Antibiotic LL-D42067.alpha. derived by aerobic fermentation of the microorganism Actinomadura madurae subspecies simaoensis NRRL 15734, useful as an antibacterial and antiparasitic agent.

SUMMARY OF THE INVENTION 
This invention relates to a new antibacterial and antiparasitic agent 
designated LL-D42067.alpha., to its production by fermentation, to methods 
for its recovery and concentration from crude solutions and to processes 
for its purification. The present invention includes within its scope the 
biologically pure culture of the antibiotic. 
The structure and relative stereochemistry of LL-D42067.alpha. have been 
elucidated by X-ray crystallography, and is shown below. 
##STR1## 
The physico-chemical characteristics of LL-D42067.alpha. are described 
below: 
(1) Molecular weight: 535 (FAB-MS); 
(2) Molecular formula: C.sub.28 H.sub.25 NO.sub.10 ; 
(3) Specific optical rotation: [.alpha.].sub.D.sup.26 =+836.sup.30 
-40.degree. (C 0.3, DMF); 
(4) Ultraviolet absorption spectra: as shown in FIG. I 
UV.sub.MAX.sup.CH.sbsp.3.sup.OH =215 nm (.epsilon. 13,200); 254 nm 
(.epsilon. 15,000); 320 nm (.epsilon. 5,100); 395 nm (.epsilon. 11,400); 
UV.sub.MAX.sup.0.1 N HCl =213 nm (.epsilon. 27,100); 253 nm (.epsilon. 
34,500); 321 nm (.epsilon. 12,200); 374 nm (.epsilon. 21,100); 389 nm 
(.epsilon. 22,900); 
UV.sub.MAX.sup.0.1 N NaOH =217 nm (.epsilon. 42,100); 253 nm (.epsilon. 
13,900); 312 nm (.epsilon. 5,700); 395 nm (.epsilon. 10,700); 
(5) Infrared absorption spectrum: as shown in FIG. II (KBr disc): 1650, 
1598, 1543, 1470, 1440, 1260, 1195, 1020 cm.sup.-1 ; 
(6) Proton nuclear magnetic resonance spectrum (CDCl.sub.3): as shown in 
FIG. III, and described in Table I; 
(7) Carbon-13 nuclear magnetic resonance spectrum (DMSO): as shown in FIG. 
IV and described in Table II; and 
(8) Proton to carbon-13 chemical shift correlation map (DMSO): as shown in 
FIG. V. 
TABLE I 
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Proton NMR Data for LL-D42067.alpha. 
.delta.* 
No. of Hydrogen** 
Multiplicity*** 
J (H) 
______________________________________ 
1.88 2 m 
2.34 2 m 
2.45 3 s 
2.58 1 m 
3.62 3 s 
3.72 1 d,d 4.64, 14.22 
3.88 3 s 
4.80 2 m 
5.08 1 m 
5.32 1 d 5.81 
5.55 1 d 5.81 
6.70 1 s 
12.76 1 s 
13.58 1 s 
______________________________________ 
*CDCl.sub.3, ppm downfield from TMS. 
**Spectrum in DMSOd.sub.6, shows two additional absorptions at 4.55(s) an 
5.91 (d) ppm. 
***s = singlet; d = doublet; t = triplet; m = multiplet. 
TABLE II 
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Carbon-13 NMR Data for LL-D42067.alpha. 
Carbon Chemical Shift (ppm)* 
Carbon Type 
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1 20.4 CH.sub.3 
2 25.4 CH.sub.2 
3 25.8 CH.sub.2 
4 29.0 CH.sub.2 
5 30.4 CH.sub.3 
6 58.5 CH.sub. 
7 61.6 CH.sub.3 
8 63.3 CH.sub. 
9 71.7 CH.sub. 
10 90.4 CH.sub.2 
11 100.0 CH.sub. 
12 109.2 q** 
13 109.7 q 
14 111.0 q 
15 113.7 q 
16 119.0 q 
17 125.8 q 
18 126.6 q 
19 134.9 q 
20 135.3 q 
21 136.1 q 
22 141.3 q 
23 147.9 q 
24 151.1 q 
25 152.5 q 
26 165.4 q 
27 165.6 q 
28 182.3 q 
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*DMSO-d.sub.6, ppm downfield from TMS. 
**q = quarternary. 
DETAILED DESCRIPTION OF THE INVENTION 
The new antibacterial agent designated LL-D42067.alpha., is formed during 
the cultivation under controlled conditions of a new strain of a new 
subspecies of Actinomadura madurae. This new strain is maintained in the 
culture collection of the Medical Research Division, American Cyanamid 
Company, Pearl River, N.Y. as culture number LL-D42067. A viable culture 
of this new microorganism has been deposited with the Patent Culture 
Collection Laboratory, Northern Regional Research Center, U.S. Department 
of Agriculture, Peoria, Ill. 61604, and has been added to its permanent 
collection. It is freely available to the public in this depository under 
its accession number NRRL 15734. 
Culture LL-D42067 was isolated from a soil sample from San Simao, Brazil. 
The culture was taxonomically characterized and was identified as a new 
subspecies of Actinomadura madurae, designated Actinomadura madurae 
subspecies simaoensis. 
Observations were made of the cultural, physiological and morphological 
features of the culture in accordance with the methods detailed by 
Shirling and Gottlieb [Intern. J. System. Bacteriol., 16:313-340 (1966)] 
and Gordon, et al. [Intern. J. System. Bacteriol., 24:54-63 (1974)]. The 
chemical composition of the cell walls of the culture was determined using 
the method of Lechevalier, et al. [Adv. Appl. Microbiol., 14:47-72 
(1971)]. Details are recorded in Tables III-V, and a general description 
of the culture is given below. Underscored descriptive colors are taken 
from Kelly and Judd [Nat. Bur. Stand., Spec. Publ., 440 (1976)] and the 
accompanying Intersociety Color Council, National Bureau of Standards 
Centroid Color Charts. 
GROWTH CHARACTERISTICS 
Table III describes the cultural characteristics of culture LL-D42067 on 
various agar media which were selected from those recommended by the 
International Streptomyces Project Committee (hereinafter referred to as 
"ISP"). 
MICROMORPHOLOGY 
Microscopic examination of the strain showed it to form short chains of 
conidia on aerial hyphae which were slightly hooked to short-spirals (up 
to three turns). The spore surfaces were smooth when observed by electron 
microscopy, distinguishing this isolate from A. verrucosopora. 
CELL WALL COMPOSITION 
Whole cell analyses showed the strain to contain meso diaminopimelic acid 
(DAP) and the sugar 3-O-methyl-D-galactose (madurose); thus it falls into 
whole cell pattern type B. The cell wall composition was of the type III 
(meso DAP, glutamic acid, alanine, muramic acid and glucosamine) and the 
phospholipid pattern of type PIV (phosphatidyl ethanolamine and/or 
methylethanolamine plus unknown glucosamine-containing phospholipids). 
These data support the assignment of the strain to the genus Actinomadura. 
The PIV phospholipid type is not typical for A. madurae, which is usually 
PI. 
PHYSIOLOGICAL REACTIONS 
The physiological reactions of strain LL-D42067 were examined using both 
the ISP system, Shirling and Gottlieb [Intern. J. Syst. Bacteriol., 
16:313-340 (1966)] and the Gordon tests, Gordon, et al. [Intern. J. Syst. 
Bacteriol., 24:54-63 (1974)]. The utilization pattern of the strain of ISP 
carbohydrate media is given in Table IV, along with those of other members 
of the genus reacting similarly. Culture LL-D42067 resembles the 
Actinomadura madurae and Actinomadura verrucosopora groups. As indicated 
above, however, it differs from Actinomadura verrucosopora in having 
smooth spore walls. A comparison of reactions in the Gordon test series of 
Actinomadura madurae (Gordon's data; see reference above) and LL-D42067, 
summarized in Table V, revealed differences only in amylase production and 
acid from glycerol and raffinose. Since amylase production and raffinose 
utilization have been found to be variable in Actinomadura madurae 
[Goodfellow, N., et al., J. Gen. Microbiol., 112:95-111 (1979)], the 
glycerol reaction remains the only physiological difference of LL-D42067 
from this taxon. 
Since strain LL-D42067 is the same as Actinomadura madurae in all 
properties evaluated except for its glycerol reaction and its PIV 
phospholipid pattern, it has been assigned to the taxon Actinomadura 
madurae as a subspecies designated Actinomadura madurae subspecies 
simaoensis. 
TABLE III 
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Cultural Characteristics of LL-D42067 Actinomadura 
madurae subspecies simaoensis on ISP Morphological Media 
Aerial Vegetative Soluble 
Agar Medium Mycelium Mycelium Pigment 
______________________________________ 
Yeast extract, Malt 
White, sparse 
Medium orange- 
None 
extract (ISP 2) brown-I53* 
Inorganic Salts 
Colorless Colorless None 
Starch (ISP 4) 
Glucose Asparagine 
Colorless Colorless None 
(ISP 5) 
Oatmeal (ISP 3) 
Sparse Light orange- 
None 
pinkish-white 
brown-I52* 
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*I = ISCC Color charts 
TABLE IV 
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Comparison of Carbohydrate Utilization Reactions of 
LL-D42067 With Related Actinomadura spp. 
A. madurae 
A. verrucosopora 
Carbohydrate 
LL-D42067 (a) (a) (b) 
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L-arabinose 
+ + + 
D-fructose 
+ + + 
I-inositol 
- variable variable 
D-mannitol 
+ + + 
raffinose 
- - - 
rhamnose + + + 
sucrose + + + 
D-xylose + + + 
______________________________________ 
(a) Goodfellow, M., et al., J. Gen. Microbiol., 112:95-111 (1979). 
(b) Nonomura, H. and O'Hara, Y., J. Ferm. Technol., 49:904-912 (1971). 
TABLE V 
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Gordon Test Reactions of LL-D42067 
A. madurae 
LL-D42067 
(Gordon Data*) 
______________________________________ 
Degradation/Transformation of 
Casein + +(98) 
Xanthine - - 
Hypoxanthine + +(98) 
Tyrosine + +(91) 
Adenine - - 
Production of 
Amylase - + 
Gelatinase + + 
Phosphatase - ND 
Nitrate Reductase + +(98) 
Urease - - 
Esculinase + +(98) 
Growth on/in 
5% Sodium Chloride 
- ND 
Salicylate - ND 
Lysozyme Broth - -(91) 
Utilization 
Acetate + + 
Benzoate - -(94) 
Citrate - +(83) 
Lactate + ND 
Malate + +(84) 
Mucate - - 
Oxalate - ND 
Propionate - ND 
Pyruvate + ND 
Succinate + +(83) 
Tartrate - - 
Growth at 
10.degree. C. - - 
45.degree. C. + -(66) 
53.degree. C. - - 
Acid from 
Adonitol + +(91) 
Arabinose + + 
Cellobiose + + 
Dextrin + ND 
Dulcitol - - 
Erythritol - - 
Fructose + ND 
Galactose + +(84) 
Glucose + + 
Glycerol - + 
Inositol - +(60) 
Lactose - +(55) 
Maltose - +(53) 
Mannitol + + 
Mannose + +(94) 
Melibiose - - 
.alpha.-Methyl-D-glucoside 
- - 
Raffinose variable - 
Rhamnose + + 
Salicin + ND 
Sorbitol - - 
Sucrose + ND 
Trehalose + +(96) 
Xylose + + 
.beta.-Methyl-D-xyloside 
+ ND 
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*Percentages of cultures showing reaction given in parentheses if not 
100%. 
ND = Not determined. 
For the production of this new antibacterial and antiparasitic agent the 
present invention is not limited to this particular organism or to 
organisms fully answering the above growth and microscopic 
characteristics, which are given for illustrative purposes only. In fact, 
it is desired and intended to include the use of naturally-occurring 
mutants of this organism as well as induced mutants produced from this 
organism by various mutagenic means known to those skilled in the art such 
as exposure to nitrogen mustard, X-ray radiation, ultraviolet radiation, 
N'-methyl-N'-nitro-N-nitrosoguanidine, actinophages and the like. It is 
also desired and intended to include inter- and intraspecific genetic 
recombinants produced by genetic techniques known to those skilled in the 
art such as, for example, conjugation, transduction and genetic 
engineering techniques. 
The in vitro antimicrobial spectrum of LL-D42067.alpha. was determined by 
the agar plate dilution method with Mueller-Hinton agar and an inoculum of 
each test organism of approximately 10.sup.4 colony forming units 
delivered by the Steers replicating device. The minimal inhibitory 
concentration (MIC) in mcg/ml was defined as the lowest concentration of 
LL-D42067.alpha. that inhibited visible growth after 18 hours incubation 
at 35.degree. C. 
The results, summarized in Table VI, show that LL-D42067.alpha. was active 
versus gram-positive bacteria and moderately active against yeasts. 
TABLE VI 
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Antimicrobial Spectrum of LL-D42067.alpha. 
Test Organism MIC (mcg/ml) 
______________________________________ 
Staphylococcus aureus 
Smith .ltoreq.0.06 
Staphylococcus aureus 
LL #14 .ltoreq.0.06 
Staphylococcus aureus 
LL #27 .ltoreq.0.06 
Staphylococcus aureus 
LL #45 .ltoreq.0.06 
Staphylococcus aureus 
ATCC 25923 .ltoreq.0.06 
Staphylococcus epidermidis 
CMC-83-56 .ltoreq.0.06 
Staphylococcus epidermidis 
ATCC 12228 .ltoreq.0.06 
Streptococcus faecalis 
ATCC 29212 .ltoreq.0.06 
Streptococcus (enterococcus sp) 
OSU-75-1 .ltoreq.0.06 
Streptococcus (enterococcus sp) 
SM-77-15 .ltoreq.0.06 
Streptococcus mutans 
ATCC 27352 .ltoreq.0.06 
Streptococcus mutans 
BHI (b) .ltoreq.0.06 
Streptococcus sanguis 
G9B (a) .ltoreq.0.06 
Micrococcus luteus 
PC 1001 .ltoreq.0.06 
Bacillus subtilis ATCC 6633 .ltoreq.0.06 
Bacillus cereus LL #4 .ltoreq.0.06 
Candida albicans CA 300 256 
Saccharomyces cerevisiae 
Y 15 32 
Escherichia coli #311 512 
Escherichia coli ATCC 25922 512 
Klebsiella pneumoniae 
AD 512 
Proteus morganii K-79-25 512 
Acinetobacter calcoaceticus 
Stfd-79-17 512 
______________________________________ 
The antibiotic LL-D42067.alpha. derives utility from its antibacterial and 
antiparasitic activities. For example, the antibiotic may be used in the 
suppression of intestinal bacterial flora, as a topical antibacterial 
agent or antiseptic against gram-positive bacteria and as a general 
disinfectant for surfaces such as instruments. It may also be useful as an 
antiprotozoal agent in the treatment of malaria. In addition to its 
antimicrobial and antiparasitic activity LL-D42067.alpha. is effective as 
an anticoccidial agent in poultry. This utility is the subject of a 
copending application for United States Letters Patent. 
In therapeutic use, the compound of this invention may be administered in 
the form of conventional pharmaceutical compositions appropriate for the 
intended use. Such compositions may be formulated so as to be suitable for 
oral or topical administration. The active ingredient may be combined in 
admixture with a nontoxic pharmaceutically acceptable carrier, which 
carrier may take a wide variety of forms depending on the form of 
preparation desired for administration, i.e., oral or topical. 
GENERAL FERMENTATION CONDITIONS 
Cultivation of Actinomadura madurae subspecies simaoensis NRRL 15734 may be 
carried out in a wide variety of liquid culture media. Media which are 
useful for the production of this novel antibiotic LL-D42067.alpha. 
include an assimilable source of carbon, such as dextrin, sucrose, 
molasses, glycerol, etc.; an assimilable source of nitrogen such as 
protein, protein hydrolysate, polypeptides, amino acids, corn steep 
liquor, etc.; and inorganic anions and cations, such as potassium, sodium, 
ammonium, calcium, sulfate, carbonate, phosphate, chloride, etc. Trace 
elements such as boron, molybdenum, copper, etc., are supplied as 
impurities of other constituents of the media. Aeration in tanks and 
bottles is supplied by forcing sterile air through or onto the surface of 
the fermenting medium. Further agitation in tanks is provided by a 
mechanical impeller. An antifoam agent such as silicone oil may be added 
as needed. 
GENERAL PROCEDURE FOR THE ISOLATION OF LL-D42067.alpha. 
The LL-D42067.alpha. antibiotic is recovered from the fermentation broth by 
filtration through diatomaceous earth, extracted into a solvent such as 
methylene chloride and purified by column chromatography on silica gel, 
using the system hexane:ethyl acetate (80:20) to remove unwanted fats and 
then methylene chloride:1% acetic acid in methanol (9:1) to give a crude 
product. 
This crude LL-D42067.alpha. is then purified by high performance liquid 
chromatography on a reverse phase column using the system 
acetonitrile:water:acetic acid (600:400:0.28).

EXAMPLE 1 
Inoculum Preparation 
A typical medium used to grow the primary inoculum was prepared according 
to the following formula: 
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Glucose 1.0% 
Dextrin 2.0% 
Yeast extract 0.5% 
N-Z Amine A .RTM..sup.1 
0.5% 
Calcium carbonate 0.1% 
Water qs 100% 
______________________________________ 
[.sup.1 A pancreatic digest of casein, registered trademark of Sheffield 
Chemical, Norwich, New York 
This medium was adjusted to pH 7.2 and then sterilized. A 100 ml portion of 
this sterile medium, in a 500 ml flask, was inoculated with mycelial 
scrapings from an agar slant of Actinomadura madurae subspecies simaoensis 
NRRL 15734. The medium was then placed on a rotary shaker and agitated 
vigorously at 210 rpm for 48-72 hours at 28.degree. C. This primary 
inoculum was then used to inoculate 12 liters of the same sterile medium 
which was then grown at 28.degree. C. for 48 hours providing secondary 
inoculum. 
EXAMPLE 2 
A fermentation medium of the following formulation was prepared: 
______________________________________ 
Sucrose 3.0% 
Soy flour 1.5% 
Corn steep liquor 0.5% 
Calcium carbonate 0.5% 
Water qs 100% 
______________________________________ 
The medium was sterilized and inoculated at the rate of 12 liters of 
secondary inoculum from Example 1 per 300 liters of medium. The 
fermentation was conducted at 28.degree. C. with a sterile air flow of 200 
liters per liter of mash per minute, agitation by an impeller operated at 
230 rpm for 135-159 hours at which time the mash was harvested and 
filtered through diatomaceous earth. 
EXAMPLE 3 
Isolation of LL-D42067.alpha. 
The fermentation filtrate from three fermentations, conducted as described 
in Example 2, were combined, making a total of 1800 liters at pH 7.5, and 
extracted with 900 liters of methylene chloride. The organic phase was 
concentrated in vacuo to give 84.1 g of residue. 
A 75.2 g portion of this residue was suspended in 300 ml of hexane:ethyl 
acetate (80:20) and allowed to seep into a glass column (2 inches.times.20 
inches) dry packed with silica gel. The column was eluted with a total of 
4 liters of the same solvent mixture in order to remove fats and silicone 
oil and was then eluted with 4 liters of methylene chloride:1% acetic acid 
in methanol (9:1) collecting 15 ml fractions. The fractions were analyzed 
by thin-layer chromatography. Antibiotic LL-D42067.alpha. appeared 
visually as a yellow spot (Rf=0.5) with the same solvent system. Fractions 
31-60, which contained most of the antibiotic, were pooled and 
concentrated in vacuo, giving 11.1 g of a red residue. 
A 5.5 g portion of the above residue was fractionated by high performance 
liquid chromatography [Prep LC System-500, Prep PAK-500/C18 cartridge, 
acetonitrile:water:acetic acid (600:400:0.28), 100 ml/minute, 5.5 g/30 
ml/injection]. Thirty 200 ml fractions were collected. Analytical high 
performance liquid chromatographic analysis of the fractions showed the 
major portion of LL-D42067.alpha. was in fraction 5. Fraction 5 was 
allowed to stand overnight. The resulting yellow crystals were collected 
by decanting off the mother liquor (which was saved), washing the crystals 
with the mobile phase and air drying, giving 11 mg of LL-D42067.alpha. as 
yellow crystals. 
The mother liquor was concentrated by slow evaporation. The resulting 
precipitate was collected by centrifugation giving 377 mg of 
LL-D42067.alpha. as a yellow amorphous solid. 
The analytical HPLC conditions were: 
______________________________________ 
Column: .mu. Bondapak C18, 3.9 mm .times. 30 cm, 
Waters Associates 
Mobile Phase: acetonitrile:water:acetic acid 
(400:600:0.28) 
Detector: UV 254 nm and UV 365 nm, 0.2 AUFS 
Flow Rate: 1.0 ml/minute 
Retention Volume of 
11.5 ml. 
LL-D42067.alpha.: 
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