Antibiotics from myxococcus

The invention relates to novel polycyclic alkaloids of formula ##STR1## in which R.sup.1 represents hydrogen or hydroxy, R.sup.2 represents hydrogen or acyl and A and B, independently of one another, each represents C.dbd.O or C--OH, and wherein the dashed line represents a C.dbd.C double bond at the position of the middle bond when A or B represents C.dbd.O, or at the position of the two outer bonds when A or B represents C--OH, a process for the manufacture of these compounds by fermentation of a novel microorganism of the species Myxococcus xanthus, the novel microorganism itself, pharmacetical preparations that contain the novel compounds, and the use of the novel compounds as antibiotics and as tumour-inhibiting agents and for the manufacture of pharmaceutical preparations.

The invention relates to novel polycyclic alkaloids having antibiotic 
properties, a process for the manufacture of these compounds by 
fermentation of a novel microorganism of the species Myxococcus xanthus, 
the novel microorganism itself, pharmaceutical preparations containing the 
novel compounds, and the use of the novel compounds as antibiotics and as 
tumour-inhibiting agents and for the manufacture of pharmaceutical 
preparations. 
The invention relates especially to compounds of the formula 
##STR2## 
in which R.sup.1 represents hydrogen or hydroxy, R.sup.2 represents 
hydrogen or acyl and A and B, independently of one another, each 
represents C.dbd.O or C--OH, and wherein the dashed line represents a 
C.dbd.C double bond at the position of the middle bond when A or B 
represents C.dbd.O, or at the position of the two outer bonds when A or B 
represents C--OH, and salts of such compounds, especially pharmaceutically 
acceptable salts. 
The compounds of formula I according to the invention are closely related 
to the compounds isolated from Streptomyces lavendulae, which are called 
saframycins. In U.S. Pat. No. 4,248,863, for example the fermentative 
preparation of the saframycins A, B, C, D and E is described and in U.S. 
Pat. No. 4,372,947 the preparation of saframycin S is described. The 
structure of the known saframycins is specified by T. Arai, Antimicrobial 
Agents and Chemotherapy 28, 5 (1985). According to this, the compounds of 
the formula I according to the invention differ from the known saframycins 
at least by the alanyl radical in the side chain, in place of which in the 
known saframycins there stands the acyl radical of pyruvic acid or another 
acyl radical, and also differ, in addition, by the meaning of the radical 
R.sup.1 and by the methoxy group at the bridged bicyclic nucleus. 
Saframycin derivatives with an alanyl radical in the side chain are 
described in European Patent Application No. EP 173 649, but differ from 
the compounds according to the invention in the meaning of the radical 
R.sup.1 and by a missing methoxy group. 
The antibiotics Y-16482 .alpha. and .beta. which are obtained by 
fermentation of a strain of Pseudomonas fluorescens, are described in 
European Patent Application No. EP 55 299. According to Y. Ikeda et al., 
J. Antibiotics 36, 1284 (1983) these compounds, also called safracins, are 
closely related to saframycins and thus also to the compounds of formula I 
according to the invention. However, the safracins differ from the novel 
compounds in their meaning of B (one of the groups B is C--H) and by the 
fact that there is no methoxy group at the bridged bicyclic nucleus. 
Other closely related compounds can be isolated from the fungus Reniera sp. 
These compounds, called renieramycins by J. M. Frinke and D. J. Faulkner, 
J. Am. Chem. Soc. 104, 265 (1982) differ from the compounds of formula I 
according to the invention at least by the side chain which, instead of 
alanylaminomethyl, is (Z)-2-methyl-2-butenoyloxymethyl (an angelic acid 
ester) in the renieramycins. 
The configuration at the chiral centers of the compounds of the formula I 
according to the invention is not known with certainty. Indications of the 
relative configuration at the various chiral centers emerge, though, from 
the "Nuclear Overhauser Effect" in proton nuclear resonance spectra and 
from the analysis of the alanine freed during hydrolysis. By analogy with 
the known absolute configuration, determined by X-ray structure analysis, 
of saframycin C (T. Arai et al., Tetrahedron Letters 1979, 2355) and of 
brominated safracin A (I. Ueda et al., Acta Cryst. C. 40, 1578 (1984), it 
is to be assumed that the compounds according to the invention have the 
spatial structure according to the formula 
##STR3## 
In compounds of formulae I and II, R.sup.2 represents hydrogen or acyl. 
Acyl R.sup.2 is especially the acyl group of a carboxylic acid or of a 
carbonic acid semiester having up to 20 carbon atoms, for example C.sub.1 
-C.sub.20 -alkanoyl, C.sub.2 -C.sub.7 -alkanoyl substituted by hydroxy, by 
oxo or by halogen, aroyl or C.sub.1 -C.sub.7 -alkoxycarbonyl. 
C.sub.1 -C.sub.20 -alkanoyl is preferably C.sub.1 -C.sub.7 -alkanoyl, for 
example formyl, acetyl, propionyl, n-butyryl, 2-methylpropionyl, 
n-pentanoyl, 2,2-dimethylpropionyl, 2-methylbutyryl, 3-methylbutyryl or 
n-hexanoyl, or straight-chained C.sub.8 -C.sub.20 -alkanoyl having an even 
number of carbon atoms, for example n-octanoyl, n-decanoyl, n-dodecanoyl, 
n-tetradecanoyl, n-hexadecanoyl, n-octadecanoyl or n-icosanoyl. C.sub.2 
-C.sub.7 -alkanoyl substituted by hydroxy, by oxo or by halo is, for 
example, trifluoroacetyl, mono-, di- or tri-chloroacetyl, glycoloyl, 
glyceroyl, lactoyl, glyoxyloyl or pyruvoyl. Aroyl is, for example, benzoyl 
or 1- or 2-naphthoyl in which the phenyl or naphthyl ring may be 
substituted by nitro, amino, halogen, for example chlorine or bromine, 
hydroxy and/or by C.sub.1 -C.sub.4 -alkoxy, for example, methoxy, for 
example 4-nitrobenzoyl, 3,5-dinitrobenzoyl, anthraniloyl, 
2,6-dichlorobenzoyl, salicyloyl, galloyl or 2-, 3- or 4-anisoyl. C.sub.1 
-C.sub.7 -alkoxycarbonyl is, for example, methoxy-, ethoxy-, n-propoxy-, 
isopropoxy-, n-butoxy-, isobutoxy- or tert.-butoxy-carbonyl. 
Preferred radical acyl R.sup.2 is C.sub.1 -C.sub.7 -alkanoyl, especially 
acetyl. 
Preferred are the compounds of formula I in which R.sup.1 represents 
hydrogen or hydroxy, R.sup.2 represents hydrogen, A represents C.dbd.O and 
B represents C.dbd.O or C--OH and the dashed line has the meaning given 
above, and pharmaceutically acceptable salts of such compounds. Also 
preferred are the compounds of formula I in which R.sup.1 represents 
hydroxy, R.sup.2 represents hydrogen, A represents C--OH and B represents 
C--OH and the dashed line has the meaning given above, and also the 
compounds of formula I in which R.sup.1 represents hydroxy, R.sup.2 
represents acetyl, A represents C.dbd.O and B represents C.dbd.O and the 
dashed line has the meaning given above, and pharmaceutically acceptable 
salts of these compounds. 
The invention relates especially to compounds of formula I in which R.sup.1 
represents hydrogen or hydroxy, R.sup.2 represents hydrogen, A represents 
C.dbd.O and B represents C--OH and the dashed line has the meaning given 
above, and pharmaceutically acceptable salts of such compounds. 
In the following, the compounds according to the invention are called 
saframycin Mx 1 (R.sup.1 =OH) and saframycin Mx 2 (R.sup.1 =H). Without 
any qualification these names indicate compounds of the formula I in which 
A is C.dbd.O and B is C--OH. With the qualification "BC" (bis-quinone) 
these names are used for compounds in which A represents C.dbd.O and B 
represents C.dbd.O. Saframycin Mx 1 BHC (bis-hydroquinone) is a compound 
of the formula I in which R.sup.1 is OH, R.sup.2 is hydrogen, A is C--OH 
and B is C--OH. The qualification "acyl" indicates corresponding compounds 
in which R.sup.2 represents acyl. 
Salts of compounds of the formula I according to the invention are 
especially pharmaceutically acceptable non-toxic acid addition salts. 
Examples of acid addition salts with non-toxic physiologically well 
tolerated acids are salts with inorganic acids, for example hydrochloric 
acid, sulphuric acid or phosphoric acid, or with organic carboxylic, 
sulphonic or sulpho acids, for example formic acid, acetic acid, propionic 
acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, 
methylmaleic acid, fumaric acid, malic acid, tartaric acid, citric acid, 
benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 
4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, 
embonic acid, nicotinic acid or isonicotinic acid, and also amino acids, 
for example .alpha.-amino acids, as well as methanesulphonic acid, 
ethanesulphonic acid, 2-hydroxyethanesulphonic acid, 
ethane-1,2-disulphonic acid, benzenesulphonic acid, 
4-methylbenzenesulphonic acid or naphthalene-2-sulphonic acid, or with 
other acidic organic compounds, such as ascorbic acid. For the purpose of 
isolation or purification it is also possible to use pharmaceutically 
unsuitable salts. 
The compounds of the formula I according to the invention and their 
pharmaceutically acceptable salts have valuable pharmacological 
properties. For example, compounds of the formula I in which A represents 
C.dbd.O are effective in low concentrations of from 0.004 .mu.g/ml to 0.3 
.mu.g/mI against various bacteria, for example against Staphylococcus 
aureus, Bacillus subtilis, Micrococcus luteus and Escherichia coli. In 
addition to antibiotic properties, the compounds also have 
tumour-inhibiting properties, for example against the experimental tumours 
leukaemia L 1210/S2, human pulmonary carcinoma MBA 9812, colon 
adenocarcinoma 26 and reticulosarcoma M5076. 
If mice with leukemia L 1210/S2 are treated with i.p. injections of 2.5 or 
5 mg/kg of saframycin Mx 2 on four successive days, then their lifespan is 
extended significantly by a factor of 1.6 or more. If treated with four 
injections of 0.625, 1.25 or 2.5 mg/kg of saframycin Mx 1 the lifespan is 
extended by a factor of 1.7 or more. Saframycin Mx 2 in 7 daily doses of 
2.5 or 5.0 mg/kg i.p. significantly reduces the weight of human pulmonary 
carcinoma MBA 9812 experimentally implanted in balb/c nude mice to 62% or 
less compared with untreated animals. Saframycin Mx 1 in 10 doses of 1.25 
mg/kg i.p. reduces the weight of colon adenocarcinoma 26 to 76% compared 
with untreated animals. After 17 doses of 1.25 mg/kg i.p. of saframycin Mx 
1 the weight of reticulosarcoma M5076 is reduced even to 33% compared with 
untreated animals. The maximum tolerable single dose of saframycin Mx 1 
and Mx 2 for mice is 12.5 mg/kg, and above 25 mg/kg, respectively. 
The invention relates also to processes for the manufacture of compounds of 
formula I. Compounds of formula I are produced by growing the strain Mx 
x48 of the species Myxococcus xanthus, or a mutant derived therefrom, that 
produces compounds of formula I, in a culture containing carbon and 
nitrogen compounds and essential inorganic salts in an easily assimilable 
form at a temperature of from 15.degree. C. to 40.degree. C. and a pH 
value of from 5 to 9 under aerobic conditions, isolating the resulting 
compounds of formula I and, if desired, converting a resulting compound of 
formula I into a different compound of formula I and/or converting a 
resulting compound into a salt and/or converting a resulting salt into the 
free compound or into a different salt. 
The invention relates also to the microorganism Myxococcus xanthus, strain 
Mx x48. It was isolated from a soil sample from Gabes Oasis, Tunisia, and 
deposited at the National Collection of Industrial and Marine Bacteria, 
Aberdeen, Scotland, under the number NCIB 12 268. 
Strain Mx x48 is able spontaneously to form natural mutants that produce 
compounds of formula I. Artificial mutants can, for example, be produced 
chemically, for example by treatment with alkylating or nitrosating 
compounds, for example N-methyl-N'-nitro-N-nitrosoguanidine or with alkali 
nitrite, such as sodium nitrite, or by irradiation, for example with 
high-energy radiation, such as ultraviolet, X-ray or radioactive 
radiation. The invention relates also to such mutants of the strain Mx x48 
that produce compounds of formula I. 
In the process for the manufacture of compounds of formula I the 
microorganism Myxocccus xanthus strain Mx x48 is grown under suitable 
conditions. 
The culture medium used for the growth must contain a carbon and nitrogen 
source and also essential inorganic salts. Suitable carbon and nitrogen 
sources are amino acids, peptides and proteins and also degradation 
products thereof, such as peptone or tryptone, and also meat extracts, 
powdered cereal, for example corn or wheat, beans, especially soya beans, 
fish meal, seeds, for example of cotton plants, distillation residues from 
the production of alcohol, yeast extracts etc. Essential inorganic salts 
contained in the nutrient solution may be, for example, chlorides, 
carbonates, sulphates, phosphates of alkali metals or alkaline earth 
metals, for example sodium, potassium, magnesium and calcium, and also 
salts of iron, zinc, manganese, molybdenum and copper. The growth is 
carried out preferably in liquid cultures, especially aqueous cultures. 
A suitable liquid culture medium is especially MD1 (peptone from casein, 
tryptically digested, 0.3%; CaCl.sub.2.2H.sub.2 O 0.05%; 
MgSO.sub.4.7H.sub.2 O 0.2%). Other suitable liquid culture media consist, 
for example, of peptone from casein 0.05%, CaCl.sub.2.2H.sub.2 O 0.05%, 
MgSO.sub.4.7H.sub.2 O 0.02%, to which there may be added, as desired, 0.5% 
single cell protein, corn steep liquor, yeast extract, amino acid 
hydrolysate of casein, or protein from fish meal. 
The culture is grown at temperatures of from 15.degree. C. to 40.degree. 
C., preferably at temperatures of from 25.degree. C. to 35.degree. C., for 
example at about 30.degree. C. Suitable pH values are from 5 to 9, 
preferably from 6.5 to 8.5. 
The growth can be carried out in stages, for example by a single or by 
several additions of nutrient solution, or can be carried out continuously 
by the continuous addition of nutrient solution. Preferably, the growth is 
in several stages, as follows: first a pre-culture is produced, for 
example in one of the mentioned culture media (inoculum), which is then, 
after about one to two days' fermentation, used to inoculate a larger 
culture, for example in a dilution ratio of from 1:10 to 1:500. This 
culture can in turn, after about two to three days' fermentation, be used 
to inoculate an even larger main culture, for example in a dilution ratio 
of from 1:10 to 1:100. 
The first pre-culture can also be obtained by several days' growth of the 
strain Mx x48 on a solid or liquid culture medium, for example agar 
containing one of the mentioned liquid culture media. Suitable culture 
media are, for example, agar 1.5%, CaCl.sub.2.2H.sub.2 O 0.1%, containing 
baker's yeast 0.5% or peptone from casein 0.3% and yeast extract 0.1%. 
The course of the fermentation can be followed analytically by sampling 
during fermentation, for example by measuring the pH value of the culture, 
which during fermentation rises from about pH 7.2 to about pH 8.0, by 
measuring the optical density which is a measure of the growth of the 
strain, gravimetrically by way of the dry weight of the biomass formed, by 
thin-layer chromatography, by reversed phase high-pressure liquid 
chromatography or by determining the antibiotic activity of the components 
contained in the culture filtrate. For example, cell-free culture 
supernatant in undiluted or diluted form can be tested in an agar 
diffusion test against Staphylococcus aureus. 
The isolation of compounds of the formula I according to the invention from 
the culture liquor is carried out according to methods known per se taking 
into consideration their chemical, physical and biological properties. 
Thin-layer chromatography, for example on silica gel with methylene 
chloride/methanol, high-pressure liquid chromatography, for example on 
reversed phase silica gel, and/or the activity against Staphylococcus 
aureus in the agar diffusion test can be used to determine the 
concentration of the compounds according to the invention in the 
individual stages of isolation. 
To isolate the compounds according to the invention from the crude 
fermentation liquor the latter is stirred, preferably for several hours, 
with macroporous non-ionic adsorber resins, for example synthetic resins 
having an aromatic basic structure, for example resins based on 
polystyrene, for example styrene/divinylbenzene copolymers. Such resins 
may be characterized by various customary statistical characteristics, for 
example pore volume, specific surface area, average pore diameter, most 
frequent pore diameter, distribution of pore sizes, distribution of 
particle sizes, and the like. Suitable adsorber resins have a pore volume 
of from about 0.5 to about 4.5 ml/g, a specific surface area of about 
100-1000 m.sup.2 /g and an average pore diameter of from about 4 to about 
130 nm, and are available, for example, under the trade names 
AMBERLITE.RTM.XAD-1, XAD-2 XAD-4, XAD-1180 and ER 180 of Rohm & Haas, 
DIAION.RTM.HP-10, HP-20, HP-21, HP-30, HP-40, HP-50 of Mitsubishi, 
DUOLITE.RTM.S-861, S-862, S-863 and ES 866 of Dia-Prosim, IMAC.RTM.Syn 46 
and Syn 72 of Akzo Chemie, KASTEL.RTM.S-111, S-112, S-114 of Montedison, 
LEWATIT.RTM.OC.1031 of Bayer and RELITE.RTM. ADS of Resindion. 
After separating the fermentation liquor from the adsorber resin, for 
example by sieving, the latter is washed with water and then eluted with 
an organic solvent or a mixture of water with an organic solvent that is 
inert towards the adsorber resin used, for example with isopropanol. The 
eluates are, if desired, treated with an organic or inorganic acid and 
concentrated in vacuo. 
It is also possible to work up the fermentation mixture in a conventional 
manner. The culture liquor is for this purpose separated from the biomass 
in a customary manner, for example by filtration or centrifugation, and 
the culture filtrate is extracted with an organic solvent that is 
immiscible or only slightly miscible with water, for example methylene 
chloride, chloroform or, preferably, ethyl acetate. Concentration by 
evaporation in vacuo yields a crude extract. By partitioning the crude 
extract in a two-phase system consisting of two organic solvents 
immiscible with one another, for example methanol/heptane, the 
fermentation products, especially the compounds of formula I according to 
the invention, become enriched in the polar phase 
The crude extracts are purified preferably by chromatographic methods, for 
example by chromatography on an ion exchanger having weakly acidic 
functional groups, for example on an ion exchanger having carboxy groups, 
by adsorption or partition chromatography and/or high-pressure liquid 
chromatography on non-polar surfaces, for example on silica gel containing 
long-chained alkyl groups ("reversed phase") or on agarose with alkyl or 
phenyl groups ("hydrophobic interaction"). Other chromatographic methods 
are also suitable, for example affinity chromatography, "fast pressure 
liquid chromatography" and the like, and also countercurrent distribution, 
for example "droplet countercurrent chromatography" or "rotational locular 
countercurrent chromatography". 
Suitable carriers for ion exchange chromatography are organic polymers, for 
example crosslinked agarose, dextran, polyacrylamide, 
styrene/divinylbenzene copolymer or cellulose. Preferably, this material 
carries weakly acidic functional groups, for example carboxy groups. 
Preferred ion exchangers are carriers of crosslinked dextran containing 
carboxyalkyl radicals, for example carboxymethyl radicals, such as are 
available, for example, under the trade name GM-Sephadex.RTM.. 
An ion exchange material having carboxy groups is pretreated in a suitable 
aqueous buffer solution, for example a buffer solution of pH 5 to pH 7, 
for example phosphate buffer pH 5. Then, the crude extracts containing the 
compounds according to the invention are added, for example by applying 
these extracts to a column containing the pretreated ion exchanger. 
Substances and impurities that have not been bound are washed out of the 
ion exchanger column with buffer solution, if necessary under pressure, 
and the desired saframycins are eluted from the column by buffer solutions 
containing an increasing concentration of salt, for example phosphate 
buffer pH 5 containing increasing concentrations of sodium chloride. The 
described ion exchange chromatography can, if desired, also be carried out 
on columns suitable for high-pressure liquid chromatography. 
Suitable carriers for adsorption and partition chromatography are, for 
example, organic polymers, for example crosslinked agarose, dextran or 
cellulose. Crosslinked dextrans that carry suitable functional groups, for 
example hydroxyalkyl groups, are preferred for the purification of the 
saframycins by adsorption and partition chromatography. Especially 
preferred is a crosslinked dextran having hydroxypropyl groups available 
under the trade name Sephadex.RTM.LH-20. 
Preferably, the extracts pre-purified by ion exchange chromatography are 
applied to a column containing a carrier having hydroxypropyl groups and 
eluted with mixtures of organic solvents of the same or a gradually 
changing composition. Preferably, methanol or mixtures of methanol with a 
further organic solvent of low polarity, for example methylene chloride, 
are used with the addition of an organic acid, for example formic or 
acetic acid, for example a methanol/methylene chloride mixture containing 
approximately 0.1% acetic acid. Like the ion exchange chromatography, it 
is also possible for the adsorption and partition chromatography to be 
carried out on columns suitable for high-pressure liquid chromatography. 
To separate compounds of formula I, extracts prepurified by ion exchange 
chromatography and/or adsorption chromatography are subjected preferably 
to a high-pressure liquid chromatography on non-polar surfaces, so-called 
"reversed phase" chromatography. The carrier used for this is preferably 
one based on silica gel carrying long-chained alkyl groups, for example 
alkyl groups of 14 to 22 carbon atoms, for example 18 carbon atoms. Such 
carriers are available, for example under the trade names 
HD-Sil.RTM.18-10-60, Nucleosil.RTM.C-18, Bio-Sil.RTM.ODS-10 or 
Hi-Pore.RTM.RP-318. The saframycin mixture to be separated is applied to a 
reversed phase high-pressure liquid chromatography column and developed in 
aqueous organic solutions containing acid, for example in mixtures of 
water with methanol, acetonitrile or tetrahydrofuran containing an organic 
sulphonic acid, for example heptanesulphonic acid, trifluoroacetic acid or 
a weak inorganic acid, for example dihydrogen phosphate. Methanol/water 
mixtures with the addition of phosphate buffer pH 6.5 are preferred. 
The purification of the crude extracts can also be carried out by 
countercurrent distribution chromatography, for example by "droplet 
countercurrent chromatography". Suitable aqueous phases are weakly acidic 
buffer solutions, for example buffer solutions of from pH 4 to pH 7, 
preferably phosphate buffer pH 5. The organic phase used is, for example, 
a chlorohydrocarbon, for example chloroform or methylene chloride, with 
which, if desired, a lower alkanol is mixed, for example n-butanol, iso- 
or n-propanol, or methanol, preferably a mixture of methylene chloride and 
isopropanol. 
A compound of formula I is converted into a different compound of formula I 
by methods known per se, for example by oxidation, by reduction or by 
acylation. 
The conversion of a hydroquinone (A and/or B represent C--OH) into the 
corresponding quinone (A and/or B represent C.dbd.O) by oxidation, and the 
conversion of a quinone into a hydroquinone by reduction, can be carried 
out, for example, analogously to the processes described in "Methoden der 
Organischen Chemie (Houben-Weyl)", 4th edition, volume VII/3a. Thieme 
Verlag Stuttgart 1977. Suitable mild oxidizing agents are, for example, 
metal salts and metal oxides, for example silver(I) oxide in diethyl 
ether, benzene or toluene in the presence of a drying agent, for example 
sodium sulphate, iron(III) salts, such as iron(III) chloride in water or 
aqueous ethanol, copper salts or thallium salts, for example thallium(III) 
trifluoroacetate. Another suitable oxidizing agent is atmospheric oxygen 
in neutral solution, for example in a buffer solution of pH 7 to pH 8. 
Suitable reducing agents are, for example, hydrogen in the presence of 
heterogeneous catalysts, for example platinum oxide or 
palladium-on-carbon, or homogeneous catalysts, for example 
tris(triphenylphosphine)rhodium(I) chloride, and also metal hydrides, for 
example borohydrides, such as sodium borohydride, or aluminium hydrides, 
such as lithium aluminium hydride, reducing salts, for example sodium 
dithionite, and also light in the presence of a hydrogen donor, for 
example normal daylight and methanol. The conversion of quinones and 
hydroquinones one into another can also be carried out by electrochemical 
methods, for example electrolysis in weakly acidic salt-containing 
solutions, for example buffer solutions of pH 4 to pH 6. 
The acylation of a compound of the formula I in which R.sup.2 represents 
hydrogen is carried out according to methods known per se, for example 
with an acid of the formula R.sup.2 --OH or with a reactive functional 
derivative of that acid. 
If a free carboxylic acid is used for the acylation, the reaction is 
customarily carried out in the presence of a suitable condensing agent, 
for example a carbodiimide, for example dicyclohexyl- or preferably 
N-ethyl-N'-3-dimethylaminopropylcarbodiimide. The condensation is 
performed in an aqueous organic solvent mixture or an aqueous buffer close 
to neutral, for example from pH 6 to pH 8, optionally while cooling or 
heating and in an inert gas atmosphere, for example under nitrogen. 
A reactive functional derivative of a carboxylic acid or of a carbonic acid 
semiester is a corresponding anhydride, for example a symmetric anhydride, 
for example acetic anhydride, a mixed anhydride of the acid R.sup.2 --OH 
with an inorganic or organic acid, for example with a hydrohalic acid, 
such as hydrochloric acid or hydrobromic acid, that is to say, for 
example, a carboxylic acid chloride or bromide or a chloroformic acid 
ester, or a mixed anhydride of a carboxylic acid with a semiester of 
carbonic acid, for example the ethyl or isobutyl semiester of carbonic 
acid. Another reactive functional derivative of a carboxylic acid or of a 
carbonic acid semiester is, for example, an activated ester, for example 
an N-hydroxyester, such as the ester with N-hydroxypiperidine, 
N-hydroxysuccinimide, N-hydroxyphthalimide or 1-hydroxybenztriazole. 
The acylation reactions with reactive functional derivatives of R.sup.2 
--OH are preferably carried out in aqueous organic homogeneous or 
two-phase solvent mixtures, if desired while cooling or gently heating, 
for example in a temperature range of from about 0.degree. C. to about 
40.degree. C., preferably at about room temperature, and optionally in an 
inert gas atmosphere, for example under nitrogen. In this case an acid 
acceptor is added, for example a suitable organic base, for example an 
amine, for example trimethylamine, triethylamine, N-methylmorpholine, 
1,5-diazabicyclo[4.3.0]non-5-ene, 1,8-diazabicyclo[5.4.0]undec-7-ene or 
pyridine, or an inorganic base, for example an alkali metal or alkaline 
earth metal hydroxide, for example sodium, potassium or calcium hydroxide, 
an alkali metal or alkaline earth metal carbonate, for example sodium 
carbonate, sodium hydrogen carbonate or calcium carbonate, or an alkali 
metal phosphate, for example sodium or potassium phosphate or hydrogen 
phosphate. Preferably, the acylation reaction is carried out in an aqueous 
buffer close to neutral, for example from pH 6 to pH 8, the buffer base 
assuming the role of acid acceptor. To inhibit the acylation of a phenolic 
hydroxy group A and/or B and, where applicable, of a hydroxy group 
R.sup.1, for example an alcohol, for example methanol or ethanol, is added 
to the aqueous organic solvent mixture. 
Salts of compounds of formula I can be manufactured in a manner known per 
se, for example by reacting the free compound with preferably 
stoichiometric amounts or a small excess of the acid forming an acid 
addition salt. 
Salts can be converted into the free compounds in customary manner, for 
example by treatment with an equimolar amount of a free base, for example 
a hydroxide, such as an alkali hydroxide, for example lithium, potassium 
orsodium hydroxide, an alkaline earth hydroxide, for example calcium 
hydroxide or magnesium hydroxide, or an ammonium hydroxide, for example 
unsubstituted ammonium hydroxide or benzyltrimethylammonium hydroxide, or 
an organic tertiary amine, for example triethylamine. It should be noted, 
however, that the free compound of formula I is only of limited stability 
and, for storage, must be converted into an acid addition salt. 
Salts of compounds of formula I are converted into other salts in a manner 
known per se. Preferably, the conversion of salts into other salts is 
carried out with ion exchangers that are charged with the desired anion, 
or by adsorption chromatography in a solvent that contains the acid of the 
desired acid addition salt in excess. 
The invention relates also to those embodiments of the process in which an 
extract obtainable at any stage of the isolation is used as starting 
material and the concluding steps are carried out, the process is 
discontinued at any stage and/or a compound obtainable in accordance with 
the process according to the invention is further processed in situ. 
The compounds of the present invention and the pharmaceutically acceptable 
salts thereof can be used, for example, for the manufacture of 
pharmaceutical preparations that contain an effective amount of the active 
ingredient preferably in admixture with a significant amount of inorganic 
or organic, solid or liquid, pharmaceutically acceptable carriers. The 
present invention relates also to pharmaceutical preparations, their 
manufacture and their use. 
The pharmaceutical preparations according to the invention are suitable for 
oral or, preferably, parenteral, for example intravenous, intramuscular or 
topical, administration. 
For example, the active ingredients of formula I of the present invention 
are used in the form of injectable, for example intravenously, 
intramuscularly, intradermally or subcutaneously administrable, 
preparations or infusion solutions. Such solutions are preferably isotonic 
aqueous solutions or suspensions which may be prepared before use, for 
example from lyophilised preparations that contain the active ingredient 
only or the active ingredient together with a carrier, for example 
dextran, mannitol or albumin. Pharmaceutical preparations for oral use 
are, if desired, sterilized and may contain adjuncts, for example 
preservatives, stabilisers, wetting agents and/or emulsifiers, 
solubilisers, salts for regulating the osmotic pressure and/or buffers. 
Also suitable are corresponding oily injection suspensions in which 
lipophilic solvents or vehicles, for example fatty oils, for example 
sesame oil, triglycerides or synthetic fatty acid esters, for example 
ethyl oleate, are used as carriers. The injectable pharmaceutical 
preparations which, if desired, may contain other pharmacologically 
valuable substances, for example other active ingredients, contain about 
from 0.1% to 100%, especially about from 1% to 50%, and in the case of 
lyophilisates up to 100%, of the active ingredient. 
Suitable carriers for oral preparations, for example dragees, tablets or 
lacquer-coated tablets, are especially fillers, such as sugars, for 
example lactose, saccharose, mannitol or sorbitol, cellulose preparations 
and/or calcium phosphates, for example tricalcium phosphate or calcium 
hydrogen phosphate, binders, such as starch pastes based on, for example, 
corn, wheat, rice or potato starch, gelatin, methylcellulose, 
hydroxypropylmethylcellulose, hydroxypropylcellulose and/or 
polyvinylpyrrolidone, and/or, if desired, disintegrators, such as the 
mentioned starches, also carboxymethyl starch, crosslinked 
polyvinylpyrrolidone, agar, alginic acid or a salt thereof, such as sodium 
alginate, or low-molecular-weight carboxymethylcellulose. Adjuncts are 
especially flow-regulating agents and lubricants, for example silica, 
talc, stearic acid or salts thereof, such as magnesium or calcium 
stearate, and/or polyethyleneglycol. 
Dragee cores are provided with suitable coatings that may be resistant to 
gastric juices. Other orally administrable pharmaceutical preparations are 
dry-fill capsules made of gelatin and soft sealed capsules made of gelatin 
and a plasticizer, such as glycerin or sorbitol. Suitable rectally 
administrable pharmaceutical preparations are, for example, suppositories 
that consist of a combination of the active ingredient with a suppository 
base material. Orally and rectally administrable preparations contain from 
about 0.1% to about 50%, especially from about 1% to about 10%, of the 
active ingredient and, if desired, other pharmacologically valuable 
compounds. 
Pharmaceutical preparations for topical uses are especially creams, gels, 
ointments, pastes, foams, tinctures and solutions that contain from about 
0.05% to about 10%, especially from about 0.5% to about 5%, of the active 
ingredient. 
The pharmaceutical preparations are manufactured in a manner known per se, 
for example by means of conventional dissolving, suspending, mixing or 
lyophilising processes described in pharmacopoeia. 
The invention also relates to the use of the compounds of formula I or 
salts thereof as drugs, for example in the form of pharmaceutical 
preparations, for the treatment of bacterial infections by gram-positive 
bacteria and for the treatment of tumours, especially pulmonary and 
gastro-intestinal tumours and leukemia, in warm-blooded animals, for 
example humans and other mammals, by enteral, for example oral, or 
preferably parenteral, administration of therapeutically effective doses. 
Depending on the species, age, individual condition, severity of the 
disease and method of administration, the daily doses are from about 0.01 
mg to about 10 mg, especially from about 0.1 mg to about 1 mg, per kg of 
body weight, as considered appropriate by the doctor prescribing the 
treatment.

The following Examples illustrate the invention but do not limit the scope 
thereof in any way. 
EXAMPLE 1 
Myxococcus xanthus Mx x48 bacterium 
a) Origin and availability of the production strain 
The bacterium Myxococcus xanthus strain Mx x48 of the Myxococcaceae family 
of the order Myxobacterales was isolated in May 1980 from a soil sample 
from Gabes Oasis, Tunisia. The Mx x48 strain was deposited at the National 
Collection of Industrial and Marine Bacteria, Aberdeen, Scotland, on 26th 
May 1986 under the number NCIB 12 268 for patent purposes in accordance 
with the Budapest treaty. 
b) Description of the production strain 
Small vegetative rods, cylindrical with abruptly tapered ends, 
cigar-shaped, 0.8.times.3.5-5 .mu.m, move on surfaces in a 
crawling-gliding manner. On suitable media, for example VY/2-agar (see 
below), the organism forms "fruiting bodies" 50-150 .mu.m in diameter in 
the form of intensely red-orange to blue-grey soft-slimy drops, vesicles 
or capituli on the agar surface. Inside them are spherical, strongly 
refracting myxospores 1.8-1.9 .mu.m in diameter. The colonies tend to 
spread like wafts as "swarms" on the agar surface and can become up to 
several centimeters in size. On some media they are intensely 
greenish-yellow to orange in color. 
c) Culture conditions 
The organism grows well on peptone agar, for example CY-agar (Casitone 
[Difco] 0.3%, yeast extract [Difco] 0.1%, CaCl.sub.2.2H.sub.2 O 0.1%, agar 
1.5%, pH 7.2) or on yeast agar, for example VY/2 agar (baker's yeast 0.5% 
based on fresh weight, CaCl.sub.2.2H.sub.2 O 0.1%, agar 1.5%, pH 7.2). In 
liquid media Mx x48 grows in homogeneous cell suspension both in shaken 
flasks (at approximately 160 revs/min) and in bioreactors. A suitable 
culture medium is, for example, MD1 1.m. (peptone from casein, tryptically 
digested [Merck, Darmstadt] 0.3%, MgSO.sub.4.7H.sub.2 O 0.2%, 
CaCl.sub.2.2H.sub.2 O 0.05%, pH 7.2). Mx x48 is strictly aerobic. All 
cultures are maintained at 30.degree. C. The generation time in MD1 1.m. 
is approximately 6.5 hours (.mu.=0.15 per hour). The maximum achieved 
optical density (623 nm, 1 cm path length) is from 1.3 to 1.5. The 
concentration of the peptone from casein can also be increased, for 
example to 0.5 or 1%. The generation time does not vary in this case, but 
the optical density, by contrast, increases to approximately 2 or 3.5 
respectively. As the concentration of peptone increases, however, the 
production of antibiotic becomes lower compared with the production in MD1 
1.m. Suitable culture media in which the strain grows with antibiotic 
formation are also the following: 0.05% casitone, 0.2% MgSO.sub.4.7H.sub.2 
O and 0.05% CaCl.sub.2.2H.sub.2 O, pH 7.2, containing either 0.5% Probion 
S (single cell protein, Hoechst) or 0.5% corn steep liquor or 0.5% 
Protaminal (fish protein concentrate, Asta) or 0.5% yeast extract or 0.3% 
casamino acids (Difco) 
d) Possible preservation methods 
1. By freezing vegetative cells of plate or liquid cultures in peptone 
solution at -80.degree. C. (viable for several years). 2. By drying 
fruiting bodies on filter paper (viable for several years at room 
temperature). 3. By drying fruiting bodies in milk, and storing under 
nitrogen at room temperature in ampoules sealed by melting (viable for 
several years). 4. By suspending vegetative cells of plates or liquid 
cultures in peptone solution and freezing in liquid nitrogen (viable for 
several years). 
EXAMPLE 2 
Manufacture of cell supernatants with antibiotic activity 
a) Conditions of production 
It is possible to detect the activity against Staph. aureus at the end of 
the logarithmic growth phase (OD 0.9-1) using the agar diffusion test in 
shaken flasks. The highest activity is achieved in the early stationary 
phase. The pH in the culture at that time is about 8 and on further 
incubation rises to about 8.5 without the optical density increasing 
further. In the late stationary phase instead the cells become clumped and 
cell lysis occurs. The antibiotic activity in this case falls again. 
b) Production in a fermenter 
1. Preculture: 50 ml of MD1 1.m. are inoculated with 0.5 ml and maintained 
at 30.degree. C. and 160 revs/min for 2 days. 
2. Prefermentation: a 25 liter bioreactor marketed by Braun, Melsungen, 
having a blade agitator system is filled with MD1 1.m. medium and 
inoculated with the preculture (No. 1). The temperature is maintained at 
30.degree. C. The speed of rotation of the agitator is 200 revs/min and 
the aeration 0.1 Nm.sup.3 /h. Without further modifications the 
fermentation is carried out over a period of 66 hours. After this time an 
OD (623 nm) of 1.1 and a pH of 7.9 are achieved. 
3. Main fermentation: A bioreactor with a capacity of 700 liters, marketed 
by Giovanola, Monthey, Switzerland and having a blade agitator system is 
filled with MD1 1.m. medium. After inoculation with the prefermentation 
product (No. 2) a speed of rotation of 150 revs/min and an aeration of 1 
Nm.sup.3 /h are set. By increasing the aeration and the speed of 
revolution the oxygen partial pressure is maintained above 60% saturation 
during the course of the cultivation. After 50 hours an OD of 1.3 and a pH 
of 8 are achieved. The culture supernatant now contains the antibiotic. In 
the agar diffusion test against Staph. aureus, a just about still visible 
inhibiting areola (approximately 7 mm in diameter) is obtained when the 
culture supernatant is diluted 1:64. 
EXAMPLE 3 
Isolation and purification of the saframycins 
a) Working up a 700 liter fermentation 
When the fermentation of Example 2b) is complete, 4.0 liters of XAD-resin 
ER-180.RTM. (marketed by Rohm & Haas) are introduced into the culture 
liquor. The mixture is stirred slowly for 5 hours. The resin is separated 
from the cells in which there is no antibiotic and from the medium using a 
seive. It is transferred into a chromatography column and eluted in one 
pass with 12 liters (1 liter/hour) of isopropanol. Over 90% of the 
antibiotic complex is eluted with the first 6 liters of isopropanol. 
Immediately 0.1% (v/v) acetic acid is added to the fractions containing 
saframycins and the whole is concentrated in a rotary evaporator in brown 
flasks. The concentrated XAD extract weighs 27 g. 
Saframycins Mx 1 and Mx 2 are basic, light-sensitive and 
oxidation-sensitive hydroquinone/quinone derivatives that are stable only 
at pH values of less than 7. Purification is therefore carried out 
substantially with the exclusion of light and at pH values around 5. 
Temporary storage of the substance-containing fractions is in brown-tinted 
glass containers, and long-term storage is at -70.degree. C. 
b) Purification of the saframycins 
The crude extract is dissolved in 300 ml of 0.07M phosphate buffer pH 5 and 
applied to an open column filled with 1 liter of swelled 
CM-Sephadex.RTM.C-25 marketed by Pharmacia in 0.07M phosphate buffer pH 5. 
First of all, any material that has not been absorbed is eluted with 2 
liters of phosphate buffer. In a further step weakly adsorbed material is 
washed down with 1 liter of 0.07M phosphate buffer pH 5/0.2M sodium 
chloride. The antibiotic complex is eluted from the column completely 
using 2 liters of 0.07M phosphate buffer pH 5/0.5M NaCl, which is visible 
by a migrating yellowish band. After concentration of the eluate and 
repeated extraction of the residue with ethanol, approximately 2.5 g of 
highly active material are obtained. This still contains residues of the 
phosphate salt. To remove the salt chromatography is carried out on a 
Sephadex.RTM.LH-20 column with methanol and 1% formic acid or acetic acid 
as eluant, phosphate being exchanged for formate or acetate, respectively. 
Basic impurities still contained in the eluate, such as 2-phenylethylamine 
and 2-(4-hydroxyphenyl)-ethylamine are eliminated by chromatography on 
Sephadex.RTM.LH-20 with dichloromethane/methanol 1:1/1% formic acid or 
acetic acid. 700 mg of a product are obtained which, according to 
analytical HPLC (Nucleosil.RTM.C-18, 7.5 .mu.m, methanol/water 
60:40/0.005M heptanesulphonic acid) contains as the main products the two 
saframycins Mx 1 and Mx 2, and also the bis-quinone derivatives saframycin 
Mx 1 BC and Mx 2 BC and the bis-hydroquinone derivative Mx 1 BHC. 
c) Separation of the saframycins 
The mixture of saframycins is separated by semi-preparative HPLC on 
HD-Sil.RTM.18-10-60 (marketed by Organogen, 25 cm.times.16 mm) with 
methanol/water 1:1/0.5% triethylammonium formate buffer pH 6.0. At a flow 
of 25 ml/min, Mx 1 is eluted after 6.4 minutes and Mx 2 after 9.2 minutes. 
Both substances are obtained in the form of viscous red-brown oils. The 
secondary component Mx 1 BHC is eluted before Mx 1, and the secondary 
components Mx 1 BC and Mx 2 BC are eluted in that order after Mx 2. 
__________________________________________________________________________ 
Example 4 
Characterisation of the saframycins 
__________________________________________________________________________ 
(a) Saframycin Mx 1 and Mx 2 
Saframycin Saframycin 
Mx 1 (R.sup.1 = OH) 
Mx 2 (R.sup.1 = H) 
__________________________________________________________________________ 
Thin-layer elution behaviour (R.sub.f values) 
Silica get 60 F 254 ethyl acetate 
0 0 
CH.sub.2 Cl.sub.2 /CH.sub.3 OH 9:1 
0.23 0.20 
CH.sub.3 OH 0.47 0.36 
RP-18 Nano-Sil C.sub.18 -100 CH.sub.3 OH/H.sub.2 O 4:1 
0.67 0.65 
Colour reaction TLC identical for both saframycins. Ninhydrin: 
red-violet, 
Dragendorff: red-brown, hydroxylamine/iron (III) chloride: intense 
yellow, 254 nm: 
fluorescence extinction. 
HPLC-retention time on HD-Sil 18-10-60 
4.97 min. 6.38 min. 
CH.sub.3 OH/H.sub.2 O 3:2/0.005 M C.sub.7 H.sub.15 SO.sub.3 H 
UV-spectrum .lambda..sub.max (log .epsilon.), c = 2 in CH.sub.3 OH 
273 nm (3.95) 273 nm (3.97) 
Optical rotation [.alpha.].sub.D.sup.25 
-70.7.degree. -119.8.degree. 
IR-spectrum as a film, .nu. (cm.sup.-1) 
3200, 2900, 3200, 2900 
(sh = shoulder) 1657, 1619(sh), 
1680(sh), 1656, 
1459, 1300, 1623(sh), 1461, 
1236, 1158. 1236, 1159, 1122. 
FAB-MS 
Negative ions range m/e 568 (MOH)H -- 
Positive ions range -- m/e 569 MH.sup.+ 
Elemental analysis monophosphate.H.sub.2 O 
monoformate.H.sub.2 O 
Emperical formula: C.sub.29 H.sub.43 N.sub.4 O.sub.14 P 
C.sub.30 H.sub.42 N.sub.4 O.sub.11 
(640.67) 
Calculated: C 49.56 H 6.17 N 7.97% 
C 56.20 H 6.60 N 8.70% 
Found: C 50.17 H 6.33 N 7.66% 
C 56.36 H 6.70 N 8.94% 
NMR-Spectrum: 15 mg of monophosphate in 0.5 ml CD.sub.3 OD 
s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = 
double doublet, 
ddd = triple doublet 
.sup.1 HNMR 
Saframycin Mx 1 Saframycin Mx 2 
Proton (ppm) Couling constants (Hz) 
(ppm) coupling constants 
__________________________________________________________________________ 
(Hz) 
1-H 4.33 s (1-4b) 3.0; (1-22a) 0.5; 
3.59 m (1-4a) 1.5; (1-4b) 3.5; 
(1-22b) 2.5; (1-22a) 3.6; (1-22b) 1.5; 
3-H 3.50 ddd 
(3-4a) 2.5; (3.14 4b) 11; 
3.12 ddd 
(3-4a) 3; (3-4b) 11; 
(3-11) 1.0; (3-11) 3; 
4-H.sub.a 3.12 dd 
(4a-4b) 17.5; 3.04 ddd 
(4a-4b) 18; 
4-H.sub.b 1.51 ddd 1.59 ddd 
11-H 4.75 dd 
(11-13) 1.0; 4.90 dd 
(11-13) 1.5; 
12-CH.sub.3 
2.84 s 2.88 s 
13-H 4.20 s (13-14) 2.8; 4.13 d 
(13-21) below 0.5; 
14-H 4.82 d 4.95 s 
21-H.sub.a 4.78 s 3.11 dd 
(21a-21b) 13; (21a-13) 3; 
21-H.sub.b -- 3.71 d 
22-H.sub.a 3.77 dd 
(22a-22b) 14.0; 
4.09 dd 
(22a-22b) 14.5; 
22-H.sub.b 3.25 dd 3.34 dd 
24-H 3.69 q (24-25) 7.0; 3.68 q (24-25) 7.1; 
25-H.sub.3 0.93 d 0.82 d 
7-OCH.sub.3 
4.02 s 4.02 s 
17-OCH.sub.3 
3.75 s 3.75 s 
14-OCH.sub.3 
3.72 s 3.69 s 
16-CH.sub.3 
2.25 s 2.26 s 
6-CH.sub.3 
1.93 s 1.93 s 
.sup.13 CNMR Saframycin Mx 1 Saframycin Mx 2 
C-Atom (ppm) (ppm) 
__________________________________________________________________________ 
1 54.63 d 61.11 d 
3 51.80 d 57.07 d 
4 25.60 t 26.19 t 
5 186.95 s 187.17 s 
6 129.33 s 128.87 s 
7 157.23 s 157.37 s 
8 182.77 s 182.91 s 
9 143.10 s 143.04 s 
10 138.43 s 137.63 s 
11 58.19 d 58.15 d 
12-CH.sub.3 42.15 q 41.39 q 
13 59.98 d 59.38 d 
14 72.98 d 75.39 d 
15 147.16 s 146.58 s 
16 123.44 s 123.36 s 
17 148.07 s 147.75 s 
18 142.74 s 143.04 s 
19 112.20 s 113.04 s 
20 119.36 s 120.75 s 
21 89.23 d 54.44 t 
22 41.39 t 39.36 t 
23 170.85 s 170.65 s 
24 50.02 d 49.85 d 
25 17.16 q 17.08 q 
6-CH.sub.3 8.76 q 8.81 q 
16-CH.sub.3 9.87 q 9.98 q 
7-OCH.sub.3 61.45 q 61.04 q 
17-OCH.sub.3 61.02 61.44 q 
14-OCH.sub.3 58.19 q 58.07 q 
##STR4## 
##STR5## 
(b) Saframycins Mx 1 BC, Mx 2 BC and Mx 1 BHC 
Saframycin Saframycin Saframycin 
Mx 1 BC Mx 2 BC Mx 1 BHC 
__________________________________________________________________________ 
Thin-layer elution behaviour (R.sub.f -values) 
Silica gel 60 F 254 ethyl acetate 
0 0 
CH.sub.2 Cl.sub.2 /CH.sub.3 OH 9:1 
0.52 0.53 
CH.sub.3 OH 0.54 0.51 
RP-18 Nano-Sil C.sub.18 -100 CH.sub.3 OH/H.sub.2 O 4:1 
0.61 0.56 
HPLC-retention time on HD-Sil 18-10-60 
7.55 min 10.86 min 3.5 min 
CH.sub.3 OH/H.sub.2 O 3:2/0.005 M C.sub.7 H.sub.15 SO.sub.3 H 
UV-spectrum .lambda..sub.max (log .epsilon.) 
264 nm (4.32) 
264 nm (4.32) 
291 nm 
.sup.1 HNMR spectrum .delta. (ppm) in CD.sub.3 OD 
4.05 and 4.04 
4.05 and 4.02 
3.78 (s,3H, 17-OCH.sub.3) 
(each s,3H, 7-OCH.sub.3) 
(each s,3H, 7-OCH.sub.3 
3.70 (s,6H, 7-OCH.sub.3 
and 17-OCH.sub.3) 
and 17-OCH.sub.3) 
and 14-OCH.sub.3) 
3.59 (s,3H, 14-OCH.sub.3) 
3.59 (s,3H, 14-OCH.sub. 3) 
2.47 (s,3H, 12-CH.sub.3) 
2.46 (s,3H, 12-CH.sub.3) 
2.95 (s,3H, 12-CH.sub.3) 
2.00 (s,3H, 16-CH.sub.3) 
2.00 (s, 3H, 16-CH.sub.3) 
2.27 (s,3H, 16-CH.sub.3) 
1.94 (s,3H, 6-CH.sub.3) 
1.94 (s,3H, 6-CH.sub.3) 
2.17 (s,3H, 6-CH.sub.3) 
1.28 (d,3H, 25-H.sub.3) 
1.29 (d,3H, 25-H.sub.3) 
1.22 (d,3H, 25-H.sub.3) 
__________________________________________________________________________ 
EXAMPLE 5 
Conversion of saframycins 
a) Oxidation of saframycin Mx 1 to Mx 1 BC with atmospheric oxygen 
10 mg of saframycin Mx 1 (in the form of the monophosphate) are dissolved 
in 2 ml of phosphate buffer pH 7.3 and the whole is stirred for 20 minutes 
in an open flask. The pH value is adjusted to about 6 with dilute 
phosphoric acid and the solution is concentrated under an oil pump vacuum. 
The residue is repeatedly taken up in isopropanol, the buffer salts 
remaining. The isopropanol solution contains 9.5 mg of saframycin Mx 1 BC. 
Saframycin Mx 2 is oxidized to saframycin Mx 2 BC in an analogous manner. 
b) Reduction of saframycin Mx 1 to Mx 1 BHC with hydrogen 
In a hydrogenation vessel, 10 mg of saframycin Mx 1 (in the form of the 
monophosphate) are dissolved in 0.5 ml of methanol and 0.5 ml of water, a 
spatula tip of palladium-on-activated carbon (10% Pd) is added and the 
whole is shaken for 10 minutes in a weak stream of hydrogen. The catalyst 
is subsequently filtered off under a nitrogen atmosphere and the solution, 
which contains 10 mg of Mx 1 BHC, is concentrated. 
Saframycin Mx 1 BHC is unstable and is re-oxidized regiospecifically to 
saframycin Mx 1 quinone/hydroquinone) in the presence of atmospheric 
oxygen. 
c) Acetylation of saframycin Mx 1 
15 mg of saframycin Mx 1 are dissolved in 2 ml of methanol and 2 ml of 
phosphate buffer pH 7.3, 0.05 ml of acetic anhydride is added and the 
whole is stirred overnight at room temperature. The reaction solution is 
concentrated, taken up in a small amount of water and applied to an XAD-2 
column. Salts are washed out with 2 bed volumes of water. The product is 
then eluted with 1 bed volume of methanol. The methanol solution 
containing 15 mg of acetyl-saframycin Mx 1 BC is concentrated in vacuo. 
UV-spectrum in methanol: .lambda..sub.max 266 nm, log .epsilon. 4.32 
FAB-MS in positive ions range: 
expected: m/e 627 (M+H).sup.+ 
found: m/e 613 (100%), [M+2xH-1x0]+H.sup.+ 
m/e 615 (30%), [M+4xH-1x0]+H.sup.+ High resolution for C.sub.31 H.sub.41 
N.sub.4 O.sub.9 cal. 613.2873, found 63.2859. 
.sup.1 H-NMR in CD.sub.3 OD: 4.05 and 4.02 (each s,3H, 7-OCH.sub.3 and 
17-OCH.sub.3), 3.55 (s,3H, 14-OCH.sub.3), 2.43 (s,3H, 12-CH.sub.3), 2.00 
(s,3H, 16-CH.sub.3), 1.95 (s,3H, 6-CH.sub.3), 1.75 (s,3H, 
acetyl-CH.sub.3), 1.0 (d,3H, 25-H.sub.3) 
EXAMPLE 6 
Determination of the minimum inhibiting concentration of saframycins Mx 1 
and Mx 2 
The test organisms, in a cell density of 10.sup.5 cells/ml (Myxococcus 
xanthus: 10.sup.7 cells/ml) each, are placed in the appropriate nutrient 
media in test tubes. 
The media used are, for Myxococcus xanthus: MD1 1.m.; for other bacteria: 
peptone from casein, tryptically digested (Merck) 0.5%, proteose-peptone 
(Difco) 0.5%, meat extract (Oxoid) 0.1% pH 7.0; for yeasts: bacto-peptone 
(Difco) 1%, yeast extract (Difco) 1%, glycerin 2%, pH 6.3. 
A series of concentrations of the saframycins Mx 1 and Mx 2 dissolved in 
methanol are added to the cell suspensions of the test organisms and the 
test tubes are incubated for 18 to 40 hours at 30.degree. C. 
The minimum inhibiting concentrations of saframycins Mx 1 and Mx 2 are 
listed in the following Table: 
TABLE 
______________________________________ 
Saframycin Saframycin 
Test organism Mx 1 (.mu.g/ml) 
Mx 1 (.mu.g/ml) 
______________________________________ 
Staphylococcus aureus GBF 16 
0.004 0.5 
Bacillus subtilis DSM 10 
0.063 1 
Micrococcus luteus DSM 348 
0.001 0.063 
Escherichia coli CG 164 
0.08 5 
Escherichia coli tol C 
0.32 20 
Proteus morganii CG 166 
1.25 &gt;20 
Myxococcus xanthus Mx x48 
20 not tested 
Saccharomyces cerevisiae GBF 26 
&gt;20 &gt;20 
______________________________________ 
Towards Staph. aureus the bis-quinones Mx 1 BC and Mx 2 BC have similar 
activities to Mx 1 and Mx 2. The bis-hydroquinone Mx 1 BHC is not stable 
under the test conditions. 
EXAMPLE 7 
Pharmaceutical preparation for parenteral administration 
5 ml portions of a sterile aqueous solution of 1% saframycin Mx 1 (in the 
form of the phosphate monohydrate) are each introduced under aseptic 
conditions into 5 ml ampoules or vials and freeze-dried. The ampoules or 
vials are sealed under nitrogen and checked. 
Solutions of saframycin Mx 2, Mx 1 BC, Mx 2 BC, Mx 1 BHC and acetyl-Mx 1 BC 
are processed in the same manner.