Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
1~4~3563 `- :
; This invention is concerned with a new member of
the acidic polycyclic ether group of antibiotics, a class of
compounds characterized biologically by their effect on
cation transport in mitochondria, This family of antibiotics
includes monen~in (J~ Amer. Chem. Soc., 89:5737, 1967)~
nigericin (Biochem. Biophys. Res. Comm., 33:29, 1968);
grisorixin ~J. Chem. SocO Chem. Commun., 1421, 1970); diane-
~ycin (J. Antibiotics, 22:161, 1969); salinomycin (J, Anti-
biotic~, 27:814, 1974)~ X-537A (J. Chem. Soc, Chem. Commun.,
967, 1972); X-206 tJ. Chem. Soc, Chem. Commun., 927, 1971)~
and A204A ~J. Amer. Chem. S~c., 95:33~9, 1973~. ;
The polycyclic ether antibiotics listed above are
activq against Gram-po~itive bacteria, fungi and protozoa~
They exhibit potent anticoccidial activity.
United States Patent 3,839,557 describes a process
for the improvemen~ of feed utilization by ruminants an~
monoga~tric animals fed on fibrous vegetable matter and ad- ;
mi~istered monensin, dianemycin, nigeriain or ather polycyclic `
ether antibiotics.
This invention is concerned with Compound 38,295,
an antibiotic having the formula:
lQ~S63
. - ~
CH3
f~
~ H C
H3C ~ ~
HO~CI~ ~ ~ CH3 ~H3 OC~3
OH H ~ ~ ~3
H3CO ~H3 H
The antibiotia i~ produced by the ~ubmerged aerobic
propagation of ~ hygroscopicus A~CC 31050 in aqueous x~
nutrient media. The antibiotic and its catlonic salts are
potent antimicrobial agents and are e~ective coccidiostats
and growth promotants in animals,
The microorgani~m useful for the preparation o ~he ;~
.
antibiotic o this invention wa~ isolated rom a soil Qample ~-
from Japan. It was grown on a number of media used for the
identification of this genus and co~cluded to be quite similar ~;to th~ descrip~ion of Strep,tomy-ces ~ygroscopicus
The culture ~Pfizer F.D, 23604) WA8 compared in a
number of feature~ on several media with a culture of
tomyces hy~ro3copiaus 482.48 obtained rom the Dutch public -
culture collection, Centraalbureau voor Schimmeloultures (CBS).
Comparisons and decisions were made on the basis of the con-
cept o the ~pecie as descri~ed in Applied Micro~iology,
4:243-250, 1956. A aomparison of the two cultures follows~
~ - chains of Pfi~er F.D. 23604
20 in ~pirals of 6-10 turns or fewer on different media re~embled
_3~
'~,
.. , . . , , , . . , ., . ,~,.. . ., ... . , ., , . , . . . . . ~
; ;:
563
the described chains of ~ y~ hygro~copicu$ CBS illus~
trated in Fig. 1 of the paper referred to above. Spores of
Pfizer F.D. 23604 examined under the scanning electron micro- `
scope looked exactly like those of the CBS strain of S. ~y~
SCOplCUS illustrated in Applied Microbiology, 18:695-696
1969 where the spores are descri~ed as having a rugo~e surface
consisting of numerous pits bordered by ridges of spore wall
material. Stained slides of spores of the two cultures looked
identical. -
Color of_colony - aerial mycelium appear~d yray on
mo~ media but the exact shade was somewhat dif~erent ~or the
two cultures. Pfiæer F.D. 23604 on Czapek-suarose agar had
a thick, irregularly folded, graying white growth whereas the ~`
; C~S strain of S. hy~roscopicus was flat, thin and gray. Pizer
~ 15 F.D. 23604 on oatmeal agar after four weeks of incubation
. .
developed a yellow cast in the previously gray aerial mycelium.
On yeast extract-malt extract agar Pfizer F,D. 23604 had yellow
spots of growth in the gray mycelium.
Melanin formation - no melanin was produced by either
culture on peptone-iron agar.
Carbon utilization - (method of Pridham and Gottlieb,
J. Bact., 56:107-114, 1948), Pfizer F.D. 23604 utilized glucose,
.
L-arabinose, dextrin, D fructose, D(+) galactose, glycerol,
inositol, inulin, lactose, maltose, D-mannitol, salic raffinosç,
rhamnose, sodium acetate, D-sorbitol, sorbose, starch, suarosa,
trehalose and D-xylose; dulcitol was not utilized. These
results are consistent with those reported for strains of
S. hygroscopicus in Applied Microbiology, 15:637-639~ 1967
and the CBS strain of S. hygrosco~icu~ reported in Industrial
3~ Microbiology, 10:183-221, 1969 with the exception that the CBS
...
., : .
.
5~;3 ;- ~
. . .
strain did not utili~e ino~l~ol. -
H2S_production - a poslti~e resul~ was ob~ained ~r : :
, , ~
: both aultures on peptone-iron agar ~lantls wlth lead aaetate ~ ~.
strips.
: 5 Milk - like re~ul~s were obtained or both cultures; :~
~ .
~: no coagulation, hydrolysis aomplete or nearly so ln 12 day~7
tan, ~oluble pigment.
Gelatin - cultures were alike in growth, color and
i; modera~e rate o~ llque~aation. :
Special charaateristics - the C~S 3~rain o S hygro-
scopicus deve~oped blaak hygroscopia areas in three week~ on
gluao~e-asparAgine agar, oatmeal agar and yeast extrac~-malt
extract agar whereas Pfl~er F.D. 23604 showed black 8pot9 only
: on yeast extract-malt extract agar. Further incubation for a ~.
week and storage o~ some Petri diah cultures at 4-10C. ~or a :-
; ~ourkh week inoreased ~he blackening of Pfizer ~.D. 23604d On ~:
oatmeal agar, C~apek-~uaro~e and yeask extraat-malt extract
agar P~izer ~.D. 23604 produced on od~ like wild onlon or
garlia with some earthy odor whereas S.. ~y~ e~ CBS pro- :
duaed only the earthy odor, and thi~ species is no~ de~crib~d
el~ewhere as having the odor o~ wild onion. Pi~er F.D. 23604
~train o~ S. hygro copl~us produaes Co~pound 38295, A new :~.
poly~yclic eth~r antibiotic, wheraas S. h~qro~copicus CBS does -~
not, nor has the production o~ ~his antibiotlc been reporked
.-~
~or other s~rain~ of S.hy~oscopicu~
This oulture ~F.D. 23604) has been depositea ln The
~erican Type Culture Colle~ion, Rockvllle, Md., and
de~ignated in the colle~tlon as A~C 31050.
Aacording ~o the presen~ inv~ntion there i9 pro- i :
vided, a proce3s ~or produoing antibiotic ~ompou~ 382~5 of ~;
-5
,
A~ ~ , ' ': "` .
the formula: ~04~563
':~ , ' '.. ,:
i CH3 . :~
~ OCN3
;i. ` :- . :'
H3 ~ 3 ~o
H ¦ I
` Ho2c ~ cA~O~ 3 CH3 ~OCH3 :`
CH3 OH N ~ H3
CH3 .:
.~, and cationic salts thereoE whlch comprises propagating the
microorganism Streptomyces hy~roscopicus ATCC 31050 at a :
temperature of 28-36C. in an aqueous culture medium contain-
ing an assimilable source of carbon, nitrogen and inorganic
, salts under submerged aerobic conditions until a level of at ~ -
. least 50 mg. per liter of said antibiotic is obtained and ~
I then separating said antibiotic from the fermentation broth ..
either as the free acid or as a cationic salt thereof.
Cultivation of Streptomyces hygroscopicus ATCC 31050 .
preferably takes place in aqueous nutrient media at a tempera-
"; ~ ,,
:
, . . ,~
.,. ' " ~"
.~ :
.~ . -5a-
.
,.
1~4~5~ij,3
ture of 28-36C., and under submerged aerobic conditions
with agitation. Nutrient media which are useful for such
purposes include a source of assimilable carbon such as
sugars, starches and glycerol; a source of organic nitrogen
such as casein, enz~ma~ic digest of casei.n, soybean meal,
cotton seed meal, peanut meal, wheat gluten, soy flour, meat
meal and fish meal. A source of growth substances such as
grain solubles and yeast extract as well as salts such as
sodium chloride and calcium carbonate and trace elements such l`
as iron, magnesium, zinc, cobalt and manganese may also be
utilized with advantageous results. If excessive foaming is
encountered during fermentation, antifoam agents such as
vegetable oils or silicones may be added to the fermentation
medium. Aeration of the medium in tanks for submerged growth
lS is preferably maintained at the rate of about 1/2 to 2 volume
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 antibiotic may
be obtained by employing growth from a slant of the culture~
The growth may be used to inoculate either shake ~lasks or
inoculum tanks, or alternatively, the inoculum tanks may be
"~ .
seeded from the shake flasks. Growth in shaken flasks will
generally have reached its maximum in 3 to 5 days whereas
inoculum in submerged inoculum tanks will usually be at the
most favorable period in 2 t~ 3 days, Substantial antibiotic
activity is obtained in the final fermentor stage in approxim~
ately 3 to 5 days. The antibiotic levels range from 50 to 500
-6-
':~
,, ,~ , .
: 1 ' . . ~ . . . . . . . . . . . .
~04~563
mg. per liter.
The process o antibiotic production is convenient-
ly followed during fermentation by ~iological assay of the
broth employing a sensitive strain of ~aphylococcus aureus
or Bacillus subtilis. Standard plate assay techni~ue i8 em-
ployed in which the zone of inhibition surrounding a filter l~ '
paper disc saturated with the broth is used as a measure o
antibiotic potency.
Thin layer chromatography employing silica gel i~
a useful tool for analyzing the antibiotic produced in er-
mentation media and the composition of crude and puriied
materials extracted from the fermentation broths. The thin
layer chroma~ograms, after development wikh ethyl acetate, are
sprayed with 3~ vanillin in ethanolic sulfuria aaid ~98.5:-
1.5%, v/v~ followed by heating at 60-80C. for a few minute3, -
The antibiotic is observed as a brilliant, pinkish-rea spot
on a white background.
Antibiotic Compound 38295 may be separated and re-
covered from clarified fermentation bxoth by extracting with
i. ~ .
an organic solvent such as chloroform, ethyl acetate or methyl
isobutyl ketone. The major portion of the antibio~ic is con-
tained in the separated mycelium, and i9 conveniently extraat-
ed therefrom by slurrying the mycelium with a water-soluble
solvent such as methanol.
The preferred method of separation and recovery of
antlbiotic Compound 38,2~5 is as follows:
The whole (unfiltered) fermentation broth is twice
extracted with about 1/5 t~ 1/2 volume of methyl iso~utyl
ketone. The solvent extract is concentrated under vacuum to
an oily residue which is then slurried in chloro~orm:hexane
--7--
',' ' ', ' ~ ' , ' "
,
~ 4~563
tl:l) and added to a silica gel column ~preferably a bed of ,~
- silica gel 60 topped with a layer of ~ilica gel PF254, both
available from E. Merck, Darmstadt, Germany). The silica gel
column is successively developed with hexane, chlorofoxm:hexan~
- S (1:1) and chloroform. The main antibiotic fraction i~ eluted
with ethyl acetate. The eluate is conce,ntrated under vacuum, ,~ -
taken up in acetone and stirred for about 30-60 minutes with
activated charcoal ~Darco G 60). The charcoal is remsved by ,'~
iltration, the solution concentrated to a small volume under
vacuum, a small amount o water added and the pH ad~usted wlth
; ~odium hydroxide to 8.0-8.5. The crystalline material that
separates is removed by filtration, washed with water and dried, ';
The antibiotic that is isolated at this stage is a
mixed sodium and potassium salt of Compound 38295 formed with
lS both sodium and potassium ions occurring in and scavenged from '~
the fermentation broth and the added sodium hydroxide. Thin
. . . . .
layer chromatograms of this material may disclo~e trace amount~
of at least two other more polar antibiotics which like Com-
. .... .
pound 38295 give a characteristic pinkish-red spot on spraying
with vanillln in ethanolic sulfuric acid~ ~
Compound 38295 may be obtained as a ~ingle enti~y '
by column chromatography on ~ilica gel PF254 made up in hexane.
The mixed sodium and potassium ~alts are adaed as a solution
in eth~l ace~ate or chloroform and the column developed under
80 p.s.i. with hexane containing increasing amounts of ethyl -~
acetate. The progress of the separation is followed by thin
layer chromatography. The appropriate fractions are combined,
evaporated under vacuum and the antibiotic cry~tallized from
acetone-watar. ,"
3~ The free acid of Compound 38295 may be derived from
-8
'
. .
~4~i63 ~:
the mixed sodium/potassium salt by adjusting the pH of an
ethyl acetate or chloroform solution to 4.5 with dilute
phosphoric acid. The organic layer is dried over anhydr~us
sodium ~ulfate, evaporated under vacuum and the residue
crystallized from chlorofoxm-hexane.
The sodium salt of Compound 38295 may be obtained
- by adjusting an ethyl acetate or chloroform solution of the
free acid to pH 8.5 with sodium hydroxide (lN). The material
i5 crystallized from acetone-water. The crystalline pota~8ium
. , .
sal~ is similarly obtained using potassium hydroxide in placa
of NaOH.
A arystalline silver salt may be prepar~d by the
addition of aqueous silver nitrate to an ethanolic solution
of the mixed sodium/potassium salt of Compound 38295. O~her
L5 readily obtainable compounds are the copper, zinc, ammonium,
calcium, magnesium and lithium salts of Compound 38295.
Because of its end use for the prevention and
treatment of coccidiosis in poultry, whole fermentation
broth containing Compound 38295 may be taken to dryness (pre-
ferably by spray-dr~ing) and incorporated in poultry feed at
the d~sired antibiotic potency level.
Compound 38295 exhibits inhibitory action against
the growth of a number of microorganisms (Table I). The
test organism is ino~ulated in a series of test tubes con-
taining nutrient medium and various concentrations of Com-
pound 38295 to determine the minimal concentration of the
antibiotic in mcg./ml, which inhi~its the growth of the
organism over a period of 24 hours.
.
1(;P4~S63 ;;
TABLE I ;;
~x~ound 38295 Co~x~nd 38295
Orgamsm ~x~d Na/K.salt) (~res acid~
St~ph. aureus OlA005 0.78 0~39
01A052 0.78 3.12 :
: OLA109 0,78 0.7S
O ~ lO 0.39 1.56 `
OlA400 ~.39 1,56 ~;
~ . . ~
S~rep. faecalis O.20 1.56 `.'
10 Stre~. Eyogenes < 0.10 < 0.10
Neisseria sicca < 0.10 ~ O.lQ
Baaillus subtilis ~ 0.10 ~ 0.10
atoba¢illus ca3ei var casei 1.56 --
Lactobacillu~ catenaorme 0.78
15 Clostridium in~x~m 0,20
Clostridlum bi_ermentans 0.78 ;~
Trep~a hyodysenteriae 0.39 - '
Compound 38295 ana its salts exhibit excellent ,-
,i'.?. ~ ,.,1 ~.
a~tivity against coccidiosis infectiQns in poultry. When in-
corporated in the diet of chickens at a level of 15 to 250 ppm, `
the compounds are effective in controlling single inection3
of Eimeria tenella, E. acervulina, E. maxima etc. and mixed
, inections of these organisms. -i~
: Efficacy data ~or Compound 38295 and its 9alt5
against coccidiosis infections in chickens are obtained in
the ollowing manner. Groups of 3-5 ten day old SPF white
leghorn coakerel ¢hick3 are fed a mash diet containing Com ,
pound 38295 or one of its alt~ uniformly dispersed therein~ ;
After being on this ration for about 24 hours, each chick is
inoculated per os with oocysts of the particular species o~ ` ;
Eimeria being tested, Other groups o~ chicks fed on an anti-
biotic-~ree mash diet are similarly infected and serve as in-
. .
~10- ';''
'
,'':
~ . : - . . . . ..
-`
:
~ 10~563 ~
fected controls. Non-infected, non-medicated chicks serve
as normal controls. The re~ults of treatment are evaluated aft2r 5 days
in the case of E. ac:rvulLna, and 6 days for all other Eimeria species.
Table II illustrates the results obtained with the
mixed sodium and potassLum salt of Compound 38295.
TABLE II
Species Ave. degree
- infection Dose (pem) of infection* Ratio*
Eimeria tenella 250 0.0 0.0
125 o,o 0 0
0.7 0.2~
1,7 0.54
lS 2.7 0.86
Eimeria acclvulLna 25Q 0.0 0.0
125 0,2 ~.1
0,0 o,o
0,4 0.2
1.2 0,6
2,0 1.0
Eimeria neca,trix 1225 0 2 0 13
0,2 0.13
Eimeria maxima 250 0.0 0.0
125 O.o o,o
0.4 0,25
0.6 0.33
0.8 0.44
1,8 0.66
Elmeria brunetti 250 0.4 0.22
125 0,4 0.22
0.8 0.44
1.2 0.66
1.6 0.88
1.6 0.88
Mixed infection '
(~occivac D, Sterwine
Laborabories, Opelika,
Alabama) 250 0.0, 0.0** 0.0, 0.0
125 0.2, 0.4 0.08, 0~18
0,2, 0.0 0.08~ 0.0
1,0, 0.6 0,38, 0.21
1,2, 1.0 0.46, 0.35
2,6, 2.0 0.85, 0.71
*The criteria used to measure anticoccidial activity consist of lesion
soores of 0 to 4 for E. ~en~lla tJ.E, Lynch~ km. J. Vet, Rss, 22:329-
326, 1961~ and 0 to 3 for other ~pecies (~, Jo~nson and W.H~ Reid~ Exp, -'~
Parasit., 28:30-36, 1970S. A c3nstant ratio is es~a~lished ~y di~iding
the lesion ~c~re of each treated group ~y ~he lesion soore of the in-
fected control.
' ' ' .. ' . :, ' ' . ,`'~,,
,~ , , ~, . . .
563 ~
Table II tcon~'d~ ;
** m~ fi~st n~r pertams t~ intestinal lesic)ns and th~ saoond nu~r
to cecal le~ions.
Similar results may be obtained with the free a~id,
S sodlum ~alt, potassium salt or dried fermentation medium con-
taining Compound 38295 in the ration at the desired antibiotic
po~enoy level.
The growth promotant properties of Compound 38295
are illustrated by the following trials with cattle.
Angus heifer calves are randomly assigned to pens,
5 Galves par pen, and administered Compound 38295 orally by
volus, in drlnking water, etc. Howeve~, the compound is ad-
van~ageously and expeditiously admini~tered, on an ac~ivity
b~si~, by inaorporation into feed compositions high in rough-
., ~ ,. .
age at levels o 11 to 33 ppm. Compound 3~295 may be in such
~arious forms as the free aaid, sodium salt, pota~sium salt,
mixed sodium and potassium salts or dried fermentation broth
produ¢ed by ~ E~Y~ a~ 2~ ATCC 31050
~,
Non-medicated and Compound 38295 feed compositions
. .
are administered to the test animals ad libitum. ~he feed
composition is a 12.5~ protein - 75:25 conc:roughage cattle
finisher ration containing corn, soybean meal, alalfa meal,
calcium phosphake, molasses, vitamins and trace minerals.
CGmpound 38295 i~ contained in a premix along with ground
aorn, mineral~ and vitamins.
''''
-12-
,, . . : :
1~4~51E;~
BASAL RATION
tl2.5% Protein - 75:25 Conc:Roughage Cattle Finisher Ration)
Ingredie~t Per cent
Ground Yellow Corn 55.6
Soybean meal, 48% protein 6.3
Alfalfa meal, 17% protein 25,0
Dried molasses* 10.0
Soybean oil 2,0
Dicalcium phosphate ~24~ Ca, 18.5% P)0.40
Trace mineral salt~* 0,50
Vitamin-mineral premix~*~ 0,20
*Cane molasses on a corn-cob fra~tion carrier: not less than
3% crude protein not more than 15% iber, not less than 42
inert sugar, maximum of 6% water.
** Contributed the following levels of trace mineral~ in
parts per million: manganese 10~ iron 6.25; copper 1.25;
iodine 0.025; cobalt 0.86; zinc 45.
*** Contributed the following levels of vitamins per pound
of ration: vitamin A 1497 I.U.; vitamin D 200 I.U,; vitamin
E 25 I.U.
PREMIX "
Ingredient Amount (4 lbs.)
` Vitamin A Supplement ~30,000 IU/gm)0,22 lb.
Vitamin D3 Supplement (200,000 IU/gm~2,00 gm~
Vitamin Supplement ~125,000 IU/lb.)0.40 lb.
Zinc Oxide (79.4~ zinc) 38.0 gm.
Cobaltous Carbonate (45~ cobalt) 1.70 gm.
Soymill run or finely ground corn 3.27 lb.
Compound 38,295 40.0 gm.
A pound of premix per ton of complete ration gives a final
Compound 38,295 level o 11 ppm.
Tabla III illustrates the results of a 28 day test
obtained with three replicate pens of 5 calves per pen,
-13-
~.
,. .
:
:~1 i4~S63
approximately initial weight of 400 lbs. per calf.
TABLE III
(mixed sodium and potassium salts of Compound 38,295)
ADG* ADG** Feed~n*~*
Treatment ~ppm) lbs. Index l~s, Index ~3~~1~dex
Control 2.07 100,0 14.2100.0 6.95 100.0
Compound 3~,295 ~11) 2.29 110,6 13,595.1 5.94 117.0
Compound 38,295 ~33) 2.42 116.9 12.487.3 S.ll 136.0
*average daily gain
**average daily feed
*** lbs. of feed per lb. of gain
Substantially ~he same results may be obtained
with the free acid, sodium salt, potassium salt or dried
fermentation broth containing Compound 38,295.
Comparable results may be obtained with other
ruminants such as sheep or monogastric animals such as
horses, pigs and rabbits.
To ackivP Compound 38,295 may be administered in an
animal feed or drink or in a premix to be used for incorpora-
tion in an animal feed or drink. A liquid antibiotic pre-
mix concentrate suitable for addition to drinking water to
inorease feed efficiency in animals may be prepared at
desired concentrations by dissolving in an appropriate sol-
vent, such as ethanol or propylene glycol Compound 38,295.
~ -
A liquid antibiotic premix concentrate suitable ;
for addition to drinking water to animals may be prepared
at desired concentrations ~y dissolving in an appropriate
solvent such as ethanol or propylene glycol the antibiotic
comprising Compound 38,295.
'
-14-
.
, : .
~4~63
EXAMPLE I
;. A sterile aqueous medium having the following compo-
sition is prepared:
Grams/liter
5 Glucose10.0
Soluble starch 2000
Yeast extract 5.0 `
Enzymatic digest of casein 5.0
Meat meal 5.0
CaC03 1. 0 .',
pH - 7~0
. Cells from a slant of Strep_om~ces h~roscopicus
ATCC 31050 are transferred tc a series of 300 ml flasks each
containing 50 ml of this sterile medium and shaken on a
15 rotary shaker for 3-4 days at 28-30C. Five ml aliquots of
this grown inoculum are transferred aseptically to 300 ml
flasks containing 100 ml of sterile medium as described above. , : ~
After shaking for 3-~ days at 28-30C., the grown inoculum is : :
transferred to four-liter ~ermentors containing two liters :
of the following sterile medium:
Grams/liter ~ .
Glucose 10.0 '~ ~ :
Corn starch 10.0 :
Soy flour 10.0 .
25 Grain solubles 5.0
NaC1 5.0
.,.,,:
. CaCO3 1,0
~H - 6.6 :
The fermentation is conducted for 90-120 hours at 28-36C.
with stirring at 1700 revolutions per minute and aeration
-15-
63
at about one volume of air per volume of broth per minute,
,
; Large fermentors containing from 80 to 10,000 gallons of medium
; may be inoculated with about 2~ of this growth. The fermenta-
tion is conducted until an antibiotic potency of at least 50 mg~
per liter is obtained ~90 to 120 hours).
One hundred liters of whole, unfiltered fermentatio~
- broth are twice extracted with one-fifth volume of methyl iso-
- butyl ketone. The separated solvent extract is concentrated
in vacuo to an oily residue (769 g.) which is dispersed on
silica gel by its addition in solution in 2 liters o chloro-
form to l kilogram of silica gel PF254, followed by removal
of the solvent under vacuum. The resultant residue is ~lurried
in 2 liters of chloroform:hexane (1:1) and added to a bed of
300 grams of silica gel 60 topped with a layer of 300 grams
' 15 of silica gel PF254. The silica gel is then washed successive-
` ly with 2 gallons of hexane, 2 gallons of chloroform:hexane
1) and 2 gallons of chloroform. The antibiotic is eluted
with 1 gallon of ethyl acetate. The ethyl acetate is removed
under vacuum and the residue (38 g.~ is dissolved in 400 ml.
; 20 of acetone. The acetone solution is stirred at room tempera-
ture for about 45 minutes with about 40 g. of activated
i charcoal (Darco G 60). Filtration is followed by concentration
to approximately 200 ml, and dilution with 100 ml, of water.
Compound 38295 separates as the mixed sodium/potassium salt on
adjustment of the pH to 8,5 with 1,~N NaOH. The crystalline
material ~13.8 g~) is dissolved in ethyl acetate and chromato-
graphed on a column of silica gel PF254 made up in hexaneq
The column i9 developed under 8~ p.s.i. with hexane contain-
ing increasing amounts of ethyl acetate, The progress of the
qeparation is followed ~y thin layer chromatography, ~he
-16-
. . . : :
104Q5i~3
appropriate fractions are combined, evaporated under vacuum
and the pure compound crystallized from acetone-water.
The pure, crystalllne mixed sodium/potassium salt
of Compound 382~5 has a melting point of 200C. and an optical
rotation of la~ D = ~36~ at a concentration of 1~ in methanol.
After drying in vacuo overnight at 70C. over phosphorus pent-
oxide, the average analysis for carbon is 60.58%~ and hydrogen
is 8.56%.
The compound is soluble in methanol, ethanol, acetone,
chloroform, methylene chloride, diethyl ether, ethyl acetate
and methyl isobutyl ketone; partially soluble in hexane; and '~
lnsoluble in water. `;
The mixed ~odium~potassium salt o Compound 38295
; possesses no aharactexistic ultraviolet light absorption
pattern.
The infrared spectrum of the mixed sodium/potassium
salt, Figure 1, is attached. A KBr pellet shows character- ;
istic absorption in the infrared region at the following wave-
lengths in microns: 2.92, 3.40, 6,27, 6.85, 7.22, 8~40, 9.00,
9.15, 9.25, 9.48, 9.98, 10.45, 10.68 and 11.45. ;
EXAMPLE II ~;
~
The method of Example I may be repeated employing
clarified fermentation broth in place of whole unfiltered
fermentation broth. ~ -
EXaMPLE III
The mycelium separated from the clarified fermenta-
tion broth of Example II is slurried several times with ;
methanol, the methanol extract concentrated under vacuum and
the residue treated by the method of Example I.
', ;'
-17-
~al4~S~ :
; EXAMPLE IV :
The fermentation process of the method of Example I
is repeated employing a fermentation medium of the following
composition:
Grams/li~er
Glucose 29,0
: Corn starch 10.0
Soy flour 10.0
. Grain solubles 5.0
: 10 Fe2~S04~3 0,2
MnC12 0.2
CaC03 1. ~
NaC1 5.0
Methyl oleate 2.0 . :
Soybean oil 2,0 :
pE 6.6 - 6.7
At the end of the fermentation cycle, the whole unfiltered
fermentation broth is taken to dryness by spray-drying.
EXAMPLE V
The mixed sodium/potassium salt of Compound 38295
i~ dissolved ln ethyl acetate and the pH adjusted to 4.5 with
dilute phosphoric acid. The solvent layer is dried over an-
hydrous sodium sulfate evaporated in vacuo and the residue
crystallized from chloroform-hexane to yield the crystalline
acid which has a melting point of 135-138C. and an optical
rotation of ~a] D = +38 at a concentration of 1% in methanol,
After drying overnight in vacuo at 70C. over phosphorus pent-
oxide, the average composition by weight is 61.6S% carbon,
8.94% hydrogen and 29.41% oxyga~ (by difference). The free
acid of Compound 38295 possesses no characteristic ultraviolet
-18-
~4~)563
light absorption pattern.
The infrared spectrum of the free acid of Compound
38295, Figure 2, is attached. A KBr pellet shows character-
istic absorption in the infrared region at the following wave-
lengths in microns: 2.92, 3.43, 5.74, 6.85, 7.25, 7.53, 8.28,
8.60, 8.80, 9.0Q, 9.15, 9,80, 10.10, 10.45, 10.68, 10.92 and
11.13.
X-ray crystal analysis of the silver salt of Compound
38295 established the structural formula of the following
representation:
.,..... ~', .
CH3
i '
H3C ~0
H3C" ~ ~
H02C~ C _ I 1 ~H3
~ ~ / ~ ~ ~ CH OCH3 -
'CH3 OH H ~3
CH3
_ - CH3 H H ~ "CH3
3 3
:- .
EXAMPLE VI
The sodium salt of Compound 38295 is obtained by the
addition of l.ON NaOH to a pH of 8.5 to an ethyl acetate solu- ~
lS tion of the free acid of Example V. The material crystallized ~-
from acetone-water has a melting point of 197-198C. and an
optical rotation of ~a]2DS = ~38 at a concentration of 1. 03~ in
methanol. After drying in vacuo overnight at 70C. over phos-
phorus pentoxide, the sodium sa}t analyzes for 60.28~ carbon
--19-- , ",
... ;, . ..
.: .
1~4~563
and 8.76% hydrogen.
The inErared spectrum of the sodium salt of Compound
38295, Figure 3, is attached. A KBr pellet shows character-
istic absorption in the infrared region at the following wave-
lengths in microns: 2.94, 3.40, 6.25, 6.85, 7.20, 7.45, 8.38,
9.00, 9.27, 9.48, 10.00, 10.45, 10.65, lû.90, 11.13 and 11.47.
EXA~IPLE VII
The potassium salt of Compound 38295 is prepared
by the method of Example VI employing potassium hydroxide in
place o~ sodium hydroxide. The material crystallized from
acetone-water has a melting point oE 196-198C. and an optical
rotation of ~c~]2D5 ~ ~38 at a concentration of 1.02~6 in
methanol. After drying in vacuo overnight at 70C. over
phosphorus pentoxide, the compound analyzes for 60,58~ carbon
and 8.70% hydrogen.
The infrared spectrum c)f the potassium salt of
Compound 38295, Figure 4, is attached. A KBr pellet shows
characteristic absorption in the infrared region at the follow-
ing wavelengths in microns: 2.94~ 3.42, 6.27, 6.84, 7,20,
8.75, 9.00, 9.14, 9.30, 9.45, 10.45, 10.92, 11.15 arld 11.47.
EXAMPLE VIII
The silver salt of Compound 38,295 is prepared by
the addition of aqueous silver nitrate to an ethanolic
solution of the mixed sodiurn/potassium salt of Example I.
The solid that separates is crystallized from methanol, It
has a melting point of 198-200C. and an optical rotation of
~] =~27~ at a concentration of 0.982% in me~hanol, The ;
D
average composition of a sample dried overnight in vacuo at
70C. over phosphorus pentoxide is 55.95% carbon and 7.89~ ,
hydrogen. The molecular weight of the crystalline silver
~20-
.
4~3
salt of Compound 38,295, determined from X-ray data, is
106V + 4 atomic mass units.
'"''' ' ''' ' ' '
~; , .
,, : '
:
-21-
..
. ~",.
