Antibiotic rubradirin B and process for preparing the same

Antibiotic rubradirin B producible by the controlled fermentation of the known microorganism Streptomyces achromogenes var. rubradiris, NRRL 3061. This antibiotic and its base-addition salts are active against various microorganisms, for example, Staphylococcus aureus, Streptococcus hemolyticus, Sarcina lutea, and Mycobacterium avium. Accordingly, they can be used in various environments to eradicate or control such microorganisms.

BACKGROUND OF THE INVENTION 
Antibiotic rubradirin and a microbiological process for its preparation, is 
disclosed in U.S. Pat. No. 3,335,057. 
BRIEF SUMMARY OF THE INVENTION 
The novel antibiotic of the invention, rubradirin B, is obtained by 
culturing Streptomyces achromogenes var. rubradiris, NRRL 3061, in an 
aqueous nutrient medium under aerobic conditions. The fermentation 
conditions disclosed in U.S. Pat. No. 3,335,057, referred to above, can be 
used to prepare rubradirin B.

DETAILED DESCRIPTION OF THE INVENTION 
Chemical and Physical Properties of Rubradirin B 
Molecular Weight: 795 (determined by field desorption mass spectroscopy). 
Elemental Analysis: Calculated for C.sub.40 H.sub.33 N.sub.3 O.sub.15 : C, 
60.38; H, 4.18; N, 5.28. Found: C, 59.25; H, 4.38; N, 5.17. 
Melting Point: &gt; 265.degree. Dec. 
Ultraviolet Absorption Spectrum 
The ultraviolet absorption maxima of rubradirin B, as reproduced in FIG. 2 
of the drawings are: 
In 0.001 N methanolic NaOH, .lambda., (.epsilon.): 248 sh (32,060), 303 
(43,450), 345 sh (17,000), 408 (11,200), 422 sh (10,550). In 0.01 N 
methanolic H.sub.2 SO.sub.4, .lambda., (.epsilon.): 240 sh (33,000), 278 
sh (25,000), 283 (25,100), 315 (33,000), 323 sh (32,500). 
Infrared Absorption Spectra 
Rubradirin B has a characteristic infrared absorption spectrum in a mineral 
oil mull as shown in FIG. 1 of the drawings. Peaks are observed at the 
following wave lengths expressed in reciprocal centimeters: 
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Band Frequency 
(Wave Numbers) Intensity 
______________________________________ 
3560 M 
3390 M 
3280 M 
3080 W 
2950 S 
2920 S 
2850 S 
2720 W 
2670 W 
2170 W 
1723 M 
1697 M 
1680 M 
1667 M 
1635 S 
1612 S 
1570 S 
1540 M 
1508 M 
1462 S 
1410 M 
1377 S 
1357 S, sh (sh=shoulder) 
1320 S 
1300 S 
1283 M 
1255 M 
1240 M 
1215 W 
1192 M 
1162 M 
1150 M 
1127 M 
1102 M 
1098 M 
1068 M 
1045 W 
1032 W 
1005 W 
967 W 
948 W 
928 W 
890 W 
880 W 
853 W 
825 W 
812 W 
805 W 
800 W 
782 W 
776 W 
760 W 
735 M 
723 W 
708 W 
698 W 
695 W 
680 W 
______________________________________ 
Key: S=Strong M=Medium and W=Weak 
Rubradirin B also has a characteristic infrared absorption spectrum when 
pressed in a KBr disc. Peaks are observed at the following wavelengths 
expressed in reciprocal centimeters: 
______________________________________ 
Band Frequency 
(Wave Numbers) Intensity 
______________________________________ 
3560 M 
3380 M 
3280 M 
3080 W 
2970 W 
2930 W 
2850 W 
2170 W 
1724 M 
1698 S 
1668 M 
1638 S 
1615 S 
1570 S 
1540 M 
1508 M 
1460 S 
1410 M 
1382 S 
1323 S 
1298 S 
1281 S 
1240 M 
1195 M 
1163 S 
1125 M 
1102 M 
1094 M 
1065 M 
1048 M 
1030 M 
1005 M 
968 M 
947 W 
927 W 
890 W 
877 W 
852 W 
825 W 
810 W 
805 W 
800 W 
792 W 
781 W 
758 M 
732 M 
708 M 
690 M 
680 M 
______________________________________ 
Solubilities 
The novel compound of the invention is soluble in aqueous bases above pH 
7.5 and insoluble in water below pH 6.0. It is also soluble in lower alkyl 
amides such as dimethylformamide and dimethylacetamide, and in 
dimethylsulfoxide and ethyl acetate which is saturated with water. It is 
very slightly soluble in lower alcohols (methanol and ethanol), 
chloroform, and tetrahydrofuran. It is insoluble in hydrocarbon solvents 
such as benzene, toluene, and the alkanes (pentane through the higher 
alkanes). 
Antibacterial Spectrum of Rubradirin B 
Rubradirin B shows the following zones of inhibition in millimeters (mm) on 
a standard disc plate assay (12.7 mm assay discs) at a concentration of 
0.5 mg/ml. 
______________________________________ 
Zone of 
Microorganism Inhibition 
______________________________________ 
Staphylococcus aureus 26 
Sarcina lutea 29 
Mycobacterium avium 25 
Bacillus subtilis 0 
______________________________________ 
On testing rubradirin B by a microplate broth dilution assay using the 
medium BHI (Brain Heart Infusion), the following spectrum was observed.) 
______________________________________ 
Minimum Inhibitory 
Microorganism Concentration (mcg/ml) 
______________________________________ 
Staphylococcus aureus 284 UC 76.RTM. 
1.5 
Staphylococcus aureus UC 570.RTM. 
3.1 
Staphylococcus aureus UC 746.RTM. 
.78 
Streptococcus hemolyticus UC 152.RTM. 
6.2 
Streptococcus faecalis UC 694.RTM. 
&gt; 100 
Escherichia coli UC 45.RTM. 
&gt; 100 
Proteus vulgaris UC 93.RTM. 
&gt; 100 
Klebsiella pneumoniae UC 58.RTM. 
&gt; 100 
Salmonella schottmuelleri UC 126.RTM. 
&gt; 100 
Pseudomonas aeruginosa UC 95.RTM. 
&gt; 100 
Diplococcus pneumoniae UC 41.RTM. 
.39 
______________________________________ 
"UC.RTM." is a registered trademark of the Upjohn Company Culture 
Collection. These cultures can be obtained from The Upjohn Company in 
Kalamazoo, Mich., upon request. 
Rubradirin B was tested in vivo in mice. Mice infected with S. aureus were 
protected subcutaneously with a CD.sub.50 of 98 mg/kg. 
THE MICROORGANISM 
The microorganism used for the production of rubradirin B is the known 
microorganism Streptomyces achromogenes var. rubradiris, NRRL 3061. This 
culture is available to the public upon request to the culture repository 
at Peoria, Ill. The characteristics of this culture are disclosed in U.S. 
Pat. No. 3,335,057. Columns 2-4. 
The new compound of the invention is produced when the elaborating organism 
is grown in an aqueous nutrient medium under submerged aerobic conditions. 
It is to be understood also that for the preparation of limited amounts 
surface cultures in bottles can be employed. The organism is grown in a 
nutrient medium containing a carbon source, for example, an assimilable 
carbohydrate, and a nitrogen source, for example, an assimilable nitrogen 
compound or proteinaceous material. Preferred carbon sources include 
glucose, brown sugar, sucrose, glycerol, starch, corn starch, lactose, 
dextrin, molasses, and the like. Preferred nitrogen sources include corn 
steep liquor, yeast, autolyzed brewer's yeast with milk solids, soybean 
meal, cottonseed meal, corn meal, milk solids, pancreatic digest of 
casein, distillers' solubles, animal peptone liquors, meat and bone 
scraps, and the like. Combination of these carbon and nitrogen sources can 
be used advantageously. Trace metals, for example, zinc, magnesium, 
manganese, cobalt, iron, and the like need not be added to the 
fermentation media since tap water and unpurified ingredients are used as 
media components. Production of the compound of the invention can be 
effected at any temperature conducive to satisfactory growth of the 
microorganism, for example, between about 18.degree. and 40.degree. C. and 
preferably between about 26.degree. and 30.degree. C. Ordinarily, optimum 
production of the compound is obtained in about 2 to 10 days. The medium 
normally stays fairly close to neutral, or on the alkaline side during the 
fermentation. The final pH is dependent, in part, on the buffers present, 
if any, and in part on the initial pH of the culture medium which is 
advantageously adjusted to about pH 6-8 prior to sterilization. 
When growth is carried out in large vessels and tanks, it is preferable to 
use the vegetative form, rather than the spore form, of the microorganism 
for inoculation to avoid a pronounced lag in the production of the new 
compound and the attendant inefficient utilization of the equipment. 
Accordingly, it is desirable to produce a vegetative inoculum in a 
nutrient broth culture by inoculating the broth culture with an aliquot 
from a soil or slant culture. When a young, active, vegetative inoculum 
has thus been secured, it is transferred aseptically to large vessels or 
tanks. The medium in which the vegetative inoculum is produced can be the 
same as, or different from, that utilized for the production of the new 
compound, as long as it is such that a good growth of the microorganism is 
obtained. 
A variety of procedures can be employed in the isolation and purification 
of rubradirin B, for example, solvent extraction, liquid-liquid 
distribution in a Craig apparatus, the use of adsorbents, precipitation 
from beer at acid pH, and crystallization from solvents. Acid 
precipitation procedures are preferred for recovery inasmuch as they are 
less time consuming and less expensive, and higher recovery yields are 
obtained thereby. 
In a preferred process, rubradirin B is recovered from its culture medium 
by separation of the mycelia and undissolved solids by conventional means 
such as by filtration or centrifugation. The antibiotic is then removed 
from the filtered beer by adjusting the pH to about 4.0 with sulfuric 
acid. The precipitate which forms is removed by filtration, using a filter 
aid such as Dicalite. The cake is then eluted with acetone or ethyl 
acetate. The cake eluates are concentrated to an aqueous mixture and 
freeze-dried. The dried material is leached with acetone or ethyl acetate 
and the solvent phase is concentrated, then diluted with four volumes of 
Skellysolve B (isomeric hexanes). The rubradirin complex is filtered off 
and dried. 
Crude preparations of rubradirin B can be subjected to silica gel 
chromatography to obtain essentially pure rubradirin B. A suitable solvent 
system in this procedure can be chloroform:methanol (97:3). Alternatively, 
essentially pure rubradirin B can be obtained by subjecting a crude 
preparation of rubradirin B to chromatography on a partition column 
consisting of diatomaceous earth buffered at pH 10 with an aqueous 
solution of 0.2 M sodium carbonatebicarbonate. The column can be developed 
with a solvent system consisting of ethyl acetate:1-butanol, buffer 
(2:2:1). 
Salts of rubradirin B are formed employing the free acid of rubradirin B 
and an inorganic or organic base. The rubradirin B salts can be prepared 
as for example by suspending rubradirin B free acid in water, adding a 
dilute base until the pH of the mixture is about 7 to 8, and freeze-drying 
the mixture to provide a dried residue consisting of the rubradirin B 
salt. Rubradirin B salts which can be formed include the sodium, 
potassium, and calcium. Other salts of rubradirin B including those with 
organic bases such as primary, secondary, and tertiary mono-, di-, and 
poly-amines can also be formed using the above-described or other commonly 
employed procedures. 
The new compound of the invention, rubradirin B, inhibits the growth of the 
following organisms: Staphylococcus aureus, Diplococcus pneumoniae, 
Sarcina lutea, Mycobacterium avium, and Streptococcus hemolyticus. 
Accordingly, the new compound can be used as a disinfectant on various 
dental and medical equipment contaminated with Staphlococcus aureus; it 
can also be used as a disinfectant on washed and stacked food utensils 
contaminated with this organism. Rubradirin B also can be used to control 
Mycobacterium avium which is a known producer of generalized tuberculosis 
in birds and rabbits. 
The following examples are illustrative of the process and products of the 
present invention, but are not to be construed as limiting. All 
percentages are by weight, and solvent mixture proportions are by volume 
unless otherwise noted. 
EXAMPLE 1 
A. fermentation 
An agar slant of Streptomyces achromogenes var. rubradiris, NRRL 3061, is 
used to inoculate a series of 500-ml Erlenmeyer flasks each containing 100 
ml of sterile seed medium consisting of the following ingredients: 
______________________________________ 
Glucose monohydrate 25 g/liter 
Pharmamedia* 40 g/liter 
Tap water q.s. 1 liter 
______________________________________ 
*Pharmamedia is an industrial grade of cottonseed flour produced by 
Traders Oil Mill Company, Fort Worth, Texas. 
The flasks are incubated for 3 days at 28.degree. C. on a Gump rotary 
shaker operating at 250 r.p.m. 
Seed inoculum (5%), prepared as described above, is used to inoculate a 
series of 500-ml Erlenmeyer flasks each containing 100 ml of sterile 
fermentation medium consisting of the following ingredients: 
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Starch 10 g/liter 
Corn steep liquor 20 g/liter 
Distiller' solubles 15 g/liter 
Mg (NO.sub.3).sub.2 .6H.sub.2 O 
3.8 g/liter 
Tap water q.s. 1 liter 
______________________________________ 
The fermentation medium presterilization pH is 7.2. 
The fermentation flasks are incubated at 28.degree. C. on a Gump rotary 
shaker operating at 250 r.p.m. The fermentation flasks are harvested after 
about 3 to 4 days. A typical shake flask fermentation is depicted below. 
The assay is against the microorganism Sarcina lutea. It is a disc plate 
assay using 0.1 M phosphate buffer, pH 7.85, as diluent. 
______________________________________ 
Day Assay, Biounit/ml 
______________________________________ 
1 trace 
2 104 
3 160 
4 64 
______________________________________ 
NOTE: One Biounit corresponds to the dilution factor of the sample to 
yield an inhibition zone of 20 mm. 
B. recovery 
Whole broth from a fermentation, as described above, is slurried with 4 
percent of its weight of diatomaceous earth and filtered. The filter cake 
is washed with 1/10 volume of water and the wash is added to the clear 
beer. The clear beer is adjusted to pH 4.0 with 6 N sulfuric acid and 
filtered with the aid of Dicalite. The spent beer is discarded. The wet 
cake is leached with ethyl acetate and the solvent phase is then 
concentrated to an aqueous phase. The latter is freeze-dried. The residue 
is dissolved in ethyl acetate and diluted with 4 volumes of Skellysolve B. 
The precipitate which is collected and dried contains a mixture including 
rubradirin and rubradirin B. 
c. purification 
A one gram quantity of crude preparation containing rubradirin B, prepared 
as described above, is chromatographed on 500 g of silica gel G (70-230 
mesh, E. Merck), buffered at pH 5.8. The first elution with 1500 ml of 
chloroform is discarded. Thereafter 20 ml fractions are collected. Tubes 
201 to 470 contain rubradirin by tlc (thin layer chromatography). The 
elution solvent is changed to chloroform:methanol (97:3). Tubes 471-510 
contain a mixture of rubradirin and rubradirin B. The solids in this 
fraction are isolated by concentration and precipitation in Skellysolve B, 
310 mg. 
The combined solids from the above chromatography and 2 similar ones, 660 
mg total, are then dissolved and suspended in 30 ml of chloroform, and 
this is stirred for 1 hour and filtered. The semicrystalline precipitate, 
160 mg, is found to be essentially pure rubradirin B by tlc. 
The tlc is run on Eastman silica gel (#6060) sheets with the solvent system 
ethyl acetate-acetone-water (8:5:1) and bioautographed on trays seeded 
with S. lutea. Approximately 0.5 .gamma. of line product preparations and 
correspondingly lesser amounts of higher purity preparations are applied 
for analyses. 
Preparations are assayed after they have been adjusted to pH 3.0 and dried 
in vacuum. Dilutions are made in methanol and a quantity of .08 ml is 
applied to 12.7 mm assay discs which are dried and placed on agar trays 
seeded with S. lutea. Assays are expressed as biounits. 
EXAMPLE 2 
Sodium Salt Of Rubradirin B 
Twenty-five mg of rubradirin B as prepared in Example 1 is dissolved in 
several drops of acetone. To this solution is added 0.5 ml of water and 1 
drop of 6 N sodium hydroxide, followed by the addition of sufficient ether 
to precipitate the sodium salt of rubradirin B. 
The tentative structure of rubradirin B can be shown as follows: 
##STR1##