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Патент USA US3091585

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United States Patent 0
3,091,575
m‘
we
‘Patented May 32-8, 1963
2
1
and sodium molybdate. The e?'ectiveness does not appear
to be governed by the ionic species since both Cr+6 and
CR+3 have demonstrated inhibition of “destructase"
3,091,575
METHOD OF PREPARING A1 STEROIDS
Louis I. Feldman, Spring Valley, and ‘Chester E. Holm
lund, Pearl River, N.Y., and ‘Norma L. Barbacci, Mont
vale, NJ., assignors to American Cyanamid Company,
New York, N.Y., a corporation of Maine
N0 Drawing. Filed June 6, 1961, Ser. No. 115,094
20 Claims. (Cl. 195-51)
activity.
The use of elements of subgroup VI of the periodic
table is not limited to introduction at the time of steroid
addition. Introduction ‘of potassium dichromate into the
formulation of the medium prior to sterilization has
proven effective and permitted a more convenient and
desirable method of inhibiting the “destructase” system.
A study of the ionic forms of compounds containing
the atoms of subgroup VI --of the periodic table shows
inhibiting the destruction of steroids in microbial systems.
that -:'Cr2'07-2 exists primarily as CrO;2 under the ‘pH
In biochemical reactions involving the use of micro
conditions existing in ‘the fermentation mash. ‘Subsequent
organisms to accomplish steroid transformations, it is
,known that destruction of the desired steroid can take 15 to sterilization by autoclaving, the greenish cast imparted
to the medium is suggestive of the chromic ion (Cr+3)
place giving lower yields or sometimes no product. This
which would be anticipated following ‘this treatment.
phenomenon may be described .as “destructase” activity
Further evidence in this direction is obtained by 1a nega~
and is manifested by the simultaneous loss of 240 mp.
tive lead acetate test for CrO;2 which demonstrates the
absorption (due to the A4-3-ketone) and blue tetrazolium
This invention relates to a new process of preparing
steroids. More particularly, it relates to a method of
absence of crow. The lead acetate test conducted on
mash to which potassium dichromate has been introduced
at the timeof steroid addition, on the ‘other hand, is posi
not the result of minor alterations brought about by re~
tive for CrO4-2. >Cr+3, in the form of C'r('NO3)3, intro
duction, isomerization or side-chain oxidation since inde—
duced at the time of steroid addition also inhibits the
pendent methods ‘to detect these changes have failed to
“destructase” activity as predicted from the above con
reveal their occurrence. Thus the “destructase” system,
at the least, simultaneously attacks both the “A” ring and
siderations.
The inhibition of ‘the “destructase” :system does ‘not
the side-chain, and the reactions involved may be consid
appear‘to be'rnerely a re?ection'ofthe redox potential im
erably ‘more profound. “Destructase” activity is very
parted by the quinonoid compounds or atoms of the sub—
wide-spread among vbiological systems and has been ob
group VI of the periodic table, sincecompounds with both
served with great vfrequency in microbiological systems.
higher and lower redox vpotentials than members of the
Microbial systems manifesting “destructase” activity have
two classes-of compounds used have failedtodemonstrate
been of great variety and’have not been limited to 1,2
dehydrogenating systems. Thus “destructase” activity is
‘this inhibition.
In carrying ‘out the process of the ‘present invention
a serious side reaction which results in a greatly decreased
yieldof steroid. With certain steroids andcertain micro 35 to produce ‘1,-2-dehydrogenation, ‘any ‘of »the organisms
described in the prior art for this purpose can be ‘used.
organisms steroid destruction is so rapid and complete
Among these organisms may be mentioned Nocardia
that no steroids can be isolated.
reactivity (due toan a ketol) in compounds containing ,
these groupings. The loss of these properties is probably
We have now vfound that the destruction of the desired
steroid can be :all but eliminated by the use of inhibitors.
corallz'nw (ATCC 999),'C0rynebacteriwm simplex (ATCC
6946), Mycol'bacterium rhvdoohrous {(ATCC 12674),
‘For example, when 1,2-dehydrogenating organisms are
Bacterium cyclooxydans '(ATCC 12673) ‘and Bacterium
mycoides (Lederle Culture No. 327). The cultivation
grown under normal fermentation conditions and at the
‘time of steroid-addition, a member of either of two classes
of compounds is introduced into the mash. The intro
and growing of these microorganisms in suitable media
is described in the prior art such as United States -Pat
duction of these additives causes inhibition of “destructase”
ents 2,822,318; 2,837,464 and the like. These media
activity resulting in a signi?cantly higher yield of the 45 comprise essentially a source of carbon, assi'milableinitro
desired 1,2-dehydro steroid. The classes of compounds
gen and trace elements. As‘pointed-tout.above,‘the.addi~
inhibiting “destructase” activity are-of the quinonoid type
tion of the claimed inhibitors preferably takes place at
or are compounds containing an atom of subgroup VI of
the periodic table.
'
V
The inhibition of destructase ‘activity appears to be a
general phenomenon manifested by quinonoid type com
pounds. Among the quinonoids found to be elfective have
been quinones, phenazines, thiazines, oxazines, benze
none-indophenols, acridines, xanthylium salts and thio
Xanthylium salts. Furthermore, the-oxidized and reduced
forms appear "equally 'eifective 'since identical results are
obtained when either hydroquinone or p-benzoquinone is
the time the steroid is added to the media although it
can take place at the time of sterilizing the media. The
amount of inhibitor to be added will, to some extent,
depend on the steroid, microorganism, media and the state
of the particular organism in a given fermentation. The
amount of inhibitor added will vary from about 2.0)(10-6
to 2.0><lt01'"1 molar. Following completion of the reac
tion, the desired steroid is recovered in the usual manner
well known in the steroid art.
The process of the present invention is useful in pre
paring A1 steroids, such as, for example triamcinolone,
prednisolone, prednisone, and almost any steroid having
--manifested generally -by~compounds containing atoms of. 60 ,a A1 double bond in ‘the ?nal product. The process of
subgroup ‘VI of the periodic table. Among the atoms
preparing'compounds, such ‘as described-in United States
shown to be eifective ‘have been-chromium, molybdenum
'Patents 2,789,118; 2,822,318, etc. 'is greatly improved
and tungsten. Compounds containing these atoms are,
‘by the 'useo'f the inhibitors of ‘the present invention. A '
for example, potassium dichromate, 'chromic vnitrate,
The following examples describe in detail ‘the results
Ichromous chloride, chromous sul?de, chromous acetate, 65 obtained with different inhibitors, ‘various microorganisms
chromic oxide, chromic sulfate, chromic phosphate, ‘fer
and steroids 'for‘varying lengths of time.
rous vchrom'ate, sodium chromate, ‘potassium chromate,
‘
Example‘ I
e
sodium dichromate, phosphotungstic acid, sodium tung
1 --used.
The inhibition of “destructase” activity appears to be
The group of organisms’ below are ‘washed aseptically
state, tungstic dichloride, tungstic tetrochlori'de, tungstic
vpentachloride, tungstic dioxide, tungstic trioxide, phos 70 from *trypticase soy agar ‘slants (Baltimore Biological
Laboratory.) with ‘sterile water into 100 ml. of sterile
‘phomolybdic acid, molybdenum disul?de, molybdenum
medium contained in a 500 ml. Erlenmeyer ?ask. The
sesquioxide, molybdenum trioxide, molybdenum chloride
3,091,575
3
:5.
also carried out with aliquots equivalent to 50 ,ug. of
steroid.
Example 11
A reaction is carried out with growing cells of N.
corallina as described in Example I. Following the pre
liminary growth period of 16 hours, at the time of addi
tion of hydrocortisone in a separate series of fermenta
?asks are incubated on a reciprocating shaker at a pre
scribed temperature for a predetermined time. The time,
temperature and medium utilized for each organism is
shown in the following table:
Organism
Time
Temp. Medium»
(hrs)
(° C.)
Nocardia corallina (ATCC 990) ___________ _.
Corynebacterium simplex (ATOO 6946) ____ __
7
7
37
33
A1
B2
Mycobacterium rhodochrous (A’I‘OO 12,674) _
7
33
A
Bacterium cycloorydans (ATGO 12,673) ____ _-
7
33
B
Bacterium mycoidcs (Lederle Culture 327)-..
7
33
A
tions, the various quinonoid compounds described below
10 are added to the fermentation in su?icient quantity to
attain the concentrations indicated.
The course of the
‘fermentation during which 1,2-dehydrogenation of hydro
cortisone (F) to prednisolone (NF), and the extent of
1 Medium A consists of 0.4% beef extract, 0.1% yeast extract, 0.4%
peptone. 1.0% glucose, 0.25% sodium chloride, pH adjusted to 7.0. _
2 Medium B consists of 0.1% yeast extract, 0.245% potassium dihy
“destructase” activity can be summarized as ‘follows:
drogen phosphate, 0.256% disodium phosphate.
pg. Observed/100 pg.
steroid added
Following the inoculum development described above,
1.0 ml. of inoculum is introduced into 100‘ ml. of the
same sterile medium, used for the inoculum develop
ment, in a 500 ml. Erlenmeyer ?ask. The ?asks are
incubated on a reciprocating shaker for 16 hours at 28°
Quinonoid (molar concentration)
3 hours
24 hours
0
A1 F
F
A1F
F
105
123
83
3
6
22
3
123
113
0
11
19
C. At this time, 200 ,ug/ml. of steroid, dissolved in
methanol at a concentration of 10 mg./ml., and the de
sired quinonoid, or compound containing an atom of
No qninonoid added to fermentation ........... _.
Phenazine methosulfate (3.3)(10-4 M) __________ __
2,6-dichlorobenzenone-indophenol (10-3 M) _____ _.
subgroup VI of the periodic table, in solution form, are
introduced into each ?ask and the incubation is allowed
to continue. At intervals 1.0 ml. aliquots are removed
aseptically from the ?asks and extracted with 8 ml. of
No quinonoid added to fermentation..-"
ethyl acetate saturated with water. Aliquots of the ethyl
acetate containing approximately 100 ,ug. of steroid is
concentrated in a tube to a dry residue.
2
2
0
Methylene blue (10-3 M) ____________ _-
83
5
92
0
No quinonoid added to fermentation
89
46
66
2
Gallocyanine (10-3 M) ____________ __
43
75
72
0
No quinonoid added to fermentation“
94
4
2
0
p~Benzoquinone (10-2 M) ______________________ -_
86
11
82
2
0
Example III
Using the reaction conditions described in Example I,
Samples are
chilled in an ice bath and 5 ml. of reagent (kept at 0°
growing cells of N. corallz'na are used to ‘ferment andro
C.) added. The reagent is prepared by dissolving 400
stenedione, progesterone, Reichstein’s Substance S (S)
mg. of isonicotinic acid hydrazide (Nutritional Biochemi
cals) per 1001 ml. of methanol containing 0.5 ml. con 35 and hydrocortisone 0F). Fermentations are carried out
in the presence and absence of phenazine methosulfate
centrated hydrochloric acid. After exactly eight minutes
(PMS) at a concentration of 33x10"4 M.
in the ice bath, the optical density of the sample is read
Conversion to the 1,2-dehydro product (A13) of each
at 410 mu against a reagent blank. The samples and
of the above (A4) compounds is summarized as follows:
blank are then heated for twenty minutes in a 60° C.
water bath, after which the optical densities are again 40
read at 410 III/L.
#g. Observed/100 pg. steroid
added
The results were computed as follows:
a=observed O.D. at 0° C. after 8 minutes
b=observed O.D. at 60° C. after 20 minutes
R1: O.D. of standard A1'4-3-ketosteroid at 0° C.
Steroid
PMS (molar
cone.)
45
O.D. of standard A1'4—3-ketosteroid at 60° C.
__O.D. of standard A4-3-ketosteroid at 60° C.
Androstenedione ________ __
2* O.D. of standard A4-3-ketosteroid at 0° C.
Al .4
A4
3 hours
24 hours
A1 ,4
A4
A1 ,4
A4
0
12
74
32
19
1
1
3. 3X10-4 M
102
3
101
3
99
11
0
3
83
11
35
2
1
Progesterone ............ __
3. 3X10-4 M
Y=O.D. of A1'4—3—ketoster0id at 60° C. corrected for
presence of A4-3-ketosteroid.
X =O.D. of N-3-ket0steroid at 60° C. corrected for the
presence of A1'4-3-ketosteroid.
1 hour
103
13
85
4
50
3
0
10
101
22
70
3
0
3. 3X10'4 M
108
7
102
4
77
5
Beichstein’s substance S- .
Hydrocortisone _________ ._
0
69
59
112
10
3
2
3. 3X10-4 M
107
10
125
6
123
11
Example IV
Using the procedure of Example I, growing cells of
Solving the simultaneous equations:
N. corallina are used to ferment hydrocortisone. Fer
mentations are carried out in the presence of various
concentrations of phenazine methosulfate (PMS) which
60
are summarized as follows:
pg. Observed/100 cg. steroid added
PMS (molar cone.)
For seventeen A4-3-ketosteroids the mean R2=1.057i.03
and for eleven A4- and A1»4-3-ketosteroid pairs the mean
1—-R1R2=0.95i0.02. The mean value for these eleven
65
pairs of the ratio
O.D. at 60° for A1'4-3—ketosteroid
=2.06 :l: 0.02
O.D. at 60° for A4-3-ketosteroid
'Ilhe constancy of this ratio permitted the construction
of standard curves for A1,4-3-ketosteroids (where stand
ards are lacking) based upon the related A4-3-ketosteroid
70
3 hours
AIF
24 hours
F
AIF
F
0 __________ __
105
3
3
0
3.3)(10-5 M
3.3)(10'4 M
1X10-3 M.--
103
125
130
2
6
7
75
123
130
0
11
10
15
85
29
71
3.3><10-a M ................ __
Example V
Using the procedure described in Example I, hydro~
cortisone is fermented in the presence and absence of
standard curve data. Paper chromatographic assays were 75 phenazine methosulfate (PMS) by growing cells of N.
3,091,575
5
6
and hydrocortisone. Fermentations are carried out in the
presence and absence of 10-‘3 M potassium dichromate
corallina, C. simplex, B. cyclooxydans and B. Mycoides.
The results obtained are summarized as follows:
, pg. Observed/100 pg. steroid added
Organism
PMS
3 hours
A1 F
Nocardia-corallina (Area 999) _________________ __
Corynebaclerium simplex (A'I‘OG 6946) __________ __
Mycobacterium rhodochrous (ATOG 12674) ______ ._
_-
,
.
.
.
.
Bacterium mycoides>(Lederle Culture 327) _______ _;
F
a
6
123
11
120
0
0
62
97
3
0
99
2
0
14
0
0
118
0
0
14
O
113
16
103
10
0
110
1
107
0
0
F
'64
15
98
125
3
A1 F
105
9
0
0
0
0
3.3)(10-5 M
102
8
111
0
100
0
O
93
0
0
O
0
0
3.3X10-4 M
109
10
115
10
110
8
introduced into the fermentation at the time of steroid
20 addition. Resuts are as follows:
The following reaction is carried out with growing
'cellsof N. corallz'naas described previously in the proce
‘dure of Example I. At the time ofsteroid addition, the
various inorganic compounds shown below are added ‘to
vthe fermentation in su?‘i'cient quantity to 'give the concen
"trations summarized:
pg. Observed/100 pg. steroid added
Potassium
dichro
Steroid
Supplement (molar concentration)
Am
KzCraO1 (LO-WM) (potassium dichromate)
.
p
24 hours
Androstenedione.--
0
'
10-3 M
AM
At- A114
A4
3 hours
24 hours
AIF
F
All‘
97
0
4
0
93
0
‘94
O
13‘
Example IX
Using the procedure previously-described in Example
119 V
4
2
0
‘I, growing-cells-o'f N. corallina are used to ferment ‘hydro
_
103
9
112
0
.
70
-8
‘21
0
cortisone. Fermentations are carried out in the presence
of the concentrations of ‘potassium dichromate as shown
No inhibitor added to fermentation"..-
011N093 (4><10-3 M) (chromic nitrate).
8 hours
Progesterone _____ __
35
No inhibitor added to fermentation ____________ __
1 hour
mate,
molar c0110.
pg. Observed/100 pg.
steroid added
24WOa.PzOs.25H2O (10--3 M) (phosphotungstic_
4 acid)
NazWO4~(10'3 M) (sodium tungstate) ____ -_'.__.-__
MOal2H3P04A8H2O (10-3 M) (phosphomolybdio
acid)
F
0
3.3)(10'4 M
0
Example VI
No inhibitoradded to'fermentation _____ _.
A1 F
96 hours
o
3.3)(10'4 M
3.3)(10'4 M
Bacterium cycloozydans (A’I‘OC 12673) __________ __
24 hours
7
N84M0042H30 (10'3 M) (sodium molybdate)_--_
‘
108
15
110
0
132
15
128
1
82 23
66
1
63
1
‘S3
16
40
below.
pg. Observed/100 pg. steroid added
, Potassium
45
Example VII
3 hours
dichromate,
24 hours
'48 hours
.molarrconc.
Using‘the‘ procedure previously described in Example
Lean-o: Housemanct-o NOQ cmowon:ug i-wlo
I, except that 10“3 M potassium dichromate'is added to
:medium A prior ‘to sterilization, N. corallina is grown in
‘medium A containingpotass'ium dichromate. The steroid
(hydrocortisone) is added and samples'handled ‘aspre
viously described, Example I. Justprior to the time of
. .r,-l
steroid addition, a lead acetate test is made on the mash.
This test is negative, indicating the absence of chromate 55
'i'on. Furthermore, the mash has afgreenish cast suggestive
of chromic ion. The results obtained upon steroid assay
on»;-
mo:7
was-
Example X
Using the procedure previously described in Example
~'I, 'hydrocor‘tisone is fermented‘ in the presence and absence
of potassium dichro'mate by ~growing'cellsof N. corallina,
are described as follows:
C. simplex,
r'hodochrous, B. cy'clooxydans and B.
mycoides. The results‘are described as follows:
pg. Observed/100 pg. ste
roid added
3 hours
pg. observed/100 pg.
steroid added
24 hours
Organism
Potassium
dichromate,
molar cone.
All?‘
Medium A ............................. -_
Medium A plus KgCTzO7 (10-3 M) - - .
__
112
97
F
AIF
9
0
F
2
102
3 hours
24 hours
NF
AlF
65
0
0
F
Nocardia corallina (ATCC 999) ____ ._
Orzjgynebacterium simplex) (ATCC
Example VIII
70 Mpcobticterium rhodochrous (ATCC
12674
Bacterium cycloozydam (ATCC
Using the procedure previously described in Example
I, growing cells of N. corallina are used to ferment an
drostenedione, progresterone, Reichstein’s Substance S 75
12673)
Bacieriu'm mycoz'des (Lederle Cul
ture 327).
0 cm
F
3,091,575
,7
We claim:
1. A method for the improvement of fermentation
selected from the oxidized and reduced forms of the
group consisting of quinones, phenazines, thiazines, oxa
processes containing a 1,2-dehydrogenating microbial
zines, benzenone-indophenols, acridines, xanthylium salts
system for the production of 1,2-dehydro steroids in
and thioxanythlium salts in a quantity su?icient to pro
which destruction of Ali4-3-keto steroids is inhibited
duce a concentration of not more than 2.0><1()-1 molar
solution.
which comprises adding as inhibitors a member selected
from the group consisting of compounds containing
chromium, molybdenum ‘and tungsten and compounds
6. A method of stabilizing Al-hydrocortisone in a fer
mentation medium in the presence of growing cells of
Nocardia corallina which comprises adding to the medium
containing the ortho and para quinonoid structure in the
oxidized and reduced state of the formulas:
10 potassium dichromate in an ‘amount suf?cient to produce
from about 2.0x l0—6 to 2.0x l0"1 molar.
7. A method of stabilizing Al-hydrocortisone in a fer
X
mentation medium in the presence of growing cells of
R
i
-R,
Nocardia corallina which comprises adding to the medium
15 phenazine methosulfate in an amount to produce from
R2
/
—R3
about 2.0><10—6 to 2.0i><10*1 molar.
8. A method in accordance with claim 3 wherein the
R1
Rs
1
chemical compound containing the quinonoid structure
and
1111 Ilia 1111
is phenazine methosulfate.
R1
R2
20
—X—Ra
R3—Y-
R2
|
R1
wherein X and Y are atoms selected from the group
9. A method in accordance with claim 3 wherein the
chemical compound containing the quinonoid structure
is p—benzoquinone.
10. A method in accordance with claim 3 wherein the
chemical compound containing the quinonoid structure
25 is methylene blue.
11. A method in accordance with claim 3 wherein
the chemical compound containing the quinonoid struc
ture is igallocyanine.
consisting of oxygen, nitrogen and sulfur, R1 is selected
from the group consisting of hydrogen, carboxylic acid,
12. A method in accordance with claim 3 wherein the
hydroxyl and halogen groups, R2 is selected from the 30 chemical compound containing the quinonoid structure
group consisting of hydrogen, hydroxyl and dimethyl
is 2,6-dichlorobenzenone-indophenol.
amino ‘and R3 when present is a member of the group
consisting of hydrogen, methyl and p-hydroxyphenyl.
13. A method in accordance with claim 2 wherein the
chromium compound is potassium dichromate.
2. A method of inhibiting the destruction of A114-3-keto
14. A method in accordance with claim 2. wherein the
steroids in substantially ‘aqueous solution in the presence 35 tungsten compound is phosphotungs-tic acid.
of 1,2-dehydrogenating microbial systems causing de—
struction of said steroids which comprises adding to the
solution an inhibitor selected from the group consisting
of chromium, molybdenum and tungsten compounds.
15. A method in accordance with claim 2 wherein the
molybdenum compound is sodium molybdate.
16. A method in accordance with claim 5 wherein the
compound of the quinonoid structure is gallocyanine and
3. A method of inhibiting the destruction of A1’4-3-keto 40 the A1,4-3-one steroid is prednisolone.
steroids in substantially aqueous solution in the presence
17. A method in accordance with claim 5 wherein the
of 1,2-dehydrogenating microbial systems causing destruc—
compound of the quinonoid structure is p-benzoquinone
tion of said steroids which comprises adding ‘an inhibitor
and the A1’4-3-one steroid is prednisolone.
to the solution selected from the group consisting of oxi
18. A method in accordance with claim 5 wherein the
dized and reduced forms of quinones, phenazines, thia 45 compound of the quinonoid structure is phenazine metho
zines, oxazines, benzenone-indophenols, acridines, Xan
sulfate and the A1’4-3-one steroid is Al-androstenedione.
thylinium salts and thioxanthylinium salts.
19. A method in accordance with claim 2 wherein the
4. In ‘a method of 1,2-dehydrogenating steroids in sub
compound containing chromium is chromic nitrate and
stantially aqueous solution in the presence of micro
the A1’4-3-one steroid is prednisolone.
organisms capable of 1,2-dehydrogenating said steroids,
20. A method in accordance with claim 2 wherein the
the improvement which comprises stabilizing the steroids
present by the addition of a member selected from the
compound containing molybdenum is phosphomolybdic
acid and the A1'4-3-one steroid is prednisolone.
group consisting of chromium compounds, molybdenum
References Cited in the ?le of this patent
compounds and tungsten compounds in an amount to
UNITED STATES PATENTS
produce from about 2.0><10-6 to 2.0><10~1 molar 55
solution.
Campbell et a1 ________ __ Mar. 31, 1959
2,880,205
5. A method of stabilizing A114-3-one steroids in a fer
OTHER REFERENCES
mentation medium in the presence of 1,2-dehydrogenating
microorganisms which comprises adding to the said me
Prescott et al.: Industrial Microbiology, McGraw-Hill
dium a chemical compound of the quinonoid structure 60 Book Company, Inc., New York, 1959, page 749.
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