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Патент USA US3037926

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“ice
3,837,916
Patented June 5., 1962
2
medium in which it is not necessary to remove chloride
3,037,916
FERMENTATION 0F TETRACYCLKNE
ions from the system. The present invention is based
upon the discovery that when a chlorination inhibitor as
hereinafter described is added to a chloride-containing
fermentation media, the activities of the microorganism
are directed from the synthesis of chlortetracycline to the
synthesis of tetracycline. The invention is of extreme
practical importance because it obviates the expense and
disadvantages of removing chloride ions from the fermen
_
Joseph Jacob Goodman, Nanuet, N.Y., assignor to Ameri
can Cyanamid Company, New York, N.Y., a corpora
tion of Maine
No Drawing. Filed Oct. 11, 1956, Ser. No. 615,220
-‘
18 Claims.
(Cl. 195—80)
This invention relates to the fermentation of certain
antibiotics of the tetracycline family and more particu 10 tation medium.
‘
larly is concerned with the inhibition of chlorination in
Thus, in accordance with one aspect of the present in
such fermentations whereby high yields of the desired
vention a chlortetracycline fermentation can be easily
tetracycline antibiotics are produced.
converted to a tetracycline fermentation by the addition to
Recently it has been discovered that microorganisms of
the nutrient medium of a relatively small amount of the
the genus Streptomyces which produce chlortetracycline, 15 novel chlorination inhibitors. For example, as little as 10
will also produce tetracycline particularly if the chloride
parts per million of some of these chlorination inhibitors,
ion concentration of the fermentation medium is kept
which when added to a standard S. aul'eofaciens chlor
low. This can be done by providing fermentation media
tetracycline fermentation, which in the absence of inhibi
from which chloride ions are excluded, either by making
tors would yield 8700 gammas per milliliter of chlortetra
up the fermentation medium with chloride-free compo 20 cycline and 555 gammas per milliliter of tetracycline,
nents or by treating the medium with agents which re—
move or sequester the chloride ions, thus making them
unavailable for the formation of chlortetracycline.
Unfortunately some of the most effective components
of fermentation media for the production of the tetra 25
have the effect of converting the fermentation to a tetra
cycline-producing fermentation and result in the forma
tion of 240 gammas per milliliter of chlortetracycline and
cycline antibiotics contain substantial quantities of chlo—
ride ions. Corn steep liquor is one of the most effective
nutrient substances for the production of the tetracycline
7540 gammas per milliliter of tetracycline. In ‘effect,
‘these novel chlorination inhibitors permit a tetracycline
chlortetracycline ratio of about 99:1 to be produced
under standard chlortetracycline fermentation conditions.
The chlorination inhibitors may also be used with equal
antibiotics as well as many other antibiotics. Apparently,
facility in converting a demethylchlortetracycline fermen
this natural material contains something that is especially 30 tation to a demethyltetracycline fermentation. These
desired by the fermenting microorganism. Highest yields
novel antibiotics form the subject matter of the copending
application of J. R. D. McCormick et al., Serial No.
of antibiotic are, therefore, obtained when a portion of
corn steep liquor is included in the aqueous nutrient me
587,518, ?led May 28, 1956, now U.‘S. Patent No.
dium.
2,878,289, entitled “New Antibiotics from Streptomyces
Corn steep liquor contains a substantial amount of 35 aureofaciens.” As described in the aforesaid application
these new antibiotics are related to tetracycline and chlor
chloride ions, however, and if formation of chlortetra
tetracycline and differ essentially therefrom in having one
cycline is to be kept at reasonably low levels, it has here~
less methyl group. The methyl group involved is most
tofore been considered necessary to reduce the chloride
likely the one occupying the 6-position on the naphtha
content of this material. The same applies to a number
cene ring. An appropriate chemical name for the tetra
of other naturally occurring nutrient materials which are
cycline analogus would be 4-dimethylamino-l,4,4a,5,5a,
advantageously used in the fermentation process.
Several means of reducing the chloride ion content of
aqueous nutrient solutions for tetracycline production
6,11,12a - octahydro - 3,6,10,12,12a - pentahydroxy - 1,11
dioxo-2-naphthacenecarboxamide. The chloro analogue
would have a chlorine atom in the 7-position and would
silver chloride is a very effective means of reducing the 45 be named accordingly. Appropriate common names for
these new antibiotics would be 6-dimethylchlortetracycline
chloride ion content. However, this is an expensive proc
have been suggested. Precipitation of the chloride ion as
ess requiring expensive raw materials, special equipment,
and 6-demethyltetracycline.
-
As described in detail in the McCormick et al. applica
tion these new antibiotics are produced by certain mutant
Ion exchange resins have also been proposed for the 50 strains of S. aureofaciens, some of which have been desig
nated as S604, S1071, V62 and B740. The morphological
reduction of the chloride ion content of fermentation
characteristics of these mutant strains of S. aureofaciens
media. These substances tend to remove some salts and
are adequately set forth in the McCormick et a1. applica
organic substances of unidenti?ed composition which are
tion and cultures of these strains have been deposited
desirable components of the fermentation media. As a
result, low yields of antibiotic may be obtained when 55 with the American Type Culture Collection in Washing
ton, DC, and have been assigned ATCC accession num
using nutrient solutions which have been pre-treated with
bers of 12551, 12552, 12553, and 12554, respectively.
ion exchange resins to remove chloride ions.
So far as the tetracycline-chlortetracycline fermenta~
The chloride deprivation systems still leave much to be
tion is concerned, I may use any chlortetracycline-tetra
desired, however, because of the capital investment re
quired, the relatively complicated means of removing 60 cycline-producing microorganism of the genus Strepto
myces. In this broader aspect of the present invention,
chloride ions from the fermentation medium, as well as
it is not necessary to use those mutant strains of S. aureo
the fact that the fermentation medium can in such in
facz'ens which are needed for the production of the de
stances be composed only of those raw materials from
and a toxic gas, hydrogen sul?de, for the recovery of
silver.
which chloride ions can be easily removed or which are
naturally low in chlorides.
methyltetracyclines described above.
Consequently, in
65 the broader aspects of this invention any microorganism
It has also been proposed to shift the equilibrium in a
tetracycline-chlortetracycline fermentation by the use of
that produces both chlortetracycline and tetracycline by
bromide which apparently represses chlortetracycline
aware, all such microorganisms are of the genus Strepto
fermentative biosynthesis may be used. Insofar as I am
myces. The species S. aureofaciens, which produces
The present invention depends for its effectiveness in 70 chlortetracycline in fermentation media in which chloride
ions are present as well as numerous natural and induced
producing high yields of tetracycline in a fermentation
formation in some respects.
3,037,916
4
mutants is preferably used and such microorganisms will,
of course, also produce tetracycline when deprived of chlo
ride ions. A number of other tetracycline-producing
microorganisms have been mentioned in the patent litera
ture as alleged distinct species ofStreptomyces such S.
viridifaciens, S. sayamaensis, VS. feafaciens, and still
others. The published morphological data onv these mi
croorganisms is insut?cient" conclusively to determine
this invention. In such instances it has been found that
even less of the organic chlorination inhibitor is required
to reduce the cblortetracycline to a given level when
bromide is added. Consequently, as little as one part per
million and in some cases even less of the azole su?ices
to reduce the chlortetracyeline to the desired degree when
bromide is added. Accordingly, bromide ion may be
used in amounts ranging from as little as one part per
whether or not they are new species or merely strains of
S. aureofaciens. Regardless of this, however, this aspect
of the present invention is not predicated upon the selec
tion of a particular species, but as indicated above, is’
,
mentation along with the Z-substituted-1,3,4-azoles of
10 million to '6000—7000 parts per million if desired.
It is a further advantage of this invention that the
' chlorination inhibitors make it possible to use a wide
variety of strains of S. aureofaeiens. That is to say, that
concerned with the use of chlorination inhibitors which
direct the activities ‘of the microorganism from the syn- .
with the chloride deprivation systems oftentimes the high
thesis of chlortetracycline to the synthesis of tetracycline. 15 est tetracycline-producing strains, that is the chloride
scavenging strains, could not be used because they also
Consequently, this aspect of the present invention in
produced a high level of chlortetracycline. With this
cludes any microorganism which produces chlortetracy
cline and tetracycline.
‘ a
invention, however, such strains may be used with facility
as the chlorination inhibitors make it possible to easily
The chlorination inhibitors of this invention for use in
shift the equilibrium in favor of tetracycline.
the fermentative biosynthesis of tetracycline and demetha
The conditions of the fermentation for both tetra
yltetracyeline are certain 2,5 -substituted-l,3,4-azoles which
cycline and demethyltetracycline are generally the same
as the presently known methods of producing tetracycline
or chlortetracycline by fermentation. That is, the fer
may be represented by the following general formula:
.lxl.
25 mentation medium contains the usual nutrients and min
eral substances. Suitable nutrient substances which may
provide those necessary substances include starch, dex
wherein X is a member of the group'consisting ‘of NH,‘
, S and O; and R1 is a member of the group consisting of I "
SH, SR, SOR, 802R, SO2NH2, bromine and chlorine,
trose, cane sugar, glucose, molasses, soybean meal, peanut
meal, yeast, meat extracts, peptone, ammonium sulfate,
urea, corn steep liquor, distillers'solubles, fish meal and
wherein R is a non-reactive, non-toxic, non-interfering 30 other conventional substances. The inorganic salts in
radical such as an alkyl radical, i.e., methyl, ethyl, pro_
pyl, butyl, etc., or a monoeyclic aryl radical, i.e., phenyl,
chlorophenyl, tolyl, etc., or a monocyclie aralkyl radical,
clude such things as calcium carbonate, ammonium sul
fate, ammonium chloride, sodium dihydrogen phosphate,
and the various trace elements such as manganese, cobalt,
i.e., benzyl, phenethyl, phenylpropyl, phenylbutyl, etc.;
zinc, copper, iron, and the like.
and wherein R2 is a member‘ of the group consisting of 35 The other general conditions of the fermentation, such
hydrogen, SH; SR, SOR, 802R, sOgNl-lgbromine and
as hydrogen ion concentration, temperature, time, rate
chlorine, and wherein R has the same meaning as above,
of aeratiompreparation of the inoculum, sterilization,
amino, lower alkylamino such as methylaminm'ethyl- "
amino, propylamino, etc., allylamino, monocyelie aryl
amino such phenylamino, chlorophenylamino, etc., mono
cyclic aralkylamino such as benzylamino, acylamino such
as acetylamino, propionylamino, butyryla'mino, etc., lower
inoculation and the like are conventional and may be
40 similar to those for the production of chlortetracycline
alkyl such as methyl, ethyl, propyl, butyl, etc., monocyclic '
aryl such as phenyl and substituted phenyl, i.e., chloro—
shown in the US. patent to Duggar No. 2,482,055, and
for the production of tetracycline shown in the U.S.
patent to Minieri et al. No. 2,734,018. For the produc—
tion of demethyltetracycline, the selection of a suitable
mutant strain of S. aureofaciens is necessary as described
phenyl, methoxyphenyl, etc., monocyclic aralkyl such as 45 hereinabove.
benzyl, phenethyl, phenylpropyl, phenylbutyl, . etc., and
Similarly, the vrecovery of the tetracycline from the
heterocyclic radicals ‘such as furyLthiazolyl and pyridyl.
fermentation liquor is conventional and need not be
Among the particularly useful compounds for carrying
described, as numerous methods of recovering tetra
out the present invention there may be mentioned,
cycline from fermentation liquors have been published.
5 - amino - 2 - mercapto - 1,3,4 - thiadiazole,
2,5 - dimer~
capto - 1,3,4 -' thiadiazoleJZ - phenyl - 5 4 merc'apto - 1,3,4
oxadiazole, 2-‘(2-furyl)-5-mercapto-l,3,.4-oxadiazole, 2
benz‘yl-mereapto-1,3,4—thiadiazole as well as various other
compounds of this invention as will be evident from an
In the examples which follow the yields of tetracycline
and chlortetraeyeline are expressedvas gammas per milli
liter (FY/ml).
In the examples that follow, the demethylchlortetracy~
. eline and demethyltetracycline content’of inhibited mashes
inspection of the detailed examples appearing herein 55 is demonstrated by three methods.
after.
i
'
'
'
i
_
i As in the case of the use of bromide ions in depressing
fermentative chlorination the inhibitors of the present '
invention may be somewhat toxic to the microorganism,
especially when used in high concentrations.‘Fortunately,
Paper chromatog
raphy using a butanol/ 0.3 M pH 3 phosphate butter sys
tem effects separation of the two antibiotics, and permits
a semiquantitative estimate of the relative amounts of
' each. Small amounts of tetracycline and chlortetraey
60 cline are usually present, but do not interfere. The sec
very small amounts of the inhibitors of this invention
ond method‘involves measurement of biological activity
depress the formation of chlortetracycline very markedly.
against both E. coli and S. aureus.
.As little as one part per million shows some effect.
demethylchlortetracycline and demethyltetracycline is
In
E. coli response to
general, it has been found that the inhibitors may be
about equal, while the response of S. aureus to demethyl
added in amounts ranging from about one part per mil‘ 65 chlortetracycline is 3—4 times the response to demethyl
lion to 500 parts per million, with the optimum range
tetracycline. Therefore, the ratio of E. coli/S. aureus
being about ?ve- parts per million to about 100 parts per
will increase as the demethyltetracyclinc/demethylehlor
million. Generally speaking, the more inhibitor that is
tetracycline ratio increases. The third assay method is
added, the less chlortetracycline will be produced. On
based on the so-called Hiscox method which is described
the other hand, as continued amounts of the inhibitor 70 in detail in the aforesaid McCormick et al. application
are added the toxic effects begin to exert themselves and
and which is speci?c for demethylchlortetracycline, and
the total yield of, antibiotic is reduced. Hence, no ad
upon a spectrophotometric assay which measures both
vantage has been’ observed in going beyond about 500 7 .demethylchlortetracycline and demethyltctracycline as
parts per million and in some cases even less.
Bromide ions may also usefully be added to' the fer
. demethylchlortetracycline.
Decrease of the Hiscox assay
of‘ demcthylchlortetraeycline is accordingly directly pro
3,037,916
portional to the decrease of demethylchlortetracycline.
The excess spectrophotometric potency is presumed to
be primarily, but not entirely, due to demethyltetracy-
Table 2-Continued
cline.
Compound
p.p.m.
The invention will be described in greater detail in con- 5
Ohlor-
Tetra~
teiiiita-
cycgltlle, Tei?ra
czfmIll‘e,
Percent
7/ -
0Y0 we
Junction with the following speci?c examples.
Z-anilino-S-mercapto-
EXAMPLE 1
.
13451555152015 ______ __
.
.
A chlortetracyolme fermentation med-rum, such as may 10
z'icePgilglilwé?-“mefcap'
Starch ______________________________ __d0____
47
2-bepzy1merc?pt0-1,3,+
com steep liquor _____________________ __do____
CaCO ____' _______________________ __ do
25 15
thladlmle ----------- -9
2-(2-thiazolyl)-5-mer~
(151151,)3 so
'
2 .
.'"' 5
2
0""
..
~
11111 chlonde
__________ __gI'amS
per Men.
5
.
‘ 1 1 ‘
tmadmmle ----------- --
2_methy1memapto_1,3,4
thiadiawle ----------- --
30
2-acetylamino-5-benzyl-
me
1
-
rcapto-1,3,4-th1ad1a-
Z019
Ohlortetra-
eycline,
cycline,
Tetra-
Percent
'y/ml-
'Y/ml-
cyclille
‘
Tetra-
-
_
0
5, 575
4,075
92.3
10
1; 175
20
445
50
215
58
6’ 628
4
1°58
100
200
110
50
4,325
4’325
07.5
97'5
6’ 122
153
6, 93g
6, Z15570
98-9
2,5aDimercapto-l,3,4~
40%
200
7 3%,”,
60
5'26“
5,560
99.0
99' 2
111155151515 _______ ._
25
’ 0
7,300
50
75
100
255
190
6, 980
7, 025
150
5, 750
_
11
-
_
2,5diazole
dibromo 1,3,4-thia
""" "
95 5
97. 4
97. 4
82: 6
94-9
2' Z53
5g‘?
2, 2305
0,250
’s52
12.1
0
6,250
852
12.1
v
'
5
2, 500
2, 410
48.1
10
25
1 005
4,920
5, 500
83.0
2
W28
5
5147(5)
» 575
m5
10
400
51200
92.9
23
5, 028
‘5’288
i812
5
3, 775
802
17.5
22
0
31.1.33
5,858
i; gig
5;?“
3212
lg-g
13
2280
1 as;
3816
25
0
5
10
5 050
four)
’
2’ 700
'
1, 150
1’445
’
2 590
740
’
3, 3410
59-1
10.5
50 3
~
73. s
620
31670
85'7
18
i’
25
3: 500
1, 020
22. 5
615
11. 5
5
11‘ 5
_
‘
5, 350
$15)
0
fonylmethane ........ ._
100
.
83'?
25
5 ‘iieigléliig??‘fis'gf
35
Control ____________ __
55.5
1'
5‘zzla‘gtgf’l‘gffg?gldrfggleu
tetracycllne contents. The results obtained are set forth
Table 1
_,
71 1
1, 310
0
--
’ 580
1, 550
7
2-be11zv1su1f0nY1-L3A-
and incubated at 26.5 “ C. on 1a rotary shaker for 96 hours.
They were then assayed for their chlortetracycline and
ppm.
7, 550
3 20 2'aégllglgi5gl?gagligrz'ole
with vegetative moculum of S. aureofaciens (strain S77)
Compound
0
18
1
1.7
The media were disP ensed
-
,
capto-1,3,4-thiadiazole-.
in appropriate amounts into ?asks, sterilized, inoculated 25
in the table below -
“1 m0
2-methyl-5-benzyhner-
To this medium were added varying ‘amounts of 2,5d1mercapto-1,3,4-thiadiazo1e.
.
7. 1
$5.;
5
mpm-ly'o'ytomdiamlenl '
'
80
Lard 011 _________________ __percent by volume__
Ammoni
°‘ 1 1
n "50"" 5 6
. .
580
1,372
13241555155015 ______ _-{
amount of chloride ion was made up ‘as follows:
_
C 0111 11our__________________ __ grams p er 11ter__ 14 , 5
Mnso 2G0‘;
7, 550
Lggg
2-allyamino-5-mereapto-
be used on large scale production, and containing a large
C 0 Cl .46H 00) ----------- --m1111grams Perdbier"
0
1g
5, 000
0
590
10. 5
Z
4, 750
5
2’ 505
2, 330
l
0
4 750
{
25
41500
835
1513
{
0
1°
4, 750
2» 15°
515
7°
11. 5
3- 15
’
49_ 2
EXAMPLE 3
EXAMPLE 2
The procedure of Example 2 was repeated except that
All of the runs reported below were carried out in 45 smaller quantities of the inhibtiors appearing below were
shaker ?asks at 26—27° C. on a rotary shaker operating
used. The results obtained are set forth in the table
at 184 r.p.m. The medium, inoculum ‘and harvest time
below:
were the same as described in Example 1, the only dif
Table 3
ference being the addition of di?erent azoles to the fer
mentation. The results obtained are set forth in the
table below.
Table 2
Compound
5-amino~2emercapto1,3,4-thlad1azole ______ __
3~n1ercapto-1,2,4—triaz0le__ {
2-(4pyridyD-5-Inereapto»1,3,4-oxadiazole ____ -_
'
2-phenyl-5-mercapto1,3,4-oxadiazole _______ --
2~p-chloropheny1-5-mercap to-1,3,4-oxadiazole _ _
p.p.n1.
Chlor-
Tetra-
tetra-
eycline,
cyeline,
7/1111
'ylml.
Percent
Tetra
cycline
g-phenyl-?-mewavm1,3,4-oxad1azole ...... __
Tetraeycline,
cyclin
'y/ml.
'y/
.
Percent
Tetra
cycline
0
8, 700
555
6.0
53
2%?
210
11533
4, 160
351%
95‘ 2
96. 5
97. 1
4
5
140
100
3, 985
3, 370
6 225
6:
94: 0
.
0
8, 700
555
6. 0
100
360
5, 356
84. 0 60 2—(2-furyl)-5-mereapto-
5
6
500
390
6, 860
v7, 180
932 2
94. B
0
7, 550
580
7. 1
20
50
1,040
475
5,850
4, 440
84. 9
90. 3
0
10g
4’
125g
7, 550
580
7. 1
5
170
6, 100
97. 2
10
40
3, 920
98. 9
0
7, 550
580
7. 1
4, 405
2, 155
32. 8
780
2, 965
79.1
g
7’ 23g
6’ 228
9;: %
Ls?madmmle ------- --
10
150
6, 505
97. 7
Z-ethylamino-S-mercapto-1,3,4~thiadiazole_ ____
40
0
5
20
7, 550
2, 540
1, 780
580
2, 130
98. 8
7. 1
45. 8
-
18
7’ 548
55
1,578
58
74.1%
50
225
3, 475
93.9
capto-1,3,4~thiadiazole__ {
55
Chlor' tetra-
7’ 375
5
2-methylam1no-5-mer-
p.p.m.
,
2g
50
10
2-(2-furyl)-5-mercapto-
Compound
7.
1,3,4-oxadiazole ______ __
2-benzylmercapto-1,3,4-
65
thiadiazole ___________ __
7
335
7,
95. 6
8
9
10
0
1
275
245
240
6, 850
5, 675
7, 520
7, 620
7, 540
805
1, 375
96. 5
96. 9
96. 9
10. 5
3
2, 605
2, 955
53. 1
5
10
1, 000
450
4, 015
5. 080
80. 5
91. 9
19. 5
EXAMPLE 4
7.0
The following is a tabulation of the combined effect
of bromide and Z-(Z-furyl)-5-mercapto-l,3,4~oxadiozole.
It can be seen that less oxadiazole is required to reduce
the chlortetracycline to a given level when bromide is
75 added.
3,037,916
POTASSIUM BROMIDE
[Grams per liter]
.
o
Oxadiazole, p.p.m.
'
'
,
1.0
2.5
4.0
'
'
CTO
T0
are
5, 200 ,
1,025
07s
3,210
3,750
4,080
3, 800
505
665
365
200
GTC
TC
2, 075
2, 050
26
2, 905
3,700
155
100
3,960
3,995
3,735
4,240
4,240
265
190
4, 260
TC
160
4, 540
90
OTC
r
1, 915
155
105
a, 100
EXAMPLE 5
as
'
3, e10
_
'
. Fermentation of S. aureofaciens stram V138 was car'
2, 890
3,000
' 3,720
3,790 -
15
'
-
TC
'-
_.
.
Spec.
.p.p.m. Z-(Z-igrgg-S‘ZIliSreaptQ-LS,47
-
-
r1ed out at 26.5“ C. ,for 120 hours in the same medium
'
‘
_
x
iaz
'
used for chloitetracycline fermentation (Example 1),
'
'
.
-
Hrmgtm ,etmgyeme
tetracycline
containing various levels of 2-pheny1-5-mercapto-1,3,4~
oxadiazole.
G-de-
laegggltélliilgg- mlggictiléglté}
'~
1 152
‘
--
Paper chromatography of the‘mashes thus obtained
-------------- "5 ------- --
5-----V-----.- ------- --. -------------------- --
'7
278
' 980
0
78°
0-56
shows the following:
this example, the, addition of KBr at levels of 1.0
0 p.p.m.+16-demethylchlortetnacycline spot larger than' 25 4.0Ingrams
per liter, with and without inhibitor, shows no
6-demethyltetracycline
effect except'a decrease of total potency.
2 p.p.m.—6-demethylchlortetracycline spot faint, 6-de
methyltetracycline spot increased
i
10 p.p.m.—-6-demethylchlortetracycline spot faint, ~6-de-»
methyltetracycline- spot increased compared to control. 30
Microbiological assays gave the following results:
EXAMPLE 7'8
Fermentation of strain $604 was carried out as in
Example}, using 50 ppm. of 2,5-dimercapto-1,3,4
thiadiazole as an inhibitor.‘ Paper strips show increased
G-demethyltetracycline and decreased 6-demethylchlor
ppm. 2-phenyl-5-meré
'y/ml.
'y/ml.
E. colt/S.
capto~l,3,4-oxadiazole
S. aureus
E. coli
aureus
tetracycline as compared to the control.
(35
405
1,095
151
641
4.2
EXAMPLE 9
2. 7 V.
Example 8 was repeated, extending the fermentation
to 140 hours. Paper'strips show G-demethylchlortetra
40 cycline and G-demethyltetracycline in the control; in
creased 6-demethyltetracycline and no é-demethylchlor
tetracycline at '50 and 100 ppm. of 2,5-dimercapto-1,3,4~
EXAMPLE, ‘6
thiadiazole.
Example 5 was repeated using 2-(2-furyl)-5-meroapto
This application is va continuation-in-part of my ap
' ' 68
61
24
'
364
5.3
V 344
5.6
124
5.2
1,3,4-0Xadiazole as the inhibitor.
7
plication Serial No. 567,440, ?led February 24,1956, now
Paper chromatographic results were substantially iden 45 abandoned.
'tical to ExampleS:
'
'
a
I claim:
a
1. In a process of producing a compound selected from
0 p.p.m.--6-demethylchlortetracycline spot larger than’
the group consisting of tetracycline and demethyltetra
'6-demethyltetracycline
5 p.p.m.--Faint 6-demethylohlortetracycline spot, 6-de 50 'cycline by aerobic fermentation of an aqueous fermenta
methyltetracycline spot larger than control
20 p.p.rn.-Faint 6-demethylchlortetracycline spot, 6-de
tion medium with a microorganism selected from the
group consisting of a tetracycline-producing microor
, methyltetnacycline spot larger than control.
tetracycline-producing microorganism of the genus Strep
tomyces, the improvement which comprises adding to
ganism of the genus Streptomyces and a demethylchlor
Microbiological assays were as follows:
said medium a small but effective amount of a
ppm. 2-(2-Iuryl)~5-
V
mercapto-l,3,4-oxadiazole
'y/ml.
71ml. ‘
E. colt/S.
S. aureus
E. 6011'
aureus
405
86
63
'
45
~
chlorination inhibitor of the formula:
I, 095
466
2. 7
5. 6
'356
5.6
192
4. 3
60
Rllzlfh V .
wherein X is a member of the group consisting of NH,
EXAMPLE 7
65 S and 0; R1 is a member of the group consisting of SH,
Example 6 was repeated, extending the fermentation
to 140 hours.
'
Paper chromatography:
0 p.p.m.—6-demethylchlortetracycline and 6-demethy1 70
tetracycline spots present
'
.2 p.p.m.'—Faint G-demethylchlortetracycline spot, '6-de
methyltetracycline spot increased
. '
SR, SOR, 802R, SO2NH2, bromine and chlorine, wherein
R is a non-reactive, non-toxic, non-interfering, radical,
and R2 is a member of the group consisting of hydrogen,
SH, SR, SOR, 802R, SO2NH2, bromine and chlorine,
wherein -R ‘has the same meaning ‘as hereinbefore de
?ned, amino, lower alkylamino, allylamino, monocyclic
arylarnino, monocyclic aralkylamino, acylamino, lower
alkyl, monocyclic aryl, monocyclic aralkyl, furyl, thiazolyl
5 ppm-Faint 6-demethylchlortetracycline spot, 6-de
‘and pyridyl radicals, which inhibits the formation of
methyltetracycline spot increased.
75 chlortetracycline and demethylchlortetracycline and
3,037,916 -
9
1%
causes the formation of substantial quantities of tetra
SH, SR, SOR, 802R, SOZNHQ, bromine and chlorine,
cycline and demethyltetracycline.
wherein R has the same meaning as hereinbefore de
?ned, amino, lower alkylamino, allylamino, monocyclic
arylamino, monocyclic aralkylamino, acylamino, lower
alkyl, monocyclic aryl, monocyclic aralkyl, furyl, thiazol
2. In a process of producing tetracycline by aerobic
fermentation of an aqueous fermentation medium with
a tetracycline-producing strain of S. aureofacz‘ens, the im
provement which comprises adding to said medium a
small but eifective amount of a chlorination inhibitor
of the formula:
yl and pyridyl radicals, which inhibits the formation of
chlortetracycline and causes the formation of substantial
quantities of tetracycline.
10. A process of producing demethyltetracycline by
N--N
Riga
10 aerobic fermentation of a chloride-containing aqueous
fermentation medium with a demethylchlortetracycline
producing strain of S. aureofaciens which comprises the
step of adding to said medium from about 1 to 500
parts per million of a chlorination inhibitor of the
formula:
15
SR, SOR, 502R, SO2NH2, bromine and chlorine wherein
R is a non-reactive, non-toxic, non—interfering radical,
and R2 is a member of the group consisting of hydrogen,
wherein X is a member of the group consisting of NH,,
S and 0; R1 is a member of the group consisting of SH,
Kiln.
SH, SR, SOR, SOZR, SOZNHZ, bromine and chlorine,
wherein R has the same meaning as hereinbefore de
?ned, amino, lower alkylamino, allylamino, monocyclic
arylamino, monocyclic aralkylamino, acylamino, lower
20
alkyl, monocyclic aryl, monocyclic aralkyl, furyl, thiazo yl and pridyl radicals, which inhibits the formation of
wherein X is a member of the group consisting of NH,
S and O; and R1 is a member of the group consisting of
SH, SR, SOR, 802R, SOZNHZ, bromine and chlorine,
wherein R is a non-reactive, non-toxic, non-interfering
chlorten-acycline and causes the formation of substantial
radical, and R2 is a member of the group consisting of
25
quantities of tetracycline.
hydrogen, SH, SR, SOR, 802R, SOZNHZ, bromine and
3. In a process of producing demethyltetracycline by
chlorine, wherein R has the same meaning as hereinbe
aerobic fermentation of an aqueous fermentation medium
with a demethylchlortetracycline-producing strain of
S. aureofaciens, the improvement which comprises adding
to said medium a small but effective amount of a chlori
. fore de?ned, amino, lower alkylamino, allylamino, mono
30
nation inhibitor of the formula:
cyclic arylarnino, monocyclic aralkylamino, acylamino,
lower alkyl, monocyclic aryl, monocyclic aralkyl, furyl,
thiazolyl and pyridyl radicals, which inhibits the forma
tion of demethylchlortetracycline and causes the forma
tion of substantial quantities of demethyltetracycline.
11. A process according to claim 9 in which the
35 amount of chlorination inhibitor is added to the extent
of from about 5 parts per millions to about 100 parts per
million.
wherein X is a member of the group consisting of NH,
12. A process according to claim 9 in which the chlori
S and 0; R1 is a member of the group consisting of SH,
nation inhibitor is 2,5-dimercapto-1,3,4-thiadiazole.
SR, SOR, 802R, SO2NH2, bromine and chlorine, where
13. A process according to claim 9 in which the
in R is a non-reactive, non-toxic, non-interfering radi 40
chlorination inhibitor is S-annno-Z-mercapto-1,3,4-thia
cal, and R2 is a member of the group consisting of
diazole.
hydrogen, SH, SR, SO11, SOZR, SO2NH2, bromine and
14. A process according to claim 9 in which the chloriq
chlorine, wherein R has the same meaning as hereinbe
nation inhibitor is Z-phenyl-S-mercapto-1,3,4-oxadiazole.
fore de?ned, amino, lower alkylamino, allylamino, mono
15. A process according to claim 9 in which the chlori
cyclic arylamino, monocyclic ara‘lkylamino, acylamino,
45 nation inhibitor is 2-(2-furyl)-5-mercapto-1,3,4-oxadi~
azole.
thiazolyl and pyridyl radicals, which inhibits the forma
lower alkyl, monocyclic aryl, monocyclic aralkyl, furyl,
16. A process according to claim 9 in which the chlori
tion of demethylchlortetracycline and causes the forma
nation inhibitor is 2-benzylmercapto-1,3,4-thiadiazole.
tion of substantial quantities of demethyltetracycline.
17. A process according to claim 9 in which the fer
4. A process according to claim 1 in which the chlori 50
mentation medium also contains at least about one part
nation inhibitor is 2,5,dimercapto-1,3,4-thiadiazole.
per million of bromide ions.
5. A process according to claim 1 in which the chlori
18. In a process for producing tetracycline by aerobic
nation inhibitor is 5-amino-2-mercapto-1,3,4-thiadiazo1e.
fermentation with a chlortetracycline-producing micro—
6. A process according to claim 1 in which the chlori
nation inhibitor is 2-phenyl-5-mercapto-1,3,4-oxadiazole. 55 organism of the genus Streptomyces, the improvement
which comprises conducting the fermentation in the
7. A process according to claim 1 in which the chlori
presence of an amount of 2,5-dimercapto-1,3,4-thiadi
azole effective to suppress the production of chlortetra
nation inhibitor is 2-(2-fury-l) -5-mercapto-1,3,4-oxadi
azole.
cycline, whereby the production of tetracycline is fa
8. A process according to claim 1 in which the chlori
vored.
nation inhibitor is 2-benzy’lmercapto-1,3,4-thiadiazole.
60
9. A process of producing tetracycline by aerobic fer
References Cited in the ?le of this patent
mentation of a chloride-containing aqueous fermentation
medium with a tetracycline-producing strain of S. aureo
UNITED STATES PATENTS
faciens which comprises the step of adding to said me
2,734,018
Minieri et a1. __________ __ Feb. 7, 1956
dium from about 1 to 500 parts per million of a chlori
nation inhibitor of the formula:
65
2,739,924
Lein et al. ___________ _- Mar. 27, 1956
316,291
Switzerland __________ __ Sept. 30, 1956
FOREIGN PATENTS
* iii.
wherein X is a member of the group consisting of NH,
S and 0; R1 is a member of the group consisting of SH,
SR, SOR, SOZR, SO2NH2, bromine and chlorine, where
70
OTHER REFERENCES
Martell et al.: Chemistry of the Metal Chelate Com
pounds, Prentice Hall, Inc., Englewood Cliffs, N.J., pp.
135 to 139, 303-305, 471.
'Sekizawa: Jour. of Biochemistry, vol. 40, No. 2,
in R is a non-reactive, non-toxic, non-interfering radical,
and R2 is a member of the group consisting of hydrogen, 75 March 1955, pp. 217-218.
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