PH26523A - A-28086d - Google Patents

Description

: ok 26573 i

Co The present patent application ims a divisional of spplication Serial No. 17244 filed oun

June 6, 1975. : The present invention relates to antibiotic

A-28080 factor DD aud to a process for the preparation thereof.

The invention provides D to antibiotic A- 20088 factor which is a white crystalline compound when crystallized trom acetone-water; which 1s =a soluble in methanol, ethanol, dimethyl formamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone and benzene; but which 1s only =»lightly

N soluble in hexane; and which ims insoluble in water; os T which melts at about 96-98°C.. and which has: 156 a) a molecular weight of 778, as : determined by high-resolution mass ’ spectrometry; bh) an approximate elemental composition } of 67.59 percent carbon; 9.38 percent } hydrogen, and 22.71 percent oxygen; ae) an empirical formula of Cy4H74014. BS determined hy high-resolution mass spectrometry; } d) a specific rotation of 658% (ec = 0.1, methanol) when determined at 26°C. 5 hf. 26522 e) an infrared absorption spectrum in ) chloroform with the following observable absorption maxima: 2.89, 3.39, 3.43, 3.50, 5.88, 6.90, 7.27, | 7.60, 7.84, 9.00, 9.26, 9.62, 10.31, 10.58, 11.10, and 11.49 microns; f) no observable ultraviolet absorption in 95 percent aqueous sthanol; g) a nuclear magnetic resonance spectrum in chloroform with the following characteristics: & 6.00, 4.20, 4.10, oC 4.00, 3.98, 3.92, 3.88, 3.83, 3.79, 3.67, 3.64, 3.67, 3.54, 2.88, 2.81, 2.71, 2.62, 2.58, 2.48, 2.43, 2.37, : 15 2.29, 2.21, 2.156, 2.10, 2.04, 1.97, 1.89, 1.83, 1.76, 1.68, 1.61, 1.58, 1.65, 1.47, 1.39, 1.30, 1.256, 1.18, a 0.95, 0.99, 0.88, 0.74, and 0.68 ppm; : ’ h) a titratable group with a pK, value of B.87 in 80% aqueous dimethylform- amide; . 1) a characteristic x-ray powder- diffraction pattern (Cut? radiation, 1.6405 >» , nickel filter) having the oo following interplanar spacings in angstroms (d):

fat. 26573 i

Relative d Intensity 12.40 100 10.20 70 8.85 90 7.80 30 6.80 10 8.3¢ 100 5.70 | 20 : 10 5.36 20 5.10 | © 20 4.990 10 4.66 20 4.46 40 156 4.20 30 3.30 10 3.156 : 10 2.99 05 . 2.77 06 . 290 2.28 o5 3) the following Rg values in the silica- : gel thin-layer chromatographic © systems indicated below, using

Bacilum subtilis ATCC 6633 as a . 26 detection organism: ]

Bf. 26523

By versus OAL VEIL. RY Hom ! : 9.26 Benzene -ethy1l acetate (3:2) 0.66 fthyl acetate-diethylamine (96:5) | : b k) the following Ry values in the paper chromatographic systems indicated below, using Bacillus subtilis ATCe : 6633 as a detection organism:

Ry Value fiolveot Svatem

Co le 9.10 Water saturated with methyl isobutyl ketone. (MIBK) . 9.26 Water maturated with MIBK - Plus 2% p-tolvenssulfonic } acid and 1% piperidine 15 . 90.38 Water:methanol :acetonse (12:3:1)- adjusted to pH 10.5 with NH,OH and then lowered to pil 7.5 with

H4PO 4 0.29 1X MIBK, 0.5% NH, OH in oo

Fo water : i 0.25 17.4 g. K,HPO,, 30 ml. ] ethanol par liter of water @.28 Henzene saturated. with : 25 } water

NE y : I ORIGINAL »

WE fh 24523 . | . i 9.09 Water s = ’ 0.64 Water: :MIBK:ethyl acetate (98:1:1) : 1) an acid function capable of forming : Bb malts and ester derivatives; and m) at least one hydroxyl group capable of esterification; and the C,-Cg-acyl ester derivatives and physiologically-acceptable palts thereof.

The invention also provides a process for preparing antibiotic A-28086 complex comprising factor A, factor B and factor D which comprises ‘cultivating Streptomvoes aureofaciens NRRL 8092 in : a oulture medium containing assimilable sources of 16 carbohydrate, nitrogen, and jnorganic salts under : submerged aeroblc fermentation conditions until a : substantial amount of antibiotic activity is produced by said organism in said culture medium.

Antibiotic A-28086 factor D is a newly- discovered member of a group of polyether antiblotics. Examples of membeps of thie group “include the earlier-discovered antibiotic A-28086 . factors A and B (moe copending U.8. patent application serial No. 477,964, filed on June 10, 1974); monensin (U.S. Patent 3,501,668); dianemycin oo | Ml, 26522 (R.L. Hamill, M.M. Hoehn, G.E. Pittenger, 3. "Chamberlin, and M. Gorman, J. Antibiotics 22, 161 (1969)1; nigericin (L.K. Steinrauf, Mary Pinkerton, . and J.W. Chamberlin, Biochem. Biophys. Ren. Coma. 6 a3, 29 (1988)]; and salinomycin (U.S. 3,867,948).

Antibiotic A-28088 factor D is produced together with factors A and B by culturing the noval strain of Straptomyvoes aureofaciens NRRL 8092 under submerged aerobic fermentation conditions until a - 19 substantial level of antibiotic activity is produced. The soproduced factors A, B, and D are

LC extracted from the fermentation broth and from the mycelium with polar organic eolvents. The co- produced factors are separated as a mixture by 16 concentrating the polar organic solvents, adding the concentrates to excesaes of petroleum other to precipitate impurities, filtering, and evaporating + the filtrates to obtain A-28086 antibiotic complex.

It should bs noted that the term “antibiotic | complex” ae used in the fermentation art and in thie . specification does not refer to a chemical complex, : but to a mixture of co-produced individual . antibiotic factors. As will be recognized by those familiar with antibiotic production by fermentation, ; 25 “ the ratio of individual factors produced in an antibiotic complex will vary, depending on the ll 96522 ) fermentation conditions ussd. The A-28086 antibiotic complex is further purified and is oo separated into individual factors A, B, and D by a merisas of chromatographic procedures.

Antibiotic A-28086 factor D of this oo invention inhibits the growth of organisms which are pathogenic to animal and plant life. In one aspect of this inhibitory activity, antibiotic A-28088 factor D is an anticoccidial agent. In addition, antibiotic A-28086 factor D is an antibacterial : | agent and increases feed-utilization efficiency in . ruminant animals. / : The infrared absorption mpactrum of antibiotic A-28088 factor D is presented in the 16 accompanying drawing. : Newly-dimcovered antibiotic A-28088 factor

D of this invention is structurally related to the

Co earlier-discovered factors A and B. A-28086 factors a, B, and D are coproduced during the fermentation and are obtained as a mixture. The factors are separated from each other, and factor D is isolated as an individual compound as hereinafter described. : Co, The following paragraphs describe the : physical and chemical properties of newly-discovered antiblotic A-28086 factor D.

ful. 6522 ! oo

Antibiotic A-28086 factor D is a white crystalline material (from water-acetons) with a melting point of about 98-98°C. A-28086 factor D has an apparent molecular weight of 778, am Ts : 5 determined by high-resolution mass spectrometry.

The elemental composition of the peak in

Co the mame spectrum of the sodium salt of A-28086 factor D was observed to he 800.5050 (Calcd. for

CqqH7301 (Na = 800.5060). In the masse spectrum of A- 28086 factor D free acid, a small peak at 778 and a larger peak at 760.5117 (calcd. for C44H72010 = - 760.6125) ware observed. The m/e 760 in the nase

IE spectrum of the free acid results from the loss of water from the molecular ion. The molecular-ion 16 composition of A-28086 factor D frees acid is,’ therefor, C4qqH7404- ©

The emprical formula proposed for A-28086 factor D ie C44H740,4. Elemental analysis of factor

D gave the following percentage composition: carbon, 67.59 percent; hydrogen, 8.38 percent; oxygen, 22.77 percent.

The theoretical percentage composition for

C44H74011 is: 67.87 percent; hydrogen, 9.B1 percent; oxygen. 22.77 percent. : oo 8.

Wt 26573 i

The infrared absorption spsctrum of A--28086 factor D in chloroform is presented in the accompanying drawing. The following absorption maxima are observed: 2.89, 3.39, 3.43, 3.50, 5.88, 6 . 8.90, 7.27, 7.60, 7.84, 9.00, 0.26, 9.62, 10.31, 10.68, 11.10, and 11.49 microns. : A-20088 factor D in 96 percent agueous ethanol shows no ultraviolet absorption. } The nuclear magnetic resonance spectrum of

A-28088 factor D in deuterochloroform showed the ' following charaterimstics: o 6.00, 4.20, 4.10, 4.00, 3.98, 95.92, 3.86, 3.83, 3.79, 9.67, 3.64, 3.87, 3.64, 2.88, 2.81, 2.71, 2.62, 2.68, 2.48, 2.43, 2.87, 2.29, 2.21, 2.16, 2.10, 2.94, 1.97, : 16 1.89, 1.83, 1.78, 1.68, 1.84, 1.58, 1.65, 1.47, ’ 1.39, 1.90, 1.26, 1.18, 6.95, 9.99, 4.80, 8.74, and 9.88 ppm. . h . Antibiotic A-28008 factor D, crystallized from acetone-water, has the following B characteristic x-ray powder-diffraction pattern (Cu** radiation, 1.5406 , nickel filter, d=interplanar spacing in —

(fat. 244523 “ § : ~N

Relative d Intensity 12.40 100 10.20 70 6 8.85 9e ; | 7.80 30 6.80 10 8.30 100 5.70 20 5.36 20 5.10 20 4.90 10 : 4.66 20 oo 4.45 40 4.20 30 3.30 10 3.16 10 2.99 es 2.77 : | 05 2.28 05 oo The specific rotation of antibiotic A-28088 factor D is -56° (c=0.1, methanol), when determined at a temperature of 25°C. oo Electrometric titration of A-28086 factor D in 80 percent aqueous dimethylformamide indicated : the premence of a titratable group with a ok, value of 8.67. ¢ oo 11 oo

Pik. 36572 [ Antibiotic A-28086 factor D is soluble in a variety of organic solvents such as methanol, . ethanol, dimethyl formamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone and benzene. A~- b 280868 factor D is only slightly soluble in nonpolar _organic solvents such as hexane and is insoluble in

Co water.

Antibiotic A-20088 factor D has an acid function capable of forming maltese and ester derivatives and at least one hydroxyl group capable of enterification.

Based on the physical characteristics hereinabove recited, a structure for antibiotic A- 28086 factor D can be proposed. Since the structure 16 determination is merely postulated, however, it is to be understood that the structure presented herein : represents merely a working hypothesis. The : tentative structure for A-28088 factor D is shown in : Formula I:

fh. 20523

H

N - . = ‘ } ’ } m . mw / \

Sy / el 3 to

Ll ~~ oa : / \ o .

NE

"mw } 5-88 o=u : Co. mM

B88 : : | z_8 ’ | 58 oo

J

} / \ o . . 1 : . ~N ™ 5-3-8

EE

3 1.4 | .

; wherein either: Ry = CHg nnd Ry = CoHg or: Ry = Cally, and R, = CHg

The R; valuss of antibiotic A-2808B8 factor

Pp in various paper chromatographic systems, using 6 Bacillua aubtilis ATCC 8833 as a detection organiem, are given in Table [. The Ry values of A-28086 factors A and B in these systems are included for rofersnca | wrposen.

- i - _ » TABLE I f | 14523

Bf Value

Kactor D factor A Factor B Solvent Syntem

Co 0.10 0.11 9.09 Water maturated with methyl isobutyl ketone , : : | (MIBK) @.26 0.41 o.18 Water saturated with " MIBK plus 2X Pp ’ toluenesulfonic acid and 1X piperidine oo 9.38 0.54 0.46 Water:methanol:acetons

Lo (12:3:1)~adjusted to pH 10.5 with NH, 0H and i then lowered to pH 7.5 16 with HaPO, : 0.29 0.48 0.36 1X MIBK, 0.5% NH 40H in water ©.26 0.16 0.33 17.4 g. K;HPO,, 30 wml. oo ethanol per liter of water ; 9.26 0.24 0.51 Benzene saturated with water - 0.909 0.24 0.11 Water Co .64 0.75 0.51 Water :MIBK: ethyl acatate (98:1:1)

Co 15 in 2523

In Table II are given the Re values for ’ antibiotic A-20088 factor D In two thin-layer chromatographic systems on silica gel (precoated platens. K. Merck, harmetadt, F-264, layer thivknens 5H 0.25 wm). again using B. subtilis ATCC 8633 as a detection organiom.

TABLE 11

Rs Value

Factor. D factor A Factor B Solvent System to e.26 0.24 9.42 Benmene-ethyl acetate : (a:2) 0.66 o_54 0.34 Rthyl acetate-disthyl- . amine (95:5) : . oo ’

. To . hd | ’ : (at. p4523 : A-28086 factor D forms acyl ester : derivatives. Easterification ocours at ome, of the ~ hydroxyl aroups of A-20088 factor D upon treatment with C,-Cg-acid anhydride or acid chloride. Buch esters ares typically prepared by reacting A-280866 factor D with, for exawple, the corresponding acid anhydride at room temperature. These ester derivatives are almo useful an antibiotics and ans agents which increase fosd-utilizatlon efficiency. 190 . Antibiotic A-280868 factor D and its C,p-Cg- acyl ester derivatives are capable of forming salts.

The physiologically-acceptable alkali-metal, : alkaline-earth-metal and amine salts of antibiotic

A-28086 factor D and of the C,-Cq-acyl-ester derivatives of A-28086 factor D are also part of : this invention.

To #simplify discussions of utility, .the term "A-28086-factor-D compound” is used to refer to :

A-28088 factor D, A C,-Cg-acyl-ester derivative of factor D or a physiologically-acceptable salt of A- 28088 factor D or of a Cy-Cg-acyl-ester derivative of factor D as above defined. : p * “Physiologically-acceptable” salts are salts which are also pharmaceutically acceptable, Te that ims, salte in which the toxicity of the compound : as 8 whole toward warm-blooded animals is not” : increased relative to the non-salt form. : 17 oo

(ok. 26572 ;

Reprenmentative and muitable alkali-metal and alkaline -marth-metal salts include the sodium, potassium, lithium, cesium, rubidium, barium, calcium, and magnesium salts. Suitable amine salts include the ammonium and the primary, secondary and tertiary C;-C4-alkylawmonium and hydroxy-Co-Cg - : alkylammonium salts. [Illustrative amine salts include those formed by reaction with ammonium hydroxide, methylamine, mec-butylamine, isopropy laminae, diethylamine, di-imopropy lamine, “ethanolamine, triethylamine, 3-amino-1l-propanol and the like.

The alkali-metal and alkaline-earth-metal

Co cationic salts of A--2080868 factor D and of the Cyp-Cg- aoyl antar derivatives of factor D are prepared according to procedures commonly employed for the preparation of catlonic salts. For example, the fres-acid form of the antibiotic factor or ester ’ | derivative is dissolved in a suitable solvent such as warm methanol or ethlianol; a molution containing } the stoichiometric quantity of the desired inorganic base in agueous methanol ie added to this aolution.

The salt thus formed can be isolated by routine : mathods,. such as filtration oe evaporation of the

Co 26 solvent. 1R

. fod. 26593. - [ : The malts formed with organic amines can be prepared in a similar manner. For example, the gaseous or liquid amine can be added to a solution of the antibiotic factor in a suitable sblvent such 6 as acetone, and the molvent and excess amine can be : removed by evaporation.

It is well known in the veterinary pharmaceutical art that the form of an antibiotic is not significant when treating an animal with the antibiotic. In momt cases, conditions within the animal change the drug to forms other than the form in which it was administered. The salt form in which it may be administered is, therefore, insignificant to the method of treatment. The salt form may, however, be chosen for reasons of economics, convenience, and toxicity.

The novel antibiotic factor of this . invention is produced by culturing an A-28088- faotor-D-producing strain of Streptomvoes aursafaciens under submerged aerobic conditions in a suitable culture medium until substantial antibiotic activity is produced. A-28086 factor D is recovered from the fermentation medium by he use of a combination of isolation and purification procedursa. The gensral methods of the isolation - and purification procedures used are well known.

od. 26572 i Te

The new organism especially useful for the preparation of the antibiotic A-28088 factor D wae derived by a peries of natural pelectlons followed by chemical mutation from the strain of fStraptomyoes aurecfaciens NRRL 6768 previously deposited for the production of A-28086 factors A and B in copending application Serial No. AT7.954, filed on June 10, 1974. After the discovery of A-280068 factor D Co produced by HS. aurecfaciena NRRL 8082, it was : 10 recognized that §. aureofaclens NRRI, 5768 also : produces A-20086 factor D as a minor factor.

The new A-28086-producing strain NRRI, 8092 which ia empecially useful for the production of A- . 28088 factor D is also classified as a strain of qfreptomvces aureafaciens Duggar, as described by E.

B. Shirling and D. aottliedb in “Cooperative pescription of Type Cultures of gtreptomyces. I11. oo Additional Species Descriptions from First and

Second gtudies,” Intern. Bull gyatenatic

Bacteriol. 18, 279-392 (1968). This classification is based on methods recommendsd for the

International Streptomyces Project [EK. B. Shirling and D. Gottlieb, “Methods for gtreptonveen species,”

Intern. Bull Systematic Bacteriol. 16, 319-349 (1968)1 along with certain supplementary tests.

Color names were assigned according to the 18CC-NBS thod (K.L. Kelly and D. B. Judd. “The 16CC-NBS

Method of Designating Color and a Dictionary of

I. 45% i

Color Names,” U.8. Dept. of Commerce, Cire. 553, 1966, Washington, D.C.). Figures in parentheses refer to the Tresner and Backus color series {Tremner, H. D. and §. J. Backus, "System of Color

Wheels for Streptomyces Taxonomy,” Appl. Microbiol. 11. 335-338 (1963)1; color tab designations are underlined. The Maerz and Paul color blocks (A. :

Maerz and M. R. Paul, "Dictionary of Color,” McGraw-

H11) Book Co., Inc., New York, N. Y_., 1650) are ‘ 10 enclosed in brackets. Cultures were grown.at 30°C. for fourteen days unless otherwise noted.

CHARACTERIZATION OF A--28086-PRODUCING . STRAIN NRRL 8090

MORPHOLOGY

On medium ISP No. 7 (tyrosine agar) the } culture produces occasional hooks, but, mainly | } produces short, straight sporophores. Spore chains are less than 10 spores per chain, usually 4-7 spores par chain. Short straight spore chaina were observed in the following media: ISP No. 3,

Czapek s-solution agar apd ISP No. 5. Abundant - coremia ware observed on Emerson s agar. Electron microscope observations were made on tyrosine agar (ISP No. 7) and glucope—asparagine agar. Spores are smooth and range in size from 1.2 to 2.8 u in length and about 1.9 v in diameter. The average spore size is 1.86 u x 1.90 u.

- 4. e522. i

Cultural Characteristics on : Medium Characteriatics

I8P Ro. 2 (yeast Growth- fair; reverse light yellow b extract-malt brown (12H8]; fair asrial extract) mycelium; poor sporulation; asrial vale grey [11A17; no soluble pigment.

ISP No. 3 Growth-sparas; roverse hyaline; {Oatmaal) no aerial mycelium; no soluble pigment.

ISP No. 4 (in- Growth-moderats; reverse greyish organic salts- yellow [11B2]; scant aerial starch agar) mycelium and sporulation; aerial pale vellow gray [1041]; no soluble pigment.

IBP No. 5 Growth-moderate; reverse pale : (glycerol yellow [10F2); fair asrial asparagine mycelium; scant sporulation; agar) aerial white [16A1}; no ®soluble ) pigment .

Tomato paste- Growth-moderate; reverses greyish oatmeal agar Breen-yellow; aerial oycelliom fair; moderate sporulation; light . 26 B © pale gray [53A21; no soluble

Co pigment . :

Glycerol-glycine Growth-abundant ; reverse greyish agar vellow [11R4}; moderate . aerial i : snycellium, white [16A1]; no ’ 30 . sporulation; no soluble pigment. :

Glucose-aspara- Growth-moderate; reverse pale gine agar yellow (10F2]; moderate serial mycelium and sporulation, white (10A1]; no soluble rigment. » 3b Nutrient agar Growth-mparse; reverse pale yellow [1682]; no . aerial mycelium; no soluble pigment .

Bennett ms agar Arowth- fair; revermm " medium “yellow pink [11A7}; very. scant

C49 aerial mycelium; nn sporulation; 99 moluble pigment. ag :

Calcium malate Growth--very scant, hyeline; no agar aerial mycelium, no soluble pigment. ' Czapek as solution Growth-very scant; reverse . agar hyaline; no aerial mycelium; no soluble pigment. .

Emerson’s agar Growth-moderate; reverss greyish yellow {1116]1; eapotty aerial i ~ mycelium; no sporulation; no 1@ soluble pigment. : Tyrosine agar Growth-moderate; revarse light vellow brown [12HE]; moderate aerial mycelium, light pale grey : margin [63A2]1, center near white, and moderate sporulation; no : : moluble pigment.

Tryptone-yeast . Growth-very scant, hyaline; no agar aerial mycelium; no soluble pigment. Co

Organlem NRRL R092 was studied for selected : physiological properties in accordance with standard : procedures. The properties observed and charateristice found were as follows:

Property Observed Characteristic

Action on milk peptonized (99X); pale-yellow : oo growth ring, cleared area pale yellow-pH reaction 4.6 b Nitrate reduction Positive Co : Nutrient gelatin 50% hydrolyzed at 14 days

Melanin pigment production on:

Tyrosine—agar Very weakly positive . 1e slants :

Tryptone-yeant- Negative - extract broth

Carrot plug ’ Abundant growth, pale yellow; no : aerial mycelium 16 Potato plug Abundant growth, grayish white; : "no aerial mycelium; no change in . i . * plug. C0 ol

Temperature re- 25°C. — Abundsnt growth; fair quirements (ISP serial mycelium; reverse wedium No. 2 1ight brown; no soluble yeast extract pigment. : . malt extract : } slants : Ye

. ~ . ‘ fo [ 565 oo 30°C - Abundant growth; fair parinl mycelimm; reverse : light brown: no soluble pigment 37°C. - Abundant growth: fair } 6 aerial mycelium; reverse brown; soluble pigment brown. 40°¢_ ~ Abundant growth; sparse asrial mycelium; reverse red brown; moluble pigment deep read brown. : 45°C _- Abundant growth; no © serial mycelium; reverne rard brown; moderate red brown pigmont.

fd 76522 .

The results of carbon utilization tests carried out with organism NRRI, BO92 are set forth below. The aymbols used to indicate growth response : are:

H + | good growth, positive utilization (+) poor to fair growth (-) faint growth, probably no utilization - no growth, no utilization

Carbon_EBource Responses

D-glucose +

Li-arabinosa + : } D-xy lone + . . D-fructone ’ ’ sucrose - 16 D-mannitol - i-~inomitol + rhamnone + raffinomse - i -C control - (No carbohydrate)

Cartain characteristics of the A-28088- producing 5. aureafaciens atrains differ from the characteristics of the organism described by

Bhirling and Gottlieb. These differences are mmmarized in Table 111:

i

Table 1] . :

Carbon oo Published

Utilization NRRL 5758 NRRL_8092 Description sucrose - - + } 5H 1-1inostitol + + - < rhamnone + : + - elatin 30% in H0X in Limited or

Liquifaction 14 daye 14 days none

Action on Milk Milk Limited and ’ Milk papto— . pepto- variable fred ized, reptonization } . while pvale- {often none); growth vel low limited growth ring growth . and coagulation i 15 ring . . | ’, ’

: a | The characterice of organism NRRL, 5768 _ which differ from the charateristics of organism

NERI, B92 are summarized in Table IV.

Table IV Bb

Charscteriatic NRRL _5756 NRRL 8082

Vegetative Color Yellow on Cream to pale— several media yellow on several media

Sporulation Some apiral Short atraight 10 . aporophores sporophores. with on tomate oncasional hooks . paste: oatmeal . and inorganic nalta-starch media drowth on

Calcium maleate Growth fair, Growth sparse, brown with clear; no clearing . clearing . 20 Inorganic maltn- Moderate Beant : aporulation; sporulation; : . aerial pur- aerial white plish white : } to gray. - 25 Bennett 8 agar Reverse pale Heverse pink : yellow oo oo ‘ | The [btreptomyces aursofacisns culturs : especially useful for the production of antibiotic

A--20088 factor D has been depoelted and made a part of the stock culture collection of, the Northern

Marketing and Nutrition Remsarch Division, U.s.

Dept. of Agriculture, Agricultural Research Service,

Peoria, 11linois, 61604, from which it is available to the public under the number NRRLI, 8092. 28 C fk 2057 i : © The culture medium employed to grow

Straplomycen aursofaciena NRRL 8092 can be any of a number of media. For economy in production, optimal yield, ease of product isolation, however, certain culture media are preferred. Thus, for exawple, preferred carbohydrate sources in large-scale fermentation are tapioca dextrin and sucrose, although glucose, corh starch, fructose, mannose, maltose, lactose, and the like can also be employed. corn oil, peanut oil, soybean oil and fish oil are other useful sources of carbon. A preferred nitrogen source is enzyme-hydrolyzed casein, although peptones, soybean meal, cotton-seed meal, amino acids such as glutamic acid, and the like are also useful. Among the nutrient inorganic salts which can be incorporated in the culture media are the customary soluble salts capable of yielding sodium, magnesium, calcium, ammonium, chloride. carbonate, sulfate, nitrate, and like ions.

Essential trace elements necessary for the growth and development of the organism should also be included in the culture medium. Such trace elements commonly occur as impurities in other constituents of the medium in amounts sufficient to mest the growth requiremnets of the organism.

Co 29 C

Wk. 26582 ! :

It may be necessary to add small amounts (1.0. 0.2 ml/1.) of an antifoam agent such an polypropylene glycol to large-scale fermentation media if foaming becomes a problem. | Although it is not essential, the antibiotic production of Streptomyces aureofacisns

NRRL 8992 is enhanced by the addition of a small : amount of oll euch as soybean oll.

For the production of substantial : ’ quantities of antibiotic A-28088 factor D, submerged : aerobic farmentation in tanke ims preferrred. Small

CL quantities of A-28088 factor D may be obtained by - shake-flask culture. Because of the time lag in antibiotic production commonly associated with 16 inoculation of large tanks with the spores form of the organism, it is preferable to ume a vegetative inoculusi. The vegetative inoculum is prepared by : inoculating a small volume. of the culturs medium with the spore form or mycelial fragments of the organism to obtain a fresh, actively growing culture oo of the organism. The vegetative inoculum is then transferred to a larger tank. The medium used for : the growth of the vegetative A can be the . ~ eame ap that employed for the larger fermentations, 256 but other media can also be employed.

Co hws

The A-20826 factor D-producing organism can SL be grown at temperaturas betwsen about 20” and about : 45°C. Optimum A-280868-factor ID production appeare to ocour at temperatures of about 279-3070. b An im customary in aerobic sulmerged culture processes, sterile air im blown through the culture medium. For efficient growth of the organism the volume of air employed in the tank production im preferably above 8.1 volume of air per volume of culture medium per minute. For efficient . production of A-20088 factor D, ths volume of air employed $n the tank production ins preferably above 9.258 volume of air per volume of culture medium per minute. High levels of dissolved oxygen do not 16 repress sntibiotic production. . . The production of A-28086 antibiotics, including factor D, can be followed during the

B fermentation by testing samples of the broth or of extracts of the mycelial solide for antibiotic activity againat organiems Known to be senaitive to : the A-28086 antibiotics. One amsay organism useful in testing the antibiotic A-28088 antibiotics, including A-28088 factor D of the present invention, is Bacillus ubtilis ATCC 6633. The biloamsay is 26 conveniently performed by paper-disc assay on agar : plates.

CO ONAl ee . - . 31 | | Co

- fuk WS}? i ’

The initial pH of the wninoculated culture medium varies with the medium used. In general, the oi should be in the range of 6.8 to 7.8. The harveat pH at the end of the fermentation is usually 8 slightly higher, in the range of 68.5 to 8.9.

Genarally. antibiotic activity ia detectable on the second day of fermentation.

Maximum production of antibiotic A-28086 factor D usually occurs between about. tha eight and the tenth . 10 days.

Following its production wnder submerged ampobic fermentation conditions, antibiotic A-20088 factor D ia recovered, togathar with. the co-produced

A--280A6 factors A and B, by methods commonly 1H employed in the fermentation art. :

The co-produced A-2n086 antibiotic factors are found in both the mycalial mars and in the oo filtered or centrifuged fermentation broth. Maximum recovery of the A-2A986 antibiotic factors ise accomplished. therefore, by oa combination of methods, including filtration, extraction, and adsorption chromatography. A preterrved solvent. for separating the co-produced A--280N06 antibiotic factors from either whole or filtered fermentation : broth is ethyl acehate, althongh other commonly used solvents are matistactory. ;

BAD ORIGINAL

Ae TT ee oo fi. 26692 : !

An empacially advantageous method ot separating A--728088 factors aA, B., and its nD is to p tower the pH of the whole fermentation broth to shout pH 3.0. This method 18 the mubject of a copending application nt Hoeck and Berg titled

ANTTRIOTIC RECOVRRY PROCKSS. U.6. Patent No. © 4,009,282, insued February 22, 1977. At about pH : : 3.9 the A-28088 {actors A. B, and U are conveniently saparatead with tha mycelial mans by filtration.

B 10 Methanol 1m a preferred mo lvent tor mepesrating the : antibiotics from the mycelial mans, but other lower : alcohols and katones are also suitable.

Co Azeotropic Aint tation con also be advantageously employad in the recovery of the A- 16 . 25986 antibiotic factorn. In thia method an organic nolvent which forma an appropriate aneotrope with ’ water is addded to the aqueous fermentation broth. : . : Thio solvent-broth mixture im subjected to = ’ : ageotropic distillation in order to remove at least : 20 | half the water from the broth, Jeaving a water- solvent mixture in which the A-28088 antibiotic factors are in solution in the organic molvent. :

Insoluble by-~productn can ber geparated by suitable : maans such an filtration or centrifugation. ‘The A 2b 28086 antibiotic factors can then be racovered from the organic mo lution by well-known — such : . i am evaporation of no Lvent, precipitation by adding a nonso lvent., or extraction. a3 BAD ORIGINAL fo. 20522 '

Organic solvents which form appropriate azeotropes with water in order to carry out such a recovery procedure include, illuatratively, butyl i ‘ alcohol. amyl alcohol, hexyl alcohol, benzyl ’ 5H aloohol, buty) acetate, amyl acetate, 1,2- dichloroethane, 3-pentanons, toluene, the xy lenen and the like. B

There is special advantage in recovery by azeotropic distillation on large-dcale fermentation processes. Hoth water and solvent taken overhead in the azeotrope can be separated by known } i techniques and thereafter recycled for further use. : : The water thus removed is free of contaminants and does not require a waste disposal process. The } solvent thum removed may be recycled to the process.

Further purification of the A-28086 antibiotic factors and separation of individual factors includes additional axtraction and 3 adsorption procedures. Admorptive materials such ams silica gel, carbon, Florist IR (magnesium silicate,

Floridin Co., P.P. Box 989, Tallahamames, Fla.) and the like can be advantageously employed. ,

Alternatively, the culture solids, : including mediim constituents and mycelium can be used without extraction or separation. but ' preferably atter removal of water, as a source of . the A-28088 antibiotic factors. Por example, after

AMOR ONE 34 .

A fuk. 0522 : 3 : i product tom of the A-2A086 antibiotic Eantors. the culture madivm can, be dried by lyophllitzatton and mixed directly into teed premix.

In another sspect, after production bi the ’ 5 A--20088 antibintic Factors itn the culture medium, : the mycelivm can be aeparested and dried to glve n } product which can be uaed directly in a teed prefix. © Under the conditions employed thus far. the

X new Streptomyces aureofacisns strain described 19 previously and designated as NRRI, 68002 produces - antibiotic factor A as the major factor. Although tha ratio of factors varies depending on the fermentation conditions used, in general factor A Co accounts for at least about H0X of the total ‘ recovered antiblotic activity. Factor D accounts for about 8-10 % of the remaining activity. Factors

A and D are recovered both from filtered broth and : trom the mynslivm. Minor factor B, which accounts for substantially the remainder of the antibiotic activity, ie present in amounts nf less than about : 1% and has thus far been observed only in the . mycelium. | : - . .

Another substance, srbitrarily designated ans A-200A8-1, 18 co-produced with antibiotic A-28088 : 26 factors A, B, and Db. Although a A-20086-1 ims not

. i ’ : : | | . oo | phos ; : ; : microbiologioally active, it ins mtructurally related i ‘to the A-26086 antibiotic factors. A-208088-1 is a i : white crystalline compound (trom acetons-water) and has » melting point otf about 166-1627¢. Comparative ’ H stuiies of tha HMR spectra and other properties of

A-200R8--1 and synthetically-prepared A-280R8 factor

A methyl ester give evidence that A-20088-1 is the methyl eater of A-28086 factor A or a closely . related compound such am a stersoisomer.

Although A-2080A68--1 initially co- : precipitates with the active A-28088 antibiotic © factors A, BH, and U, it is readily separated from i } | them by siticea gel chromatography. A-28086--1 has an approximate Wg value of 0.53 on silica anl thin- 1H lnyar chromatography with athy] acetate an the . saluting solvent and using vanillin spray resgent (3% : vanillin in methanol + 2.5 ml. cone. Hp50, par 100 ml. solution) for detection. After spraying with vanillin and heating, A-28086-1 gives a blue spot while the A-28086 antibiotic factors A, B, and D given bright pink mpots which quickly turn dark. : Co, Antibiotic A-28086 factors A, B, and 1) are separated from each other and are 1solated as. individual compounds by the use of wall-knovn : :

Co 2% methods such as column chromatography, thin-layer chromatography and the like. For exomple, column chromatography over silica ael is used to separate

Bes i factors A, B, and D by luting the column with . varying solvent mixtures, such asm benzene-ethy) ee acetate. ising benzene-—-aethyl acetate solvent . mixtures over a silica ge) column, A-28088-1 is 6b eluted firat: factor NB ia eluted next; and factors A and D sre alnted together later. Additional chromntography using tine-mesh silica gel effects a asparation of factors & and Db. ‘Thin-layver . chromatography, as dascribad hereinabove, is a | conveniant. method for monitoring elution progress. ,

Am in the came with A-2B0ORE factors A and ) B, the newly-discovererl A-200B6- Factor-D compounde : of this invention also inhibit the growth of bacteria and fungi which are pathogenic to animal and plant life. One measure bf the antimicrobiel activity of A-28088 factor D was made using the conventional dinc-ditfusion method (6-mm pads were dipped in solutions containing | mg. of A-28088 i” factor D per ml. of solution; pads were placed on agar plates seeded with tent organism). The results of these tentn ara summarized in Table V. | :

. . . :

BL 76572 i

TABLK V

Tost Zune af

Organism Inhibition (mm)

Staphylogocous. aureus 22 6 Bacillus subtilis

ATCC 6833 3

Saraina lutea

ATCC 8341 28

Saccharomyces pastorianum

ATCC 2368 20 . Neurospora cragsa 12 . 10 Candida alblcana | 19 : In another aspect of thelr antimicrobial } activity, A-280868-fsctor-D compounds inhibit the growth of anaerobic bacteria. The minimal - inhibitory concentrations (MIC ms) at which A-28086 156 factor D inhibits various snasrobic bacteria were determined by standard agar-dilution assay.

Endpoints were read after 24-hour incubation. The ] results of these tests pre swmmavized in Table VI. i . . 3A tnt 26573 . : TABLE VI

Taat Urguniem MIC _ tng/ml)

Actinomyces bovis | EL +

Clostridium inecunm «0.5 b lostridium perfringens <®. 5

Clostridium ramomum «0.5

Clostridium asp icum «2.5 :

Cloatvidivm seapticum having 6

Bubacterium serolaciens <0.5 : 19 Baptococcun onasrobiua “0.5

Paptostreptaccuas intermediua “@.6 ~ Brovionlbacterium acnes 14 <@_5

Praplonicbactecium acnes 79 «9.6

Bacteroides fregilin ssp. . 15 fragilis 1877 4.9

Bauterolides fragilis map. fragilie 1936H 6.0

Bacteraides fragilis sep. thetalotaomicron 4.0 26 Bacteroides fragilis snp. : ~ yulgratis No. 1563 . 4.9

Baqterciden fragilin sap. yulgatia 1211 1.0 .

Fusobacterium. avabiosun 2.0

Fumobacteciun necrorhorum 16.0

Yelllonella alcalescena 2.0 » 3a .

Ane

: fod. 26522 ‘ h Tha A-20008-factor-D-compounds are also antiviral agante. A 20088 factor D ie active against Maryland B virua, type 111 poliovirus, (OK " virua, herpes virus and Semlikl Forest virus, as : 5- demonstrated by ip vitro plaque suppreasion tests, : similar to that described by Sminott, Applied

Miccebiclugy 9 LU], 86-72 (1961). A-2HOBH factor D, im also active against Transmimaihle Uantro- enteritis virus, Nevicanl: Lo Digeana, Virus, and 19 Infections Bovine Rhinotrocheltin vivus, an demonstrated by aimllar timsue -culture tests. | ’

The fact that the A-2A088-factor--D . | componnde have activity hoth against viruses and against suaerchic bacleria makes thess compounds 6 particularly bensficial for the treatment or : - prevention of enteritis in chickens, swine, cattle and sheep. The A-28086-faotor 1) compounds are also . useful tor the treatment of entero-toxemia in rmainants - } oo 20 Anticoctidal activity ie an jmportant property of the A 28086-factor-D compounds of this - invention Rxperiments ip vitro demonstrate the oo | ankicoceidal activity of A-280A8 factor D ‘mgninst

Eimeria tenella. The following ma thod was used [for

Co 26 further detatls, mee L. R. McDougald and R. B. talloway In Exper. Parasitology 34, 188-186 (1873) 1: : 44 - gAB SRIGINAL

Ce ee—

B fukrisr 4 : . : Host call enltures are prepsred by conventional techniques uming Leighton tuben, plastic dishes or plates, or other suitable culture vamsaeln. Following 2-3 days growth in sony suitable : b - oulturas medium (Kogle nn minimal essential medium, lactalbumin hydrolysate in Earle ms or Hank's balanced saline. Madium 199. otc.) a eunitable nono layer usually forms and is ready for infection and drug treatment. Primary cultures of chick 190 kidney celle were usad (or theme studies.

Viable oooystn of Eimoris_Lenalla are obtained from the fecen of infected chickens and are : : clenned and mterilized prior to use. The oocysts a. are sporulated by incubation at room temperature while bubbling air through the suspension or by shaking gently on a gyrotory shaker or by othar suitable means. Potassium dichromate or sulfuric acid (or other chemicals) may be employed to prevent bacterin] or mold growth doring sporulation.

Excystation of infective sporozoites from ) the oocysts im accomplished by a combination of machanieal disruption of the oocyst wall, followed by treatment with trypmin and blle salts to induce the sporozoites to free theme ves from the sporooyats.

BAD ORIGINAL

41 oo fol 26522 . ! Cell culbures are infected by the introduction of viable sporozoites into culture vessel. Test chemicals can he intvoduced at this time ov suhsaquent. ly. The teat chemical im

H introduced in the desired connentration in 10% dimethy [formamide io physiological saline. The oo endpoint of the Leal is ilohibition of asexual } deve lopment stages, and ia datermined by microscopic examination of cultures fixed and stained 968 hrs. 19 postinfection. Results are recorded as active (A), inactive (HN) or cyLoboxic (0) depending on the presence or ahmsence of seconl-generstion schiaonte and any appareunt koricity to the call monolayer.

The anticovecidal activity of A-28088 factor 16 D dwmimnatrated by thie test {es muwmarized in Table

VII.

Co - : Le :

fuk. pés22

Po } . TABLR VI]

Anticocoidial Activity of

Antibiotic A-208088 Factar D

Concent, ration .

H of A-2B0R68 Factor I {meg/ml) : Rating : 5h ¢ 1 ¢ 0.2 C : 9.4 Act ' 0.03 A oo 0.02 A : CY tA 0.008 : N 16 * Results of two tests

Co : oo . ’r . . 4.3

Bl. 76523 [

Another useful teatures of the A-28086- factor-D compounds ia thelr ability to {wprove_ feed- utilization efficiency in ruminant animals which have nn developed rumen function. The mechanism for utilization of the major mtritive portion (carbohydrates) of ruminant, fesds im wall known.

Microorganisms in the rumen of the animal degrade carbohydrates to produce monosaccharides and } then convert there monosaccharides to pyruvate 1@ compounds. Pyruvaltas are metabnlized by microbiological procensess to form acetates, butyrates or proplonates, collectively known as : volatile fatty acide (VFA) For a more detalled dircunsion. naa. fang in "Physiology of Digestion and | Metahol lam tn the Rominent.” Phillipson mt al..

Co Edm... Orie} Presa, Newcant le wpon Tyne, England, : . -197@, ep 490-410. . The relative eftleciency of VFA utilization im discussed by McCullough in Feadatuffs, June 19, 1971, paan 19, Rekeland at al. in J. An. Sol. 83, 282 (9711: and Church at al. In “Digestive } Physiology and Nutrition of Ruminants,” Vol. 2, 1971. pp 622 and 625. Although scatstes and butyrate are utilized, proplonales are utilized 26 with greater efficiency. Furthermore, when too . little propionate ie avsilable, animals may develop ketoris. A beneficiel compound, therefore, stimulates animals to produce a higher proportion of : a4 .

Jol e523 . i . propionates from carbohydrates, thersby inoreasing carhohydrate-ntilization efficiency and also reducing the incidence of ketosis.

The activity of anch a bona fiotal compound } 5 can be detarmined by onbrarving 11e affect on the to rroduction and concentration ot rroploanate compounds ; in the rmmen. waiung the totlowing methods: «

Homen 1 lueid is obtained Fram n mtrer with a : mirglical ly installed fistula opening into the rumen.

Po The steer is maintained on a high-grain ration. A samples of ramen flold ir atralned through four layara of chasmacioth, aud the filtrate is | oo aagl lected The particulate matter retained by the - cheansscloth ia ceasuapendnd in enough physiological buffer to return 1t to the original volume of the rumen fluid, and this sauspenmion is strained again.

The buffer used .has the teillowing composition: . . . an CL

GAVE ee " A . BAD ORIGINAL ~) ~—

fod. 26523 ingredient B/liter

Ha TP, : aaa

KH, PO, BOIHY

CMa yy 280

KC or

Hatt @¥onh

Meth, a 1p

Chi, 2HL,0 a nn

Festi), 7TH,0 A eon 19 Mnti, 0 © ong

Znt0 qT Q. (1a

Cui), 5H, 0 002

Cott, BHO 9.001

Cam described by Uhenp et al in J. Dalry Sei. 34, 1226. 1230 (VUES)

The two (1 1teataes are combined and allowed to stand until pirticeninke matkar separates tn the top The clenr bayer in eeparated, dilated with the nama tmfter (1:1) and then adiustad to hetween pli 8.8 btn 7 O

Tha diluted rHmen {intd (10 ml ims placed in 265-ml. flak with 46 mg. of the above-described faad, no additional 1 wy of ‘aoybean protein, and © the compound to be teatead "Wor raplteate flasks vaad per teeatment Wien rate of four conbrol $F lasks aach are also employed A oxero-bima control and an 1

BAD ORIGINAL

: . . I eat”

Joke 2573 oC i incubated 18-hour control ere used. All temt flasks : : are incubated for 18 hours at 30°. After i ‘ incubation the pH ia measured, and 20 parcent metaphoephoric acid (2 ml.) ta added to each flask. bH The samples are allowed to settle. and the supernatants are analyzed by gas chromatography for propionate, acetate, and butyrate compounds. Active compounds significantly increase propionate production over that oft controls.

Teast-compound results are statistically

To compared with control results. Table VIII shows the ratio of volatile-fatty—ac id concentrations in A-- : 2808a-tactor-I-treated flasks to concentrations in control flasks. ; 15 TABLE V1)

A-28086 I) Molar ¥ Molar ¥ Molar ¥ Total VFA mg/ml. Acetate _Butvrate _Provionate. mM/l 1.0 0.0715 0.7973 1.7981 1.1979 0.3 0/976 0.8726 1.6871 1.0630 - - » . } 47 vv

Ok. 26472 ; oo

The A- 20086 factor-b compounds of this invention are typically affactive in increasing the efficiency of fasd-utilization when administered to ruminants orally at rates of from about 0.06 mg. /kg./day to about 5.8 mg. / keg. /day. Host : beneficial vermults are achieved at rates of from : : about 8.1 me./kg./day to about “4.5 wma./kg./day. A . : prafarred method of administration of an A-28086- factor- compound is by mixing it with the animals’ feed; howaver, it can he administered in other waye. for example, tahlets. drenches, boluses, or capsules. Formulation of these various dosage forms can be accomplished by methods well kaown in the vaetarinary pharmaceutical art. Rach individual dosage anit should contain a guantity of an A- 20086 factor-D compound directly ralatad to the i proper daily dose for the animal to be treated. . This invention furthar ralates to feed ’ } compositions adapted to fatten cat ble comprising 29 ‘cattla fead and from 1 to 30 grams per ton of an A- 2pea6-factor-0 compound. in order to iL luntrate more fully the operation of this invention, the following examp lem are provided. ’ oo an

Joi. 24522 i

TO KXAMPLE

A. Bhake- (lasek Fermentation nf A-20086 ’ : A culture of Gtreptomyces aureofacisns

NRRL: 8992 was prepared and maintained on an agar 5] slant having Lhe following compoattion: * 5

AL wsa3 } i.

Inaredient Amount

KoHPO 4 2 a.

MeB0, TH, 0.25 g.

NH NO 2 a

CaC0g 2.5 ga.

FeS0,. TH,0 | 8.001 ga.

MnCl, 7H,0 2.001 a.

ZnB0 4 . TH, | 0.001 a.

Glucoss | le g. 19 Agar 2a pg.

Maionized water-g. 8. | 1 liter rf tonadinsted) 1.1

The slant was inoculated with Btreptomycen aursofecienn NRRL. A092, aud the inoculated slant wan incubated at 3070. for abont seven days. The mature glant culture was covered with sterile beet serum and was scraped with a sterile loop tv prepare a ‘ ) spore and myrelial suspension trom the slant culture. ‘he resulting suspension was lyophilized nto A ma Umum of six pellets. . : | me of the Lyophile pe) lets thus prepared was used to inoculate 68 ml. of a vegetative medium having the following composition: :

Fie) .

i. fot 26623 } i :

Ingredient Amos b oo

Glucone 26 gpg. . Soybean flour : 15 g. ; Corn steep liquor 19 gu.

CaCO | | oop

Tap water q.8. 1 Jitar ' wl adjusted to pH 8/5 with di) NaOU

The inoculated vagetative medimm, In a 25Q- nl. Erlenmeyer flask, waa incobated at 399°C. for 48 . i . 190 | hours on a rotary shaker at 250 rpm with a two-inch arc. ’ : This incubated vegetative medium described above (0.5 ml., | percent) was used to inoculate 6e@ ml. of a fermentation medium having the following 16 compoaition: . . > » *

AETHER . . i

HA 26523 j . tngradicot © Amount . Tapioca dextrin? 60.0 ga. . Enzyme--hydrolyzed cngein a. ga.

Knsymat. ic hydrolyante af sanein®*¥ ne gag.

BH Cag 2.0 g.

MgB0, THO 8.5 g. . oo Blacketrap mr lansas ) 165.0 da.

Refined poybean otl 5.9 ml.

Tap water q.m. 1 liter rH {noadjusted) a.68 * grpley Dextrin No. iL, AK. fitaley Co., Decatur, tiv. ' 5X pnber RUC, Amber Laboratories, Juneau, Wimc. *XX gz amine A, Shetfleld Chemical Co., Norwich,

N.Y.

B. rank Fermentation of. A-20006 :

The initial procedures Amancribed in (A) for the ahake-flask fermentation of A-YA0REB was also used for tank fermentation. In order to produce a 29 | larger volume of jfnocalum, 1@ ml. of the incubated vegetative medium was umad to inoculate 400 ml. oft a second-stage vegetative madium having the same composition an that of the vagatative medium. This ascond-atape medivm, in a 2-13ter Erlenmeyer flask, was incubated at 397C. for 24 hours on a rotary i shaker at 250 rpm with a two-inch arc:

BAD ORIGINAL

Fil me———

oo | fil. 26523 : . This tneubated nmecond-stage vegetative . © medium (800 ml) was wmed to inoculate 100 liters of sterile fermantation madium having the following composition: 6b Inarediont Amount:

Taploca dextrin' Ae. 0 g/l. - Enzyms-hydrolyaed canain®® 6.6 a/l.

Enzymatic hydrolysate of name int ¥* 2.9 p/l. ’ (:aC0g 2.6 asl. . 19 Matic, TH.,0 . 20.5 asl. } Bleckstrap mol npasen ha g/l.

Ref ined soybean oil Ee wl/1.

Tap water 3.8, 1 titer os pH (unadjusted) 6.8 * Staley Dextrin No. 11, A.R. Staley Co., Decatur,

FEL. } x Ambar FHC, Awber laboratories, Junasw, Wisc.

AX NZ Amine A, Sheffeield Chemical Cn., Norwich,

N.Y.

Cove The ¢ll of the medium wae 8.8% 0.1 after sterilization by autoclaving at 121°C. for 30 minutes at 15-20 pounds pressure. ln eo 165~1liter fermentation tank, the inoculated production medium wan allownd to ferwent for 10-12 days at 2A + 1°0, } »

The fermentation medium wae asrated with sterile air at the rata of ©.4 volumag of air pear volume of i © ocultare medium per minute. The madiue wae stirred with conventional agitators at 306 rpm. . . . fi} , : . ' y .

Jak ese i

EE KXAMPLE 2

The A-280R8 antibiotics were produced according to the process of Example 1, but using a mhake-flask/tank production medium having the b following composition:

Ingredient. Amount . Taplooa dextrin® 0. g/l.

Glucone i.e g/l. :

Enzyme - hydrolyzed cansin*® 3.0 g/l. ; : 10 Enzymatic hydrolysate of cage in? ** 1.0 g/l. : Yeast extract 2.5 g/l}.

CaCOg 2.0 g/l.

MgB0,4.TH,0 1.6 g/l. : Blackstrap molassas 16.0 g/l. 16 Refined soybean oil 5.0 ml/l.

Tap water q.8. 1 liter . pH (unadjusted) 6.6 ¥ gtaley Dextrin No. 11, A.K. Btajey Co., Decatur, ir. * xu Amber EHC, Amber Laboratories, Juneau, Wisc.

XE go pine A, Bheffeield Chemical Co., Norwich,

N.Y. ”

The pil of the medium wae 6.4 after sterilization by antoclaving as describad in Example : 26 1. :

Jaf. 26623 i : KXAMPLE 3 . teparation of the A-20086 Factors A. B. aod D

Whole fermentation broth (80 liters), obtained by the method described in Example 1, wasms b adjuated to pH 3 hy the addition of dilute HCL. The resulting solution wam filtarad useing a filter ald (Hyflo Super-cel, a diatomacenus narth, Johns-

Man.ille Products tlorp.}. The separated mycelial cake wan extracted with 30 litters of mathanol, adding 1 6568 kg. of NaHCO, to the extrant with "stirring. After peparating of thie extract, the mycelial cake was again extracted with another 30 liters of methanol. The two methanol sxtracts were combined and concentrated under vacuum to remove the 16 methanol. The remaining aqueous msolution (about 7 liters) was adjumted to pH 7.56 with dilute HCl. The resulting solution was extracted twice with ethyl acetate (7-liter portions). The sthyl acetate extracte were combined and concentrated under vacuum to give an oily residue. This oily residue wae dissolved in 1500 ml. of scetone. Water (1500 ml) was added to the acetone solution. The resulting solution was adjusted to pH 3 with dilute HCl and : was stirred one hour. The precipitate which had formed was separated by filtration and then wan dissolved in acetone (1599 m)): water (400 ml) was added to this solution. The resulting asnlution was

Jat. 26523 po. allowed to stand for 18 houre for crystallization to occur. The crystals formed were separated by £11tration and dried under vacinm to give 74 g. . crude crystalline product containing A-28086 factors

D and A and other crystalline impurities.

This crude crystalline product (40 fq.) was dissolved in about 256 ml. of benzenas. The benzene ‘molution was then applied to a silica gel column (9- x 120-cm column; Grace-Davideson grade 82 silica gol). The colusm was eluted successively with 40

Liters of each of the following: : 1) benzene 2) benzene: ethyl acetate (9:1) 3) benzene: ethyl acetate (4:1) 4) banzens: ethyl acetate (7:3) 6) benzene: ethyl acetate (1:1) 8) anthy) acetate 7) methanol

One-liter frantions were collected. Each fraction was checked by ammay against Bacilua mubtilis and by thin-layer chromatography to identify the eluted compounds. A--28086-1 was aluted with benzena:ethyl acstate (4:1). A-28088 factor B was meparated during elution with benzene:athyl acetate (7:3).

A-2B0R6 factors A and D werms eluted in the fractions obtainerd with benzene: ethyl acetate (7:3 and 1:1), fractions 119-158. These fractions were 8 Co

Jaf. 6533 . : i . , combined and evporated to dryness winder vacuum. The residus thus obtained was dissolved in acetone (500 i : ml.) Writer (6500 ml). was added to the acetone : solution, and the resulting solution was adjusted to pH 3 with dilute HCL and was stirred for one hour.

The precipitate which formad was separated hy filtration and was crystallized from acetone (500 : ml. )-water (180 ml). “The cryslalas thus formed were neparated by filtration and dried under vacuum to give 20.1 pg. of a mixture of A-20088 factors A and n. ; 57 Lo

Jaf. 20592 i

RXAMPLR 4

Geparation ond Purification of Individual Factors... end

The crystalline mixture of A-26088 factors 6 A and D obtailnedd 1n Example 3 (18.8 g.) was oo dissolved in benzene (53 ml). The benzene solution was applied to a silica-gel column (1- x 188-cm column; F. Merck gerade 80 silica gel, finer than 230 mash ASTM). The column was eluted, at a flow rate of 96 ml. per hour, successively with: . 1) 12 liters of benzene : | 2) 12 liters of benzene:ethyl acetate oo (9:1) i | 3) 12 liters of benzene:ethyl acetate 165. (4:1) 4) 32 Jiters of bhenzene:ethyl acetate (7:3) ; 6) 10 liters methanol oo : | Thin- layer cellulose chromatography (Merck

Darstadt cellulose on aluminum support) was followed by B. subtilis bloautography to monitor the elution . procedure. The following solvent system was used: : water:mesthanol:ecatone (12:3:1). adjusting the solution first to pH 18.5 with NI OH and then to oH 7.5 with HCL. 6a :

Jaf #4503

I

One- to two-lLiter fractions were collected until activity was rdetectsd; then 200-ml. fractions were collected. The fractions containing only A- 28088 tactor D were combined and evaporated under i 5 vacuum to an rasidue. This residue was crystallized from acetone-water (1:1). ‘The orystals were separated and dried under vacuum to give 140 mg. of crymtal line A-28088 factor 12. : The fractions contalning A-200868 factor D : with a trace of A-200088 factor A were treated in the mame manner to give an additional 150 mg. of crystalline A--280608 factor D containing a mall > amount of A-280A08 factor A. ’ The fractions contaluing only A-28086 4 . - 1 factor A were alno treated in the same manner to oT give 4.7 g. of crystalline A~26088 factor A.

RE EXAMPLE 5 - A-28086 Factor D Acetvl Ester Lerivative

Antibiotic A-28088 factor D ls dissolved in pyridine. A stoichiometric amount of acetic anhydride ie added to this solution. The resulting solution im mixed thovoughly and then is allowed to atand overnight at room temperature.

. \ : : | ~ , « ’ (iA. 26522

An excess of water 4s then added, mixing thoroughly and allowing the mixture to stand for several hours at room temperature. The precipitate which forms is paparated by filtration. washed with water, and dried. The resulting solid im dissolved in acetone and evaporated to dryness under vacuum to

CL . give A--280H8 fantor I) acetyl =ster derivative.

EXAMPLE 8-0

Antiblotic A-20006 factor D propionyl ester } 10 derivative, prepared by reacting A-20086 factor D with propionic anhydride in the presence of pyridine according to the method of Example BH. . Antibiotic A-200R8 factor ND n-butyryl ester . : derivative, prepared by reacting A-208088 factor V ’ 16 with n-butyrie anhydride In the presence of pyridine 3 socording to the method of Rxample 6.

N . :

Ps . ; Antibiotic A-28088 factor D n-caproyl ester derivative, prepaved by reacting A-26086 factor D . with coaproic anhydride in the prossnce of pyridine ) according to the method of Example 6. ’ Antibiotic a-28088 fagtor D p-valeryl ester derivative, prepared by reacting A-206086 factor D with valeric anhydride in the presence of pyridine . according to the method of Example 5. . : Be fot. 72522 i -

RXAMPLE 10 : Preparation of A-26086 Factor D Sodium Salt ’ Antibiotic A-20086 factor D is dissolved in acetons. An equivalent amount of water ia added to : this solution. and suffcient 5 N sodium hydroxide is ot added to bring the pH of the =olution to about pH 11. The resulting solution l= stirred for about an hour and then is extracted wilh ethyl acetate. The athyl acetate extract is evaporated under vacuum to : 10 give A-2080368 factor D sodium malt.

EXAMPLES 11-13

Antibiotic A-280868 factor 1) potassium salt, prepared from A-28086 factor B and 5 N potassium : hydroxide, using the method of Example 10.

Antibiotic A-28088 factor D bariuh salt, prepared from A-28088 faclor D and saturated barium hydroxide, using the method of Example 190. - Antibiotic A-280868 factor D cesium salt, : prepared from A-280868 factor D and 1 N cesium hydroxide, using the method of Example 10. ’ . ’ r

Jaf. 26573 ;

EXAMPLE 14

Chick Ration Containing A-28086 Factor D for Coccidioaia Control

A balanced, high-energy ration adapted to 6 fead chicks for rapid weight gain is prepared by the following recipe: i .

Pr

- -— ) © Ingredient x lbn

Ground yellow corn He ‘ 1,006 . . Soybean meal, solvent - extracted dehulled, finaly ground, 50 per cant. protein 31.09 621.8

Animal fak (beet fa Low) 8.5 130 ried fimh meal, with aolubles (80% protein) 5.0 190 19 Distillers solubles from corn 4.9 9

Dicalciom rhosphate, fred grade 1.8 a6

Calcium carbonate 0.8 14 / 16 Vitamin premix (representing vitamins a,

D, RB, RK, and Bio, choline, niacin, pantothenic acid : riboflavin, biotin, with

Alucore bulking agent) 2.5 10 . Trace mineral premix (representing Mn&0,4, zn,

Kl, Fel0,. CatCD,) 9.2 4 4 3 . 2-Amino-4-hydroxybutyric acid (hydroxy analog of methionine) a.1 . 2

A-28088 Factor b 0.01 0.2 fait 20572 1 - .

These substances are mixed in accordance with atandard feed-mixing techniques. Chicka fed such a ration, with water ad libitum, are protected against exposure to coccidioning weight gains are fH comparable to those of cocetdinnis-free chicks fed a aimi tar, unmedicatad diet.

EXAMPLE 5

Bao. Cattle Ration Containing A-26008_A-28086 Factor D

A balanced high-grain haaf-cattle ration is prepared as follows: ” »

Aad

BA. 76522 . i : ingredient x lhe } Finely groun corn : | 87.8 1368

Ground corn cob 10 200

Dehydrated alfalfa meal,

A 17 percent protein 6 100

Delntl lad soyhean meal, solvent extracted, 590 percent protein 9.99506 199.912

Cane molasses fa 100. 0 19 - Urea a8 12.0

A-20088 Factor D 9.0044 2.008

Dicaleium phosphate, ’ feed grade a.8 10.9

Calcium carbonate 0.5 10.0 1b Bodium chloride 6.3 6.0

Trace mineral premix @.03 0.8

Vitamin A and D, premix® @_o7 1.4 - Vitamin R premix** 0.05 1.0 ’ Calcium propionate a.16 3.0 {) : * Containing per pound: 2,000,000 I.U. of To vitemin A; 227,200 [.U. of vitamin Dy and

Co 385.7 g. of soybean feed with 1X oll added ** Corn distillers dried grains with solubles containing 20,000 [.U, of d-alpha- 26 . tocophery)l acetates por pound

Jat: 26523 i ’ -

The mixed fead was compressed into pellets.

At an average daily ingestion rate of 16 pounds of feed per animal, this feed supplies approximately 300 mg. of A-20086 factor D par animal per day. .

Claims (1)

1. - ) Jad, 28523 i : We claim: ) = : CL A method of lncreasing fomd- ‘ . | utilization efficiency in ruminant animals which comprises orally administering to animals sald an 6 effective amount of a compound selectad from the group conginting of antiblotic A-20000 factor D which is a white crystalline compound when crystallized from acebtons-wnter; which is n soluble in methanol, ethanol, dimethyl formamide, dimethyl ! 19 rulfoxide, ethyl acetate, chloroform, acetone and i benzens; but which is only slightly soluble in hexane; which is tusoluble in water; which melts at ’ about 96-90°C_, and which has: : a) a molecular welght of 778, as 16 datermined by high-resolution mans . ' : apasctrometey; b) an approximate elemental composition

   - . "of 67.69 parcent carbon; 9.88 percent oo hydrogen, and 22.77 pavcent oxygen; Co | a) an empirical formmla of CyaH7401y, am ’ determined by high-resolution hose ) Po spectrometry; | - ! | , . d) a specific rotation of -56° (6 = 0.1, oo methanol) when determined at 2BY¢. ; : 26 a) an infrared absorptidn spectrum in : ) chloroform with the following = a7 oo

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   0.10 Water maburated with mehyl : trotmtyl ketone (MIBK)

   9.26 Water saturated with MIBK : 6 plus 2% p-toluenesulfonic acld nnd 1% piperidine i

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