The fermentation of a substrate selected among erythronolide B, erythronolide A and erythronolide A oxime with a novel mutant, Streptomyces antibioticus ATCC 31771, obtained from an industrial stock for the production of oleandomycin, said novel mutant being incapable of producing the same oleandomycin, permits novel macrolide antibiotics to be produced, having not only an activity range like that of erythromycin, but characterized by a greater stability in acidic environment, whereby for the administration of antibiotic it is no longer necessary to have recourse to esters and/or salts highly toxic for the organism.

The present invention relates to novel antibiotics of the macrolide family. 
More specifically the present invention relates to novel macrolide 
antibiotics obtained both starting from erythronolide B having the 
formula: 
##STR1## 
namely a natural substrate which is the biosynthetic precursor of the 
erythromycin A, and from semisynthetic substrates such as erythronolide A, 
having the formula: 
##STR2## 
and erythronolide A oxime, having the formula 
##STR3## 
Another object of the present invention is a novel microorganism, 
Streptomyces antibioticus ATCC 31771 obtained by mutagenic treatment from 
Streptomyces antibioticus ATCC 11891, producer of oleandomycin. It is 
known that the Streptomyces antibioticus ATCC 11891, is capable of using a 
semisynthetic substrate (erythronolide A oxime) (LeMahieu et al, J. 
Antib., 1976, (7), 728), but not the aforesaid erythronolide B, which is a 
substrate which can be obtained by direct fermentation in relevant amounts 
and thus at economically feasible costs using micro-organisms producers of 
erythromycin and their mutants. 
On the other side it is known that erythromycin is highly unstable in 
acidic environment, whereby the administration thereof must take place in 
form of esters and/or salts which, even if improve the stability, give 
place to well known properties of the active principle, objectionable and 
unfavourable from the therapeutical point of view, such as hepatic 
toxicity, low tolerability and others. 
The oleandomycin by itself is known for its high toxicity. 
The main purpose of the present invention is that of providing novel 
antibiotics having activity like that of the erythromycin, but devoid of 
the afore mentioned disadvantages of this antibiotic and particularly 
stable in acidic environment and administerable without the necessity of 
having recourse to toxic esters and/or salts. Another purpose of the 
present invention is that of providing a process for obtaining these 
antibiotics starting from a novel micro-organism, capable of using natural 
and semisynthetic substrates, particularly erythronolide B. 
A further purpose of the present invention is that of providing a 
composition having antibiotic activity for the treatment of diseases 
induced by germs and bacteria sensitive to the action of erythromycin and 
of like antibiotics. 
These purposes are achieved by means of the novel macrolide antibiotics and 
the related preparation process according to the present invention which 
is characterized in that for the fermentation, carried out in a per se 
known manner, a substrate selected among erythronolide B, erythronolide A 
and erythronolide A oxime and, as the fermentation agent, Streptomyces 
antibioticus ATCC 31771 are used. 
Another feature of the present invention resides in the preparation of the 
aforesaid fermentation agent, namely the Streptomyces antibioticus ATCC 
31771 which is derived from the Streptomyces antibioticus ATCC11891 by 
mutagenic treatment with ultraviolet rays, with an emission maximum at 254 
nm and at a dosage such as to kill about 99.6% of the spores, the desired 
micro-organism being furthermore characterized by the incapability of 
producing oleandomycin. 
By considering in a greater detail the novel macrolide antibiotics of the 
present invention, they shall be hereinafter individuated on the basis of 
the substrate from which they are prepared and on the basis of the 
characteristics of chromatographic analysis (the own reference of the 
Applicant being indicated in brackets). 
(1) 3-O-oleandrosyl-5-O-desosaminyl-(8S)-8-hydroxyerythronolide B (P 
15148), having the formula: 
##STR4## 
It is obtained from erythronolide B by means of the micro-organism of the 
present invention; at the thin layer chromatografic analysis (TLC) has 
R.sub.st of 0.8 with respect to the oleandomycin and R.sub.st of 0.75 with 
respect to the erythromycin A. By using the anisaldehyde reagent (by which 
the oleandomycin takes violet colour and the erythromycin A brown colour) 
this antibiotic takes a purple colour. 
Under HPLC (high pressure liquid phase chromatography) the peak 
corresponding to P 15148 has a retention time with respect to erythromycin 
A of 0.69 and with respect to oleandomycin of 1.08. 
(2) 3-O-oleandrosyl-5-O-desosaminyl-erythronolide B (P 15149), having the 
formula: 
##STR5## 
It is also obtained through fermentation of erythronolide B and has 
R.sub.st of 0.9 with respect to oleandomycin and R.sub.st of 0.85 with 
respect to erythromycin A. In the anisaldehyde test it is violet coloured. 
Under HPLC the peak corresponding to P 15149 has a retention time with 
respect to erythromycin A of 0.79 and with respect to oleandomycin of 
1.22. 
(3) 3-O-oleandrosyl-5-O-desosaminyl-15-hydroxyerythronolide B (P 15150), 
having the formula: 
##STR6## 
It is obtained by fermentation with Streptomyces antibioticus ATCC 31771 
from erythronolide B as the substrate and has R.sub.st of 0.47 with 
respect to the oleandomycin and R.sub.st of 0.44 with respect to the 
erythromycin A. At the anisaldehyde test it is purple coloured. Under HPLC 
the peak corresponding to P 15150 has a retention time with respect to 
erythromycin A of 0.52 and with respect to oleandomycin of 0.80. Although 
the structure of the novel macrolide antibiotic P 15150 has not been 
certainly assessed, the chemical and physical data obtained suggest the 
above reported structure. 
(4) 3-O-oleandrosyl-5-O-desosaminyl-(8S)-8,19-epoxyerythronolide B (P 
15153), having the formula: 
##STR7## 
It is obtained by fermentation with the micro-organism of the invention 
from erythronolide B; at the thin layer chromatographic analysis (TLC) 
shows R.sub.st of 0.8 with respect to the oleandomycin and R.sub.st of 
0.75 with respect to erythromycin A. By using the anisaldehyde reagent 
this antibiotic appears violet coloured. 
Under HPLC the peak corresponding to P 15153 has a retention time with 
respect to erythromycin A of 0.64 and with respect to oleandomycin of 
0.99. For the above four antibiotics, which are obtained in admixture from 
erythronolide B, after 100 hours of fermentation and before the isolation, 
the total antibiotic activity, expressed as activity of erythromycin A, is 
about 120-150 mcg/ml. 
(5) P 15151. It is obtained from the fermentation with Streptomyces 
antibioticus ATCC 31771 of erythronolide A and has R.sub.st of 0.88 with 
respect to oleandomycin and Rf of 0.8 with respect to erythromycin A. 
From the analysis of the culture broth, after 90 hours of fermentation, 
there is found an antibiotic activity, expressed as activity of 
erythromycin A, of about 70 mcg/ml. 
(6) P 15152. It is obtained from erythronolide A oxime, has Rf of 0.47 with 
respect to the oleandomycin and of 0.44 with respect to the erythromycin 
A. 
From the analysis of the culture broth, after 90 hours of incubation, there 
is detected a total antibiotic activity of about 50 mcg/ml (expressed as 
activity of erythromycin A), such an activity being however referred to 
the mixture of P 15152 and of another antibiotic, which is concomitantly 
produced, which is the same already described in J. Antib. (1976), 29, 
728. 
Turning now to an examination of the novel macrolide antibiotics produced 
according to the present invention, they are all characterized by a 
bacteriostatic spectrum like that of erythromycin and by the stability in 
acidic environment, whereby their direct administration by oral route is 
possible, without having recourse to esters and/or salts, showing the 
afore mentioned disadvantages and problems. The above is confirmed by the 
following Table 1, wherein the MIC values are reported, indicating the 
bacteriostatic power, and by the Table 2, giving the stability values in 
acidic environment. Without having to be construed in a limiting sense, it 
seems plausible that the greater stability of the macrolide. antibiotics 
of the present invention can be attributed to the presence in the molecule 
of the antibiotic of the neutral sugar oleandrose (J. Antib. (1976) XXIX 
(7), 728). 
TABLE I 
__________________________________________________________________________ 
Bacteriostatic power in solid medium of Erythromycin A, Erythromycin B, 
oleandomycin, P 15149, P 15148, P 15153, P 15150 
on aerobic and anaerobic bacteria, Gram positive and Gram negative 
minimum inhibiting concentrations expressed on mcg/ml. 
Eryth- 
Eryth- 
olean- 
romy- 
romy- 
domy- 
Microorganism Product 
cin A 
cin B 
cin P 15149 
P 15148 
P 15153 
P 
__________________________________________________________________________ 
15150 
AEROBIC 
A. Gram positive 
Staphylococcus aureus ATCC 6538 P 
LRP 39 
0.049 
0.097 
0.39 
0.78 1.56 0.195 
3.12 
Staphylococcus aureus LRP 14.degree..degree. 
0.049 
0.195 
0.39 
0.78 1.56 0.195 
3.12 
Staphylococcus aureus ATCC 14154 
LRP 78.degree. 
&gt;25 &gt;25 &gt;25 &gt;25 &gt;25 &gt;2 &gt;25 
Streptococcus faecalis 
LRP 7 
0.097 
0.195 
1.56 
0.78 0.78 0.78 6.25 
Streptococcus faecalis 
LRP 61 
0.195 
0.195 
1.56 
0.78 0.78 0.39 3.12 
sub. zymogenes ATCC 12958 
Streptococcus pneumonieae ATCC 6303 
LRP 35 
0.012 
0.024 
0.195 
0.097 
0.195 
0.049 
0.195 
Streptococcus pneumonieae 
LRP 52 
0.012 
0.024 
0.195 
0.097 
0.195 
0.049 
0.195 
Streptococcus pneumonieae 
LRP 53 
0.012 
0.024 
0.195 
0.097 
0.097 
0.024 
0.39 
Streptococcus pyogenes ATCC 8668 
LRP 34 
0.012 
0.024 
0.195 
0.097 
0.195 
0.049 
0.097 
Streptococcus pyogenes 
LRP 197 
0.012 
0.024 
0.195 
0.097 
0.195 
0.049 
0.097 
Corynebacterium diphteriae 
LRP 24 
0.006 
0.012 
0.097 
0.049 
0.097 
0.024 
0.097 
Micrococcus luteus ATCC 9341 
LRP 6 
0.006 
0.006 
0.049 
0.097 
0.049 
0.024 
0.195 
Micrococcus luteus ATCC 15957 
LRP 193.degree. 
&gt;25 &gt;25 &gt;25 &gt;25 &gt;25 &gt;25 &gt;25 
Bacillus subtilis LRP 25 
0.049 
0.049 
0.39 
0.039 
0.78 0.195 
3.12 
B. Gram negative 
Haemophilus influenzae ATCC 19418 
LRP 213 
3.12 
6.25 
&gt;25 6.25 &gt;25 12.5 &gt;25 
Neisseria Gonorrhoeae LRP 2149424 
0.049 
0.097 
0.195 
0.195 
1.56 0.195 
0.195 
Escherichia coli LRP 50 
6.25 
25 &gt;25 &gt;25 &gt;25 25 &gt;25 
Klebsiella pneumoniae LRP 54 
25 25 &gt;25 &gt;25 &gt;25 &gt;25 &gt;25 
Proteus vulgaris ATCC 6380 
LRP 13 
&gt;25 &gt;25 &gt;25 &gt;25 &gt;25 &gt;25 &gt;25 
Pseudomonas aeruginosa 
LRP 9 
&gt;25 &gt;25 &gt;25 &gt;25 &gt;25 &gt;25 &gt;25 
Salmonella typhi LRP 8 
12.5 
25 &gt;25 &gt;25 25 25 25 
Shigella sonnei LRP 5 
12.5 
&gt;25 &gt;25 &gt;25 &gt;25 &gt;25 &gt;25 
Acholeplasma laidlawii ATCC 23206 
LRP 204 
0.097 
0.097 
6.25 
12.5 6.25 1.56 6.25 
Mycoplasma hominis I ATCC 14097 
LRP 211 
&gt;25 &gt;25 &gt;25 &gt;25 &gt;25 &gt;25 &gt;25 
ANAEROBIC 
Clostridium perfrigens ATCC 3624 
LRP 206 
1.56 
1.56 
3.12 
12.5 25 3.12 6.25 
Bacteroides fragilis ATCC 23745 
LRP 205 
0.195 
0.195 
0.39 
3.12 6.25 6.25 &gt;25 
Fusobacterium necrophorum ATCC 27852 
LRP 210 
1.56 
6.25 
6.25 
&gt;25 &gt;25 25 &gt;25 
__________________________________________________________________________ 
Legenda: 
.degree.Erythromicina resistant 
.degree..degree.Penycilline resistant 
TABLE 2 
______________________________________ 
Stability in acidic environment at 25.degree. C. of the novel 
antibiotics in comparison with erythromycin A 
______________________________________ 
ERYTHRO- 
MYCIN A 
pH 2.0 3.0 4.0 
t 1/2 (minutes) 
2 6 120 
P 15148 
pH 2.0 3.0 4.0 
t 1/2 (hours) 
27 &gt;100 &gt;100 
P 15149 
pH 2.0 3.0 4.0 
t 1/2 (hours) 
18 &gt;100 &gt;100 
P 15150 
pH 2.0 3.0 4.0 
t 1/2 (hours) 
&gt;100 &gt;100 &gt;100 
P 15153 
pH 2.0 3.0 4.0 
t 1/2 (hours) 
65 &gt;100 &gt;100 
______________________________________ 
Note: 
t 1/2 represents the half life time of the antibiotics as determined by 
high pressure liquid phase chromatography (HLPC).

The following examples illustrate, without limiting sense, the preparation 
of the Streptomyces antibioticus ATCC 31771 and of the novel antibiotics 
of the invention. 
EXAMPLE 1 
Preparation of mutant Streptomyces antibioticus ATCC 31771 
A suspension of spores of Streptomyces antibioticus ATCC 11891, producer of 
oleandomycin, has been subjected to mutagenic treatment with U.V. rays 
(emission maximum at 254 nm) at a dosage such as to kill about 99.6% of 
the spores (about 3000 erg/sp. cm.). 
The surviving spores have been seeded on a nutrient medium and the 
resulting colonies have been analized looling for their uncapability of 
producing oleandomycin, using the method described by A. Kelner (1949) J. 
Bact. 57, 73. 
The mutants blocked in the oleandomycin synthesis (about 2% of the 
surviving organisms) have been then analized with respect to their 
capability of recognizing and converting the substrates erythronolide B, 
erythronolide A and erythronolide A oxime, to novel compounds having 
antibiotic activity. 
EXAMPLE 2 
Preparation of the antibiotics 
3-O-oleandrosyl-5-O-desosaminyl-(8S)-8-hydroxyerythronolide B (P 15148), 
3-O-oleandrosyl-5-O-desosaminyl-erythronolide B (P 15149), 
3-O-oleandrosyl-5-O-desosaminyl-(8R)-8.19-epoxy-erythronolide B (P 15153) 
and 3-O-oleandorsyl-5-O-desosaminyl-15-hydroxyerythronolide B (P 15150). 
Method A 
The mutant Streptomyces antibioticus ATCC 31771 is maintained on agarized 
culture slants of the following medium: 
______________________________________ 
peptone 0.2 grams per 100 mls 
glycerine 0.5 grams per 100 mls 
sugar can molasses 
0.5 grams per 100 mls 
K.sub.2 HPO.sub.4 
0.1 grams per 100 mls 
MGSO.sub.4.7H.sub.2 O 
0.05 grams per 100 mls 
NaCl 0.5 grams per 100 mls 
FeSO.sub.4.7H.sub.2 O 
0.001 grams per 100 mls 
CoCl.sub.2.6H.sub.2 O 
0.002 grams per 100 mls 
agar 2.0 grams per 100 mls 
distilled water 
to volume 
pH corrected to 7.0 
______________________________________ 
With a spore amount taken from one slant a 500 ml Erlenmeyer flask is 
seeded, containing 50 ml of cultivation medium having the following 
composition: 
______________________________________ 
dextrose 1.5 grams per 100 mls 
soy bean meal 3.0 grams per 100 mls 
yeast autolysate 
0.1 grams per 100 mls 
MgSO.sub.4 7H.sub.2 O 
0.1 grams per 100 mls 
calcium carbonate 
1.0 grams per 100 mls 
soy bean oil 0.6 grams per 100 mls 
distilled water 
to volume 
______________________________________ 
The flask is incubated at 28.degree. on a rotary shaker at 220 rpm for 24 
hours. After this time, a 10 liter glass fermentor containing antibiotics 
of the present invention can be 5 liters of the aforesaid medium is seeded 
with 15 mls of this culture. The fermentor is incubated at 28.degree. C., 
under 800 rpm stirring and with an air flow of 0.6 v/v/min. for 16 hours. 
At the end of this period, 3.5 liters of the above culture are used for 
seeding a 200 liter steel fermentor containing 100 liters of the following 
medium: 
______________________________________ 
dextrose 5.0 grams per 100 mls 
soy bean meal 2.0 grams per 100 mls 
dried baker yeast 
0.4 grams per 100 mls 
corn meal 1.6 grams per 100 mls 
calcium carbonate 
3.0 grams per 100 mls 
NaCl 0.3 grams per 100 mls 
fat oil 1.0 grams per 100 mls 
tap water to volume 
______________________________________ 
The incubation is carried out at 28.degree. C. with 250 rpm stirring and an 
aeration of 1 v/v/min. 
After 32 hour growth the culture is added with 75 g of erythronolide B and 
the incubation is continued for further 68 hours. 
At the end of this time a sample of the culture broth is filtered and 
extracted at pH 8.5 with an equal volume of CH.sub.2 Cl.sub.2. 
The extract is analyzed by TLC on silica gel plates developed for 2 hours 
with the system CH.sub.2 Cl.sub.2 : 95% methanol: conc. NH.sub.4 
OH(90:10:1). 
By this plate a bioautography is effected against Micrococcus luteus 
(Sarcina lutea) ATCC 9341 or alternatively the plates can be sprayed with 
an anisaldehyde reagent (0.5% v/v of anisaldehyde in a mixture of 
methanol: glacial acetic acid: conc. sulfuric acid, (85:10:5)) and heated 
to 120.degree. C. for 5 minutes. 
Under the above fermentation conditions and parallel to the use of the 
erythronolide B, four novel substances having antibiotic activity and 
indicated by the references P 15148, P 15149, P 15150 and P 15153 are 
produced. Under TLC these substances show R.sub.st values of 0.8, 0.9, 
0.47 and 0.8 with respect to the oleandomycin. With respect to 
erythromycin these R.sub.st values are 0.75, 0.85, 0.44 and 0.75. 
By using the anisaldehyde reagent, the products P 15148, P 15150 and P 
15153 appear purple coloured, whereas P 15149 is violet coloured (the 
oleandomycin is violet, whereas the erythromycin is brown). 
A sample of the organic extract is also controlled under HPLC. 
To this end it is evaporated to dryness, taken with acetonitrile and 
injected in column (RP810 .mu.m 25 cm; mobile phase 0.01 M pH 7 phosphate 
buffer and acetonitrile (36:64); flow 2 mls/min; column temperature 
40.degree. C.). 
For the novel substances P 15148, P 15149, P 15150 and P 15153 peaks are 
detected with retention times with respect to erythromycin A of 0.69, 
0.79, 0.52 and 0.64 and with respect to oleandomycin of 1.08, 1.22, 0.80 
and 0.99. 
After 100 hours of fermentation the total antibiotic activity, expressed as 
activity of erythromycin A, is about 120 to 150 mcg/ml. 
The stirring is reduced to a minimum by stopping the air feeding and the 
temperature is lowered to 15.degree. C. A fermenting culture of 
Streptomyces erythreus NRRL 2338 is prepared according to the conditions 
described in the German open application (OS) No. 1900647 of Aug. 13, 
1970. 
By adding 4% v/v n-propanol, after 144 hours of fermentation, in the 
culture broth about 500 mcg/ml of erythronolide B and traces of 
erythromycin (less than 20 mcg/ml) are obtained. 
At this time the culture is centrifuged and the clear supernatant is 
sterilized by filtration. 
A vegetating culture of Streptomyces antibioticus ATCC 31771 is prepared 
according to the procedure described in the method A. 
A second growing passage is carried out under the same conditions by 
seeding with 1 ml of vegetating culture a second 500 ml Erlenmeyer flask 
containing 50 mls of the culture medium of example 2. 
With 1.0 mls of second growth culture a 500 ml Erlenmeyer flask is seeded, 
containing 30 mls of the following culture medium: 
______________________________________ 
dextrose 5.0 grams per 100 mls 
soy bean meal 2.0 grams per 100 mls 
baker yeast 0.2 grams per 100 mls 
maize meal 0.3 grams per 100 mls 
calcium carbonate 
2.0 grams per 100 mls 
tap water to volume 
______________________________________ 
The flask is incubated at 28.degree. C. on rotary shaker at 250 rpm. After 
32 hours of incubation the flask is added with 10 mls of the afore 
described clear supernatant and the incubation is continued for further 64 
hours. 
The culture is analized according to the procedure described in the example 
1. A total antibiotic activity (expressed as activity of erythromycin A) 
of about 30 mcg/ml is obtained; by HPLC the presence of the four novel 
antibiotics P 15148, P 15149, P 15150 and P 15153 as well as traces of 
erythromycin A are detected. 
In the control flasks, for which no addition of the filtrate containing 
erythronolide B took place, the antibiotic activity is absent. 
EXAMPLE 3 
Purification of the antibiotics 
3-O-oleandrosyl-5-O-desosaminyl-(8S)-8-hydroxyerythronolide B (P 15148), 
3-O-oleandrosyl-5-O-desosaminylerythronolide B (P 15149), 
3-O-oleandrosyl-5-O-desosaminyl-(8R)-8,19-epoxyerythronolide B (P 15153) 
and 3-O-oleandrosyl-5-O-15-hydroxyerythronolide B (P 15150). 
To the final fermentation mixture, reported in the example 2 (method A), 
there are added under stirring 30 liters of a 10% w/v ZnSO.sub.4 solution 
and 30 liters of a 2% w/v NaOH water solution. 
After addition of a filtration aid, the solid content is removed by 
filtration through a rotary filter under vacuum after continuous washing 
of the solid on the filter cake. The washed solids are disposed and, at 
the end of the filtration, 150 liters of clear filtrate are recovered. 
The filtrate is then salted with a suitable salt, such as NaCl, Na.sub.2 
SO.sub.4, (NH.sub.4).sub.2 SO.sub.4 and the like added in an amount of 15 
to 25% w/v in order to facilitate the subsequent extraction with a 
suitable solvent not miscible with water. The pH of the filtrate is 
corrected to between 5.4 and 6.2 by means of diluted acids, such as for 
instance phosphoric or acetic acid. 
By this operation the inactive solids are precipitated and then removed by 
filtration. 
A suitable solvent not miscible with water such as butyl acetate, 
chloroform, methylisobutylketone, methylene chloride or dichloroethane is 
contacted, by means of a continuously operating centrifugal extractor, 
with the clear filtrate, in a ratio of between 20 and 50% v/v. 
The operation is repeated twice and the organic phases are combined. 
The combined organic phases prevailingly contain P 15149. In the aqueous 
phase P 15148, P 15150, P 15153 and residual P 15149, not completely 
extracted from the organic phase, are present. 
The organic phase (about 100 liters) is distilled under vacuum at low 
temperature to a volume of about 10 liters. 
Under vigorous stirring there are added 3 liters of 10.sup.-4 M acetate 
buffer at a pH of about 4.0 and the phases are then separated. 
The operation is repeated three times and at the end all the P 15149 is 
present in the acqueous phase; the solvent, containing much impurities, is 
disposed. 
The combined aqueous phases are brought to a pH of about 8.0 with diluted 
NaOH and then extracted three times with 3 liter fractions of an organic 
solvent such as methylethylketone or butyl acetate. The water phase is 
disposed and the organic phases are combined and dried onto anhydrous 
Na.sub.2 SO.sub.4. 
The dried solution is distilled under vacuum at low temperature to a volume 
of 500 mls. 
To 5 liters of a non polar solvent, such as hexane or petroleum ether 
heated to about 40.degree. C. there are added, under stirring, 500 mls of 
the concentrated solution; the addition is completed over about 1 hour. 
At the end of the addition the heating is stopped and the mixture is cooled 
slowly to +5.degree. C. 
After about 2 hour standing at that temperature, the precipitation of P 
15149 which already began at about 18.degree. C. is completed. 
The white and fluffly precipitate is filtered, washed with solvent and 
dried, giving place to 4.2 g of P 15149 which at HPLC analysis show a 
purity of 85%. 
In order to obtain n an analitically pure sample it is necessary to further 
purify the solid by means of column chromatography on silica gel according 
to the method indicated by N. L. Oleinick, J. Biol. Chem., Vol. 244, N. 3, 
pag 727 (1969). 
The 25-80 fractions, containing 
3-O-oleandrosyl-5-O-deoxaminyl-erythronolide B (P 15149) are combined, 
evaporated to dryness under vacuum at 50.degree. C., and crystallized from 
acetone-n-hexane, giving 1.835 g of P 15149 having the following 
characteristics: 
m.p.: 128.degree.-131.degree. C. 
[.alpha.].sub.D.sup.20 -68.2.degree. (C=1 in methanol); UV (methanol) 290 
nm (.epsilon.59.8); IR (KBr) 3490, 1720, 1700 (shoulder), 1460, 1380, 
1335, 1275, 1250 (shoulder), 1180, 1160, 1140, 1105, 1075, 1050, 1000, 
945, 920, 890, 830 cm.sup.-1 (FIG. 1). 
The analysis for C.sub.36 H.sub.65 NO.sub.12 gave the following values: 
calculated (%): C 61.43; H 9.30; N 1.99. found (%): C 61.34; H 9.38; N 
2.05. 
The aqueous phases containing P 15148, P 15150, P 15153 and P 15149 (not 
completely extracted) are made alkaline with diluted NaOH, extracted 
several times with an equal volume of CH.sub.2 Cl.sub.2. Before each 
extraction the pH of the aqueous phase is brought again to 9. 
By TLC and HPLC control it is possible to individuate the extracts 
containing only the antibiotic P 15150 (more soluble than the others in 
water phase) and the extracts still containing P 15148, P 15149, P 15150 
and P 15153 together. 
The organic phases containing only P 15150 are combined and evaporated 
under vacuum at 50.degree. C. to dryness, giving 3.4 g of P 15150 with the 
following chemical and physical characteristics: 
[.alpha.].sub.D.sup.20 -55.degree. (C=1 in methanol); UV (methanol) 288 nm 
(.epsilon.42); IR (KBr) 3460, 1715, 1700 (shoulder), 1455, 1375, 1330, 
1300, 1270, 1255, 1240, 1170, 1105, 1070, 1045, 1000, 990, 980, 940, 915, 
885, 825 cm.sup.-1 (FIG. 2). 
The corresponding hydrochloride has melting point 160.degree.-163.degree. 
C. (acetone). 
The analysis for C.sub.36 H.sub.65 NO.sub.13 gave the following results: 
calculated (%): C 57.17; N 8.80; N 1.85; Cl 4.69. found (%): C 56.92; H 
8.77; N 1.91; Cl 4.59. 
The other organic phases, still containing P 15150 and the remaining 
antibiotics are evaporated to dryness under vacuum at 50.degree. C. and 
the obtained residue is purified in a silica gel column according to the 
aforesaid method of N. L. Oleinick. 
By carrying out the elution with the solvent indicated in this method 
fractions are collected containing only P 15149 (fractions 52 to 110), P 
15148 (fractions 130 to 185), P 15153 (fractions 260 to 450). When from 
the TLC and HPLC control it appears that the eluted fractions no longer 
contain P 15153, the elution solvent is changed. By eluting with isoropyl 
alcohol-acetone (1:1.5) the fractions containing only the antibiotic P 
15150 are collected. 
The fractions 52 to 110, combined and evaporated to dryness under vacuum at 
50.degree. C. gave 0.425 g of 
3-O-oleandrosyl-5-O-desosaminyl-erythronolide B (P 15149), which after 
crystallization from acetone n-hexane shows chemical and physical 
characteristic equal to those reported for the P 15149 previously 
isolated. 
The fractions 130 to 185, combined and evaporated to dryness under vacuum 
at 50.degree. C., gave 3.2 g of 
3-O-oleandrosyl-5-O-desosaminyl-(8S)-8-hydroxy-erythronolide B (P 15148), 
after crystallization from acetone, characterized by: 
m.p. 204.degree.-206.degree. C. 
[.alpha.].sub.D.sup.20 -48.35 (C=1 in methanol); UV (methanol) 280 nm 
(.epsilon.39.2) IR (KBR) 3590, 3520, 3440 (broad), 3280 (broad), 1725, 
1455, 1380, 1365, 1305, 1280, 1270, 1260, 1175, 1155, 1095, 1060, 1050, 
1020, 1005, 970, 935, 915, 885, 875, 820 cm.sup.-1 (FIG. 3). 
The analysis for C.sub.36 H.sub.65 NO.sub.13 gave the following values: 
calculated (%): C 61.43; H 9.30; N 1.99. found (%): C 61.34; H 9.38; N 
2.05. 
The fractions 260 oto 450, combined and evaporated to dryness under vacuum 
at 50.degree. C., gave 10.3 g of 
3-O-oleandrosyl-5-O-desosaminyl-(8S)-8,19-epoxy-erythronolide B (P 15153), 
after crystallization from acetone/n-hexane, with the following chemical 
and physical characteristics: 
m.p. 125.degree.-130.degree. C. 
[.alpha.].sub.D.sup.20 -27.7.degree. (C=1 in methanol); UV (methanol) 288 
nm (.epsilon.42) IR (KBr) 3485, 1720, 1460, 1380, 1330, 1305, 1270, 1170, 
1140, 1110, 1075, 1050, 1000, 930, 920, 890, 830 cm.sup.-1 (FIG. 5). 
The analysis for C.sub.36 H.sub.63 NO.sub.13 gave the following values: 
calculated (%): C 60.23; H 8.85; N 1.95. found (%): C 60.12; H 8.75; N 
1.79. 
By evaporation under vacuum of the fractions collected with isopropyl 
alcohol-acetone (1:1.5) and subsequent crystallization from 
acetone/n-hexane, 15.4 g of P 15150 are obtained with the same properties 
previously indicated. 
EXAMPLE 4 
Preparation of the antibiotic P 15151 
To a fermenting culture of Streptomyces antibioticus ATCC 31771 prepared in 
a flask according to the procedure described in the example 2, there are 
added, after 30 hours of incubation, 15 mg of erythronolide A prepared 
starting from erythromycin A according to the method described in J. Med. 
Chem. (1974), 17, 953. 
After further 60 hours of incubation, the culture broth is analyzed as 
described in the example 2 and an antibiotic activity of about 70 mcg/ml 
(expressed as activity of erythromycin A) is found. 
By TLC analysis a novel active substance, P 15151, is found, having Rf 0.8 
with respect to the erythromycin A and 0.88 with respect to the 
oleandomycin. This compound is furthermore different from the antibiotics 
P 15148, P 15149, P 15150 and P 15153. 
EXAMPLE 5 
Preparation of the antibiotic P 15152 
To a flask containing a culture of Streptomyces antibioticus ATCC 31771, 
prepared according to the procedure described in the example 4, there are 
added, after 30 hours of incubation, 15 mg of erythronolide A oxime, 
chemically prepared according to the method described in J. Med. Chem. 
(1974), 17, 953. 
After further 60 hours of incubation, the culture broth is analized as 
described in the example 2 and a total antibiotic activity of about 50 
mcg/ml (expressed as activity of erythromycin A) is revealed. 
By TLC analysis there are found two active substances, one of which is the 
same described in J. Antib. (1976), 29, 728, (Rf 0.73 with respect to the 
oleandomycin), whereas the second one is novel and is indicated by the 
reference P 15152, having Rf 0.47 with respect to the oleandomycin and 
0.44 with respect to erythromycin A. It is to be pointed out that, within 
the scope of the present invention, there are contemplated also the other 
possible uses of the Streptomyces antibioticus ATCC 31771 as regards the 
introduction in the molecule of precursors, either novel or already known, 
of macrolide antibiotics, the oleandrose meant as an agent giving 
stability in acidic environment and thus promoting the administration by 
oral route.