon
radicicola ATCC 1011, Enicoccum humicola ATCC 12722,
Gonaronella butleri ATCC 22822, Hynomyces chzysospermus, .
Mortierella isabellina ATCC 42613, Mucor mucedo ATCC
4605, Mucor ctriseo-cyanus ATCC 1207A, Myrothecium
verrucaria ATCC 9095, Nocardia corallina, Paecilomyces
carneus ATCC 46579, Penicillum patulum ATCC 24550,
Pithomyces atro-olivaceus IFO 6651, Pithomyces
15~ cvnodontis ATCC 26150, Pvcnosporium sp. ATCC 12231,
Saccharonolvspora ervthrae ATCC 11635, Sepedonium
chrysospermum ATCC 13378, Stachvlidium bicolor ATCC
22672, Streptomyces hvaroscot~icus ATCC 27438,
Streptomyces purnurascens ATCC 25489, Svncephalastrum
racemosum ATCC 18192, Thamnostvlum piriforme ATCC 8992,
Thielavia terricola ATCC 13807, and Verticillium
theobromae ATCC 12474.
Additional organisms that may be expected to
show activity for the 11a-hydroxylation include
Cephalosporium aphidicola (Phytochemistry (1996), 42(2),
411-415), Cochliobolus lunatas (J. Biotechnol. (1995),
42(2), 145-I50), Tieahemella orchidis (Khim.-Farm.Zh.
(1986), 20(7), 871-876), Tieahemella hyalospora (Khim.-
Farm.Zh. (1986), 20(7), 871-876), Monosporium olivaceum
(Acta Microbiol. Pol., Ser. B. (1973), 5(2), 103-110),
Aspercrillus ustus (Acta Microbiol. Pol:, Ser. B. (1973),
5(2), 103-110), Fusarium crraminearum (Acta Microbiol.
Pol., Ser. B. (1973), 5(2), 103-110), Verticillium
glaucum (Acta.Microbiol. Pol., Ser. B. (1973), 5(2), 103-
110), and Rhizopus niQricans (J. Steroid Biochem. (2987),
28(2) , 197-201) .
CA 02553378 1996-12-11
27
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. 4rhatever
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.
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
CA 02553378 1996-12-11
28
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 S$ 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 O.S~ 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 1o 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 5o 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:
1. Solid Medium #1: 1% glucose, 0.25% yeast extract,
0 . 3 ~ KZHP04 and 2 ~ agar ( Bacto ) ; pH
adjusted to 6.5 with 20o NaOH.
2. Solid Medium #2: 2~ peptone (Bacto), 1~ yeast
extract (Bacto), 2~ glucose and 2~
agar (Bacto); pH adjusted to 5 with
10 ~ H3 P04 .
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 . S$ KHZPOQ, pH
adjusted to 5.8.
5. Difco Mycological agar (low pH).
CA 02553378 1996-12-11
29
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 I5 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 N2 or other
cryogenic liquid.
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
18540). The seed stock so produced is then introduced
into the production fernienter together with the substrate
of Formula XIII. The fermentation broth is agitated and
CA 02553378 1996-12-11
aerated for a time sufficient for the reaction to proceed
to the desired degree of completion.
The medium for the seed ferrnenter preferably
comprises an aqueous mixture which contains: between
5 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 0.050 and about 0.5~ by weight of
a phosphorus source, e.g., an ammonium or alkali metal
phosphate such as ammonium phosphate monobasic or
10 dipotassium hydrogen phosphate, betv~ieen 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 1o and about 2o by weight agar or other non-
digestible polysaccharide. A particularly preferred seed
15 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 5g by weight
glucose, and between about 0.050 by weight and about 0.5~
20 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
S~ by weight corn steep liquor, between about 0.25% and
25 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,
30 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
CA 02553378 1996-12-11
31
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:
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.
Spores of the microorganism are introduced into
this medium from a vial typically containing in the
neighborhood of 109 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' 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 35% to 45%. Since the cycle in the
seed fermentation vessel (or any vessel of a plurality
which comprise a seed fermentation train) depends on the
2~ 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
t mperature in the range of about 23° to about 37°C,
preferably in range of between about 24° and about 28°C.
Culture from the seed fermentation system is
CA 02553378 1996-12-11
32
introduced into a production ferrnenter, 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 11a-hydroxylation reaction, the particle size of the
Formula XIII substrate is reduced by passing the
substrate through an off line microriizer 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
5o 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 2% 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
CA 02553378 1996-12-11
33
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:
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 D.21% ammonium phosphate
monobasic and 3% glucose; pH adjusted to
6.5 with 20% NaOH.
Non-sterile canrenone and sterile nutrient
solutions are chain fed to the production fermenter in
five to twenty, preferably ten to 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%
CA 02553378 1996-12-11
34
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
15~ 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 HC1 and/or between about 0.001% and about
0.003% cefalexin, again based on the total amount of
broth.
Typically, the production fermentation batch
cycle is in the neighborhood of 80-160 hours. Thus,
portions of each of the Formula XIII substrate and
nutrient solutions are typically added every 2 to 10
hours, preferably every 4 to 6 hours. Advantageously, an
antifoam is also incorporated in the seed fermentation
system, and in the production fermenter.
CA 02553378 1996-12-11
Preferably, in the process of Fig. 1, the
inoculum charge to the production fermenter is about 0.5
to about 70, more preferably about 1 to about 2%, by
volume based on the total mixture in the fermenter, and
5 the glucose concentration is maintained between about
0.01% and about 1.0%, preferably between about 0.025% and
about 0.50, more preferably between about 0.050 and about
0.25% by weight with periodic additions that are
preferably in portions of about 0.05% to about 0.25% by
10 weight, based on the total batch charge. The
fermentation temperature is conveniently controlled
within a range of about 20° to about 37°C, preferably
about 24°C to about 28°C, but it may be desirable to step
down the temperature during the reaction, e.g., in 2°C
15 increments, to maintain the packed mycelium volume (PMV)
below about 600, more preferably below about 500, and
thereby prevent the viscosity of the fermentation broth
from interfering with satisfactory mixing. If the
biomass growth extends above the liquid surface,
20 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 35o to 450, within the first 24 hours of the
25 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
30 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
35 fermenter at between about atmospheric and about 1.0 bar
gauge, most preferably in the neighborhood of about 0.7
CA 02553378 1996-12-11
36
bar gauge. Agitation rate may also been increased as
necessary to maintain minimum dissolved oxygen levels.
Advantageously, the dissolved oxygen is maintained at
well above 10%, in fact as high as 50o 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 necessazy 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. re~iaining 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.
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
500, 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 80g, 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.
A particularly preferred process for
preparation of a compound of Formula VIII is illustrated
in Fig. 2. Again the preferred microorganism is
AsperQillus 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 0.5o and about 5~ by weight glucose, between about
0.1~ and about 3~ by weight yeast extract, and between
about 0.050 and about 0.5~ by weight ammonium phosphate.
CA 02553378 1996-12-11
37
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 30o 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.
Preferably, in operation of the process of Fig.
2, the inoculum is introduced into the production
fermenter in a proportion of between about 0.5o and about
7%, the fermentation temperature is between about 20° and
about 37°C, preferably between about 24°C and about 28°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
CA 02553378 1996-12-11
38
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,
CA 02553378 1996-12-11
39
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.
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 750 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).
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 200 ~ and at least 75~ by
weight of the particles are smaller than 240 ~. 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
CA 02553378 1996-12-11
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
5 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
10 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
15 the reaction cycle.
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.
20 Alternatively, the product can be extracted from the
entire reaction broth with organic solvents. Product of
Formula VIII is recovered by solvent extraction. For
maximum recovery, both the liquid phase filtrate and the
biomass filter or centrifuge cake are treated with
25 extraction solvent, but usually z9B% 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
30 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,
35 preferably in large excess relative to the initial charge
of substrate, e.g., 50 to 100 ml. solvent per gram of
CA 02553378 1996-12-11
41
initial canrenone charge, and the resulting suspension
preferably refluxed for.a period of 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 to separate. Formula
VIII product is recovered by 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 0° to 20°C, preferably about 10° to about
15°C for a time sufficient for crystal precipitation and
growth, typically 8 to 12 hours.
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
control of process parameters such as pH, temperature and
dissolved oxygen. Moreover, sterile conditions are
readily preserved without resort to antibiotics.
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 100, by volume, sufficient to
provide energy for the reaction and assure conversion of
the glucose to C02 and H20, 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.
CA 02553378 1996-12-11
42
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 ~,, and
at least 75~ by weight of the particles are smaller than
240 fit. 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 ferznenter 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 inlcude, for example,
DMF, DMSO, C6-C12 fatty acids, C6-C1, 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.
A second option is to initially dissolve the
substrate in a water miscible solvent such as acetone,
methylethyl ketone, methanol, ethanol, or glycero3 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
CA 02553378 1996-12-11
43
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.
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 3o to about 5o by weight, can be used to
advantage.
A third option is to solubilize the substrate
in an aqueous cyclodextrin solution. Illustrative
cyclodextrins include hydroxypropyl-~i-cyclodextrin and
methyl-~i-cyclodextrin. The molar ratio of
substrate:cyclodextrin can be about 1:1 to about 1:1.5,
substrate:cyclodextrin. The substrate:cyclodextrin
mixture can then be added aseptically to the
bioconversion reactor.
11a-Hydroxycanrenone and other products of the
11a-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.
Preferably., the compounds of Formula VIII
correspond to Formula VIIIA in which -A-A- and -B-B- are
CA 02553378 1996-12-11
44
-CHz-CH2-, R3 is hydrogen, lower alkyl or lower alkoxy,
and R$ and R9 together constitute the 20-spiroxane ring:
o
"" XXXI I I
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
R3 R8
1 v R9
B
8
C'
NHZ VII
wherein -A-A-, R3, -B-B-, Re and R9 are as defined above.
Where the substrate corresponds to Formula VIIIA, the
product is of Formula VIIA
Y~
_a ,
~ CHZ~ 2 C-X
V
i
NHp VIIA
wherein -A-A-, -B-B-, R3, yl, y2, and X are as defined in
Formula XIII.
C~anidation of the 11a-hydroxyl substrate of
CA 02553378 1996-12-11
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.
5 Alternatively, cyanidation can be effected without a
cyanohydrin by using an alkali metal cyanide in the
presence of an acid.
'In the ketone cyanohydrin process, the reaction
is conducted in solution, preferably using an aprotic
10 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
15 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%
20 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
25 triethylamine is used as the base, the reaction is
advantageously conducted in the range of about 80°C to
about 90°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
30 reaction is conducted at 105°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
35 from this solution, and is thereafter washed with
distilled water and air dried. Liberated HCN may be
CA 02553378 1996-12-11
46
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 HC1. 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°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.
Alternatively, the reaction may be conducted in
an aqueous solvent system comprising a 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 HC1. See U.S. patent
3,200,113.
According to a still further alternative, the
reaction may be conducted in a water-miscible solvent
such as dimethylformamide, dimethylacetamide, ~-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° to 10°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.
According to a still further alternative, the
CA 02553378 1996-12-11
47
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 C1 to CS
carboxylic acid, sulfuric acid being particularly
preferred. Anomalously, no discrete proton source need
be added where the cyanidation reagent is commercial LiCN
in DMF.
Cyanide ion 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°C, typically 60°C to
100°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.
CA 02553378 1996-12-11
48
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
CHz-CHZ-, R3 is hydrogen, lower alkyl or lower alkoxy, and
.R8 and R9 together constitute the 20-spiroxane ring:
r
Q
I11 XXXI I I
Most preferably the compound of Formula VII is
5'R(5'a),7'(3-20'-Aminohexadecahydro-11'3-hydroxy-
10'a,13'a-dimethyl-3',5-dioxospiro[furan-2(3H),I7'a(5'H)-
[7,4]metheno[4H]cyclopenta[a]phenanthreneJ-5'-
carbonitrile.
In the next step of the Scheme 1 synthesis, the
enamine of Formula VII is hydrolyzed to produce a
diketone compound of Formula VI
R3 nB
R9
9
'.
VI
where -A-A-, R3, -B-B-, Re and R9 are as defined in
Formula VIII. 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 a lower alkanol, is preferably used as a
cosolvent. The acid should be present in proportion of
at least one equivalent per equivalent of Formula VII
CA 02553378 1996-12-11
49
substrate. In an aqueous system, the enamine substrate
VII can be substantially converted to the diketone of
Formula VII in a period of about 5 hours at about 80°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.
Preferably, the enamine substrate of Formula
VII corresponds to Formula VIIA
Y2
[ CH2] ~ C=X
L
1 O N H z VI IA
and the diketone product corresponds to Formula VIA
Y2
Y1
~..~~[ ~H2~ 2 C-X
VIA
in each of which -A-A-, -B-B-, Y1, YZ, and X are as
defined in Formula VIIIA.
At the end of the reaction period, the solution
is cooled to 0° and,25°C to crystallize the product. The
product crystals may be recrystallized from a suitable
solvent such as isopropanol or methanol to produce a
CA 02553378 1996-12-11
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 -CH2-CH2-, R3 is hydrogen, lower alkyl or lower
alkoxy, and Re and R9 together constitute the 20-spiroxane
ring: '
0
,inn XXXIII
Most preferably, the compound of Formula VI is
4'S(4'a),7'a-Hexadecahydro-11'ot-hydroxy-10'~i,13'~-
dimethyl-3',5,20'-trioxospiro[furan-2(3H),17'~i-
[4,7]methanol17H]cyclopenta[a]phenanthrene]-5'~i(2'H)-
carbonitrile.
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
indeed added to mixture at the end of the cyanidation
reaction, and the proportion of acid added is sufficient
CA 02553378 1996-12-11
51
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 greater
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°C, more preferably in the range of about 90°C to
about 100°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°C and about 15°C,
advantageously in an ice bath to about 5°C to about 10°C,
for precipitation of the product diketone of Formula VI.
The solid product may be recovered, as by filtration, and
impurities removed by washing with water.
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, cleave the bond between the carbonyl group and
the 4-carbon, and form an a-oriented alkanoyloxycarbonyl
substituent at the 7 position and eliminating cyanide at
the 5-carbon. The product of this reaction is a
hydroxyester compound corresponding to Formula V
R3 Re
R9
H 0////
B
8
A
,/ V ////R1 V
3 0 where -A-A- , R3 , -B-B- , R6 and R9 are as def fined in
CA 02553378 1996-12-11
52
Formula VIII, and R1 is lower alkoxycarbonyl or
hydroxycarbonyl. The metal alkoxide used in the reaction
corresponds to the formula R1°OM where M is alkali metal
and R1°O- corresponds to the alkoxy substituent of R1.
Yields of this reaction are most satisfactory when the
metal alkoxide is potassium 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 R1°OH where R1° 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 and R1°OM is
present in a proportion of between about 0.5 and about 4
moles 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.
In the conversion of the diketone of Formula VI
to the hydroxyester of Formula VI, by-product cyanide ion
can react with the product to form 5-cyanoester. Because
the equilibrium is more favorable at low concentrations,
the reaction is preferably run at rather high dilution,
e.g., as high as 40:1 for reaction with Na 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.
In accordance with the invention, it has been
further discovered that the reverse cyanidation reaction
CA 02553378 1996-12-11
52a
may be inhibited by taking appropriate chemical or
CA 02553378 1996-12-11
53
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 an
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.
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.
Preferably, the diketone substrate of Formula
VI corresponds to Formula VIA
Y2
1
HO H3C Y ~wC CH2~ p C-X
CH3 ~e
I ~CN
VIA
CA 02553378 1996-12-11
54
and the hydroxyester product corresponds to Formula VA
Y2
HO H3C wC CHZ) 2 C-X
CH3 B
i A Bi
A
~~~i~R 1
VA
in each of which -A-A-, -B-B-, Y1, Y2, and X are as de-
fined in Formula XIIIA and R1 is as defined in Formula V.
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 -
CH2-CH2-, R3 is hydrogen, lower alkyl or lower alkoxy, and
RB and R9 together constitute the 20-spiroxane ring:
0
0
XXXIII
Most preferably, the compound of Formula V is Methyl
Hydrogen l1a,17a-Dihydroxy-3-oxopregn-4-ene-7a,21-
dicarboxyhate, -y-Lactone.
The compound of Formula V may be isolated by
acidifying the reaction solution, e.g., with concentrated
HC1, 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 0° to 25°C and the product
solid is isolated by filtration.
CA 02553378 1996-12-11
54a
According to a preferred mode of recovery of
CA 02553378 1996-12-11
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
5 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
10 crystalline product without extraction operations.
In accordance with a further alternative, the
reaction solution containing the product of Formula V may
be quenched with mineral acid, e.g., 4N HC1, after which
the solvent is removed by distillation. Removal of the
15 solvent is also effective for removing residual HCN from
the reaction product. It has been found that 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
20 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
25 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
30 with a dessicant such as sodium sulfate. The crude lla-
hydroxy-7a-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 good
leaving group at the 11 position thereby producing a
35 compound of Formula IV:
CA 02553378 1996-12-11
56
R°
IV
where -A-A-, R', -B-B-, Re and R9 are as defined in
Formula VIII, R1 is as defined in Fozmula V, and R2 is
lower arylsulfonyloxy, alkylsulfonyloxy, acyloxy or
halide. Preferably, the 11a-hydroxyl is esterified by
reaction with a lower alkylsulfonyl halide, an aryl
halide or an acid anhydride which is added to the
solution containing the intermediate product of Formula
V. Lower alkylsulfonyl halides, and especially
10~ methanesulfonyl chloride, are preferred. Alternatively,
the 11-a 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 lla-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 K or Na 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
2S 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
CA 02553378 1996-12-11
57
-10°C and about 10°C.
Preferably, the hydroxyester substrate of
Formula V corresponds to Formula VA
Y2
1
H0~ H3C Y ~w~ CH2) p C-X
~~i
CH3 B
A
Ai
o ~ ~ R' VA
and the product corresponds to Formula IVA
Y2
H3~ ~W ~~CHZ)2 C=X
R ,
CH3 B
A
A~
iiii R ~
IVA
in each of which -A-A-, -B-B-, Yl, YZ, and X are as
defined in Formula XIII, R1 is lower alkanoyloxycarbonyl
or hydroxycarbonyl, and R~ is as defined in Formula IV.
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 ZA. Preferably, the compounds flf Formula IV
correspond to Formula IVA in which -A-A- and -B-B- are -
CH2-CHI-, R3 is hydrogen, lower alkyl or lower alkoxy, and
RB and R9 together constitute the 20-spiroxane ring:
0
0
'n XXX I I I
Most preferably, the compound of Formula IV is Methyl
Hydrogen 17a-Hydroxy-lla-(methylsulfonyl)oxy-3-oxopregn-
4-ene-7cr,21-dicarboxylate, 'y-Lactone.
CA 02553378 1996-12-11
58
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 HC1. 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.
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.
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
CA 02553378 1996-12-11
59
process, which is removal of the 11a-leaving group from
the compound of Formula IV, thereby producing an enester
of Formula II:
R3 .,e
R9
II
where -A-A-, R3, -B-B-, R8 and R9 are as defined in
Formula VIII, and R1 is as defined in Formula V. For
purposes of this reaction, the R2 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 formic acid
and potassium formate a relatively high ratio of 9,11 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
CA 02553378 1996-12-11
60
R3
~8
R9
3
0
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.
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
CA 02553378 1996-12-11
61
preferably about 20% by weight, based on the
trifluoroacetic acid eliminating reagent.
Alternatively, the lla-leaving groups from the
compound of Formula IV, may be eliminated to produce an
S enester of Formula II by heating a solution of Formula IV
in an organic solvent such as DMSO, DMF or DMA.
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:
Ra Ra
~ \ _R9
B
i
B
Aco ~ ~ ~~~~R' IV (Z)
of the compound of Formula IV. Alternatively, the 3-enol
ester can be formed by treatment with an acid anhydrides
and base such as acetic acid and sodium acetate. Further
alternatives include treatment with ketene in the
presence of an acid to produce 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 D-9,11 enol acetate of Formula
IV (Y)
Rs Ra
~ \ _Ra
'B
AiA ~ B
A c O \ \ ~~~~ R ~
IV (Y)
which can then be converted to the enester of Formula II
CA 02553378 1996-12-11
62
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.
Preferably, the substrate of Formula IV
corresponds to Formula IVA
Yz
Y~ I
~ ~..WC CH2] 2 C-X
~ ~ ~~,,R1 IVA
and the enester product corresponds to Formula IIA
Y2
Y~ I
~ ~..WC CH2] 2 C~X
CH3)
AiA B
yiiA ~
IIA
in each of whicr -A-A-, -B-B-, Y', Yz, and X are as
defined in Formula XIII and R' is as defined in Formula V.
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
CA 02553378 1996-12-11
63
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. patent
4,559,332
. or more preferably by the novel reaction using
a haloacetamide promoter as described below.
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 HC1
scavenger such as imidazole is added. Mixing of base
with the solution 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., 0°C to room
temperature or slightly above, and reacted for a period
of typically 1 to 4 hours. After reaction is complete,
the solvent is stripped, preferably under high vacuum
(e. g., 24" to 28" Hg) conditions at -10° to +15°C, more
preferably about 0° to about 5°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.
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 C0, so the addition
rate must be controlled accordingly. To promote the
CA 02553378 1996-12-11
64
removal of water, the temperature of this reaction is
preferably maintained in the range of 60° to 90°C, most
preferably about 65° to about 75°C. This reagent is then
added to the product solution of the compound of Formula '
IV to effect the elimination reaction. After 4-8 hours,
the reaction mixture is preferably heated to a
temperature of at least about 85°C, but not above about
95°C until all volatile distillate has been removed, and
then for an additional period to complete the reaction,
typically about.l to 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.
It has further been found that the enester of
15~ 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.
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 lloc-hydroxy group of the Formula V
hydroxyester is replaced by halogen, and the Formula II
enester is then formed in situ by thermal dehydro
halogenation. 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
3S tetrahydrofuran and cooled to 0°C to -70°C. The sulfuryl
halide is added and the reaction mixture is warmed to
CA 02553378 1996-12-11
moderate temperature, e.g., room temperature, for a time
sufficient to complete the elimination reaction,
typically 1 to 4 hours. The process of this embodiment
not only combines two steps into one, but eliminates the
5 use of: a halogenated reaction solvent; an acid (such as
acetic); and a drying reagent (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
10 drying reagent. '
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
15 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
20 aqueous solution of inorganic salt, e.g., 10~ by 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.
25 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
30 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
35 recovery of residual sulfonic or dicarboxylic ester
therefrom. The combined eluate and wash fractions are
CA 02553378 1996-12-11
66
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 lloc-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 dzy 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. patent 4,559,332.
In an especially preferred embodiment of the
invention, the solvent is removed from the reaction
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.
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
CA 02553378 1996-12-11
67
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.
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.
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 K2HP04 or
K2HP04/KHzP04 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
CA 02553378 1996-12-11
68
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 KZHP04) may be
only a small catalytic concentration. Generally, it is
preferred that HP04 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.
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
15~ toluene and acetonitrile have also been found
satisfactozy. 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.
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 as to be as effective
as the halogenated solvents or toluene in carrying out
the conversion of this step of the process.
While trichloroacetamide is a highly preferred
CA 02553378 1996-12-11
69
reagent, other trihaloacetamides such as
trifluoroacetamide can also be~used.
Trihalomethylbenzamide, and other compounds having an
arylene moiety between the electron withdrawing
trihalomethyl group and the carbonyl of the amide, may
also be useful. 3,3,3-Trihaiopropionamides may also be
used, but with less favorable results. Generically, the
peroxide activator may correspond to the formula:
R°C ( 0 ) NHZ
where R° is a group having an electron withdrawing
strength (as measured by sigma constant) at least as high
as that of the monochloromethyl group. More
particularly, the peroxide activator may correspond to
the formula:
x' o
I (I
X~-C-RP-C-NH=
la
x
where X', X', and X3 are independently selected from among
halo, hydrogen, alkyl, haloalkyl and cyano and
cyanoalkyl, and Rp is selected from among arylene and -
(CX'XS) ~-, where n is O or 1, at least one of X1, XZ, X3, X'
and X' being halo or perhaloalkyl. Where any of Xl, XZ,
X3, X' or XS 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 X1, X2 and X3 are halo; or in which all of X1, X2, X', X4
and X5 are halo or perhaloalkyl. Each of Xl, X2 X3, X4 and
XS is preferably C1 or F, most preferably C1, though mixed
halides may also be suitable, as may perchloralkyl or
perbromoalkyl and combinations thereof.
Preferably, the peroxide activator is present
in a proportion of at least about 1 equivalents, more
CA 02553378 1996-12-11
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 '
5 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
10 particular theory, it is believed fihat the reaction
mechanism involves formation of an adduct of the
activator and OOH-, that the formation of this reaction is
reversible with the equilibrium favoring the reverse
reaction, and that a substantial initial excess of
15 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 0° to 100°C.
The optimum temperature depends on the selection of
20 solvent. Generally, the preferred temperature is between
about 20°C and 30°C, but in certain solvents, e.g.,
toluene the reaction may be advantageously conducted in
the range of 60°-70°C. At 25°C, reaction typically
requires less than 10 hours, typically 3 to 6 hours. If
25 needed additional activator and hydrogen peroxide may be
added at the end of the reaction cycle to achieve
complete conversion of the substrate.
At the end of the reaction cycle, the aqueous
phase is removed, the organic reaction solution is
30 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, at with least a mild
to moderately alkaline wash, e.g., sodium carbonate.
35 Preferably, the reaction mixture is washed successively
with: a mild reducing solution such as a weak (e.g. 3~ by
CA 02553378 1996-12-11
71
weight) solution of sodium sulfite in water; an alkaline
solution, e.g., NaOH or KOH (preferably about 0.5N); an
acid solution such as HC1 (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.
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 olefinic carbons are
tetrasubstituted and trisubstituted, i . a . , RaR'C=CR~Rd and
RaRbC=CR'H where Ra to Rd 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 t.etrasubstituted double bonds.
Exemplary substrates for this reaction include 0-9,11-
canrenone, and
~o
TO
mmnCp2CHs
O
CA 02553378 1996-12-11
72
o
0
0 0
M
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.
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.
The improved process has been shown to be
particularly advantageous application to the preparation
of
CA 02553378 1996-12-11
73
~o
O
yrrrur C02CH3 IB
/0
:'O
o
by epoxidation of:
0
IC
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 provides tight
regiocontrol for epoxidation across trisubstituted double
with disubstituted and a,f~-keto olefins in the same
molecular structure. Thus, reaction yield, product
profile and final purity are substantially enhanced. It
has further been discovered that the substantial excess
CA 02553378 1996-12-11
74
oxygen generation observed with the use of
trihaloacetonitrile is not experienced 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, e.g., using m-chloroperoxybenzoic acid or
other peracids and the reagent is inexpensive, readily
available, and easily handled.
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:
CA 02553378 1996-12-11
LICE D11F, EtyN.
(eotono eyanoAyddn,
~Hydreayl~flsn 16 ~ C. 4-16 h
Canronono
11~-Hyd~o:yclnnlnpnl
t. 11~C~ Et'N, t. NaOCHS. CH~OH,
CH=Cit. Atlyx
-10' C t0 10~ C
Nwylato Hydra:y~~Hr
HCOOH. HGOOK. AciO.
70~C, 1~ h
Enoal~r
Scheme 2
ChC-C(O)-NHt, H=O:. CI
t0~C.24h
EPo:yIthlurnaHt
0
The second of novel reaction schemes (Scheme 2)
of this invention starts with canrenone or other
5 substrate corresponding to Formula XIII
R3 nA
R9
XIII
HC1. CH~OH. H=O.
10 ' C. 6n
CA 02553378 1996-12-11
76
where -A-A-, R3, -B-B-, R8 and R9 are as defined in
Formula VIII. In the first step of this process, the
substrate of Formula XIII is converted to a product of
Formula XII
Rs
G
XII
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
Y2
1
r. Y GH2~ 2 C-X
XIIIA
and the enamine product corresponds to Formula XIIA
R3 .,8
CA 02553378 1996-12-11
77
Y2
.. Y~ C-X
CHZ) 2
' XIIA
in each of which -A-A-, -B-B-, Y', Y2, and X are as
defined in Formula XIII.
In the second step of scheme 2, the enamine of
Formula XII is hydrolyzed to an intermediate diketone
product of Formula XI
R3
08
R9
3
'.
~.
XI
where -A-A- , R3 , -B-B- , Re and R9 are as def fined in
Formula VIII, 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
CA 02553378 1996-12-11
78
Y2
CH2) Z C-X
' XIZA
and the diketone product corresponds to Formula XIA
Yz
Y~
\~yCCHp72 C-X
CH3 B
"iA B
XIA
in each of which -A-A-, -B-B-, Y1, YZ, and X are as
defined in Formula VIIIA.
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,
R3 Re
R9
B
8
0 ~~~R' X
in each of which -A-A-, R3, -B-B-, Rg and R9 are as
defined in Formula VIII. R1 is as defined in Formula V.
The process is carried out using substantially the same
CA 02553378 1996-12-11
79
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
Y2
Y
H3 IC ~..v~ CH2) z C-X
B
8
u'
~ XIA
and the intermediate product corresponds to Formula XA
Y2
Y~
H3C \~~~C CH2) 2 C-X
CH3 B
A
Ai
Q/ ~ ''~~R~
XA
in each of which -A-A-, -B-B-, Y1, Y2, and X are as
defined in Formula XIIIA. R1 is as defined in Formula V.
The compounds of Formula X are next 9a-
hydroxylated by a novel bioconversion process to yield
products of Formula IX
R3 Re
I ~ R9
A ,'~0 H / B
Ai B
''i~ R '1
IX
where -A-A- , R3, -B-B- , RB and R9 are as def fined in
Formula VIII, and R1 is as defined in Formula V. Among
the organisms that can be used in this hydroxylation step
CA 02553378 1996-12-11
are Nocardia conicruria ATCC 31548, Nocardia aurentia
ATCC 12674, Corynespora cassiicola ATCC 16718,
Streptomyces hvdroscopicus ATCC 27438, Mortierella
isabellina ATCC 42613, Beauvria bassiana ATCC 7519,
5 Penicillum purpuro e~ ATCC 46581, Hypomyces
chrysospermus IMI 109891, Thamnostylum piriforme ATCC
8992, Cunninghamella blakesleeana ATCC 8688a,
Cunninghamella echinulata ATCC 3655, Cunnincthamella
elegans ATCC 9245, Trichothecium roseum ATCC 12543,
10 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, Nocardia canicruria, Norcardia restrictus
15 ATCC 14887, Pseudomonas testosterone ATCC 11996,
Rhodococcus ectui ATCC 21329, Mycobacterium fortuitum
ATCC-6842, and Rhodococcus rhodochrous ATCC 19150. The
reaction is carried out substantially in the manner
described above in connection with Figs. 1 and 2. The
20 process of Fig. 1 is particularly preferred.
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%
25 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:
soybean meal: between about 0.5% and about 3% by weight
30 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
560.5% by weight of a yeast derivative such as autolyzed
yeast or yeast extract; between about 0.05% and about
35 0.5% by weight of a phosphate salt such as K2HP04; pH = 7;
CA 02553378 1996-12-11
81
peptone-yeast extract-glucose: between about 0.2% and
about 2o by weight peptone; between about 0.05% and about
0.5% by weight yeast extract; and between about 2o and
about 5o by weight glucose;
Mueller-Hinton: between about 10% and about 40% by
weight beef infusion; between about 0.350 and about 8.750
by weight casamino acids; between about 0.15% and about
0.7o by weight starch.
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 Na forsnate for
biotransformations) or in Mueller-Hinton broth.
The production of 11~-hydroxymexrenone from
mexrenone by fermentation is discussed in Example 19.
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 IXA in which -A-A- and -B-B- are
-CHZ-CHI-, R' is hydrogen, lower alkyl or lower alkoxy,
and Re and R9 together constitute the 20-spiroxane ring:
0
,iun XXXI I I
In the next step of synthesis scheme 2, the
product of Formula IX is reacted with a dehydration
reagent to produce a compound of Formula II
CA 02553378 1996-12-11
82
R3 RB
R9
B
i
A/A B
0/ / i,~~R1
II
wherein -A-A-, R3, -B-B-, Re and R9 are as defined in
Formula VIII, and R1 is as defined in Formula V. Where
the substrate corresponds to Formula IXA, the product is
of Formula IIA
Y2
1 (
H3C Y \\\\~ CHZ~ 2 C-X
CH3 \~~OH ~B
A
A/ ~B
0/ / ''~,R1
IXA
Y2
Y1 I
-C-X
H31 ~.w~ CH2~ 2
CH3~ ~ ~B
A
A/ B
1
R IIA
in each of which -A-A-, -B-B-, Y1, Y2, and X are as
defined in Formula XIIIA and R1 is as defined in Formula
V.
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. patent 4,559,332; or preferably by the novel
epoxidation method of the invention as described
hereinabove.
In a particularly preferred embodiment, the
overall process of Scheme 2 proceeds as follows:
CA 02553378 1996-12-11
83
0 0
Illll
LOCI, DMF, Et3N,
acetone tyanohyarm, ~C4 CH30H, Hz0
/ BS' C, 8-15 h 0' C. Sh
0
Canrenone
NHZ 0
0
1. Na0CH3. CH30H, tAflux
0 2. i7ecrystalize Irom CHZCIZ,
IIII Toluene
'\~0 H
0 - 0
0 / x~x~C00CH3 C
9-c-Hyoroxymexrenone III
Novel
8ioconversion
Ph$OCI, Or CI503H
D 0 / ~~~~COOCH3
0 "."",.,3
Enester Mexrenone
C13C-C(O)NHZ, HZOz, CHZCl2,
10' C. 24 h
:H3
11-B-HyCroxymexrenOne
EPoxymexrenone
Scheme 3
The synthesis in this case begins with a
substrate corresponding to Formula XX
R3
O
\H$C ~
CH9~ ( ~B
A/A B
R2e0 \ \
XX
where -A-A- and R3 are as defined in Formula VIII, -B-B-
is as defined in Formula VIII except that neither R6 nor
CA 02553378 1996-12-11
84
R' is part of a ring fused to the D ring at the 16,17
positions, and R26 is lower alkyl, preferably methyl.
Reaction of the substrate of Formula XX with a sulfonium
glide produces the epoxide intermediate corresponding to
Formula XIX
R3\
\H3C 0.
CH3) ~
A
Ai B
8260 \ \
XIX
wherein -A-A-, R3, -B-B-, and Rz6 are as defined in
Formula XX.
In the next step of synthesis scheme 3, the
intermediate of Formula XIX is converted to a further
intermediate of Formula XVIII
0
R3 C02 Et
HaC O
iiiii
CH3 ~ ~B
A
Ai B
XVIII
wherein -A-A-, R3, and -B-B- .are as defined in Formula XX_
In this step, Formula XIX substrate is converted to
Formula XVIII intermediate by reaction with NaCH(COOEt)2
in the presence of a base in a solvent. Exposure of the
compound of Formula XVIII to heat water and an alkali
halide produces a decarboxylated intermediate compound
corresponding to Formula XVII
CA 02553378 1996-12-11
spirolactone moiety are essentially the same.
Reaction of the intermediate of Formula XVII
wherein -A-A-, R', and -B-B- are as defined in Formula XX.
The process for conversion of the compound of Formula XX
to the compound of Formula XVII corre~ponds essentially
5 to that described in U.S. patents 3,897,417, 3,413,288
and 3,300,489. While the substrates differ, the reagents,
mechanisms and conditions for introduction of the 17-
with a dehydrogenation reagent yields the further
intermediate of Formula XVI.
XVII
0
R3 O'
~~ii~
B
A~ ~ Q
/ /
v
XVI
where -A-A-, R' and -B-B- are as defined above.
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 carbon-6 followed by
dehydrohalogenation reaction.
The intermediate of Formula XVI is next
converted to the enamine of Formula XV
CA 02553378 1996-12-11
86
0
c
NHp
wherein -A-A-, R3, and -B-B- are as defined in Formula XX.
Conversion is by cyanidation essentially in the manner
described above for the conversion of the 11a-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.
The products of Formula XV are novel compounds,
which may be isolated by chromatography. These and other
novel compounds of Formula AXV have substantial value as
intermediates for the preparation of compounds of Formula
Z, and especially of Formula IA. Compounds of Formula
AXV correspond to the structure
-9
R~
v
NHS AXV
where -A-A-, -B-B-, R3, R8 and R9 are as defined above.
In the most preferred compounds of Formula XV, and -A-A-
and -B-B- are -CHz-CH2- .
In accordance with the hydrolysis described
CA 02553378 1996-12-11
87
above for producing the diketone compounds of Formula VI,
the enamines of Formula XV may be converted to the
diketones of Formula XIV
0
° xIv
wherein -A-A-, R', and -B-B- are as defined in Formula XX.
Particularly preferred for the synthesis of
epoxymexrenone are those compounds of Formula XIV which
also fall within the scope of Formula VIA.
The products of Formula XIV are novel
compounds, which may be isolated by precipitation. These
and other novel compounds of Formula AXIV have
substantial value as intermediates for the preparation of
compounds of Formula I, and especially of Formula IA.
Compounds of Formula AXIV correspond to the structure
Ra
AXIV
where -A-A-, -B-B-, R', R8 and R9 are as defined above.
In the most preferred compounds of Formula AXIV and XIV,
-A-A- and -B-B- are -CH2-CH2- .
The compounds of Formula XIV are further
CA 02553378 1996-12-11
88
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
XXXI
0
p3
~3~ O
IIIII
B
B
iiii R 1
XXXI
before further conversion to a product of Formula XXXII
0
H3C
R3 0
1 n
v
0
CH3 ~~ B
A
Ai B
iii, R 1
XXXII
wherein -A-A-, -B-B- and R3 are as defined in Formula XX.
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. patent 4,559,332. In a
particularly preferred embodiment, the overall process of
Scheme 3 proceeds as follows:
CA 02553378 1996-12-11
89
0
0 0 0 ~COZEt
s\ /\
rllrl
Sunonlum NaCH(COOEt)z
M20 \ \ YIiC~ M80 \ \ O /
Heet
NdOI
DMSO
0 0
O IIIII
. KCN, Pyrr01iCm8. Et3N
. TnmemyquaniCine~ 0 ar Cmaranu
MeOH 0 0 /
HOI, OH30H, HBO
80 ' C, Sh
1. NaOCH~, CH30H, reltuz ,I
2. ReCry5181iteC Irpm CHZCIZ ~ Pv~~ C13C(0)-NHZ. Hz0=. C!
Toluene 1 rl ~ ~0' C. 2~ h
/~
u' ~ " ~GVVl.H3
EpOxYme><r8nOn8
0
Scheme 4
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.
Thereafter, the intermediate of Formula XVII is
epoxidized, for example, using the process of U.S. patent
4,559,332 to produce the compound of Formula XXIV
CA 02553378 1996-12-11
0
H3C
R3 0
1111
i,
C H ~~p 8
A 3 ~~~
A~
0
XXIV
wherein -A-A-, R3, and -B-B- are as defined in Formula XX.
However, in a particularly preferred embodiment of the
invention, the substrate of Formula XVII is epoxidized
5 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
10 conditions and proportions of reagents for this reaction
are substantially as described for the conversion of the
Formula-II enester to epoxymexrenone.
It has been found that the epoxidation of the
substrate of Formula XVII can also be effected in very
15 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
20 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,
25 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
CA 02553378 1996-12-11
91
H3C
a3 ae
~ R9
B
B
"_ ~ ' XXIII
wherein -A-A-, -B-B- and R3 are as defined in Formula XX.
Particularly preferred compounds of Formula XXIII are
those in which -A-A- and -B-B- are as defined in Formula
XIII.
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
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° to
about 100°C, conveniently at atmospheric reflux
temperature in a solvent such as carbon tetrachloride,
acetonitrile or mixture thereof. However, reaction from
4 to 1D 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.
A suitable dehydrohalogenation reagent, e.g.,
CA 02553378 1996-12-11
92
1,4-diazabicyclo[2,2,2]octane (DABCO) is added to the
solution, along with an alkali metal halide such as Liar,
the solution heated to a suitable reaction temperature,
e.g., 60° to 80°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.
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. In the most
preferred compounds of Formula XXIII, -A-A- and -B-B- are
- CHI - CHI - .
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
CA 02553378 1996-12-11
93
H3C 0
R3
0
i~~~
CH3\'~0 ,g
g
CN
0 / ' _/ v,
~C
NH2 XXII
wherein -A-A-, R3, and -B-B- 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.
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 -CHZ-CH2-
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.
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 -CH2-CHI-.
Compounds of Formula XXI are converted to
compounds of Formula XXXII using the epoxidation process
CA 02553378 1996-12-11
94
described hereinabove or the process of U.S. patent
4,559,332. In a particularly preferred embodiment, the
overall process of Scheme 4 proceeds as follows:
0
0
0 0
I ~~~~~ °
I I
\~~, °
4 Steps cyc-cco~-~~Z.~ZOZ.c~sc~=
\ \ ~ / 10- 7. n
Me0 0 0
ODO or Cm oranll
-° ° 1 °
i~~ HC~. CH~OH, H. aetew crsnonyerin
° 80'C- 5h 'TMG, OMF
s--~-
CN / /
0 0
0
naOCH~.CH30H,
Irafy x
Epoxymexrenone
Scheme 5
The process of scheme 5 begins with a substrate
corresponding to Formula XXIX
CA 02553378 1996-12-11
R3
H,C
CH3 B
A
XXIX
wherein -A-A-, -B-B- and R3 are as defined in Formula XX.
This substrate is converted to a product of Formula
XXVIII
R3
n
B
5 "~ a XXV I I I
by reaction with trimethylorthoformate.
wherein -A-A-, R3, and -B-B- are as defined in Formula XX.
Following the formation of Formula XXVIII, the compounds
of Formula XXIX are converted to compounds of Formula
10 XXVII using the method described above for conversion of
the substrate of Formula XX to Formula XVII. Compounds
of Formula XXVII have the structure:
0
H3C
R3 0.
III
CH3
A/A B
~ / °RJ
XXVII
wherein -A-A-, -B-B- and R3 are as defined in Formula XX,
15 and R" is any of the common hydroxyl protecting groups.
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
CA 02553378 1996-12-11
96
0
H3C
R3 0
till
CH3 8
A
i = ~B
OR"
XXVI
wherein -A-A-, -H-B- and R3 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.
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. In the most
preferred compounds of Formula XXVI, -A-A- and -B-B- are
-CHZ-CHZ- .
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
0
H3C
R3 0
ilili
CH3 ~~OR" ~B
A
Ai ~B
CN
0 / ~~.
C
NHZ
XXV
wherein -A-A-, R3, and -B-B- are as defined in Formula XX.
CA 02553378 1996-12-11
97
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. In the most preferred
compounds of Formula XXVI, -A-A- and -B-B- are -CHZ-CHZ-.
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
XIV. 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.
In a particularly preferred embodiment, the
overall process of Scheme 5 proceeds as follows:
CA 02553378 1996-12-11
98
0
i~l~l
_ v - v 4 Steps -_
OR' CMeO)3CH_ OR -- OR"
0 / Me0 \ \ 0
ODO or Chloran~l
0 0
HCI, CN30H, HzO,
Ketone cyanOtlyOrm
80~ C. 5 n
'MG. DMF
~~ri~
Q NHZ
Na0CH3, CH30H,
relluz
0
t \
0 ,
Illllf C13CCN, HzOz, CH~CIZ,
1D' C. 24 h
0 / ~~~~COOCH3
a
EDO%ymel(renOne
Scheme 6
Scheme 6 provides an advantageous method for
the preparation of epoxymexrenone and other compounds
corresponding to Formula I, starting with lla-
hydroxylation of androstendione or other compound of
Formula XXXV
R~
n
B
XXXV
CA 02553378 1996-12-11
99
wherein -A-A-, R3, and -B-B- are as defined in Formula
XIII, producing an intermediate corresponding to the
Formula XXXVI
R3
.,
a
XXXVI
where -A-A-, R3, and -B-B- are as defined in Formula XIII.
Except for the selection of substrate, the process for
conducting the 11a-hydroxylation is essentially as
described hereinabove for Scheme 1. The following
microorganisms are capable of carrying out the 11a-
hydroxylation of androstendione or other compound of
Formula XXXV:
Asperctillus ochraceus NRRL 405 (ATCC 18500);
As~erQillus niQer ATCC 11394;
Asperctillus nidulans ATCC 11267;
Rhizopus oryzae ATCC 11145;
Rhizopus stolonifer ATCC 6227b;
Trichothecium roseum ATCC 12519 and ATCC 8685.
11a-Hydroxyandrost-4-ene-3,17-dione, or other
compound of Formula XXXVI, is next converted to 11x-
hydroxy-3,4-enol ether of Formula (101):
R3
O
6
B
' \ \
101
where -A-A-, R3, and -B-B-, are as defined in Formula XIII
and R11 is methyl or other lower alkyl ( C1 to C4 ) , by
CA 02553378 1996-12-11
100
reaction with an etherifying reagent such as trialkyl
orthoformate in the presence of an acid catalyst. To
carry out this conversion, the lla-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,
l0 preferably at about 0°C to about 15°C. The mixture is
then warmed and the reaction carried out at a temperature
of between 20°C and about 60°C. Preferably the reaction
is carried out at 30° to 50°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° to 15°, preferably about 5°C,
and the solvent removed under vacuum.
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:
R$
O~
N 0,~~~
B
AiA B
R~~O
102
where -A-A-, R3, R11, 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
CA 02553378 1996-12-11
101
metal alkoxide to form the five membered spirolactone
ring and produce the intermediate of Formula 103
0
R~ O C02Rt2
FIO~~~~ _ _
B
AiA B
R~~O
103
where -A-A-, R', R~1, and -B-B- are as defined in Formula
101, and Rl~ is C1-C4 lower alkyl. 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:
0
R$
O
B
B
RI~O~~/~/
104
where again -A-A-, R3, R11 and -B-B- are as defined in
Formula XIII.
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 Re and R9 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
CA 02553378 1996-12-11
102
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 Liar 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.
The compound of Formula VIII is then converted
to epoxymexrenone or other compound of Formula I by the
steps described hereinabove for Scheme 1.
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 -CH2-CHz- and R3 is hydrogen, lower alkyl or
lower alkoxy. Most preferably, the compound of Formula
101 is 3-ethoxy-lla-hydroxyandrost-3,5-dien-17-one, the
compound of Formula 102 is 3-ethoxyspiro[androst-3,5-
diene-17~,2'-oxiran]-lla-ol, the compound of Formula 103
is ethyl hydrogen 3-ethoxy-lla-17a-dihydroxypregna-3,5-
diene-21,21-dicarboxylate, gamma-lactone, and the
compound of Formula 104 is 3-ethoxy-lla-17a-
dihydroxypregna-3,5-diene-21-carboxylic acid, gamma-
lactone.
In a particularly preferred embodiment, the
overall process of Scheme 6 proceeds as follows:
CA 02553378 1996-12-11
103
0 o p
HO~~ HO,~ii
CH(OR)s
1 t ~hydroxyladon / ----~ \
O O geld utalyat O
Blooomrerslon (t Ot )
11 ~a ~Hydroxyandrostendlone
Androatendione (AD) . (CH~S'X
KOH, DMSO
O 0
O \'COzEt O~
H O,~
NaCI, DMF ~,, ~~~~~ CH2(COpEt)s
heat
NaOEt
\ \ (103) RO \ \ (102)
RO
3 methods
possIDH
O
Teehnobpy
shown In Seheme 1
1 t ~a ~Hydroxyunnnone Epoxymexnnone
Scheme 7
Scheme 7 provides for the synthesis of
epoxymexrenone and other compounds of Formula I using a
starting substrate comprising i3-sitosterol, cholesterol,
stigmasterol or other compound of Formula XXXVII
.,
D
Rts
) ' 14 'R 1 a
R
XXXVII
where -A-A-, R3, and -B-B- are as defined in Formula XIII,
D-D is -CH2-CHz- or -CH=CH-, and each of R13, R14, R15 and
Rls is independently selected from among hydrogen or C1
CA 02553378 1996-12-11
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to C4 alkyl.
In the first. step of the synthesis lla-
hydroxyandrostendione or other compound of Formula XXXVI
is prepared by bioconversion of the compound of Formula
S XXXVII. The bioconversion process is carried out
substantially in accordance with the method described
hereinabove for the lla-hydroxylation of canrenone (or
other substrate of Formula XIII).
In the synthesis 11~-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. patent 3,759,791. Thereafter, 4-
androstene-3,17-dione is converted to lla-
hydroxyandrostenedione substantially in accordance with
the method described hereinabove for the lla-
hydroxylation of canrenone (or other substrate of Formula
XIII) .
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:
CA 02553378 1996-12-11
105
O O
HO~~~~ HO,~~~
Et
CH(OR)D
Slseonvenlon / --. \ \
H ~ --~ O Edp eatlly.t O
(10t)
t t -a-nraroxrwarostrnawn~
sho..rroi
(CH~eS~X
KOH. DIISO
O O
3 nrthee.
Pe.Nlle
O/ \'CO=Et p
1lr
HO
N~C4 D N F H 0~~~~ ~~ ~ ~ ~i~~
CH=(Cn=Et~
-" w--
helt
NlOEt
\ \ (103) R \ \
a (102)
0
TeohnebaY
.hpvrn 1n Scheme 1
11.~.Hrdre:yetrnlMne EPexrme:rlnene
The methods, processes and compositions of the
invention, and the conditions and reagents used therein,
are further described in the following examples.
Example 1
Slants were prepared with a growth medium as
set forth in Table 1
TABLE 1 - Y P D A
(medium for slants and plates)
yeast extract 20 g
peptone 20 g
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glucose 20 g
agar 20 g
distilled water, q.s. to 1000 ml
-pH as is 6.7
-adjust at pH 5 with H3P04 10 0
w/v
Distribute
-for slants:
7.5 ml in 180 x 18 man tubes
-for plates (10 cm of
25 ml in 200 x 20 mm tubes
-sterilize at 120C for 20 '
minutes
-pH after sterilization:5
To produce first generation cultures, a colony of
AsperQillus-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°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.
The first generation slant cultures were
suspended in a sterile solution (4 ml? containing Tween
80 nonionic surfactant (3o 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 2
(for second generation and routine
slants)
malt extract 20 g
peptone 1 g
glucose 20 g
agar 20 g
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distilled water q.s. to 1000 ml
-pH as is 5.3
-distribute in tubes (180 x
18 mm) ml 7.5
-sterilize at 120°C for 20
minutes
The second generation slants were incubated for 10 days
at 25°C, producing a heavy mass of golden-colored spores;
reverse pigmented in brown orange.
A protective medium was prepared having the
composition set forth in Table 3.
TABLE 3 - PROTECTIVE
MEDIUM
Skim milk 10 g
distilled water 100 ml
In a 250 ml flask
containing 100 ml
of distilled water
at 50C, add skim
milk. Sterilize at
120C for 15
minutes. Cool at
33C 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 100x10 man tubes for lyophilization. These
were pre-frozen at -?0° to -80°C in an acetone/dry ice
bath for 20 minutes, then transferred immediately to a
drying room pre-cooled to -40° to -50°C. The pre-frozen
aliquots were lyophilized at a residual pressure of 50 ~t
Hg and <-30°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.
To obtain mother culture slants suitable for
CA 02553378 1996-12-11
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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°C. At
the end of incubation, the culture developed on the
slants was preserved at 4°C.
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.
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 (3o 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 4
(for primary and transformation
flask
culture and round bottomed flask)
glucose
20 g
peptone
20 g
yeast autolysate 20 g
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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°C x 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°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 x 5
min . ) .
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°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 10o and 15%. Accordingly,
the culture was deemed suitable for transformation of
canrenone to 11a-hydroxycanrenone.
Canrenone (1 g) was micronized to about 5 ~, and
CA 02553378 1996-12-11
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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 HC1 (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 5
Indicative
Additions
of Steroid
and~Solutions
(additives
and antibiotics)
in the
Course
of Bioconversion
of Canrenone
in Shake
Flask
Reaction Steroid glucose
Suspension yeast anti-
time solution auto- biotic
hours ml approx. ml lised solution
mg. sol. 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
?2 3 150 1 0.5 1
90
As reaction proceeded, the reaction mixture was
periodically analyzed to determine glucose content, and
by thin layer chromatography to determine conversion to
lloc-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
CA 02553378 1996-12-11
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solution, autolyzed yeast solution and antibiotic
' solution is set forth in Table 5. The transformation
reaction continued for 96 hours at 25°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
lla-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 6
Time Course
of Bioconversion
of Canrenone
in Shake
Flask
Time Transformation Ratio
hours Canrenone Rf. llahydroxy
Canrenone
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 -10 -90
Example 2
A primary seed flask culture was prepared in
the manner described in Example 1. A nutrient mixture
CA 02553378 1996-12-11
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was prepared having the composition set forth in Table 7
TABLE 7
For Transformation Culture
in 10 1 glass fermenter
quantity g/1
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 1
-sterilize the empty
fermenter for 30
minutes at 130C
-load it with 3 1 of
deionized water,
heat at 40C
-add while stirring
the components of
the medium
-stir for 15 minutes,
bring to volume of
3.9 1
-pH as is 5.1
-adjust of 5.8 with
NaOH 20% w/v
-sterilize at 120C
x 20 minutes
-pH after
sterilization 5.5
-5.7
An initial charge of this nutrient mixture (4L) 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
CA 02553378 1996-12-11
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the vessel were 210 mm high and extended radially
inwardly 25 mm from the interior vertical wall of the
vessel.
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°C,
and an aeration rate of 0.5 1/1-min. at a pressure of 0.5
kg/cm2. At 22 hours, the PMV of the culture was 20-25%
and the pH 5 to 5.2.
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 8
Indicative
Additions
of Steroid
and Solutions
(additives
and antibiotics)
in the
Course
of Bioconversion
of Canrenone
in
10 1 Glass
Fermenter
Reaction Steroid glucose yeast anti-
time Suspension solution autolyzed biotic
hours ml approx ml solution solution
gr ml m1
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
CA 02553378 1996-12-11
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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 I2.5 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
11a-hydroxycanrenone. Based on TLC analysis of reaction
broth samples as described hereinbelow, additional
canrenone was added to the reaction mixture as canrenone
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/cm2. The temperature was
maintained at 28°C until PVM reached 45%, then decreased .
CA 02553378 1996-12-11
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to 26°C and maintained at that temperature as PVM grew
from 45o to 600, and thereafter controlled at 24°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 11a-
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.
After all canrenone additions had been made,
the reaction was terminated when TLC analysis showed that
the concentration of canrenone substrate relative to 11a-
hydroxycanrenone product had dropped to about 5$.
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°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.
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°C
and kept under agitation for about one hour. The
resulting suspension was filtered to remove the
CA 02553378 1996-12-11
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crystalline product, and the filter cake was washed with
ethyl acetate (40 ml). After drying, the yield of 11a-
hydroxycanrenone was determined to be 60 g.
Example 3
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 x 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°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
I5 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 100.
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 man;
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 msn 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.
CA 02553378 1996-12-11
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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 130C
-load it with 6 L of
deionized water;
heat at 40C
-add while stirring
the components of
the medium
-stir for 15 minutes,
bring to volume of
95 L
-sterilization at
121C for 30 minutes
-post sterilization
pH is 5.7
-add sterile
deionized water
to 100 L
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/cm2. The temperature was controlled at 28°C until PMV
reached 250, and then lowered to 25°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 10
Time Course for Mycelial Growth in
Seed Culture Fermentation
CA 02553378 1996-12-11
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Fermentation pH packed dissolved
period h mycelium oxygen
volume (pmv)~
(3000
rpms5min)
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 t 5
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'. 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 11
For Bioconversion Culture
in 1000 L Fermenter
Quantity g/L
glucose 16 kg 23
peptone 16 kg 23
yeast autolysed 16 kg 23
CA 02553378 1996-12-11
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antifoam SAG 471 0.080 Kg traces
deionized water q.s. to 700 L
-sterilize the empty
fermenter for 1 hour
at 130C
-load it with 600 L
ofdeionized water;
heat at 40C
-add while stirring
the components of
the medium
-stir for 15 minutes,
bring to volume of
650 L
-sterilization at
121C 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.
The nutrient mixture containing inoculum was
incubated for 24 hours at an aeration rate of 0.5L/L-min
at a head pressure of 0.5 kg/cm2. The temperature was
controlled at 28°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 12
Time Course
for Mycelial
Growth in
Ferrnenter of
the Transformation
Culture
Fermentation pH packed dissolved
period h mycelium o
xygen
volume (pmv)
o
(3000 rpmx5
min)
0 5.6 0.2 100
CA 02553378 1996-12-11
120
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.
To the transformation culture thus produced was
added a suspension of canrenone (1.250 kg; micronized to
5 ~,) 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 13 ADDITIVE SOLUTION
(for transformative culture)
quantity
dextrose 40 Kg
yeast autolysate 8 Kg
antifoam SAG 471 0.010 Kg
CA 02553378 1996-12-11
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deionized water q.s. to 100 1
-sterilize a 150 1 empty
fermenter for 1 hour at
130°C
-load it with 70 1 of
deionized water; heat
at 40°C
-add while stirring the
components of "additive
solution
-stir for 30 minutes,
bring to volume of 95 1
-pH as is 4.9
-sterilize at 120°C
I5 x 20 minutes
-pH after sterilization
about 5
Bioconversion was carried out for about 96 hours with
aeration at 0.5 L/L-min. at a head pressure of 0.5 kg/cm2
and a pH of ranging between 4.7 and 5.3, adjusted as
necessary by additions of 7.5 M NaOH or 4 M H3P04. 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°C, lowered to 26°C when PMV reached
45~, and lowered to 24°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 t.o the batch. Disappearance of
canrenone and appearance of 11a-hydroxycanrenone were
also monitored during the reaction by HPLC. When at
least 90% of the initial canrenone charge had been
converted to 11a-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
CA 02553378 1996-12-11
122
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 11a-hydroxycanrenone produced. The .
schedule for addition of canrenone, sterile additive
solution, and antibiotic solution is as shown in
Table 14.
TABLE 14
Additions
of the
Steroid
and Solutions
(additives
and antibiotics)
in. the
Course
of Bioconversion
of Canrenone
in Fermenter
Reaction C A N R Sterile anti- volume
time E N O N additive biotic liters
hours E solution solution about
in suspension liters liters
Kg Progress
-ive Kg
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
CA 02553378 1996-12-11
123
76 0
80
84
88
92
Total
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 11a-
hydroxycanrenone in the harvest broth, and was therefore
eliminated. The mycelia were suspended in ethyl acetate
(1000 L) in an extraction tank of 2 m3 capacity. This
suspension was heated for one hour under agitation and
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°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
CA 02553378 1996-12-11
124
filter. The product was dried under vacuum for 16 hours
at 50°C. Yield of llot-hydroxycanrenone was 14 kg.
Examt~le 4 '
Lyophilized spores of AsperQillus ochraceus
NRRL 405 were suspended in a corn steep liquor growth
medium (2 ml) having the composition set forth in
Table 15:
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 121C for
20 minutes.
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 16 - GYPA
(Glucose/Yeast Extract/Phosphate
Agar for Plates)
Glucose (charge after sterilization) 10 g
Yeast extract 2.5 g
3 0 KZHP04 3 g .
Agar 20 g
distilled water, q.s, to 1000 ml
adjust pH to 6.S
sterilize 121C for 30 minutes
A 0.2 ml aliquot of the suspension was transferred onto
CA 02553378 1996-12-11
125
the surface of each plate. The plates were incubated at
25°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 27:
TABLE 17 - GYP/Glycerol
(Glucose/Yeast Extract/
Phosphate/Glycerol
medium for stock vials)
Glucose (charge after sterilization) 10 g
Yeast extract 2.5 g
KZHPOQ 3 g
Glycerol 20 g
Distilled water, q.s. to 1000 mL
Sterilize at 121C 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 11x-
hydroxycanrenone. The vials comprising the master cell
bank were stored in the vapor phase of a liquid nitrogen
freezer at -130°C.
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°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.
CA 02553378 1996-12-11
126
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°C.
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°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.
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°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 110c-
hydroxycanrenone.
Transformation was conducted in a Braun E
Biostat fermenter configured as follows:
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 x
1.4 cm each
Impeller spacing: 65.5, 14.5 and 25.5 cm from bottom
CA 02553378 1996-12-11
127
of tank
Baffles: four 1.9 x 48 cm
Sparger: 10.1 cm diameter, 21 holes -1 ~n
diameter
Temperature control: provided by means of an external
vessel jacket
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 18
(Growth medium for bioconversion
culture in 10 L fermenter)
Quantity AmountlL
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 ?.5L
sterilize 121C 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°C for 30 minutes in the
Braun E fermenter. The pH after sterilization was
6.3~0.2, and the final volume was ?.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
CA 02553378 1996-12-11
128
8.0 L by addition of 50% sterile glucose solution.
Fermentation was carried out at a temperature of 28°C
until the PMV reached 50~, then lowered to 26°C, and .
further lowered to 24°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 50o 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.
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 11a-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.
CA 02553378 1996-12-11
129
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°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°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°C.
Example 5
Lyophilized spores of AsperQillus 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
CA 02553378 1996-12-11
130
phase of a liquid nitrogen freezer at -130°C.
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°C.
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°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 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 121C for 30 minutes
Cultivation of a secondary seed culture was
initiated by introducing 8L growth medium having the
composition set forth in Table 19 into a 14L 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.
The inoculated mixture was cultivated at 28°C
for 18-20 hours, 200 rmp agitation, aeration rate was 0.5
CA 02553378 1996-12-11
131
vvm. At the end of the propagation, the culture
exhibited the same properties as described above for the
primary seed.
Transformation was conducted in a 60L
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 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 121C for 30 minutes
Example 6
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
CA 02553378 1996-12-11
132
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.
CA 02553378 1996-12-11
133
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CA 02553378 1996-12-11
134
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
1Q canrenone to the subject t<o the reaction.
Canrenone proved stable at sterilization
temperature (121°C) but aggregated into chunks. A Waning
blender was employed to crush the lumps into fine
particles, which were successfully converted to product.
Example 7
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. Lumps of canrenone formed on
sterilization were delumped through a Waning blender
thus producing a fine particulate suspension entering the
CA 02553378 1996-12-11
135
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.
CA 02553378 1996-12-11
136
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CA 02553378 1996-12-11
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CA 02553378 1996-12-11
138
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
The description and results of Example 8 are
summarized in Table 24. Four fermentation runs were made
in which lla-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 lloc-
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.
CA 02553378 1996-12-11
139
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CA 02553378 1996-12-11
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CA 02553378 1996-12-11
141
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
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.
Four bioconversion runs were carried out
substantially in the manner described for run R4B of
Example 8, except as described below. In run RSB, the
top turbine disk impeller used for agitation in the other
runs was replaced with a downward pumping marine
impeller. The downward pumping action axially poured the
broth into the center of the ferznenter and reduced cake
formation. Methanol (200 ml) was added immediately after
inoculation in run RSD. 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.
CA 02553378 1996-12-11
142
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CA 02553378 1996-12-11
143
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
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
the runs of this Example are set forth in Table 27:
CA 02553378 1996-12-11
o -x
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CA 02553378 1996-12-11
145
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
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 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 -
NH4H2 P04 3
Glucose 15
OSA 0.5 ml
pH adjusted to 6.5
with 2.SNNaOH
Canrenone charge 160 g canrenone 160 g canrenone
was sterilized was sterilized
&
blended outside in the fermenter
the fermenter
CA 02553378 1996-12-11
146
Medium charge Glucose feeding; Glucose feeding;
canrenone was no other
added with 1.6L 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 89.50, indicating that no significant substrate
loss or degradation in bioconversion. Mixing was
determined to be adequate for both runs.
Residual glucose concentration was above the
desired 5-10 gpl 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
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 800 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 dzyness 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 29 - Recovery of 11a.-Hydroxycanrenone
at 1 Liter Extraction (~ of Total)
CA 02553378 1996-12-11
147
Run Number 1st 2nd 3rd Supernatant
Extract Extract Extract
R5A 790 16~ 2% 2~
R5A 84~ 120 2% 20
R4A 72~ 20g 4~ 4~
R4A 79~ 14~ 2~ 50
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 11a-
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
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
CA 02553378 1996-12-11
148
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.
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 30
- Particle
Distributions
of
Three Different
Canrenone
Samples
Sample 45- <180 mean Run #: o
125 ~ ~. size 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)
Sterilize 24.7 65.1 157.4 R4B:
_
d Sample ~ 97.6 (120 h)
RSB:
93.8 (120 h)
From the data in Table 30, it will be noted that
agitators and shear pump were effective to reduce the
CA 02553378 1996-12-11
149
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
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 40o PMV. Its pH was
5.4 and 14.8 gpl glucose remained unused.
A transformation growth medi~un (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 30o by weight glucose
and 10% by weight yeast extract. pH of the feed was
adjusted to 5.7.
Using this medium, (Table 20), two
bioconversion runs were made for the conversion of
canrenone to lloc-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.
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
CA 02553378 1996-12-11
150
carried out at a temperature of 28°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 20o 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.
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 82 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.
CA 02553378 1996-12-11
151
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CA 02553378 1996-12-11
152
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CA 02553378 1996-12-11
153
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CA 02553378 1996-12-11
154
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ei c~1 01 O O C1 O M C~
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f
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01 t!1 Lf1 CD r-I e-W i ll1 I~ ri
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i
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ri e1 r-I e-i '-1 .y -~1 .-i ri v-~1
~
e-I e-i e-i .-i ri e~ r-i e-i rl e-i
01 tD tff 01 N N v-1 lG I~ e~-i
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CA 02553378 1996-12-11
155
Example 15
Various cultures were tested for effectiveness
in the bioconversion of canrenone to lla-hydroxycanrenone
according to the methods generally described above.
A working cell bank of each of Asperaillus
ni er 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°C for about 20
hours. The incubator was agitated at a rate of 200 rpm.
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°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 o.f 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.
Samples of the broth were taken to analyze for the extent
CA 02553378 1996-12-11
156
of bioconversion. After three days, the fermentation
using ATCC 11394 provided conversion to 11a-
hydroxycanrenone of 80 to 90~; ATCC 11145 provided a
conversion of 50~; and ATCC 6227b provided a conversion
of 80 to 90~.
Example 16
Using the substantially the method described in
Example 15, the additional microorganisms were tested for
effectiveness in the conversion of canrenone to 11x-
hydroxyc~anrenone. The organisms tested and the results
of the tests are set forth in Table 33:
CA 02553378 1996-12-11
157
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p t I ca t o, o, 0 0 0 * ao ~I o, o, v
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o
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in
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CA 02553378 1996-12-11
158 v
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CA 02553378 1996-12-11
159
Example 17
Various microorganisms were tested for
effectiveness in the conversion of canrenone to 9a.-
hydroxycanrenone. Fermentation media for the runs of this
Example were prepared as set forth in Table 34:
TABLE 34
Soybean Meal:
dextrose 20 g
soybean meal 5 g
NaCl 5 g
yeast extract 5 g
KHZ PO9 5 g
water to 1 L
pH 7.0
Peptone/yeast extract/glucose:
glucose
40 g
bactopeptone 10 g
yeast extract 5g
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.
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 15o crossing volume
- and the latter incubated for 24 to 48 hours before
addition of steroid substrate for the transformation
CA 02553378 1996-12-11
160
reaction.
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°C and
60% humidity.
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°C.
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.
Reaction product was analyzed first by thin
layer chromatography on silica gel (250 Nzn) fluorescence
backed plates (254 nm). Ethyl acetate (500 ~,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
CA 02553378 1996-12-11
161
spectrometry. Plates were developed in a 95:5 v/v
chloroform/methanol medium.
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 NovaPakTMC-18 (4 ~n particle size) RadialPak
4 mmcartridge. 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.
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.
Mass spectrometric fragmentation patterns
proved useful in sorting from among the hydroxylated
substrates. The two expected hydroxylated ~anrenones,
11a-hydroxy- and 9a-hydroxy, lost water at different
frequencies in a consistent manner which could be used as
a diagnostic. Also, the 9a-hydroxycanrenone formed an
ammonium adduct more readily than did lloc-
hydroxycanrenone. Set forth in Table 35 is a summary of
the TLC, HPLC/W and LC/MS data for canrenone
fermentations, showing which of the tested microorganism
were effective in the bioconversion of canrenone to 9a-
CA 02553378 1996-12-11
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hydroxycanrenone. Of these, the preferred microorganism
was Corynesyora cassiicola ATCC 26718.
CA 02553378 1996-12-11
163
TABLE 35 - Summary of TLC,
HPLC/W, and LC/MS
Data for Canrenone Fermentations
Evidence
for 9ocOH-canrenone
Culture TLC spot HPLC-peak MS: 357
at 9aQH- at 9aOH- (M + H),
AD canrenone 339(-H20)
w/W & 375
( +NH4 )
Absidia coerula ATCC n y y/n
6647
Absidia alauca ATCC n
22752
Actinomucor eleQans ATCC tr y tr
6476
AsperQillus flavipes tr
ATCC 1030
AsperQillus fumictatus tr y n
ATCC 26934
Asperctillus nidulans tr y y
ATCC 11267
AsperQillus niQer ATCC n y y
16888
AsperQillus nicrer ATCC n y n
26693
Asperaillus ochraceus n y n
ATCC 18500
Bacterium cvclo-oxvdans n tr n
(Searle) ATCC 12673
Beauveria bassiana ATCC tr y y
715
Beauveria bassiana ATCC y y y
13144
Botryost~haeria obtusa y tr tr
IMI 038560
Calonectria decora ATCC n tr y
147 67
Chaetomium cochiiodes tr tr y/n
ATCC 10195
Comomonas testosterone tr tr n
(Searle) ATCC 11996
Cozynespora cassiicola y y y
ATCC 16718
CA 02553378 1996-12-11
164
Cunninahamella y y y
blakesleana ATCC 8688a
Cunnincrhamel la y y y
echinulata ATCC 3655
Cunninahamella eleQans y y y
ATCC 9245
Curcularia clavata ATCC n y y/n
22921
Curvularia lunata ATCC y n n
12071
Cylindrocarnon tr n n
radicicola (Searle) ATCC
11011
Epicoccum humucola ATCC y y y
12722
E~icoccum orvzae ATCC tr tr tr
12724
Fusarium oxvsporum ATCC tr
7601
Fusarium oxvsnorum f.sp. n
ce ae ATCC 11171
Gibberella fuiikuroi tr y y
ATCC 14842
Gliocladium deliauescens y tr tr
ATCC 10097
Gonctronella butieri ATCC y y W? y
22822
Hyt~omyces chnrsospermus y y y
Tul. IMI 109891
Lipomvces linofer ATCC n
10792
Melanos~ora ornata ATCC tr n n
26180
Mortierella isabellinav y y n
ATCC 42613
Mucor crrisco-cvanus ATCC n
1207a
Mucor mucedo ATCC 4605 tr y y
Mycobacterium fortuitumn
ATCC 6842
Myrothecium verrucaria tr tr y
ATCC 9095
CA 02553378 1996-12-11
165
Nocardia aurentia n tr n
(Searle) ATCC 12674
Nocardia cancicruria y y n
(Searle)
Nocardia corallina ATCC n
19070
Paecilomyces carneus n y n
ATCC 46579
Penicillium chrvsocrenum n
ATCC 9480
Penicillium patulum ATCC y y y/n
24550
Penicillium purnurocrenum tr y y
ATCC 46581
Pithomvces atro- tr y tr
olivaceus ATCC 6651
Pithomyces cvnodontis n tr tr
ATCC 26150
Phvcomvces blakesleeanus y y y/n
Pvcnosporium sp. ATCC y y y/n
12231
Rhizo oaon sp.
Rhizonus arrhizus ATCC tr y n
11145
Rhizopus stolonifer ATCC n
6227b
Rhodococcus eaui ATCC n tr n
14887
Rhodococcus ecrui ATCC tr tr n
21329
Rhodococcus s n n n
Rhodococcus rhodochrous n tr n
ATCC 19150
Saccharopolvspora y y y
a thaea ATCC 11635
Sepedonivm ampullosporum n n n
IMI 203033
Sepedonium chnrsospermum n
ATCC 13378
Septomvxa affinis ATCC n y W? y/n
6737
CA 02553378 1996-12-11
166
Stachvlidium bicolor y y y/n
ATCC 12672
Streptom n
~ces
,
cal~fornicus ATCC 15436
Streptomvces n
cinereocrocatus ATCC
3443
Stre~tomvces coelicolor n
ATCC 10147
Streptomvces flocculus
ATCC 25453
Streptomvces fradiae n
ATCC 10745
Strentomvces ariseus n
subsp. ariseus ATCC
13968
Streptoimrces ariseus n
ATCC 11984
Streptomvces hvdroQenans n
ATCC 19631
Streptomvces y y y
rosco icus ATCC.27438
Streptomvces lavendulae n
Panlab 105
Stre~tomyces n
paucisporocaenes ATCC
25489
Stre~tomvces n tr tr
urourascens ATCC 25489
Streptomyces
roseochromoQenes ATCC
13400
Streptomvces spectabilis n
ATCC 27465
Stvsanus microsyorus
ATCC 2833
Svncephalastrum n
racemosum ATCC 18192
Thamnidium
electans ATCC
_
18191
Thamnostylum piriforzne y tr ~ y
ATCC 8992
Thielavia terricolan n '
ATCC 13807
CA 02553378 1996-12-11
167
Trichoderma viride ATCC n
26802
Trichothecium roseum tr y y/n
ATCC 12543
Verticillium theobromae y tr tr
ATCC 12474
Example 18
Various cultures were tested for effectiveness
in the bioconversion of androstendione to 11x-
hydroxyandrostendione according~to,the methods generally
described above.
A working cell bank of each of Asr~ercrillus
ochraceus NRRL 405 (ATCC 28500); AsperQillus niQer ATCC
11394; Ast~erQillus nidulans ATCC 11267; Rhizopus orvzae
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°C for about 20 hours. The incubator was agitated at a
rate of 200 rpm.
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 crissolved in
methanol (6 ml) at 36°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
CA 02553378 1996-12-11
168
having a chromatographic mobility similar to that of 11a
hydroxy-androstendione standard (Sigma Chemical Co., St.
Louis) was present. The results are shown in Table 36. ,
Positive results are indicated as "+". '
TABLE 35
Bioconversion of androstendione
to 11 alpha-
hydroxy-androstendione
Culture ~ ATTC# media TLC
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 +
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
11a-hydroxyandrostendione standard.
AsnerQillus 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 inThatman K6F silica gel
60I~, 10x20 size, 250 thickness. The solvent system was '
methanol: chloroform, 5:95, v/v. The crystallized product
CA 02553378 1996-12-11
169
and 11a-hydroxyandrostendione standard were both analyzed
by LC-MS and NMR spectroscopy. Both compounds yielded
similar profiles and molecular weights.
Example 19
Various microorganisms were tested for
effectiveness in the conversion of mexrenone to 11~3-
hydroxymexrenone. Fermentation media for this example
were prepared as described in Table 34.
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:
11a-hydroxymexrenone and 11a-hydroxycanrenone have the
same chromatographic mobility. 11a-hydroxycanrenone and
9a-hydroxycanrenone exhibit the same mobility pattern as
11a-hydroxyandrostendione and 11(3-hydroxyandrostendione.
Therefore, 11~i-hydroxymexrenone should have the same
mobility as 9a-hydroxycanrenone. Therefore, compounds
extracted from the growth media were run against 9a-
hydroxycanrenone as a standard. The results are shown in
Table 36.
TABLE 37
Summary of TLC Data for
11(3-hydroxymexrenone Formation
from Mexrenone
Spot
Microorganism Mediums Character
Absidia coerula ATCC 6647 M,S strong
Asperaillus niQer ATCC S,P faint (S)
16888 ? ( P)
Beauveria bassiana ATCC P strong
7159
Beauveria bassiana ATCC S,P ?,?
13144
Botzvosphaeria obtusa IMI faint
038560
CA 02553378 1996-12-11
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Cunnincthamel la
blakesleeana ATCC 8688a S,P strong '
echinulata ATCC 3655 S,P strong
elegans ATCC 9245 S,P strong
Curvularia lunata ATCC S strong
12017
Goncxronella butleri ATCC S,P strong
22822
Penicillium patulum ATCC S,P strong
24550
Penicillium nurnurocrenum S,P strong
ATCC 46581 '
Pithomvces atro-olivaceus S,P faint
IFO 6651
Rhodococcus ecrui ATCC M faint
14887
Saccharopolvspora ervthaea M, SF faint
ATCC 11635
Strentomyces hyaroscopicus M, SF strong
ATCC 27438
Streotomvces purnurascens M,SF faint
ATCC 25489
Thamnidium elecrans ATCC S,P faint
18191
Thamnostvlum piriforme S,P faint
ATCC 8992
Trichothecium roseum ATCC P, S faint (P)
12543 ? (S)
1 M = Mueller-Hinton
P = PYG (peptone/yeast extract/glucose)
S = soybean meal
SF = soybean meal plus formate
? - questionable difference from no substrate control
These data suggest that the majority of the
organisms listed in this table produce a product similar
or identical to 11~-hydroxymexrenone from mexrenone. '
CA 02553378 1996-12-11
171
Examule 20
Scheme 1: Step 1: Preparation of 5'R(5'a),7'~3-
20'-Aminohexadecahydro-11'(3-hydroxy-10'a,13'a-dimethyl-
3',5-dioxospiro[furan-2(3H),17'a(5'H)-
[7,4]metheno[4H[cyclopenta[a]phenanthrene]-5'-
carbonitrile.
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°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°C, transferred to a 200
gallon glass-lined reactor with quenched with 130 kg of
6.7~ sodium hypochlorite solution slowly. The product
was filtered and washed with 3 x 40 L portions of water
to give 21.4 kg of the product enamine.
o v
n
v v
LiCI. DMF, EhN,
Ae~ton~ eyanohydrin
i5'C, i:15 h
Stop 1
Example 21
Scheme 1: Step 2: Preparation of 4'S(4'a),7'a-
Hexadecahydro-11'a-hydroxy-10'~i,13'(3-dimethyl-3', 5, 20'-
trioxospiro [ furan-2 (3H) , 17' (3-
[4,7]methanol17H]cyclopenta[a]phenanthrene]-5'(3(2'H)-
CA 02553378 1996-12-11
172
carbonitrile.
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 75L of methanol. The
mixture was heated to 80°C for 5 hours, cooled to 0°C for
2 hours. The solid product was filtered to give 36.5 kg
of dry product diketone.
O
HC1, CH~OH, Hp0
a0° C, Sh
Stop 2
Example 22
Scheme 1: Step 3A: Preparation of Methyl
Hydrogen lloc,l7a-Dihydroxy-3-oxopregn-4-ene-7a,21-
dicarboxylate, 'y-Lactone.
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
2S minute period. After the addition was complete, the
CA 02553378 1996-12-11
173
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%.
Example 23
Scheme 1: Step 3B: Conversion of Methyl
Hydrogen 11a,17a-Dihydroxy-3-oxopregn-4-ene-7a,21-
dicarboxylate, ~-Lactone to Methyl Hydrogen 17a-Hydroxy-
11a-(methylsulfonyl)oxy-3-oxopregn-4-ene-7a,21-
dicarboxylate, y-Lactone.
A 5-L four neck flask was equipped as in the
above example, except that no trapping system was
CA 02553378 1996-12-11
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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°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°C. Stirring was continued for l,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 HC1, 1N NaOH, and saturated
15~ 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°C overnight, to
give 144.61 g (88~) nearly white solid, m.p. 149°-150°C.
Material prepared in this fashion is typically 98-990
pure by HPLC (area a). In one run, material having a
melting point of 153°-153.5°C was obtained, with a
purity, as determined by HPLC area, of 99.5.
CA 02553378 1996-12-11
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Example 24
Scheme 1: Step 3C: Method A: Preparation of
Methyl Hydrogen 17a-Hydroxy-3-oxopregna-4,9(11)-diene-
7a,21-dicarboxylate, y-Lactone.
A 1-L four neck flask was equipped as in the
second example. Formic acid (250 mL) and acetic
anhydride (62mL) 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°C (this,was later repeated at
70°C with better results) for 16 hours. After 16 hours,
the mesylate 5 was added and the internal temperature was
increased to 100°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 as a mixture of 82.1% 6 7.40 8
and 5_7q 9 (by HPLC area ~).
Example 25
Scheme 1: Step 3C: Method B: Preparation of
Methyl Hydrogen 17a-Hydroxy-3-oxopregna-4,9(11)-diene
7x,21-dicarboxylate, y-Lactone.
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°C. The mesylate (123.65 g) was added,
CA 02553378 1996-12-11
176
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°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
gu~r~y 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 Q9-11
enester 6, 22.3 of the Q1,12 enester 8, 10.8 of the 7-
a,9-a-lactone 9 and 5.7$ unreacted 5.
Example 26
Scheme 1: Step 3D: Synthesis of Methyl Hydrogen
9,11a-Epoxy-17a-hydroxy-3-oxopregn-4-ene-7oc,21-
dicarboxylate, 'y-Lactone.
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°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
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9~1°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°C.
Example 27
Scheme 1: Step 3: Option 1: From 4'S(4'a),?'a-
Hexadecahydro-11'a-hydroxy-10'~,13'~-dimethyl-3',5,20'-
trioxospiro[furan-2(3H).17'~-
[4,7]methano[17H]cyclopenta[a]phenanthrene]-5'~(2'H)-
carbonitrile to Methyl Hydrogen 9,11x-Epoxy-17a-hydroxy-
3-.oxopregn-4-ene-7a,21-dicarboxylate, ~-Lactone.
Diketone (20 g) was charged into a clean and
dried reactor followed by the addition of 820 ml of MeOH
and 17.5 ml of 25~ NaOMe/MeOH solution. The reaction
mixture was heated to reflux condition (-67°C) for 16-20
hours. The product was quenched with 40 mL of 4N HC1.
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
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chloride and cooled to 0°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.
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°C for 4 hours.
The formic acid mixture was added to the
concentrated mesylate solution 5 prepared above. The
mixture was heated to 95-105°C for 2 hours. The product
mixture was cooled to 50°C and the volatile components
were removed by vacuum distillations at 50°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.
The product was dissolved in 180 mL of
methylene chloride and cooled to 0 to 15°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°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 10o 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
CA 02553378 1996-12-11
179
of eplerenone.
Epoxrn~xronon~
Example 28
Scheme 1: Step 3: Option 2: Conversion o
1'S(4'a),7'a-Hexadecahydro-11'a-hydroxy-10'~i,13'~i-
dimethyl-3 ' , 5 , 2 0 ' -trioxospiro [ furan-2 ( 3H ) ,17 ' ~i-
[4,7]methanol17H]cyclopenta[a]phenanthrene)-5'(3(2'H)-
carbonitrile to Methyl Hydrogen 9,11a-Epoxy-17a-hydroxy-
3-oxopregn-4-ene-7a,21-dicarboxylate, Y-Lactone without
intermediate.
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)
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
reflex for 20 hours and 175 mL 4 N hydrochloric acid was
added dropwise while refluxing continued. (Caution, HCN
evolution during this operation') The reflex condenser
was replaced with a takeoff head and 1.6 L of methanol
was removed by distillation while 1.6 L of aqueous 10~
DI kit one
CA 02553378 1996-12-11
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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 (0.50 of the total
was removed for analysis). ,
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°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°C. Stirring was continued for 2
hours, while the reaction mixture warmed to ambient. A
column.consisting of 250 g Dower 50 W x 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 x 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 20 of this was removed
for qc. The remainder was further reduced to a final
CA 02553378 1996-12-11
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volume of 150 mL (crude mesylate solution).
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°C for 16 hours. The temperature was
then increased to 100°C and the crude mesylate solution
was added over a thirty minute period via the addition
funnel. The temperature dropped to 85°C as methylene
chloride was distilling out of the reaction mixture.
After all of it had been removed, the temperature climbed
back to 100°C, and was held there for 2.5 hours. The
reaction mixture was cooled to 40°C and the formic acid
was removed under pressure until the minimum stir volume
had been reached (-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).
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°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°C throughout the addition and on
continued stirring overnight (23 hours total). The
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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 3o 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 27.10 EPX.
Example 29
11a-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 5°C.
Triethyl orthoformate (334.5 g) was added to the solution
over a 15 minute period at 0° to 15°C. After the
tristhyl orthoformate addition was complete the reaction
mixture was warmed to 40°C and reacted at that
temperature for 2 hours, after which the temperature was
increased to reflex and reaction continued under reflex
for an additional 3 hours. The reaction mixture was
cooled under vacuum and the solvent removed under vacuum
to yield 3-ethoxyandrosta-3,5-dime-17-one.
Examole 30 - Formation of Enamine from
11a-hvdroxvcanrenone
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183
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 11a-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°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°C overnight to yield the 11a-hydroxy
enamine, i . a . , 5'R (~' a) ,'7 '~i-20' -aminohexadecahydro-11' ~-
hydroxy-10'a,13'a-dimethyl-3',5-dioxospiro[furan-
2(3H),17'a(5'H)-[7,4]metheno [4H]
cyclopenta[a]phenanthrene]-5'-carbonitrile (4.9 g).
Example 31 - Conversion of 11a-hydroxycanrenone to
Diketone
CA 02553378 1996-12-11
184
0 0
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
dimethyformamide] (9 mL) was added followed by
11a-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°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°C. The solid was isolated
by filtering through a sintered glass filter followed by
washing twice with water (20 mL). The solid diketone,
i . a . , 4 ' S ( 4 ' a ) , 7 ' oc-Hexadecahydro-11' oc-hydroxy-10' $ ,13 ' ~-
dimethyl-3',5,20'-trioxospiro[furan-2(3H),17'~-
[4,7]methanol17H]cyclopenta[a]phenanthrene]-5'~(2'H)-
carbonitrile was dried in a vacuum oven to yield 3.0 g of
a solid.
Example 32
A suspension of 5.0 g of the diketone produced
in the manner described in Example 31 in methanol (200
mL) was heated to reflex and a 25~ solution of potassium
CA 02553378 1996-12-11
185
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
HC1 (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
min. The temperature of the solution was raised to
10 81.5°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
15 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°C (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
l1a,17a-dihydroxy-3-oxopregn-4-ene-7x,21-dicarboxylate,
~-lactone by weight. The yield was 56.1%.
Example 33
Diketone 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°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°C). The addition rate
was controlled to prevent the pot temperature from
exceeding 30°C. The mixture was thereafter heated to
CA 02553378 1996-12-11
186
reflex conditions (about 67°C) and continued under reflex
for 16 hrs. A sample was then taken and analyzed by HPLC
for conversion. The reaction was continued under reflex
until the residual diketone was not greater than 3% of
the diketone charge. During reflex 4 N HC1 (120 mL) was
charged to the reaction pot resulting in the generation
of HCN which was quenched in a scrubber.
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°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°C
range. An aqueous solution containing optionally 5% by
weight sodium bicarbonate (1200 mL) at 25°C was charged
to the cooled slurry and the resultant mixture then
cooled to 0°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°C for 2 hrs. to complete the precipitation
and crystallization after which the solid product was
recovered by filtration and the filter cake washed with
water (100 mL). The product was dried at 80-90°C under
26 " mercury vacuum to :constant weight . tnlat-er content
after dzying was less than 0.25 by weight. Adjusted
molar yield was around 77-80% by weight.
Example 34
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
CA 02553378 1996-12-11
287
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.
Example 35
The hydroxyester prepared as by Example 34
(1.97 g) was combined with tetrahydrofuran (20 mL) and
the resulting mixture cooled to -70°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 enester
(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°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 was sulfuryl chloride causes the
lloc-hydroxy group of the hydroxy ester of Formula V to be
replaced by halide and this is followed by
dehydrohalogenation to the D-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 36
Hydroxyester (20 g) prepared as by Example 34,
CA 02553378 1996-12-11
188
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°C
using an ice bath. Methanesulfonyl chloride (5 mL) was
added to the solution of CHZC12 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°C. The reaction was very exothermic;
therefore cooling was necessary. The reaction mixture
was stirred at about 5°C for 1 h. When the reaction was
complete (HPLC and TLC analysis), the mixture was
concentrated at about 0°C under 26 in Hg vacuum until it
became a thick slurry. The resulting slurry was diluted
with CHZC12 (160 mL), and the mixture was concentrated at
about 0°C under 26 in Hg vacuum to obtain a concentrate.
The purity of the concentrate (mesylate product of
Formula IV wherein R'=H and -A-A- and -B-B- are both -CHZ-
CHZ-, i.e., methyl hydrogen 11a,17a-dihydroxy-3-oxopregn-
4-ene-7a,21-dicarboxylate, y-lactone to methyl hydrogen
17a-hydroxy-11a-(methylsulfonyl)oxy-3-oxopregn-4-ene-
7a,21-dicarboxylate, 'y-lactone was found to be 82~ (HPLC
area g). This material was used for the next reaction
without isolation.
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°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
CA 02553378 1996-12-11
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the amount of water present in the reaction (formic acid
and potassium formate contained about 3-5o 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 <0.1o 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°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°C. Upon completion
(TLC and HPLC analysis; <0.1~ starting material) the
content was cooled to 50°C and vacuum distillation was
started (26 in Hg/50°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°C water bath.
The resulting enester, solid product i.e., methyl
hydrogen 17a-hydroxy-3-oxopregna-4,9(11)-diene-7a,21-
dicarboxylate, y-lactone weighed 92 g (77o mol yield).
Example 37
Hydroxyester (100 g; 0.22 mol) prepared as by
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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.
Methylene chloride (1 L) was charged to the
flask and the hydroxyester dissolved therein under
agitation. The solution was cooled to 0°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
15~ was highly exothermic. Addition time was around 10 min.
under agitation and cooling. The charge mixture was
cooled to 0°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°C for an additional
min. and checked again for reaction completion.
25 Analysis showed the reaction to be substantially complete
at this point; the solvent methylene chloride was
stripped at 0°C under 26" mercury vacuum. Gas
chromatography analysis of the distillate indicated the
presence of both methane sulfonyl chloride and
30 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°C. The solvent was again stripped at 0-5°C
under 26" mercuxy vacuum yielding the mesylate of Formula
IV wherein R3 is H, -A-A- and -B-B- are -CHZ-CHZ- and R1 is
methoxy carbonyl. The purity of the product was about
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90-95 area o.
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°C during addition of acetic anhydride.
The elimination reagent mixture was heated to 70°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°C and
volatile distillate collected until no further distillate
was generated. Distillation ceased at about 90°C. After
distillation was complete, the reaction mixture was
stirred at 95°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°C and the formic acid and
solvent removed from the reaction mixture under 26"
mercury vacuum at 50°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
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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°C and the product recovered by filtration.
The final product was a foamy brown solid when dried.
Further drying at 45°C under reduced pressure for 24 hrs.
yielded 95.4 g of the enester product which assayed at
68.8%. The molar yield was 74.4 corrected for both the
starting hydroxy ester and the final enester.
Example 38
The procedure of Example 37 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 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.
TABLE
38
Factor Set pointPurpose of Experiment Key results
Basic 2 g/125 Treating the reaction mixtureThe yield
g
3 alumina product with basic alumina to removewas 93%
0
Et,N.HCl salt and to
eliminate the iN NaOH and
1N
HC1 washes
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Basic 2 g/125 Treating the reaction mixtureThe yield
g
silica product with basic silica which was 95~
is
cheaper to remove Et,N.HCl
salt and eliminate 1N NaOH
and 1N HC1 Washes
~ Exampla 39
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°C. The solution was then heated
to 30°C for 30 min. to provide an elimination reagent
useful for converting the mesylate of Formula IV to the
enester of Formula II.
The preformed TFA/TFA anhydride elimination
reagent was added to a previously prepared solution of
the mesylate of Formula IV. The resulting mixture was
heated at 40°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 1-
neck flask and concentrated to dryness under reduced
pressure at room temperature (22°C). Ethyl acetate 1137
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
24o 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 24o 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
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phase addition, the mixture was stirred for 10 min. and
allowed to stand for 15 min. for separation. The
resulting solution was concentrated to dxyness under ,
reduced pressure at 45°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, 2.45 area ~ of the
11,12-olefin, 1.5~ of the 7,9-lactone, and 1.1~ of
unreacted mesylate.
Example 40
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 °C with stirring. After 5 hours the
mixture was cooled to 25 °C and concentrated in vacuo at
10 mm of Hg. The residue was dissolved in CHZClZ (20 ml)
and washed with 5$ aqueous NaHC03. The CH2C12 layer was
concentrated in vacuo to give 1.47 g of a tan oil. This
material was recrystallized from CH2C12/Et20 to give 0.50
g of enol acetate of Formula IV(Z).
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 °C with stirring. After 60
minutes the mixture was cooled to 25 °C and diluted with
CHZC12 (20 ml). The solution was washed with water (20
ml) and dried over MgSDq. 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, IV(Y). The crude
product contained less than~2~ of the 7,9-lactone
impurity.
Example 41 - Thermo elimination of Mesvlate in DMSO.
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a
sol vsnt
A mixture of 2 g of mesylate and 5 ml of DMSO
in a flask was heated at 80 °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.
Example 42
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). A clear solution was obtained.
Hydrogen peroxide (30o 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 50 of unreacted enester remained
relative to epoxymexrenone in the mixture.
CA 02553378 1996-12-11
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Example 43
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°C and a 30% hydrogen peroxide
(25 g) was added over a ten min. period. The reaction
mixture was allowed to come to 20°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.
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 HC1
(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 9~.2%_
Example 44
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 74.40 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°C '
after which HPLC showed a 90o conversion of enester to
CA 02553378 1996-12-11
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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
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°C for 18 hrs. The
residual enester was less than 2~. After work up, 5.71 g
of epoxymexrenone resulted.
Example 46
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°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
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°C and the solution of dipotassium hydrogen
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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
solution 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°C for 18.5 hrs.
No oxygen evolution was observed throughout the course of
the reaction. The reaction mixture was diluted with
water (69.4 mL) and the mixture stirred at about 250 rpm
for 25 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°C being
acceptable). The aqueous and organic layers were allowed
15~ to separate and the lower methylene chloride layer was
removed.
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%) indicating that only
0.0434 mol of hydrogen peroxide were consumed in the
reaction was 0.0307 mol of olefin. Back extraction with
a small amount of methylene chloride volume was
sufficient to insure no loss of epoxy~nexrenone in the
aqueous layer. This result was confirmed with the
application of a second large methylene chloride
extraction in which only trichloroacetamide was
recovered.
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
CA 02553378 1996-12-11
199
observed at the end of the stir period.
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.
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°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.
The methylene chloride phase was washed once
with 0.1 N hydrochloric acid (61 mL) for l5 min. under
250 rpm agitation at a temperature irr the range 15-25°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°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°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.
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°C as
the product crystallized. Stirring was continued at 50°C
for 1 hr. The temperature was then lowered to 20-25°C
CA 02553378 1996-12-11
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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°C as the product crystallized; and
stirring was continued at 50°C for 1 hr. The temperature
was then lowered to 20-25°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%.
Examule 48 - Epoxidation of Formula IIA using toluene
The enester of Formula IIA was converted to
eplerenone 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 °C
and was complete in 4 hours. The resulting three phase
mixture was cooled to 15 °C, filtered, washed with water
and dried in vacuo to yield 2.5 g of product.
Example 49 - Epoxidation of 9,11-Dienone
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A compound designated XVIIA (compound XVII
wherein -A-A- and -B-B- are both -CH2-CHZ-) (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°C after which
30% Hydrogen peroxide (200 g) was slowly added over a
period of 1 hour. The reaction mixture was stirred at
12° 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 - Epoxidation of Compound XVIIA Usina
m-chloro~erbenzoic acid
Compound XVIIA (18.0 g) was dissolved in 250 mL
of methylene chloride and cooled to 10°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
CA 02553378 1996-12-11
202
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 - Epoxidation of Compound XVIIA usincr
Trichloroacetamide
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.
Examz~le 52
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 R3 is H
and -A-A- and -B-B- are each -CHz-CHZ-) (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 m~nol; 2.00 eq.) was charged to the flask and
the resulting mixture heated at reflux, 80 to 85°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°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°C. 104.6 g of the 3-methyl enol ether D-5,6,9,11,-17-
CA 02553378 1996-12-11
203
oxirane steroid product was isolated.
Example 53
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°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°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-D5,6,9,11,-17-spirolactone-21-methoxycarbonyl
intermediate was isolated.
Example 54
The 3-methyl enolether-~5,~6,9,11,-17-
spirolactone-21-methoxycarbonyl intermediate (Formula
XVIII where R3 is H and -A-A- and -B-B- are-each
-CHZ-CHZ-; 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°C for 3 hrs. after which
the reaction mixture was checked for completion of the
reaction by HPLC. Water was thereafter added to the
CA 02553378 1996-12-11
204
mixture over a 30 min. period while the mixture was being
cooled to 15°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
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°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 X4,5,9,12,-17-spirolactone was isolated
(compound of Formula XVII where R3 is H and -A-A- and -B-
B- are -CHZ-CH2-; 95 . 9 % yield) .
Example 56
Compound XVIIA (1 kg; 2.81 moles) was charged
together with car3~on tetrachloride t 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°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 5g sodium bicarbonate
CA 02553378 1996-12-11
205
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°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°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°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°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°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
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
CA 02553378 1996-12-11
206
and treated with tetramethyl guanidine (45.49 g; 49.6 mL;
395.0 irnnol; 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 °C.
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 nuxture was ,
then poured into ice water (2 L) with stirring. The
stirred suspension was further treated with 10~ aqueous
HC1 (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 x 800 mL); and the
organic layers were combined and back extracted with
water (2 x 2 L). The methylene chloride solution was
concentrated in vacuo to give 61 g of a dark red oil.
After the aqueous wash fractions were allowed
to sit overnight, a considerable precipitate developed.
This precipitat~e~was collected by filtration and
determined to be pure product enamine (14.8 g).
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
9,11-epoxyenamine (4.600 g; 0.011261 mol; 1.00
CA 02553378 1996-12-11
2 07
eq.) as prepared in accordance with Example 57 was
introduced into a 1000 mL round bottom flask. Methanol
(300 mL) and 0.5o by weight aqueous HC1 (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 (81.30 .
Example 59
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°C on a rotary evaporator and the residue
partitioned between methylene chloride and 3.0 N HC1.
Concentration of the organic phase yielded a yellow solid
(193 mg) which was determined to be 22% by weight epoxy
mexrenone. The yield was 200.
0 0
D
to oq. NaOMo
MoOH
t
.i2 p. NsOMo
N~OH
~pl~r~non~
CA 02553378 1996-12-11
208
Example 60
To 100 mg of the diketone suspended in 2.5 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 reflex. 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
reflex for 23 hours at which time the major product was
eplerenone as judged by HPLC.
o
CH$OH
~~thrlaminr
Example 61
To 2 g of the diketone suspended in 30 ml of
dry methanol was added 0.34 mL of triethylamine. The
suspension was heated at reflex for 4.5 hours. The
mixture was stirred at 25 ~C for 16 hours. The resulting
suspension was filtered to give 1.3 g of the 5-cyanoester
as a white solid.
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 reflex for 4 hours and was stirred at25x
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
CA 02553378 1996-12-11
209
give 3.1 g of crystalline material which was homogeneous
by HPLC.
In view of the above, it will be seen that the
several objects of the invention are achieved and other
advantageous results attained.
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.
