Method for the separation of antibiotic macrolides

This case relates to a novel process which aids in the isolation and purification of novel compounds which are produced by the microorganism, Streptomyces avermitilis. The process described utilizes a column containing a hydroxyalkylated dextran gel as a step in the separation of the desired compounds. The compounds which are isolated and purified are described generically as C-076 and have significant parasiticidal activity.

SUMMARY OF THE INVENTION 
This invention is directed to a process for isolating the novel chemical 
compounds C-076 which are produced by the fermentation of a nutrient 
medium with a strain of the microorganism, Streptomyces avermitilis. 
The compounds, to which the novel techniques of isolation and purification 
of this invention are directed, are described in co-pending U.S. 
application Ser. No. 772,601 of G. Albers-Schonberg, R. Burg, T. Miller, 
R. Ormond and H. Wallich. Said application teaches the use and 
characterization of the C-076 compounds as well as the utilization and 
characterization of the microorganism, Streptomyces avermitilis. Said 
application is hereby incorporated by reference in this application. 
More particularly, this invention is comprised of a novel technique wherein 
it is an object of this process to aid in the isolation of the 
parasiticidal active compound in a substantially purified form. Further 
objects of this invention will become apparent from the following 
description. 
DESCRIPTION OF THE INVENTION 
In accordance with this invention, novel techniques of extraction and 
fractionation are utilized to isolate and purify substances generically 
described herein as C-076. These substances are prepared by growing under 
controlled conditions strains of microorganisms of Streptomyces 
avermitilis. These substances are described as C-076 A1a, A1b, A2a, A2b, 
B1a, B1b, B2a, and B2b. 
Based on taxonomic studies, the microorganisms capable of producing these 
C-076 compounds are of a new species of the genus Streptomyces, which has 
been named Streptomyces avermitilis. One such culture, isolated from soil 
is designated MA-4680 in the culture collection of MERCK & CO., Inc., 
Rahway, New Jersey. A C-076 producing sample of this culture has been 
deposited in the permanent culture collection of the Fermentation Section 
of the Northern Utilization Research Branch, U.S. Department of 
Agriculture at Peoria, Illinois, and has been assigned the accession 
number NRRL 8165. A sample of NRRL 8165 has also been deposited, without 
restriction as to availability, in the permanent culture collection of the 
American Type Culture Collection at 12301 Parklawn Drive, Rockville, 
Maryland 20852, and has been assigned the accession number ATCC 31,267. 
However, the present invention also embraces mutants of the above described 
microorganism. For example, those C-076 producing mutants which are 
obtained by natural selection or those produced by mutating agents 
including X-ray irradiation, ultraviolet irradiation, nitrogen mustard or 
like treatments are also included within the ambit of this invention. 
One example of such an organism is a strain of Streptomyces avermitilis MA 
4848 which was isolated after irradiation with ultraviolet light of 
Streptomyces avermitillis MA 4680. A lyophilized tube and a frozen vial of 
this culture has been deposited in the permanent culture collection of the 
American Type Culture Collection, and they have been assigned the 
accession numbers 31272 and 31271 respectively. Slightly higher 
fermentation yields of C-076 have been obtained using this frozen stock as 
inoculum. 
In particular, this invention is directed to a method for the separation of 
C-076 B1 B2 components wherein a solution containing C-076 B1 B2 
components is chromatographed on a column containing hydroxyalkylated 
dextran gel and eluting said column. The solvent utilized in the elution 
step is a mixture of lower boiling hydrocarbons:monoaromatics:lower 
alcohols in the proportions of about 30-90%:10-60%:5-25% and recovering 
said components. 
This invention also includes the process wherein C-076 A1, A2 components 
are separated by chromatographing on a column containing hydroxyalkylated 
dextran gel and eluting said column. The solvent utilized in the elution 
step is a mixture of low boiling hydrocarbons:monoaromatics:lower alcohols 
in the ratio of about 5-7:0.5-1.5:0.5-1.50 and recovering said components. 
By the term, "lowering boiling hydrocarbons" is meant hydrocarbons having 
from 5 to about 8 carbon atoms wherein such carbon atoms may be a straight 
or branched chain. Illustrative of such low boiling hydrocarbons are 
pentane, hexane, heptane, octane, isopentane and the like. 
By the term, "monoaromatic" is meant monocyclic aromatic compounds which 
may contain one or more lower alkyl substituent. Illustrative of such 
monoarmomatic compounds are toluene, benzene, xylene and the like. 
By the term, "lower alcohol" is meant alcohols having from 1 to about 3 
carbon atoms. Illustrative of such lower alcohols are methanol, ethanol, 
propanol, isopropanol and the like. 
Although the elution solvent mixtures and proportions may be utilized over 
a fairly wide range in the separation of the B1 and B2 components, a more 
narrow range is usually utilized for optimum separation of the components. 
Thus, the case of the separation of B1 and B2 components, a more preferred 
solvent mixture and proportions are hexane:toluene:methanol in a ratio of 
about 3:1:1. 
Also, in the separation of the A1 compound from the A2 component, a 
preferred solvent mixture and ratio will result in a more desirous 
separation. Thus, in the case of A1 A2, a more preferred solvent mixture 
and ratio is hexane:toluene:methanol in a ratio of 6:1:1. 
The C-076 compounds are produced during the aerobic fermentation of 
suitable aqueous nutrient media under conditions described hereinafter, 
with a producing strain of Streptomyces avermitilis. Aqueous media such as 
those used for the production of many antibiotic substances are suitable 
for use in this process for the preparation of C-076. 
Such nutrient media contain sources of carbon and nitrogen assimilable by 
the microorganism and generally low levels of inorganic salts. In 
addition, the fermentation media may contain traces of metals necessary 
for the growth of the microorganisms. These are usually present in 
sufficient concentration in the complex sources of carbon and nitrogen 
which may be used as nutrient sources, but can, of course, be added 
separately to the medium if desired. 
In general, carbohydrates such as sugars, for example dextrose, sucrose, 
maltose, lactose, dextran, cerelose and the like, and starches are 
suitable sources of assimilable carbon in the nutrient media. The exact 
quantity of the carbon source which is utilized in the medium will depend, 
in part, upon the other ingredients in the medium but it is usually found 
that an amount of carbohydrate between about 0.5 and 5% by weight of the 
medium is satisfactory. These carbon sources can be used individually or 
several such carbon sources may be combined in the same medium. 
Various nitrogen surces such as yeast hydrolysates, yeast autolysate, 
soybean meal, casein hydrolysates, yeast extracts, corn steep liquors, 
distillers solubles, cottonseed meal, meat extract and the like, are 
readily assimilable by Streptomyces avermitilis in the production of the 
C-076 compounds. The various sources of nitrogen can be used alone or in 
combination in amounts ranging from about 0.2 to 6% by weight of the 
medium. 
Among the nutrient inorganic salts which can be incorporated in the culture 
media are the customary salts capable of yielding sodium, potassium, 
magnesium, ammonium calcium, phosphate, sulfate, chloride, carbonate and 
like ions. Also included are trace metals such as cobalt, manganese, iron 
and the like. 
The fermentation employing the C-076-producing microorganisms can be 
conducted at temperatures ranging from about 20.degree. C. to about 
40.degree. C. For optimum results, it is most convenient to conduct these 
fermentations at a temperature in the range of from about 24.degree. C. to 
about 30.degree. C. Temperatures of about 27.degree. C.-28.degree. C. are 
most preferred. The pH of the nutrient medium suitable for producing the 
C-076 compounds can vary from about 5.0 to 9.0 with a preferred range of 
from about 6.0 to 7.5. 
Small scale fermentations are conveniently carried out by placing suitable 
quantities of nutrient medium in a flask employing known sterile 
techniques, inoculating the flask with either spores or vegetative 
cellular growth of a C-076-producing strain of Streptomyces avermitilis, 
loosely stoppering the necks of the flask with cotton, and permitting the 
fermentation to proceed in a constant room temperature of about 28.degree. 
C. on a rotary shaker for about 3 to 10 days. For larger scale work, it is 
preferable to conduct the fermentation in suitable tanks provided with an 
agitator and a means of aerating the fermentation medium. The nutrient 
medium is made up in the tank and after sterilization is inoculated with a 
suitable source of vegetative cellular growth of a C-076 producing strain 
of Streptomyces avermitilis. The fermentation is allowed to continue for 
from 1 to 8 days while agitating and/or aerating the nutrient medium at a 
temperature in the range of from about 24.degree. C. to 37.degree. C. The 
degree of aeration is dependent upon several factors such as the size of 
the fermentor, agitation speed and the like. Generally, the larger scale 
fermentations are agitated at about 95 to 150 rpm and about 2 to 20 cubic 
feet per minute of air. 
The substances of this invention, which are generially referred to herein 
as C-076, are found primarily in the mycelium on termination of the 
Streptomyces avermilitilis fermentation, and may be recovered and 
separated from one another as described below. Four major and four minor 
components of the C-076 as elaborated by Streptomyces avermitilis have 
been isolated. The eight different compounds are identified herein as 
C-076 A1a, A1b, A2a, A2b, B1a, B1b, B2a and B2b. 
Based on experimental data, the C-076 compounds are believed to have the 
following planar structural formula: 
##STR1## 
wherein R is the .alpha.-L-oleandrosyl-.alpha.-L-oleandroside of the 
structure: 
##STR2## 
and wherein the broken line indicates a single or a double bond; R.sub.1 
is hydroxy and is present only when said broken line indicates a single 
bond. 
R.sub.2 is propyl or butyl; and 
R.sub.3 is methoxy or hydroxy. 
In the foregoing structural formula, the individual compounds are as set 
forth in Table IV. 
TABLE IV 
______________________________________ 
R.sub.1 R.sub.2 R.sub.3 
______________________________________ 
Ala Double bond butyl 
OCH.sub.3 
Alb Double bond propyl 
OCH.sub.3 
A2a 
OH butyl 
OCH.sub.3 
A2b 
OH propyl 
OCH.sub.3 
Bla Double bond butyl 
OH 
Blb Double bond propyl 
OH 
B2a 
OH butyl 
OH 
B2b 
OH propyl 
OH 
______________________________________ 
The major C-076 compounds are not produced in equal amounts by the 
fermentations described herein. In general, it has been found that the A1 
compounds comprise about 20 to 30% by weight of the total C-076 complex 
produced, the A2 compounds about 1 to 20 % and the B1 and B2 compounds 
each about 25 to 35%. 
The separation of the C-076 series of compounds from the whole fermentation 
broth and the recovery of the individual components is carried out by 
solvent extraction and application of chromatographic fractionations with 
various chromatographic techniques and solvent systems. 
The C-076 compounds have slight solubility in water, but are soluble in 
organic solvents. This property may be conveniently employed to recover 
them from the fermentation broth. Thus in one recovery method, the whole 
fermentation broth is filtered and the aqueous filtrate discarded. The wet 
mycelial cake is then extracted with an appropriate organic solvent. While 
any organic solvent may be employed, it is preferable to use a water 
miscible solvent such as acetone, methanol, ethanol and the like. 
Generally, several extractions are desirable to achieve maximum recovery. 
The solvent removes the C-076 active components as well as other 
substances lacking the antiparasitic activity of C-076. If the solvent is 
a water miscible one, the water is also removed from the wet mycelia. The 
extracted mycelia may be discarded. The solvent extracts are evaporated to 
remove the organic solvent and extracted several times with a second 
solvent. When the first extraction employs a water miscible solvent, the 
second extraction preferably employs a water immiscible solvent such as 
chloroform, methylene chloride, carbon tetrachloride, ethylacetate, 
methylethyl ketone, methylisobutyl ketone and the like. These latter 
extracts are dried and concentrated using known techniques to afford a 
residue comprising C-076 admixed with other materials. This fraction is 
then conveniently chromatographed in order to separate the active C-076 
compounds from other material and also to separate and isolate the 
individual C-076 compounds. The chromatographic techniques which may be 
employed to purify the C-076 compounds are generally known to those 
skilled in this art. Examples of such techniques are column 
chromatography, using such media as silica gel, aluminum oxide, dextran 
gels and the like, and elution of such columns with various solvents, 
and/or a combination of two or more solvents, in varying ratios. Liquid 
chromatography is employed for the detection of the C-076 compounds, and 
high pressure liquid chromatography may be employed to isolate purified 
fractions containing one or more of such compounds. Likewise, thin layer 
chromatography may be employed to detect the presence of, and to isolate 
the individual C-076 compounds,. The use of the foregoing techniques as 
well as others known to those skilled in this art, will afford purified 
compositions comprising the C-076 compounds as well as the individual 
C-076 compounds themselves.

The following examples are capable of wide variation and modification and 
any minor departure or extension is considered as being within the skill 
of the artisan and as falling within the scope of this invention. 
EXAMPLE 1 
A 250-ml. baffled Erlenmeyer flask containing 50 ml. of the following 
medium: 
Lactose: 2.0% 
Distiller's Solubles: 1.5% 
Autolyzed yeast, Ardamine pH: 0.5% 
pH--before sterilization: 7.0 
is inoculated with the contents of one frozen vial of Streptomyces 
avermitilis MA-4848 and incubated on a rotary shaker at 28.degree. C. for 
24 hours at 150 rmp. 
Ten ml. of the above fermentation medium is employed to inoculate 500 ml. 
of the same medium as above in a 2-liter baffled Erlenmeyer flask. The 
fermentation medium is incubated at 150 rpm on a rotary shaker at 
28.degree. C. for 24 hours. 
All of the foregoing medium is employed to inoculate 467 liters of the 
following medium in a 756-liter stainless steel fermentor: 
Lactose: 2.0% 
Distiller's Solubles: 1.5% 
Autolyzed yeast, Ardamine pH: 0.5% 
Polyglycol 2000: 0.32 ml./liter 
pH--before sterilization: 7.0 
The fermentation medium is incubated at 28.degree. C. for 40 hours with an 
air flow of 10 cubic feet per minute and an agitation rate of 130 rpm. 
230 Liters of the above medium is employed to inoculate 4,310 liters of the 
following medium in a 5,670-liter stainless steel fermentor: 
Dextrose: 4.5% 
Peptonized Milk: 2.4% 
Autolyzed yeast, Ardamine pH: 0.25% 
Polyglycol 2000: 2.5 ml./liter 
pH--before sterilizaton: 7.0 
The fermentation continues for 144 hours at 26.degree. C. with an air flow 
rate of 54.3 cubic feet per minute and agitation rate of 120 rpm. 
The fermentation medium is filtered and the mycelial filter cake washed 
with about 550 liters of water, the filtrate and washings are discarded. 
The filter cake is agitated with about 1500 liters of acetone for about 
one hour and filtered. The filter cake is washed with a mixture of about 
150 liters of acetone and 40 liters of deionized water affording about 
2000 liters of extract. 
The foregoing fermentation and extraction is repeated on the same scale 
affording a further 2000 liters of acetone extract which is combined with 
the first extract and evaporated to a volume of about 800 liters. The pH 
of the concentrate is adjusted to about 4.7 with concentrated hydrochloric 
acid and combined with about 800 liters of methylene chloride. The 
combined solvents are agitated for about 4 hours and separated. The 
aqueous layer is combined with an additional 800 liters of methylene 
chloride and agitated for about 4 hours. The layers are separated and each 
methylene chloride extract separately treated with about 10 kilograms of 
Super-Cel and filtered. Both extracts are evaporated to a combined volume 
of about 60 liters. 
EXAMPLE 2 
The 60-liter solution of C-076 in methylene chloride of the previous 
example is concentrated to dryness in vacuo and the residue in combined 
three times with 60 liter portions of methanol and evaporated to dryness 
to remove any residual methylene chloride. The final methanol concentrate 
volume is approximately 36 liters. The methanol solution is stored 
overnight and filtered. The filter cake is washed with 40 liters of fresh 
methanol and the methanol filtrates and washings are combined. The 
methanol solution is combined with 95 liters of ethylene glycol and 130 
liters of heptane. The 2-layer solution is agitated for 5 minutes and the 
lower layer (ethylene glycol and methanol) is separated. The heptane 
solution is washed with a mixture of 20 liters of ethylene glycol and 6.3 
liters of methanol. After 5 minutes of agitation, the lower layer is 
separated and combined with the first ethylene glycol/methanol extract. An 
equal volume of water (approximately 150 liters) containing 79 g. of salt 
per liter is added to the ethylene glycol/methanol extracts. This solution 
is extracted with 150 liters of ethyl ether with agitation for 5 minutes. 
The ether layer is washed with 75 liters of water (1/2 volume) and 
agitated for 5 minutes and the layers separated. This procedure is 
repeated an additional 2 times (the final water wash contains 20 g. of 
salt per liter) affording a final ether layer volume of 110 liters. The 
ether layer is concentrated in vacuo, to a minimum volume, keeping the 
temperature less than 25.degree. C. Forty liters of methylene chloride is 
added to the residue and the solution is evaporated to dryness. This 
procedure is repeated and the final residue concentrated in vacuo at 
50.degree. C. to dryness. 
EXAMPLE 3 
A sample of 150 g. of crude C-076 is dissolved in 3 liters of a solvent 
mixture of hexane:toluene:methanol in the ration of 3:1:1. The solution is 
passed through a column of Sephadex LH-20 (having a 30 centimeter 
diameter) in the above solvent taking fractions at the rate of 250 ml. per 
minute. After two 20-liter portions of the solvent mixture are collected 
and discarded, a forecut of 10 liters is taken and discarded. Then 30 rich 
cuts of 2 liters each are taken. Fractions 1-13 and 25-30 are discarded. 
Fractions 14-16 are combined an contain 20 g. of predominately C-076 B1a. 
Fractions 22-24 are combined and contain 6.7 g. of approximately 30% C-076 
B1b. Fractions 17-20 contain a mixture of C-076 B1a and B1b. 
Fractions 17-20 above are combined and concentrated and passed through a 
Sephadex LH-20 column with the same solvent system as above. Three 
20-liter forecuts are taken and discarded. Rich cuts are then taken as 
follows: 5 cuts of 2 liters each (fractions 1-5); 20 cuts of 1 liter each 
(fractions 6-25); and 10 cuts of 2 liters each (fractions 26-35). 
Fractions 1-15 are discarded; fractions 16-21 contain 13.5 g. of C-076 B1a 
and 0.4 g. of C-076 B1b; fractions 22-26 contain 44 g. of C-076 B1a and 
0.31 g. of C-076 B1b; fractions 27-30 contain 10.2 g. of C-076 B1a and 0.8 
g. of C-076 B1b. 
EXAMPLE 4 
A column of Sephadex LH-20 is washed with hexane:toluene:methanol 3:1:1 
overnight at 0.28 ml./min. to equilibrate the resin. Final bed volume is 
53 ml. (1.3 cm. diam. .times.40 cm. height). A 62-mg. sample of mixed 
C-076 B1 and B2 is dissolved in 1 ml. of solvent mixture, applied to the 
column and developed at 0.28 ml./min. 
The composition by high pressure liquid chromatography (HPLC) assay is: 
B2a: 52.4% 
B1a: 44.7% 
B1b: 2.7% 
B2b: 0.2% 
Column effluent is collected in fractions, the first 25 ml., then every 5 
minutes automatically. A 2-.mu.l. aliquot of each fraction is spotted on a 
TLC plate containing phosphor to determine which fractions contain UV 
absorbing compounds. Fractions 12 through 26 are selected for further 
investigation by TLC and total solids. A 5-.mu.l. aliquot of each fraction 
is spotted on a 20.times.20 cm. TLC plste (Silica-Gel 60-F254, E. M. 
Laboratories Inc., 500 Exec. Blvd., Elmsford, NY 10523). The spots are 
dried and the plate is then developed with chloroform:ethyl 
acetate:methanol 9:9:1. The plate is examined under short-wave UV light 
and identification of the components in each fraction is made by 
comparison to a reference sample run on the same plate. 
Fractions 12 through 17 contained C-076 B1, while fractions 21 through 26 
contained C-076 B2. Fractions 18 through 20 contained mixtures of B1 and 
B2, however, total solids determinations on each fraction indicate that 
these fractions contain only a small portion of the total amout charged. 
The recoveries and elution volumes of the two major components are 
tabulated below. 
______________________________________ 
Elution Volume 
Recovery 
______________________________________ 
C-076 B1 0.87 column volume 
89% 
C-076 B2 1.05 column volume 
68% 
______________________________________ 
EXAMPLE 5 
A sample of 200 mg. of crude C-076 is dissolved in 0.8 ml. of a solvent 
mixture of hexane:toluene:methanol in the ratio of 6:1:1. The solvent 
mixture is chromatographed on a column 1.27 cm..times.65 cm. containing 
Sephadex LH-20 as the packing. The column is eluted with a solvent mixture 
of hexane:toluene:methanol in a 6:1:1 ratio at a flow rate of 2 ml. per 5 
minutes with 2-ml. cuts being taken. Five (5) microliters of each cut is 
applied to E. Merck silica-gel 60-F254 plate and the C-076 components 
determined by thin layer chromatography (tlc) with a 10:10:1:2 system 
(chloroform:ethyl acetate:methyl alcohol:methylene chloride). Based on the 
tlc results, the cuts are pooled as shown in Table I as assayed using high 
pressure liquid chromatography assay techniques. 
TABLE I 
______________________________________ 
HPLC Assay (%) 
Sample No. 
Volume Yield (mg.) Ala Alb A2a 
______________________________________ 
Feed 0.8 ml. 200 71.9 12.3 13.7 
1 60-116 ml. 164.4 82.8 15.2 0.9 
2 128-146 ml. 
22.4 1.4 0.4 95.7 
______________________________________ 
Sephadex is a tradename given to dextran gels which are natural polymers of 
glucose made by fermentation and cross-linked with epichlorohydrin. 
Although the hydroxy propylated dextran gels are the preferred packing 
utilized in this invention, other derivatives that might be utilized are 
the nitro alkyl, cyano alkyl or other derivatives of the alkyls that would 
exhibit similar properties as the hydroxy propylated dextran gel.