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. 19580)

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 '60007000 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
benzyl-mereapto-1,3,4thiadiazole 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 contentof inhibited mashes

inspection of the detailed examples appearing herein 55 is demonstrated by three methods.

after.

'

'

'

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 methodinvolves 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 34 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

czfmIlle,

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'icePgilglilw?-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

0""

..
~
11111 chlonde
__________ __gI'amS
per Men.

.
1 1

tmadmmle ----------- --

2_methy1memapto_1,3,4
thiadiawle ----------- --

30

2-acetylamino-5-benzyl-

me

rcapto-1,3,4-th1ad1a-

Z019

Ohlortetra-

eycline,

cycline,

Tetra-

Percent

'y/ml-

'Y/ml-

cyclille

Tetra-

5, 575
4,075

92.3

10

1; 175

20

445

50

215

58

6 628

158

100
200

110
50

4,325
4325

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

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

6,250

852

12.1

'

2, 500

2, 410

48.1

10
25

1 005

4,920

5, 500

83.0

W28

5147(5)

575

m5

10

400

51200

92.9

23

5, 028

5288

i812

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

1445

2 590
740

3, 3410

59-1
10.5
50 3
~
73. s

620

31670

85'7

18

25

3: 500

1, 020

22. 5

615

11. 5

11 5

5, 350

$15)

fonylmethane ........ ._

100

83'?

25

5 iieiglliig??fis'gf
35

Control ____________ __

55.5

1'

5zzlagtgflgffg?gldrfggleu

tetracycllne contents. The results obtained are set forth

Table 1

_,

71 1

1, 310

--

580

1, 550

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'agllglgi5gl?gagligrz'ole

with vegetative moculum of S. aureofaciens (strain S77)

Compound

18

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.;

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"

1g

5, 000

590

10. 5

4, 750

2 505

2, 330

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 2627 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-1,2,4triaz0le__ {
2-(4pyridyD-5-Inereapto1,3,4-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

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

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

7, 550

580

7. 1

20
50

1,040
475

5,850
4, 440

84. 9
90. 3

10g

125g

7, 550

580

7. 1

170

6, 100

97. 2

10

40

3, 920

98. 9

7, 550

580

7. 1

4, 405

2, 155

32. 8

780

2, 965

79.1

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

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 ___________ __

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

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]

.

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

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-SZIliSreaptQ-LS,47

r1ed out at 26.5 C. ,for 120 hours in the same medium

'

iaz

'

used for chloitetracycline fermentation (Example 1),

'

'

Hrmgtm ,etmgyeme

tetracycline

containing various levels of 2-pheny1-5-mercapto-1,3,4~

oxadiazole.

G-de-

laegggltlliilgg- mlggictilglt}
'~

1 152

--

Paper chromatography of themashes thus obtained

-------------- "5 ------- --

5-----V-----.- ------- --. -------------------- --

'7

278

' 980

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

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-mer

'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 -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:

'

'

I claim:

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-

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 -

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. aureofaczens, 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, noninterfering 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 aralkylamino, 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-benzylmercapto-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.