Note: Descriptions are shown in the official language in which they were submitted.
556~
The phenomenon of synergism has been ext~nsively
reported in antibiotic literature: The Journal o Anti-
; biotics 25, No. 6, 371 ~1972); J. Chem. Soc. l9C, 1653 (1966);
Bull. Soc. Chim. Belg. 68, 716 (1959); J. Amer. Chem, Soc. 82,
S 4414 (1960); Tetrahedron Lettexs 2687 (1971); J. Antibiotic~,
Ser. A 14, 14 (1961); Nature 187, 598 (1960); J. Chem. Soc.
2286 (1960); Antimicrobial ~gents & Chemotherapy 360-365
(1964); Tetrahedron Letters 4231-4238 (1966); Oxganic Mass
Spectrome~ry 6, 151-166 (1972); Tetrahedron Letters 369-372
(1966) and J. Chem. Soc. 19C, 1669-1676 (1966~.
The new ~ynergi~tic mixtures of antibiotics of the
present invention ~oin the family of other reported synerglstic
mixtures~ mikamycin, pristinamycin, ostreogrycin, strepto-
gramin, P.A. 114, vernamycin and ~irginiamycin.
lS This invention is concerned with a mixture of ant~-
biotics produced by the submerged aerobic propagation of
Actino~lanes auranticolor ATCC 31011 in a~ueou3 nutrient
media. Thi5 mixture, containing macrocyclic lactone3 and
depsipeptides, may be separated and recovered ~rom fermenta-
tion broth by solvent extraction, counter-current di~tribu-
tion, column chromatograph~ or combinations thereo~. The
ind~vidual antibiotic component~ exhibit signiflcant anti-
biotic activlty. The crude anti~iotic mixture or combin~-
tions of a pure =acrocycl~c lactone and a pure depsipep~id~
25~ obtained from the crude mixture ~emon~trate marked synergi~ti~
-2
~LS56~
antibiotic ac~ivity. The crude an~iobiotic mixture, the pure
individual antibiotic components and mixtures of pure macro-
cyclic lactones and depsipeptides are ef~ective chick and
swine growth promotants and therapeu~ic agents in the control
of swine dysentery.
The microorganism useful or the preparation o~ the
antibiotics of this invention was isolated from a soil sample
in Egypt. It was grown on potato-carrot agar and found to
belong to the class of actinomycetes producing sporangia like
those of the genus Actinoplanes. It was grown, therefore,
. .
on a number of media used for the study of this genus. Sus-
; pensions of the culture were prepared by crushing pieces of
the culture, derived rom agar slants, in small tu~es each
containing about 0.2 ml. sterile distilled water, rinsing
out the contents a~d combining the aontents with addi~ional
~terile water to make a volume of about 5 ml. for the culture.
~hese suspen~ions were used to plan~ the culture in tubes, ;~
slants or petri dishes o~ the various media. The incubation
temperature was 28C. except where otherwise noted. Readings
of results were made at intervals up to 22 days for some
tests but most result~ were recorded after 14 days. ~he
colors of the aulture are those of Maerz and Paul, Dictionary
of Colors, 2nd edition, l9S0, as we~l as personal descrip-
tive terms. ~his new culture (Pfizer F.D. 24090) was sub-
mitted to the American Type Cul~ure Collection in Rockville,
.
Maryland on March 11, 1974 and given the designation
Aatinoplanes auranticolor ATCC 31011. The permanency of the
; deposit and ready accessibility thereto by the publia are
aforded in the event the patent ls granted.
--3--
-,~
5561~
Identiication media used for the characterization
of the culture and refe~ences for ~heir composition are as
follows:
1. 2% Tap water agar.
2. Potato-Carrot Agar. M.P. Le~hevalier, J. Lab. &
Clin. Med. 71, 934-944 (1968~. Usle only 30 g.
potatoes and 2.5 g. carrot~ bu~ 20 g. agar~
3. Czapek-Sucrose Agar. Waksman, S.A., The Adtino-
; mycetes 2, 32% (1961) Medium No. 1, p. 328.
4. Glucose-Asparagine Agar. Waksman, as above, Medium
No. 2, p. 328.
5. Yeast Extract-Malt Extract Agar. Antibiotics Ann.
1956/1957, pp. 947-953.
6. Hickey and ~resner Agar. ~. Bact. 64, 891-892
(1952).
7. Potato-Glucose Agar. Peel, cu~ up and steam 100 g~
potatoes in S00 ml. water, filter through chease
oloth, add 10 g. glucose, 20 g. agar and enough
water to make one liter.
8. Starch Agar. J. Bact~ 73, 15-27 (1957).
9. Gelatin. J. Bact~ 73, 15-27 (19S7).
10~ Tyrosine Agar. 3. Bact. 69, 147-15n (1955).
11. ~ifco Pep~oné Iron Agar.
12. Difco Skim Milk.
13. Dextrose Nitrate Broth. Waksman, S.A., The Acti-
nom~cetes 2, 328 (1961).
Medium Mo. 1 with 3 0 g. glucose in place of sucrose
''~
'' .
5~i61~
and without agar.
14. Organic Nitrate Broth. J. Bact., 73, 15-27 (1957).
15. ATCC Medium 172. American Type Culture Catalogue,
10th edition, p. 235 (1972).
16. Carbon Utilization. J. Bact., 56, 107-114 (1948).
The description of this new culture is as follows:
Tap Water Agar - growth poor, thin, flat, near 9D2 (very
pale pink); no aerial mycelium; substrate mycelium colorless
to 9D2; no soluble pigment.
Czapek-Sucrose Agar - growth moderate to good~ flat, near
9G6 (pale orange); no aerial mycelium; substrate mycelium
near 9G6; no soluble pigment.
Glucose-Asparagine Agar - growth moderate, raised, rough-
ened, near 9L9 (light orange); no aerial mycelium; no soluble
pigment. , ~;
Yeast Extract-Malt Extract Agar - no growth.
Hickey and Tresner Agar - growth moderate to good, slight-
ly raised and roughened, near 9F9 (dull orange); ~aint whitish
bloom on surface; substrate mycelium near 9I8; pale brownish
soluble pigment.
Potato-Glucose Agar - growth moderate, raised, roughened,
near 9L9 (light orange); no aerial mycelium; substrate
mycélium near 9L9; no soluble pigment Tyrosine Agar - growth
; poor to moderate, ~lat, near 13A10 (dull reddish orange);
no aerial mycelium; substrate mycelium near lODll; brown
soluble pigment.
Gelatin - growth moderate, flat, near 9K12 (reddish
orange); trace of whiti~h bloom: suhstrate mycelium near
9K12; no soluble pigment.
~tarch Agar - growt~ moderate to good, rai~ed, near
'
. '
.. . ..
~ ~ 5 S~ ~
9klO (orange); light whitish bloom; substrate mycelium near
9klO; pale yellow solu~le pigmsnt.
Starch was weakly hydrolyzed; glelatin liquefaction
was strong; nitra~es were no~ raduced to nitrites in either
nitrate medium even in 22 days (growth was very poor in
dextrose nitrate ~rot~ ~ut good in organic nitrate ~roth);
H2S was weakly produced; there was no soluble pigment in
peptone iron agar; there was no coagulation or hydrolysis
of milk even in ~2 days; tyrosine was not diges~ed; growth
on ATCC Medium 172 occurred at 21 to 37C. with best growth
at 28 and 37C.; there was no growth at 45C. ~rabinose,
fructosa, glucose, mannitol, ra~finose, rhamnose, sucrose and
xylose were utilized; lnositol was not utilized. There was no
odor on any medium.
Sporangia were produced only on ~he potato-carro~
agar. They formed a palisade layer. Mea3urements were
5.5-11 x 4.5-8 microns in width and breadth and 9-12 microns
in height. Th~y were quite numarous, irregular in shape and
set ~pores free by gradual softenlng. Sporangia ~rom potato-
carror agar after threa weeks incubation released spores in a
few houxs at about 21C. when pieaes o~ the growth were sub-
merged ln a ~mall amoun~ of a solution o~ 1 g. of glucose
and 1 ml. of Tween 80 ~"Tween" is a trade mark) ln 1 liter
o~ water ~a modification of a solution used by M.L. Higgins,
J. ~act~, 94, 49$-498, 1967). The spoes were in chains of
; irregular shape in ~he sporangia but when set ree were sub-
globose and 1.6 microns wide to broadly elliptical, 1.6-2.2 x
1,1-1.6 microns. Almosk all were motil~.
~ tenta~i~e identiication led to a comparison o
this cul~ura with A.auranticolor ATCC 15330~ The new
--6--
~ .
L5S6~3
strain A. auranticolor ATCC 31011 and A. auranticolor ATCC
15330 looked essentially alike in morphological traits, color
and soluble pigment on Bennett's Agar, Nutrient Agar, Yeast
Extract A~ar, Glucose Asparagine Agar, Glycerol-Asparagine
Agar, Calcium Malate Agar and Tyrosine Agar.
Neither culture reduced nitrate to nitrite; both
produced H2S weakly and failed ~o produce melanin on peptone-
iron agar; both hydrolyzed starch. Ao auranticolor ATCC
15330 caused no change in skim milk tubes whereas the new
~ 10 culture caused clearing in three of the six tubes of milk used
; and after 21 days produced a yellow-cream soluble pigment.
A. auranticolor ATCC 31011 utilized glucose, arabi-
nose, fructose, mannitol, raffinose, rhamnose, sucrose and
xylose. A. auranticolor ATCC 15330 utili2ed all these sugars
with the exception of ra~finose. Sporangia and spores of the
two cultures were similar with the spores of A. auranticolor
15330 more rod shaped.
Most importantly, A. aurantlcolor ATCC 15330 did
not produce any antibiotic activity under the fermentation
conditions in which A. auranticolor ATCC 31011 produced the
_ _
mixture of antibiotics of the present invention.
Cultivation of A. auranticolor ATCC 31011 prefer-
ably takes place in aqueous nutrient media at a temperature
of 28-36C., and under submerged aerobic conditions with
agitation. Nutrient media which are useful or such purposes
include a source of assimilable carbon such as sugars~ starch
and molasses; a source of organic nitrogen such as casein,
enæ~matic digest of casein, soybean meal, cottonseed meal,
peanut meal and wheat gluten. A source of growth suhstances
such a~ distillers' solubles, ish neal and yeast extract as
.~,
,,".
~S56~
well as salts such as sodium chloride and calcium carbonate
and trace minerals such as iron, magnesium, zinc, cobalt and
manganese may also be utilized with advantageous results.
If excessive foaming is encountered during fermentation, anti-
foam agents such as vegetable oils or silicones may be addedto the fermentation medium. Aeration of the medium in tanks
for submerged growth is preferably maintained at the rate o
about 1/2 to 2 volumes of free air per volume of broth per
minute. Agitation may be maintained by means of agitators
generally familiar to those in the fermentation industry.
Aseptic conditions must, of course, be maintained through the
transfer of the organism and throughout its growth.
Inoculum for the preparation of the antibio~ic mix-
ture may be obtained by employing growth from a slant of the
culture on ~ medium such as ATCC Medium 172 to which previous
reference was made. The growth may be used to inoculate either
shake flasks or inoculum tanks, or alternatively, the inoculum
tanks may be seeded from the shake flasks. In shaken flasks
growth will generally have reached its maximum in about 4 days
whereas inoculum in submerged inoculum tanks will usually be
at the most avorable period in 2 to 3 days. Substan;tial
antibiotic activity is obtained in the final fermentor stage
in approximately 20 to 30 hours.
The process of antibiotic production is conveniently
followed during fermentation by biological assay of the broth
-employin~ a sensitive strain of Staphylococcus aureus.
Standard plate assay technique is employed in which the zone
o inhibition surrounding a filter paper disc saturated with
the broth is used as a measure o antibiotic potency. After
the ermentation broth has reached a desired level o~ anti-
-8-
. ~ . , .
~ ;S~i8
biotic potency, the products are isolated from either wholebroth or filtered broth. In the latter case, the mycelium
is removed by filtration or centrifugation. Various types
of equipment such as filter presses, centrifuges, etc. may
be employed.
Thin layer chromatography employing silica gel is
a useful tool for analyzing the antibiotic mixture produced
in fermentation media and the composition of crude and puri~
fied materials extracted from fermentation broths. The re-
solution of the components of the antibiotic mixture is im-
portantly dependent on antibiotic loading of the system.
~oo little antibiotic potency fails to reveal minor anti-
biotic components; too much antibiotic potency results in a
dragging effect with resulting poor resalution.
The developing system for the thin layer chromato-
graphy is chloroform-ethanol (9:1~. The thin layer chromato-
grams, after developmen~, may be observed under ultraviolet
light at 254 m~ and 366 m~. Bioautographic detection of the
antibiotic components may be accomplished by means of an
overlay of a thin layer o~ nutrient agar seeded with a sensi-
tive strain of Staphylococcus aureus or other sensitive
organism.
The primary components in the antibiotic mixture
produced by A. auranticolor ATCC 31011 include a number of
macrocyclic lactone and depsipeptide antibiotic components.
The appearance or non-appearance or percentage composition
of these antibiotic components varies from fermentation ~o
fermentation and is a ~unction of time, pH, med:ia composition,
etc. Under sets of conditions given in the examp:Les herein-
after, major antibiotic components in the antibiotic mixture
_g_
4SS~3
are Compounds 37,277 (depsipeptide) and 36,926 ~macrocyclic
lactone) while the minor antibiotic components are Compounds
37,932 (depsipeptide) and 35,763 (macrocyclic lactone).
The infrared spectra o~ Compounds 37,277, 36,926,
35,763 and 37,932 are shown in Figs. 1 to 4 respectively of
the accompanying drawings.
The components of the antibiotiLc mixture may be
separated and recovered ~rom fermentation broth by a number
of different procedures including solvent extraction, Cra~g
counter-current distribution, column chromatography or combi
nations thereo~. Various organic solvents such as chloro-
form, ethyl acetate and methyl isohut~l ketone are u~eful in
extracting the antibiotics from broth. Solvent extraction
i8 preferably carried out by twice extracting the broth at
about pH 7 with a volume of solvent approxlmatel~ equal to
about 1/3 to 1/2 the volume o~ broth from which it is desired
to recover the antibiotic mixture. Depending on volumes o~
broth involved, various pieces o~ equipment such as separatory
funnles, stirred tanks and mechanical extracting devices
~uch as centrifugal separators are helpul ~or extraction
purpose3.
The preferred method o~ separation and recovery of
the components o~ the antibiotic mixture is as follows: either
whole or clarified bxoth is ad~usted to about pH 7 and twice
extracted with about 1/3 to 1/2 volume of methyl isobutyl
ketone. The solvent extract is concentrated under ~acuum
and the concentrate defatted by extraction with heptane or
petroleum ether. The deatted sol~ent concentrate is then
taken to dryne~ under vacuum. The solids are subjected to
Craig counter-current distrihution t6 plates) utilizing
toluen~, 5 parts: ethanol, 2 part~: aqueous phosphate buf~er,
pH 4.5, 3 part~. ~he separated layers furnish the upper and
lower phases of the counter-current distribution system.
'' ~' ~:''
- ~4S~i6~
After distribution, the layers are monitored by thin layer
chromatography. The separated fract:ions are taken to dry-
ness under vacuum.
The solids containing the depsipeptides are dis-
solved in chloroform, treated with activated charcoal, filter-
ed and evaporated ln vacuo. The resi.due obtained on evapora-
tion of the chloroform is dissolved in acetone. The solids
precipitated by the addition of heptane are dissolved in a
small amount of chloroform and applied to a column of pH 6
buf~ered silica gel made up in chloroform: n-propanol
(99:1% v/v). The column is developed with the same solvent
system under 80 psi. The column cuts are monitored by thin
layer chromatography. The cuts containing separated depsi-
peptides are combined, evaporated in vacuo and crystallized
; 15 from acetone-heptane.
The counter-current fractions containing the macro-
cyclic lactones are combined, evaporated ln vacuo and the
solids taken up in ethyl acetate. The solution is stirred
with silica gel, filtered and the solvent removed ln vacuo.
The residue is taken up in ethyl acetate and precipitated
wikh hexane. The solids are dissolved in a small amount of
chloroform and chromatographed under 80 psi on a pH 6.0 buf-
fered silica gel column made up in ethyl acetate. The develop
ing system is ethyl acetate-tetrahydrofuran-hexane (80:20:20)
saturated with aqueous pH 6.0 phosphate buffer. The coIumn
cut~ are monitored by thin layer chromatography. The cuts
containing separated macrocyclic lactones are combined and
evaporated In vacuo. The individual fractions are separately
worked up by further Craig counter-current distribution and/or
~olumn chromatography utilizing different developing sys ems.
~SS6~
Careful monitoring at every purification stage locates the
individual macrocyclic lactones sufficiently isolated so
that the solvent fractions can be taken to dryness to yield
: the pure compounds.
A. auranticolor ATCC 31011 produces at least four
depsipeptides and at least four macrccyclic lactones. How-
ever, the primary components are the depsipeptides Compounds
37,277 (major) and 37,932 (minor) and the macrocyclic lactone
Compounds 36,926 tmajor) and 35,763 (minor).
Crude antibiotic mixtures obtained directly from
broth and purified individual components possess wide anti-
bacterial spectra. Among the organisms failing to propagate
in the presence of the antibiotics are Salmonella ~y~
Shigella dysenteriae, Escherichia coli, Klebsiella pneumoniae,
Staphylococcus aureus, Streptococcus pyogenes, Streptococcus
faecalis, Diplococcus pneumoniae, Bacillus subtilis,
: .
Corynebacterium diphtheriae, Clostridium septicum, Brucella
abortus, Neisseria sicca, Iactobacillus acido~hilus and
Pasteurella multocida.
The antibiotics of this invention, either as a
crude mixture or in the form of the purified individual com- ;~
ponents or mixtures thereof, may be employed in the treatment
of various infections in man and animals. In general, these
`~ antibiotics are most desirably administered in daily oral
doses of 0.5-1 gram or parenteral doses of 100 to 500 mg.,
depending on the type and severity of the infection and
weight of the subject b~ing treated.
The compounds of this invention may be administered
alone or in combination with pharmaceutically-acceptable
carriers, and such administration can be carried out in both
-12- ;
'' / ~'''~''
.. . . ~ . ~ . . .. .
~ 55~
single and multiple doses.
For purposes of oral administration, tablets con
taining various excipients such as sodium citrate, calcium
carbonate and dicalcium phosphate may be employed along with
various disintegrants such as starch, alginic acid and certain
complex silicates together with binding agents such as poly-
vinylpyrrolidone, sucrose, gelatin and gum acacia. Additional-
ly, lubricating agents such as magnesium stearate, sodium
lauryl sulfate and talc are often useful for tableting pur-
poses. Solid compositions of a similar type may also be empaoyed as fillers in soft and hard-filled geIatin capsules;
preferred materials include lactose as well as high molecular
weight polyethylene glycols. When aqueous suspensions and/or
elixirs are desired for oral administration, the essential
active ingredient therein may be combined with various sweet-
ening or flavoring agents as well together with such di-
luents as water, ethanol, propylene glycol, glycerol and
various combinations thereof.
Solutions of these antibiotics in sesame or peanut
oil or in aqueous propylene glycol may be employed for paren-
teral administration.
It is of interest that the individual antibiotics
` of this invention exhibit antimicrohial activity which is
largely bacteriostatic in nature. However, crude antibiotic
mixtures or mixtures of a purified depsipeptide and a puri
~ied macrocyclic lactone exhibit synergistic activity which
is largely bactericidal in nature.
When major macrocyclic lactone CompouncL 36,926 (A)
and major depsipeptide Compound 37,277 (B) were assayed in-
dividually and in combination by the tube dilution method
~ . ~
-13-
, .
, . . .
. . .
iS6~3
versus the following organisms, the following minimal in-
hibitory concentration in micrograms/ml. (M.I.C.) were found:
Crude
Organism A B A + BMixture
Staph. aureus 005 1.56 25 0.10 ~< 0.10
Staph. aureus 400 3.12 12.5 0.78 ~'~ 0.10
Strep. faecalis 100 1~.5 1.560.20
Neisseria sicca 0.39 50 0.10~ 0.10
TreE~ema hyody~senter1ae ~ -- 0.19
Stre~. p~ogenes 0.39 12.5 0.10~ 0.10
Bacteroides fragilis 35614 25 50 0.78 0.78
Clostridi_m inocuum 6.25 ~100 0.200.39
Lactobacillus casei var.
casei 3.12 6.25 0.200.39
Comparable results were obtained when minor macro-
cyclic lactone Compound 35,763 (A') and minor depsipeptide
Compound 37,932 (B') were assayed individually or in appro-
priate combinations, i.e. A' A, B' B while (A' ~ B')
(A' + B') (A + B') (A + B).
Maximum synergistic activity of cornbinations of
purified macrocyclic lactone and depsipeptide is obtained
over a range of about a ratio of 1~2:1. Approximately such
ratios occur in ermentation broths of A. auranticolor ATCC
31011 and in crude antibiotic mixtures isolated therefrom.
2~ This finding is in contrast to other reported synergistic ~ -
antibiotic mixtures where the synergistic factors occur
in fermentation broths and crude mixtures at sub-optimal
ratios.
In vivo protection data provided by oral and sub-
cutaneous administxation in mice experimentally infected
--1~--
". ,.
551~8
with a strain of Staphylococcus ureus are shown in Table I.
Table I
PD50 Values (mg./kg.)
S. aureus 01A005
Oral Subcutaneous
Compound 36,926 ~2n0 ~200
Compound 37,277 > 200 ~200
; Compounds 36,926 + 37,277 210 60
~, , (1:1)
Crude antibiotic mixture 150-200 72-120
(Lot 1, p. 25)
The antibiotics of the present invention may be
regarded to be of special interest as growth promotants in
poultry and animals because of their wide antibacterial
lS spectra and for the treatment of swine dysentry because of
marked activity against Treponema hyodysenteriae, an
anaerobic spirochete implicated in this disease.
The growth promoting activity o ~ crude antibiotic
mixture ~Lot 1, p. 21~ was determined in young feeder pigs
in a 40-day trial. Average daily gain, feed consumption and
efficiency were significantly improved (p ~ 0.01) over the
~; non-medicated control (Table II).
Table II
Average Average FePd
Treatment Daily Gain(kg) Daily Feed(kg) Efficiency*
Non-medicated 0.35 0.91 2.57
- Lot 1, (p. 25)
50 ppm 0.63 1.35 2.15 ;~
~; * lbs. of feed per lb. of weight gain
Comparable results may be obtained wil:h the other
. "'!
crude antibiotic mixtures of the compositions described on
page 21 over a range of 10 to 100 ppm.
.
, ' ,
~9LS~
Comparable results may also be obtained by ad-
ministration of the individual pure Compounds 36,926, 37,932
and 37,277 or mixtures of the pure compounds approximating
in composition those of the crude antibiotic mixtures
described on page 21.
Growth promoting efficacy was demonstrated in a
chick battery feed trial. Significant improvements
(p~-0.01) in weight gains over the non-medicated controls
were observed for the chicks on an antibiotic-feed diet
(Table III). -
Table III
Average Average Feed
Weight Gain Consumed Feed
Treatment (grams) (grams~ Efficiency
; 15 Non-medicated 566 939 1.66
' Lot l, (p. 25~ 10 ppm 608 949 1.56
'.''' '" ''"
; Comparable results may be obtained with the other
, crude antibiotic mixtures of the compositions described on
page 21 over a range of 10 to lO0 ppm.
Comparable results may also be obtained by ad-
ministration of the individual pure Compounds 36,926, 37,932
and 37,277 or mixtures of the pure compounds approximating
in composition those of the crude antibiotic mixtures
described on page 21.
;, 25 The prophylactic efficacy of the antibiotics of
this invention was determined in swine experimentally in-
- fected with infectious material causing swine dysentery.
Colonic content and mucosal scrapings were obtained from a
clinically diagnosed field outbreak of swine dysentry.
Normal pigs were infected with this material by direct
-16-
.
~L~455~
inoculation. Antibiotic-containing feed was administered over
a 28 day period. The results are shown in Table IV.
- Table IV
Averaye
Treatment Morbidity (%) Mortality (~) Daily Gain (kg)
_ _ _
Non-medicated 100 40 -0.13
Lot 1, (p. 25)
50 ppm 0 0 0.66
37.5 ppm 0 0 0.57
. .
25 ppm 0 0 0.68
12.5 ppm 0 0 0.61
6.25 ppm 50 10 0.47
Comparable resuits may be obtained over a range of
10 to 100 ppm with the other antibiotic mixtures of page 21,
the pure individual Compounds 36j926, 37,932 and 37,277, or
mixtures of the pure compounds approximating in composition
those of the crude antibiotic mixtures described on page `21.
EXAMPLE I
~ A sterile aqueous medium having the following com-
- 20 position is prepared:
Gra~s/liter
Glucose 10.0
Soluble starch 20.0
Yeast extract 5.0
i~ 25 Enzymatic digest of casein 5.0
CaC03 1. 0
pH - 7.0
Cells from a slant of A. auranticolor ATCC 31011
grown on ATCC medium 172 are transferred to a series of
300 ml.-Erlenmyer flasks each containing 50 ml. of this
-17-
: . :
~ ' ' .. ' ~
~g~4556~3 :
medium and shaken on a rotary shaker for 3-4 days at 28-30C.
Aliquots of 5 ml. of the grown inoculum are transferred to
300 ml. - Erlenmyer flasks each containing 100 ml. of the
sterile medium described above. After shaking for 3-4 days
at 28-30C., 5-10% v/v of the grown i.noculum is transferred
to a four liter fermentor containing two liters of the
following sterile medium: `
; Grams/liter
Yeast extract 2.0
Glucose 10.0
Corn Steep Liquor 1 ml.
Enzymatic digest of casein S.0
Cobalt Chloride 0.002
pH - 7.0
The fermentation is conducted for 20 to 30 hours at 28-36C.
with stirring at 1700 revolutions/minute and aeration at
about one volume of air per volume of broth per minute.
The whole broth is adjusted to pH 7 if necessary,
and twice extracted with 1/3 to 1/2 volume methyl isobutyl
ketone. The solvent extract is concentrated under vacuum and
defatted by extraction with petroleum ether. The activity
in the broth, solvent extract and subsequent fractions is
followed by silica gel thin layer chromatography using a
- developing s~stem of chloroform-ethanol ~9:1) and observa-
tion under ultraviolet light a~ 254 and 366 m~.
The defatted solvent concentrate is taken to dryness :
; under vacuum. The solids are subjected to a 6 plate Craig
counter-current distribution utiliziny toluene, 5 parts:
I ethanol, 2 pa-rts: aqueous phosphate buffer, pH 4.5, 3 parts.
~ 30 The separated la~vers furnish the upper and lower phases of
1 : :
18-
. ....
the counter-current distribution system. After distribution,
tha layers are monitored by thin layer chromatography.
The depsipeptide, Compound 37,277, is concentrated
in the upper layer, primarily in plate 0. A second depsi-
peptide, Compound 37,932, is recovered from the upper layersof plates 1, 2 and 3. The macrocyclic lactone, Compound
35,763, is found primarily in the lower layers of plates 0
and 1. Compound 36,926 is concentrated in the lower phases
of plates 2, 3, 4 and 5.
The upper phase of plate 0 containing Compound
37,277 is taken to dryness under vacuum, dissolved in
chloroform and stirred for about 30 minutes with activated
charcoal. The ~olution is filtered and evaporated in vacuo.
The re~idue is dissolved in acetone and the solids precipitated
by the addition of heptane. The precipitated solids are dis-
solved in a small amount of chloroform and chromatographed
on a column o pH 6 buffered silica gel made up in chloroform:
n-propanol (99:1% v/v). The column is developed with the same
system under 80 psi. Column cuts are monitored by thin layer
chromatography. The fractions containing separated Compound
37,277 are combined, evaporated in vacuo and the compound
crystallized from acetone-heptane.
Compound 37,277 does not have a definitive melting
point. Decomposition commences at 140-150C. It is insoluble
in diethyL ether, hexane, heptane and water. It is slightly
;~ soluble in acetone and benzene and readily soluble in
methanol, ethanol, chloroform and methylene chloxide.
Analysis of Compound 37,277 gives the following
average proportions:
~.
19-
:
Carbon 60.91
Hydrogen 5.98
Nitrogen 10.45
Oxygen (by difference) 22.66
Compound 37,277 is optically active having a rota
tion of ~]25 = + 11 (c = 1.0, EtOH). I~s ultraviolet light
absorption maxima in ethanol occur at 225~ 274, 282, 303
and 355 m~ with El cm values of 309.3, 36.67, 45.01, 70 and
20, respectively.
The infrared spectrum of Compound 37,277, Figure 1,
is attached. A chloroform solution shows characteristic ab-
sorption in the infrared region at the following wavelengths
in microns: 3.05, 3.40, 5.70, 5.77, 5.93, 6.07, 6.62, 6.82
and 7,67.
; 15 The counter-current fractions containing Compounds
35,763 and 36,926 are evaporated in vacuo, the residue taken -~
up in ethyl acetate and the solution stirred with silica gel.
The filtered solution is evaporated in vacuo, the residue ;~
taken up with ethyl acetate and the solids precipitated by
~, 20 the addition of hexane. T~e precipitated solids are dissolved
in a small amount of chloroorm and chromatograph~d on a
column o~ pH 6.0 buffered silica gel made up in ethyl
acetate. The column is developed with ethyl acetate: tetra-
hydrofuran: hexane ~80:20:20) saturated with aqueous pH 6.0 -
phosphate ~uffer under 80 psi. The first 17 column cuts each
of 20 ml. contain a yellow oil which is discarded. Cuts -
26-40 are rich in Compound 36,926. Cuts 41-50 are largely
a mixture of Compounds 36,926 and 35,763. Cuts 26-~0 are
combined, concentrated ln vacuo and again chromatographed on
sil1ca gel with 20 mlO cuts being collected. The first 15 -
-20-
., .
5~3
cuts contain a yellow oil which is discarded. Cuts 16-30 are
primarily Compound 36,926. Cuts 31-40 contain a mixture of
Compounds 36,926 and 35,763. Cuts 16-30 are evaporated ln
vacuo, the residue dissolved in a small amount of chloroform
and applied to a column of pH 6.0 buffered silica gel made
up in chloroform. The column is developed with chloroform:
ethanol (95.5:4.5% v/v) under 130 psi., and 160 cuts of 6 ml.
collected. The cuts 60-90 assay for only Compound 36,926.
They are combined and evaporated ln vacuo. The residue is
dissolved in the minimum volume of ethanol and precipitated
with ether to give pure, amorphous Compound 36,926.
-Compound 36,926 is soluble in methanol, ethanol,
chloroorm and methylene chloride. It is insoluble in di-
ethyl ether, hexane and heptane.
~ompound 36,926 does not have a definitive melting
point. Decomposition commences at about 100C. Analy~is
gives the following average proportions:
Carbon 57.89
Hydrogen 6.78
Nitr~ogen 8.04
Oxygen (by difference) 27.29
The molecular weight by high resolution mass spectrum is 501,
and the molecular formula C26H35N3O7.
Compound 36,926 is optically active having a rota-
...
tion of [~]25 = _ 130 (c = 1.0, EtOH). Its ultraviolet
light absorption maximum in ethanol is 214 m~ with El%Cm f
723.8.
The infrared spectrum of Compound 36,926, Figure
2, is attached. A KBr pellet shows characterist:ic absorption
in the infrared region at the following wavelengths in
-21-
''
. ~ . ...
i6E~
micronsO 2.95, 3.40, 5.75, 5.g8, 6.23, 6.58, 6.87, 7.45,
8.25, 8.38, 8.80, 9.08, 10.15, 10.35, 11.10 and 13.30.
This compound may be similar to or indistinguish- .
able from antibiotic A2315B reported at the Fourteenth
Interscience Conference on Antimicrobial Agents and Chemo- :
therapy, September 11-13, 1974.
Compound 35,763 is isolated and purified in much
the same way as Compound 36,926. The pure compound is solu-
ble in methanol, ethanol, chloroform and methylene chloride.
It is in~oluble in diethyl ether, hexane and heptan~e. There ~ :
is no definitive melting point. Decomposition commences at
about 100C. Analysis gives the following average propor~
tions:
Carbon 61.29 :~
Hydrogen 6.73
Nitrogen 8.83
Oxygen (by difference) 23.15
Molecular Weight by high resolution mass spectrum is 503, :
and the molecular formula C26H37N3O7. ~.
Compound 35,763 is optically active having a rota-
tion of E~]25 ~ - lI4 (c = 1.0, EtOH). Its ultraviolet
light absorp~ion maximum in ethanol is 218 m~ with El%m of
668.9.
The infrared spectrum of Compound 35,763, Figure 3,
is attached. A KBr pellet shows characteristic absorption
~; in the in~rared ~egion at the following wavelengths in
microns: 2.95, 3.38, 5.73, 6.00, 6.23, 6.60, 6.88, 7.23,
. 8.25, 8.38, 8.83 and 10.20.
Compound 35,763 may be similar to or indistinguish-
able from antibiotic A-2315 described in Dutch Patent
-22-
.. ~ .
.
-.
, .. , . , ..... . : .. :.. ... . ., .. .. . , . : , ,
~-- ;
~4556~
7,310,613. ~-
The counter-current fractions containing Compound
37,932 were taken to drypess under vacuum, the residue
triturated with petroleum ether and chromatographed on a
silica gel column as described above. The appropriate frac-
tions were combined and crystallized from acetone:heptane
to afford Compound 37,932.
Compound 37,932 is soluble in methanol, ethanol,
chloroform and methylene chloride. It is insoluble in di-
1~ ethyl ether, hexane, heptane and water. The compound doesnot have a deinitive melting point. Decompo~ition commences
at approximately 185C. Elementary analysis gives the follow-
ing average proportions:
; Carbon 59.41
; 15 Hydrogen 6.01
Nitrogen 10.66
~ Oxygen (by difference) 23.92
`~ Compound 37,932 is optically active having a rota-
tion of [~]25 = ~ 5.0 (c = 0.25, CHC13). Its ultraviolet
light absorption maxima in ethanol occur at 226~ 276, 283,
305 and 355 m~ with El~ values of 304.4, 36.8, 43.49, 70.2S
and 20.07, respectively.
The infrared spectrum of Compound 37,932, Figure
4, is attached. A KBr pellet shows charactsristic absorp-
tion in the infrared region at the following wavelengths in
~microns: 2.96, 3.05, 3.38, 5.68, 5.73, 5.93, 5.98, 6.13, 6.50,
6.5~, 6.88, 7~40, 7.65, 8.08, 8.45, 8.60, 9.03, ~.45, 9.70,
~ 9.98, 10.55, 11.00, 11.25, 11.60, 12.35 and 13.25.
-~ EXAMPLE II
The method of Example I may be repeated with com-
-23-
,: , ~' '
: '
, , .
~5~i68
,i `
parable results employing a fermentation medium of the ollow-
ing composition:
Grams/liter
: Glucose 8.Q
Tryptose 3.0 :~
Enzymatic digest of :
casein 1.0
Glutamic acid 1.0
Soy flour 0.5
NaCl 8.3
K2HPO4 2.4 ~:
KH2PO4 2.0
MnC12 0.02
EXAMPLE III
The method of Example I may be repeated with com~
parable results employing a fermentation madium having the
following composition:
Grams/liter
Glucose 10.0
Soy flour 10.0
; Corn steep liquor 1 ml.
EXAMPLE IV
` The method of Example I may be repeated with com-
parable re~ults employing a fermentation medium having the
.: 25 follow1ng composition:
Grams/llter ~. "
.~' Molasses 10,0
I Enzymatic digest of casein 1.0
i. Yeast extract 2.0
Corn steep liquor 1 ml.
. Casein 3.0 .
-24-
:;',`'''`. ~ -~ '
"
- - , ~ , . ; . . . . .
~55i6~
EXAMPLE V
The method o Example I was repeated. The methyl
isobutyl ketone extract of the finished fermentation broth
was taken to dryness under vacuum and the residue triturated
with petroleum e~her. The friable mat:erial was milled and
the antibiotic components were quantit:atively dete~mined
by high pressure liquid chromatographic assay. Representative
bulk lots of crude antibiotic mixtures from separate fermenta-
tion runs assayed (per cent) as follows:
Lot No. 35~76336,92637,932 37,277 Misc. Com~onent~
1 8.5 32.0 8.7 19.3 1.3
2 3.1 39.5 7.8 15.9 1.2
3 6.0 38.6 10.7 25.5 2.~
4 9.4 47.8 0.9 26.9 0.5
lS 5 a.4 31.9 2,2 20.3 1.4
,~
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