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3,837,916
Patented June 5., 1962
2
medium in which it is not necessary to remove chloride
3,037,916
FERMENTATION 0F TETRACYCLKNE
18 Claims.
(Cl. 19580)
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
and 6-demethyltetracycline.
Consequently, in
3,037,916
4
.lxl.
2,5 - dimer~
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
liter (FY/ml).
In the examples that follow, the demethylchlortetracy~
. eline and demethyltetracycline contentof inhibited mashes
'
'
'
Paper chromatog
In
E. coli response to
. demethylchlortetracycline.
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.
Ohlor-
Tetra~
teiiiita-
cycgltlle, Tei?ra
czfmIlle,
Percent
7/ -
0Y0 we
EXAMPLE 1
.
13451555152015 ______ __
z'icePgilglilw?-mefcap'
47
2-bepzy1merc?pt0-1,3,+
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
_,
71 1
1, 310
--
580
1, 550
2-be11zv1su1f0nY1-L3A-
ppm.
7, 550
3 20 2'agllglgi5gl?gagligrz'ole
Compound
18
1.7
capto-1,3,4-thiadiazole-.
1 m0
2-methyl-5-benzyhner-
7. 1
$5.;
mpm-ly'o'ytomdiamlenl '
'
80
1 1
n "50"" 5 6
. .
580
1,372
Mnso 2G0;
7, 550
Lggg
2-allyamino-5-mereapto-
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
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
3,037,916
POTASSIUM BROMIDE
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
'
2, 890
3,000
' 3,720
3,790 -
15
'
TC
'-
_.
Spec.
.p.p.m. Z-(Z-igrgg-SZIliSreaptQ-LS,47
'
iaz
'
'
'
Hrmgtm ,etmgyeme
tetracycline
G-de-
laegggltlliilgg- mlggictilglt}
'~
1 152
--
'7
278
' 980
78
0-56
EXAMPLE 7'8
Fermentation of strain $604 was carried out as in
'y/ml.
'y/ml.
E. colt/S.
capto~l,3,4-oxadiazole
S. aureus
E. coli
aureus
(35
405
1,095
151
641
4.2
EXAMPLE 9
2. 7 V.
61
24
'
364
5.3
V 344
5.6
124
5.2
7
plication Serial No. 567,440, ?led February 24,1956, now
Paper chromatographic results were substantially iden 45 abandoned.
'tical to ExampleS:
'
'
I claim:
mercapto-l,3,4-oxadiazole
'y/ml.
71ml.
E. colt/S.
S. aureus
E. 6011'
aureus
405
86
63
'
45
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
'
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
. '
3,037,916 -
1%
quantities of tetracycline.
10. A process of producing demethyltetracycline by
N--N
Riga
Kiln.
20
alkyl, monocyclic aryl, monocyclic aralkyl, furyl, thiazo yl and pridyl radicals, which inhibits the formation of
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
65
2,739,924
316,291
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,
70
OTHER REFERENCES
Martell et al.: Chemistry of the Metal Chelate Com