PH27073A - Antibiotic A80777 and process for its production - Google Patents

Description

- ee ——————————————— ee — 3755 71°73

THE DIRECTOR OF THF. PATENT OFFICE:

BE IT KNOWN THAT ROTERT L. "AMILL CITIZEN OF THE

UNITED STATES NF AMERICA NOMICTIEN IN GREENWINM, TNDTANA

WHOSE POST OFFICE ANDRESS IS 617 BROOKVIEW DRIVE ANN BE IT

KNOWN THAT RAYMON™ CHE=-FONG YAO CITTZRN OF THE UNITED STATES

OF AMERICA DOMICILED IN CARMEL, INDIANA WHOSE POST OFFICE

ADDRESS IS 987 HAWTHORNE TRIVE HAVE TNVENTED A MEW AND USEFUL

ANTIBIOTIC AB0S77 AND PROCESS FOR ITS PROMUCTION OF WHICH

THE FOLLOWING TS THE SPRCTIFTCATIN:

BAD Ohiwen po 21°78 7 ’ 4] .

ATTY Tn ANITA PRONEAs

EORTTE PROCIeTTN od This invention relates tn the new prlycther antibiotic i mo ARD577 and £9 a new strain of Actinomadura sp., Mil 19273,

E : 5 vhich prohicrs this antibintic. The structure of AIIS77 is

Lo 2 shown in Formula 1 - te oo oo : it :

RE $y wre >» — SE ire

RE . i CHo &\. : i H .

Lid : — H 0 H O H MW

A | 0 Me -_f" 0 Ho Lo “ie. Ho ono re wom Jane si . " CH, 5 0] HoR,C! H So

Le i Ha aw OCH 4 +

FN 15 H . HL a . fie ~ # ‘ 0 4 - “a * oo

Eo n° .

Hol 0 el oo ws wr] | Co iid . o i 1 To

From 20 -

HE This invention also relates to acyl ester and alkyl : aly he go LL a jo ] ether derivatives of A%0577 and tn the salts of A577 and of Ea a

Sil "

TO the derivatives. So - Another aspect of this invention is a process for Co

I. | * bois, wr : Pn

Co -1- EL

Sd EH

Ca BAD opyg 2 sh \ vot “i a INAL 2) “in ; fd :

Ad . N

= I fos a

EL hi

EE SE

“i CE ~ CE do bree od Fa producing antibiotic AIIS77 by cultiveting Actinomadura sn., :

NRRL 19276, or an A%OS77-producing variant, rutant or reeom- ul binant thereof, in a enlture medium containing assimilable id ;

HH sources of carbon, nitroren and inorpanic salts -nder submerged wh 3 5 aerobic fermentation emditions until antibiotic ANT? is i: . produced, A3DS77 is extracted from the fermenlatinsn birath and from the rmycelinm with orpanie solvents. A577 is separated and further parified by techniques such as colwm chrontse raphy.

Se ~ 10 tecanse Actinomadura sp., ‘MT 127%, is a newly : : = discovered sirain, this invention further provires a biolo-

Ce fically pure cultnre of this microsrpanism or a rmtant, variant! i a . or recorbinant thereof which nroduces antibiotic ATI577, - : ; ADS77 is a useful antibacterial arent. Tt improves

Li 15 feed=utilization efficiency in rminants and acts as a rrowth ¢ - promotant in ruminants aad in monosastric animala. Tn addition, = | A0577 has parasiticidal activity an? 1s useful as an fono- } \/ phore, Therefore, the invention also relates tn a feed ecnn- &ri i pogition for increasing feed utilization efficiency in riminant 3 20 animals comprising animal feed and an effective amount of i antibiotic AN0577, its acyl ester or alkyl cther derivatives,

Ee © or a salt of A577 or of the derivatives; an? in a process for increasinr feed utilization cfficiency vhich corprizes # orally administering to an animal an effective amoint of these 2 -2- 3 g PAD ORiGiNaL 9

——————eeeeees _—m——_—

F073 corny fa,

The Adraginen shou Lhe follorine infrared atsarntion spectra in ehloroform: pure 1 ANGTT (Ha alt)

Vipire 2 TMSTT (VY Salt)

Trproved antibiotics eontinme to be needed in the veterinary field, Tnhancing grouth pronation iu aniimls is one desired feature of such antibioties. rowth promotion ean he achieved by redneinp discase and hy increasing foed-ntilizalion efficiency. "f particular interest is ;rowth promotion in riminants, such as cattle,

The mechanism for utilizing the naj sr nutritive. portion (earbohyirates) of rmminant feeds is well noun, Vicros orranigms 1 the rumen of the animal leprade earbohyeirstes to produce monosaccharides and then eonvert these mnonnsaccharides to pyruvate compounds, Perwvates are metabolized by mierahio- logizal processes to form acetates, butyrates or propionates, collactively known as volatile fatty acida (ra),

The relative efficiency of +A utilization is connected to overall efficiency. Thns, althongh acetates and buterates are used, propionates are used with greater efficiency. Also, the fermentation efficiency of propionate production is creater than that of butyrate or acetate, This is in adtitinng ta the utilization afficicun,, A bennficinl compowd, therefore, -1-

BAp . :

Oem gg)

TT —

7°73 stirmlates animals to produce a hirher pronortion of prapionates from carbohydrates, thereby inereasine carbohydrate-ntilization s nfficiency. 20577 is a new member of the pronp of polyether antibiotics. estley {John I. Westley, "Polycther Antibiotics:

Maturally “Occurring Acid Tonophores, Vol. 2, Chemistry, "iarcel

Pelzer, lew Yori, 1983; Journal of daturnl froducls, he(1), [12877) has separated existing polyethers by .elass and type, "ging Vestley's system, A30577 is a new member of the Class > 10 proup of polyethers, This pronp includes tetronomyein, which ‘ is fescribed in J. Antibiotics, 142 (1982), and 11179403, which is described in J. Chem. joc., Chem. Comm,, 1073 (1971) and 1,5. Patent No. 11,279,349.

ATS77 {in its sotinm salt form) hag the follow ine characteristics:

State: white crystals (from acetone-water) mp: (Na Salt) 276-279°C, (x Salt) 270-2720, llolecnlar weight: 732 by field desorplion 290 rnass spectrometry (FMS): 760 for acid form,

J 7%: -19,03 (c 5, tel) lo titratable groups (467 THF)

Fmpirical formula: Chole pM

IV max: LThanol nentral 252 nm ( € = 17,735), 277% aw l= ne “lan 9

Woe

(+ =10,M3 acidic 2%0 nm (¢ = 1,715), 289 nm (r =17,91) basic 20% nm (& = 192,722), 2% nm (+ = 15,969), 276 nm (& = 9,605)

In (Va salt, CitC1,): 018, 2966, 2231, 1750, 1611, 1453, 1439, 1219, 1022 and 1012 cm™L (sce Fig, 1)

In (Kk salt, CHC14) 3023, 3019, 2967, 296L, 2930, 287, 1750, 1650, 1h51, 1,37, 122L, 121), 1210, 1079, 1059, 1222 and 1012 em”! (see Fim. 2)

Solubility: Not very soluble in water; soluble in . dimethyl sulfoxide, dimethylformamide, lower alcohols such as mathanol, ketones such as acetone, esters such as ethyl acetate, halopenated hydrocarbons such as chloroform and hydrocarbons such as diethyl ether, benaegene, toluene and warm hexane.

Based on its physical characteristics A80577 is believed to have the structure shown in formula 1. As is appa- rent from its structure, ABO577 is a charged molecule and is capable of forming salts. AB0577 also has up to four hydroxyl groups which can be esterified or which can form ether deriva- tives. The acyl ester and the alkyl ether derivatives of A%0577, -5-

BAL Cia, 3

Fr

: 7°73 and the pharmaceutically-acceplable salts of AID577 and of these derivatives are also useful as antibiotics and as agents which increase feed-utilization efficiency.

The term "an AB0577 compomd" 1s used herein to designate antibiotic AB0577 (formula 1), an acyl ester or an alkyl ether derivative of antibiotic AS80577 or a pharmaceuti- cally acceptable salt of antibiotic AB0577 or of its acyl ester or alkyl ether derivatives.

Antibiotic ARO577 is produced by a eulture of an

A30577-producing strain of Actinomadura sp., NRRL 18236, as described herein. The antibiotic is produced under submerged aerobic conditions in a suitable culture medinm; it can be re- } covered from the culture medium by using various isolation and purification procedures understood in the art,

A culture of the ARO577-producing organism has been deposited and made part of the stock culture collection of the

Midwest Area Northern Regional Research Center, Agricultural

Research Service, '".3. Tepartment of Agriculture, 1A15 North

University Street, Peoria, Illinois 6160L, from which it is available to the public under the accession number NRRL 18236.

Taxonomic studies of this organism were carri.d out by Frederick P. Merte of the Lilly Research Laboratories. Rased on these studies, the organism belongs to the genus Actinomadura [Techevalier and lechevalier, 1970, In Prouser, H. (ed.). The

Actinomycetales. VEB Gustav Fischer Verlag, Jena, pp. 393-h05 7.

Gm

—- - —_— —7¢73

The methods recommended by the International Streplo- myces Project (ISP) for the characterization of Streptoryces species [.B. Shirling and ND, Gottlieb, "llethnds for Charac- terization of Streptomyces Species", Int. J, Syst. Bacteriol. 16(3), 213-00 (1966) 7 have been followed.

Melanoid pipment production (chromorenicity) was determined with TSP No. 1 (tryptora-yeast extract broth), 15p

No. 6 (peptone-yeast extract iron agar), and ISP Mo. 7 (tyro- sine agar).

Starch hydrolysis was determined by testing for the ’ presence of starch with iodine on ISP No. Ii (inorranic salts- starch apar) plates, l'orphology was studied using an optical lirht micro- scope. A scanning electron microscope (3il1) was used to study the spore surface ornamentation, }

IC35-NDS Centroid Color Charts, standard sample Ho, 2106 (National Bureau of Standards, 195%, "1.5. Tepartment of

Commerce, Waghington, M.C.) and the Color Harmony Manual (Lith ed., Color Standards "epartment, Container forporation of

America, Chicago, Illinois, 1958) were used to assim color names to the reverse side and aerial spore mass respectively,

The isomers of diaminopimelic acid (NAP) and the carbohydrates in hydrolysates of whole cells were established by the chromatographic methods of Becker et al, [1 Becker,

M.P, Lechevalier, N.F. Gordon, and N.A, Techevalier, "hapid / N

777° mM.fferentiation between Hocardia and Jtreptomyces by Faper : Chromatography of ‘hole-cell Hydrolysates', Appl. "icroblol, 12, Lh21-h23 (1961) 7 and of lachevalier / 1. P. lechevalier, and H. lechevalier, "Chemical Composition as a Criterion in the Classification of Aerobic Actinorycetes”, Int, J. Syst.

Bacteriol. 20, li35-Lk3 (1070) 7.

Resistance to antibiotics was measured by padding antibiotic sensitivity discs onto the surface of seeded I5P lo, 2 apar plates. o 10 Cultural Characteristics oo Culture A%D577 rrew well on both complex and defined } } media. The aerial spore rass was moderately formed, and was white. The raverse side was yellowish white to pale yellow. lo distinctive pigments wore observed. lo soluble pigments were obgerved, except for a light yellow browm pigment in several media. These cultural characteristics are riven in

Table 7, l=

27°73

Table I

Cultural Characteristics of A30577 at 30°C 18 Jays incubation

G: Abundant Ge: Vair

ISP Rr: A8.d.Y Czapek's Rt: 92.y White

No. 2 C: Trace: a Yhite Am: Fair: a ‘hite

Sp: lone tn l,y Vr Sp: tone

Gs: Tair Tomato Gs: Poor

IP Re 92,y Yhite Paste R: AB,4,Y

No. 3 Am: Fair: a White natmeal Am: Toor: a White : Sp: None Apar Sp: None

Gt: (ood G: Good

Isp Re 92.y Vhite Potato Rt A9.p.Y

Hos I Am: Good a White Carrot Am: Good: a 'hite / Sp: lone Agar Sp: lone

GG: Abundant O: Poor

T3P Rt 89.p.Y Jensen's RN: 92,y white

Nos 5 Am: Good: a ‘hite Arar Am: Poor: a “hite

Spt lone Sp: None “Oa

. TT —_—— “I°R

G: Good G: Oood 15P BR: 90.py.Y Glucose I: 89.p.Y

Noe 7 Am: lone Asparagine Am: Good: a ‘hite

Sp: lone Sp: None a

G: Good G: Trace only

No. R: A9,p.Y 0Olycerol R: - 172 Am: Good: a white Glycine Am: -

Sp: Mone Spe - ’ G: Hot grown G: Abundant

Tap Rs - Yeast Re A7.m.Y

Water Am: - lextrose Am: Fair: a White

Agar 5p: - Agar Sp: None to l.yBr

G: Abundant :

Emerson's R: B7.m.Y agar Am: Trace: a White

Sp: None to l.,yBr }

G = Growth; R = Reversey Am = Aerial mycelium; Sp = Soluble Pigment

- —_— 2173 lorphological Characteristics

Culture AN0577 produces an extensive substrate mycelium,

Voderately formed aerial hyphae produce clusters of tightly packed short chains, arranred in Rectus-flexibilis (NF) morpho-

S logy. This morphology is typical of the penns Actinomadura. The apore shape is spherical, spore slizo averapes 0.8 ym, ani the spore surface has a Mstinctive rranular appearance, The spore chain contains less than 10 spores per chain.

Physiological Characteristics / 10 Malvais of hydrolyzed whole cells indicates the pre- sence of meso-diaminopimelic acid. Madurose waa detedted in the whole cell extracts. Galactose, pglucose, mannose and ribose vere also detected, The cell wall is type JIT and the supar pattern is type DB. Galactose is not atypical of type I sugar pattern.

Culture AAD577 utilized the following carbohydrates: adonitol, N and Learabinose, cellobiose, duleitol, ethanol, j-erythritol, n-fructose,D-galactose, glucose, plycerol, rly=- cogen, i-inositol, N-mannitol, D-mannose, N-relizitose, N- melibiose, l-rharmose, D-ribose, sucrose, N-trehalose N-xylose and sodium butyrate, It was nnable to utilize: cellulose, dextrin, inulin, D-lactose, N-maltose, L-methyl-D-glucosie,

Neraffinose, salicin, sorbitol, l-sorbose, and xylitol. Control plates with no carbohydrates supported a minimal growth, wll

Culture AB0577 grew in a temperature range of 15-45°C, optimum growth appeared to be between 30 and 37°C. AB0577 tole- rated up to 4% NaCl, produced catalase and reduced nitrates. It did not hydrolyze starch.

AB0577 was resistant tot bacitracin 10 units, cepha-

Jjothin 30 ug, lincomycin 2 ug, oleandomycin 15 ug, penicillin

G 10 units, rifempin 5 ug, and tetracycline 30 ug, It was sensitive to: gentamicin 10 ug, neomycin 30 ug, streptomycin ug, tobramycin 10 ug and vancomycin 30 ug. 10 The chemotaxcnomic properties and the general cultural and morphological characteristics of ABO577 support the assign- ment of this strain to the genus Actinomadura. No attempt to determine the species was undertaken. Therefore this strain is classified as Actinomadura sp. A80577.

As is the case with other organisms, the characteristics of the A80577-producing culture Actinomadura sp., NRRL 18236, are subject to variatifm. Recombinants, mutants or variants of the strain may be obtained by methods known in the art. For example, mutants can be obtained by treatment with various known physical and chemical mutagens such as ultraviolet light,

X-rays, gamma rays and chemicals such as N-methyl-N%-nitro-N- nitrosoguanidine. All natural and induced variants, mutants and recombinants of this Actinomadura sp. strain which retain the characteristic of A80577 production are part of this invention.

The culture medium used to grow Actinomadura sp.,

NRRL 18236, can be any one of a mumber of media. For economy im

-_—_—— — —- _— 27°73 production, optimal yield, 2nd eage of product isnlation, hou. ever, certain cnlture media are preferred, For example, a pro- ferred carbohydrate source in larpn-secale fermentatinn ia rlncose, althourh blackstrap molasses, starch and the like ean / S also be nsad,

A preferred nitrosen source is enzymehydrolyzed canein, although other nitrogen sources shoul: also be useful,

Among the nutrient inorganic salts which may advanta- reously be incorporated in the enlture media are the customary soluble salts capable of yleldinm zine, sediw., rapnesium, rnlelum, armonium, chloride, carbonate, sulfate, nitrate and like ions.

F.sgsential 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 substituents of the medi'm in amounts sufficient to meat the prowth requirements of the organism. If foaming is a problem, small amownts (1.e, 0.2m1/L.) of an antifoam agent such as polypropylene glycol mar he added to large scale fermentation media 1f neaded,

For production of gubatniinl quantities of antibiotic

A%0577, submerged aerobic fermentation in tanks is preferred,

Small quantities of A577 may be obtained by shake-flask culture.

Because of the time lag in antibiotic production commonly associated with inoculation of larre tanks with the spore form of the organisn, it is preferable to nse a vegetative inoculrm,

The vegetative inoculum is prepared by inoculating a small volume of culture medium with the spore form or mycelial frag- ments of the organism to obtain a fresh, actively growing culture of the organism. The vegetative inoculum is then transferrcd to a larger tank, The vegetative inoculum medium can be the same as that used for larper fermentations, put 6ther madia are also suitable,

A%0577 4s produced by Actinomadura sp., when grown at temperatures between about 260 and about 37°C. A good tem- perature for 210377 production appears to be about 30°C,

As is customary in submerged aerobic culture pro- cesses, sterile air is blown into the vessel from the bottom while the medium is stirred with ronventional tinrbine $1ellinrs,

In general, the aeration rate and apitation rate should be sufficient to maintain the level of dissolved oxygen at or above 1307 of saturation.

Production of antibiotic ABO577 can be followed during the fermentation by testing samples of the broth for antibiotic activity against organisms known to be senéitive to antibiotic. One assay organism ngeful in testing AS30577 4g Pacillus subtilis ATCC 6633. The bioassay is convenicntly performed by the apar-well plate test.

Following its production under submerged aerobic fermentation conditions, A13577 can be recovered from the ==

— —_— 27°73 / fermentation medium by methods used in the fermentation art.

The antibiotic activity produced during fermentation of the

ARO577-producing organism occurs both in the mycelia and the broth, Maximum recovery of ARO577 is accomplished, therefore, by initially filtering the medium to separate the broth from the mycelial mass, The filtered broth and the mycelial mass can then be purified separately to give their respective portion of AB0577. A variety of techniques may be used in this puvrifi- cation.

A preferred technique for purification of the filtered broth involves adjusting it to a pl of about 9 and extracting with a suitable solvent such as, for example, ethyl acetate.

The extracting solvent can then be evaporated under vacuum to glve the broth portion of A80577.

A preferred method of purifying the mycelial mass is to extract the separated mycelial filter cake with a suit- able solvent such as, for example, acetone. The extracting solvent is then evaporated under vacuum to give a concentrated aqueous solution, This aqueous solution is then adjusted to a pH of about 9 and 1s extracted with a suitable solvent such as, for example, ethyl acetate. The extracting solvent 1s then concentrated wnder vacuum to give the mycelial portion of

AROST7.

The broth and mycelial portions of the A%0577 complex are further purified by similar procedures. A preferred procedure involves silica pel chromatography.

Separation of antibiotic AR0577 can be followed by thin-layer chromatography (TLC) or high performance liquid chromatography (MPIC). Convenient silica rel TLC solvent systems are ethyl acetate, ethyl acetate acetonitrile ammonium hydro=- xide and tolnene:acetonitrile:acetic acid. Ireferred silica rol solvent sysicms are ethyl acetate, ethyl acetatesacetonitrile: ammonia (9039:1) and toluene:acetonitrile:acetle ncid (h0:59:1),

Polyamide plates also may be conveniently used with an acetone: water:ammonia solvent system, preferably in the ratio 30:69:1.

The antibiotic can be detected by bioautography using, for example, Facillus subtilis or by other methods such as, for example, vanillin-sulfuric acid spray reagent,

Alternatively, the culture solids, inelnding medium constituents and mycelium can be uscd without extractior or separation, but preferably after removal of water, as a source of AB0577. For example, after production of AS80577, the whole fermentation broth can be dried by lyophilization, by drun- drying, or by azeotropic distillation and drying. The dried "broth is then mixed directly into feed premix.

The salts of AROS77 and of its derivatives are usefnl for separating and purifying the antibiotics. The pharmiceuti- cally-acceptable salta are particularly useful, Examples of salts are the alkali-metal and alkaline-earth-netal salts of

AB0577 and of its derivatives,

—— _——— 21 °/ 3

Representative and suitable alkali-metal and alkaline- carth metal salts of A12577 include the sodium, potassium, 1ithium, cesium, rubidium, barium, caleimm and magnesium salts,

Tt 1s well known in the veterinary pharmaceutical art

S that the form of an antibiotic is not ordinarily of preat sirni- ficance when treating an animal with the antibiotic. In most cases, conditions within the animal change the drug to 8 form other than that in which 1t was administered. The salt form in which it may be administered is, therefore, not of preat sieni- ficance. The salt form may, however, be chosen for reasons of economy, convenience, and toxicity.

The alkali-metal and alkaline-earth-metal cationic

Co salts of AR0S77 are prepared according to procedures commonly used for the preparation of cationic salts. For example, the free acid form of A%0577 is dissolved in a suitable solvent guch as acetone; about 1/2 volume of -water 4s added and this solution is adjusted to a pil of about 9 to 10 with the hase of the desired cationic salt (e.g. Nal, vn), The salt thus formed can be isolated by routine methods, such as filtration 29 or evaporation of the solvent,

A preferred method of forming salts is to dissolve

ABOS77 (acid form) in a solvent such as tetrahydrofurany add an equal volume of waters; adjust the mixture to pil 10 with the corresponding cationic base (e.g. NaOH, KOH, etc.); and extract with a water immiscible solvent such as diethyl ether oo or ethyl acetate. The separate: organic phase 1s washed with water and concentrated to dryness. The residue is lyophilized fron dioxane. The salt can be crystallized from an appropriate solvent, such ag acetone.

The alkyl ether derivatives of ARDS77 are those com= pounds wherein one or more of the hydroxyl groups has been re- placed by a YR group wherein?

Y represents 0 or Sg and

I represents (1=Cp=alkyl,

C1=C},=alkoxy=Cp=Co-alkyl, 01-C),~a1koxycarbonyl-Co=Cy-alkyl, amino-Cp-Cg-alkyl, mercapto-Co=Cg=-alkyl, hydroxyalkyl, haloalkyl, or (Rt), ~pheny1 (Cli) } wherein R' represents Cq=C),-alkyl, Cq~C),-alkoxy, or hydroxy rt represents 0-23 and n represents 0-3.

The term "alkyl" means a Cy to Cq straight or branched chain hydrocarbon, preferably a Cqto Cy hydrocarbon, €.F., methyl, ethyl, propyl, isopropyl, n-butyl, etc.

The term "alkoxy" means a Cy to Cq lower alkyl group having an oxygen function substituted therein, such as methoxy,

Ie = ethoxy, propoxy and the like.

The term "hydroxyalkyl" refers either to a monohydroxy-

Cy=Cg-alkyl moiety or, when Y is 0, to the 2, 3=ihydroxyprop-l- yl moiety.

The term "haloalkyl" refers to a Cp-Cg-alkyl moiety v having from one to three halogen substituents, selected fron the group consisting of bromine, chlorine, and fluorine. ‘hen the alkyl moiety is dihalo- or trihnlo-substituted, the halo- substituents must be the same halogen moiety.

Preferred AB0577 ether -lerivatives are those compounds wherein Y represents 0 and R represents Cq-"g-nlkyl, The ethor derivatives are prepara: by reacting A30577, or a salt thereof, with a corresponding primary alcohol or thiol,

With some of the starting alcohols or thiols it may be necessary to add an acid catalyst to the reaction, Snitohle catalysts include hydrochloric acid, ‘sulfuric acid, perchloric acid, methanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, selenium dioxide, and boron trifluoride.

A solvent such as, for example, water, acetone, ben=- zene, ether, tetrahydrofuran, or dioxane may bo added to faci- 1itate the reaction, Reactions generally occur at room terpe~ rature, although higher temperatures may be used,

The acyl ester derivatives of A%0577 are those cor.- pounds wherein one or more of the hydroxyl groups has been replaced by a radical of the formula

0

I

Ry=C-0~ wherein Ry is Cy to Cg-alkyl or hydrogen.

A10577 acyl ester derivatives are preoa&red by treating

S ABOS77 with a corresponding acid anhydride or acid chloride.

Esterification occurs at one of the AJUST7 hydroxyl groups. Such esters are typically prepared by reacting AT0577 with, for example, the corresponding acid anhydride at room temperatwvre,

Although ordinary reaction work-up procedures are sometimes sufficient, additional prrification may be required to obtain the compounds of this invention. Such purification may be accomplished by well-known methods, such as, for example, colum chromatography, thin-layer chromatography, fractional crystallization and the like.

The ARD577 compounds inhibit the growth of bacteria which are pathogenic to animal life, For example, Table ITI shows the minimal inhibitory concentration (MIC) at which

ABOS77 inhibits certain organisms. The if1C's in Table III were determine by conventional agar-dilution assays.

. 9 72

Table 111: Antibacterinl ‘ctlivity of 10577 (a aalt)

Test Orranism ne (Onep/rid) eo ————————————

Staphylococcus aureus X1.1 045 " “7 0.5 " " Yh" 2.5 u " 5170 0,5

Staphylococcus epidermidis "P11 0.5 " " 222 0.5

Streptococcus pyogenes C203 0.125 " pneumoniae Farkl 0.125 " " X66 0.5 " " 2701 0.5

Haemophilus influenzae 32-128

Other Cram-nepative bacteria tested > 128

Veterinary Organisms

Staphylococcus 8p. ¢ 0.05

Streptococcus sp. ¢ 0,05

Pasteurella multocida 1.56

Pasteurella hemolytica 5 SO

Bordetella bronchiseptica > 50

Mycoplasma gallisepticum 0,79

Mycoplasma hyopneumoniae 1.56

Escherichia coli >» 50

Salmonella typhimurium > 50

The ARDS77 compounds also are active apainst anaerobic bacteria. Table IV ghows the MIC's at which ARDS77 inhibits vori- ous anaerobic bacteria, as determined by ataniard apar-dilntion assay. Fnd points were read after 2hi-hour incubation.

Table IV: Susceptibility of Anaerobic Bacterial Tsolates to 730577 (Na salt)

Ce ——————————————————————

Anaerobic Bacteria ne (mep/mh) ee

Clostridium difficile 299L <0.5

Clostridium perfringens 91 < 0,5

Clostridium septicum 1127 {045 fubacteriun cercfaciens 12135 < 045

Peptococcus asaccharolyticus 1302 40.5

Peptococcus prevoti 1281 < 7.5

Peptostreptococcus anaerobius h23 {2.5

Peptostreptococcus intermedius 162 < 0.5

Propionibacterium acnes 79 < 0.5

Bacteroides fragilis 111 >128

Bacteroides fragilis 1877 >128

Bacteroides fragilis 1936B >128

Nacteroides thetaiotaomicron 1L33 lL

Racteroldes melaninopenicus 1956/23 1.0

Bacteroides melaninogenicus 2736 16

Bacteroides vulpatis 1211 h ge

Anaerobic Bacteria 116 (meg/ml)

Bacteroides corrodens 137h >121

Fusobacterium symbiosum 1170 0,5

Fusobacterium necrophorum A0ShA {0.5

The acute toxicity of A80577 in mice, when administered by intraperitaneal injection and expressed as Les was 52,3 mp/ ge oo

Another important property of the A%0577 compomndes is the ability to improve feed-utilization efficiency in animals,

For example, the ABOS77 compownds improve feed-utilization efficiency in ruminants which have a daveloped rumen function,

The efficiency of feed use can he monitored by observing the production and concentration of propionate com- pounds in the rumen using the method described by Arthur P, Raun in U.S. Patent 3,794,732 (see especially Example 5), Table V shows the effect of compound AS80577 on ruminant feed-utilization efficiency, Table VA. shows the relationship between dosare of

AB0577 and propionate production, Table VB, shows the ratio of volatile-fatty-acid (VFA) concentrations in AB0577-treated flasks to concentrations in control flasks in this test. 2 J

Table V: Sffect of A8D577 (Ma Salt) on Ruminant Feed-'"tilization nfficiency

A, 'roplonate

Froduetion, mM/d g “osape, ppm Observations U'ean Std. ev, 0 2€ 0.6 1.4 0. 1h 1.2 1.7 0.2 1 11.9 1.) 1,0 1h 22.2 3. 5.0 1h 21.0 2.7

B.

Ratio of Treated to Control Values

Nosage Molar 4 Molar 7 tolar ~ Total wrA i5 meg/mL Propionate Acetate Butyrate mit/L 1.0 1.532 0.045 0.75h 0,858 2.5 1.159 0.751 0.827 0,823

S.0 1,562 0,799 0.839 0.773 10.0 1.709 0.772 0.729 0.876 1503 two-tailed t-test; significant at PgR. 0; C3>99 percent upper confidence limit i] Fp

2747 g

Tha ANOGT7 compounds are typically effective ir increasing propionate and, thereby, improve the ef ficiency of feed utilization when administered to ruminants orally at rates of from about 0,01 mg/kg/day to about 1.0 mp/kp/day. Preferable rates of administration are Crom about 0,06 ne/kg/day to abont 0.35 mp/kg/ day. A preferred method of administration is tn mix the compound with the animals! feed, Feed compositions adapted to increase feed utilization in ruminant animals typically comprise a feed ration and from 0,5 to 50 rrams of an AROSTT compound per ton of feed, preferably 2 to 15 rrams per ton.

As described supra, ARDS77 compounds are active apainst anaerobic bacteria, incluwiing Clostridium perfringens. AROS TT compounds should, therefore, be beneficial in the treatment of (which includes prevention of ) enteritis in chickens, swine, cattle aad sheep. R30577 coupourrls shold also ve useful in the treatment of cnterotoxemia in ruminants.

Ihe ABOS77 compounds can he administered to animals orally or parenterally. The moat practical way to administer the ARNST7 compounds is by formulation into the feed supply. A variety of feeds, inelnding the common ry feeds, liquid feeds, and pelletod feeds, may be vwsed. Although the preferred method of administration is by mixing 1t with the animals! feed, it can also be a‘ministered in other ways, for example, tablets, drenches, boluses, or capsules, hach individnal doanpe mnit should contain a quantity of AR0577 compornd directly related «25 — — \gro ORIGINAL ))

Loan

: _—_—— 21073 to the proper dnily cose for the amimad to be treated,

The methods of farmalating rugs into animl feeds are well knowm. A preferred mathad is to make a concentrated rap premix which in turn ie used to prepare meticated feeds. Typical prenizes may contain from about 1 to abont. 200 rrama of rng par pownd of premix, Promixes mov be either liquid or solid prepa- rations.

The final formulation of feeds for animals will Aepend upon the amount of drug to be administered, The common methods of formulating, mixing, nnd pelleting feeds may bo used to pro- pare feeds containing an ABO577 compound.

The AR0577 compounds may be formulated for parenteral a‘ministration by methods recognized in the veterinary phnrma- ceutical art. Tffective injectable compositions containing the oF 15 AR0S77 compounds may be in either suspension or solution form,

In the solution form, the A30577 compomnd is dissolved In a physiologirally acceptable carrier. Such carriers comprise a suitable solvent, preservatives such an benzyl aleohol, if needed, and buffers. V'sefnl solvents include, for example, alcohols, glycols, or inert oils such as veretable oils or highly refined mineral oils.

Injectable suspension compositions are prepared using a nonsolvent for the compound with adjuvants, as a earrier, The nonsolvent can be, for example, water or a glycol such as poly- 2s ethylene glycol. “26m - ORIGINAL d

VL ee -_—— 217

Suitable physiologically-acceptable adjuvants are necessary to keep the compoimd suspended in anspension compond- tions. The adjuvants may ha chosen from among thickeners anch as carboxymethyleellulose, polyvinylpyrrolidone, pclatin, and the alpinates. any surfactants are also useful, for engpending the compounds, lecithin, alkylphenol polyethylene oxida adducts, naphthalenesulfonates, alkylhenzenesnlfonates, and thn palyovy- ethylene sorbitan esters are naeful saapendine agants in 1iauid nonsolvents. l'any substances which affect the hydrophilieity, density, and surface tension of the liquid nonsnlvent can assist in making injectable suspensions in individnal cases, For axample, silicone antifoams, glycols, sorbitol, and sumars can be nseful suspending agents,

In order to illustrate more fully the operaticn of this invention, the following examples are provided: 27= fi ORIGINAL 9 — .

Lxarple 1

Treparation of Antibiotic ABSTY

Ising Actinomadura sp.,

A. Shake-flask Fermentation of Actinomadura sp., co The culture Actinomadura sp., MRI 11235, either as a lyophilized pellet or as a suspension maintained in liquid nitrogen, is used to inoculate a vegetative redinm having the following composition:

Vegetative or Sced ledium

Ingredient Amount (g/L)

Nlucose 10.0

Soluble starch ’ 20.0

FEnzyme-hydrolyred casein 5.0

Yeast extract 5.0

Cato, 1.0 "elonized water N.8. 1 liter 11-7 Amine A, Humko~theffield Chemieal, . 20 Horwich, MJ. «28a ‘BAD ORIGINAL 9

F073

Slants or plates are prepared hy addins 2,57 apnar to the seed medium, The inoculated slant 1s incubated at n°c, for from about 10 to abont 1h Arye, The mature slant culture is scraped with a sterile tool to loosen the spores and removn and macerate the mycelial mt, About one-fourth of the lnosnned spores and culture growth thns obtained is used to inoculate 50 ml of a first-stape seed medium,

The inoculated [irst-stape medium is Inecnbated in a 250-mL Erlenmeyer flask at 20°C, for about 72 hours on a shaker orbiting in a two-inch (5.0% cm) circle at 250 rpm, . This incubated first-stage medium (1,20 mL) 1s nned to inoculate 50 ml of a production medium having the follouing composition:

Ingredient Amount (g/1)

Glucose 25,0

Plackstrap molasses 15.0

Yeast extract 5.0 “nzyme-hydrolyegnd caseinr 3.0

MESO), (anhydrous) 1.0

Catnq 7.0)

Tap water 9.8. 1 liter mMNeZ Amine A, «?0uw 5

BAD ORIGINAL GH ro TE

The Tranednind © rotabiog ce ba fa inesbnted Taos 250ml, wlneronth Nelo over (Taal pf ne, tar 9 Lo 10 dares on a ghaler orbiting in a teeoinen cirele at 250 rpm,

RB. Tank Fermentation of Actinomadura sp.,

UU —

In order to provide a large volume of inoculvm, 10 nl of incubated first-stage medium, prepared as described in Section

A, 4s used to inoculate h0O ml of a sccond-stage growth medium having the same composition as that of the first-stape mediim, - This second~stare vegetative medimn is incubated in a two-1ilay wide-mouth rlenmeyer flask for about Lf hours at 20°C, on a ghaker orbiting in a two-inch circle at 250 rpm.

Tncubated second-stage vegetative medium (400 ml) thus propared is used to inoculated 100 liters of sterile production medium, prepared as deseribed in Section A except that 1-200 (0.1 m1/1) and Sag L71 (0,2 g/1.) antifoam agents are added, The inoculated production medium is allowed to ferment in a 165- - liter stirred fermentation tank for li to & days at a temperature of 30°C. The airflow (0.5-0.6 v/v/m) in the stirred vessel (200- 250 rpn) is adjusted to maintain a dissolved oxygen level shove 30% of air saturation, - a ano ORIGINAL 9 ee 4

277 3

Example 2

Isolation of A%0577 Sodium Salt

Combined fermentation broth (187 1) from two 100 L fermentations was filtered with the aid of 3% Hyflo Supercel.

The mycelial filter cake was extracted twice with 0 I acetone.

The acetone extracts were combined and concentrated in vacuo to remove the acetone. The concentrate (20 1.) was combined with the broth filtrate (162 L), the solution was adjusted to pH 0,0 with 50% NaOil and extracted with 125 L ethyl acetate, The ethyl acetate extract was concentrated in vacuo to a residue which crystallized upon standing at 59¢, The crystals were washed sequentially with pentane, acetonitrile and diethyl ether and dried in vacuo. A yield of 36.4 grams of white crystalline A0577 sodium salt (m.p. 275-2780C) was obtained.

The crystals were recrystallized by dissolving 1,0 in 100 ml acetone, adding 100 ml i,0 and allowing to stand at 50¢ for 72 hours to erystallize. Crystallization was completed with the further addition of 100 ml of wateriacetone (3:1). The crystals were filtered off and dried to yield 788 mg (m.p. 276- 278°C).

Further recovery of ANOS77 sodium salt from the com- bined above washes and mother liquor was achieved by concen- trating the solution to dryness, dissolving in 250 ml toluene and applying to a colum containing 2 I, of silica pel {Grace ik

Grade 62) packed in toluene. The column was washed w ith 10 1, toluene and then developed sequentially with 10 L toluene ethanol (98:2) and 10 L toluene:ethanol (96:1) collecting 1 L fractions.

The elution was followed by TIC and binassay using Bacillus subtilis. Fractions 13-18 containing AB0577 were combinad and no concentrated in vacuo to a residue which was washed with dinthyl ether and dried to yield 2.23 5 of amorphous AB30577 Na.

Example 3

Preparation of A30577 Free Acid

A80577 sodium salt (1 g) was dissolved in 100 ml ace- tone and 0.1 TIC1 (100 ml) ms added. The solution was stirred for 15 minutes. The solutioh was extracted twice with ethyl acetate (100 mL each). The ethyl acetate extracts were combined and concentrated in vacuo to an nlly residue, The residue vas dissolved in dioxane (50 mL) and freeze-dried to yleld 0,9 g of

A30577 acid.

Example ly

Preparation of AB0577 Potassium Salt

AB0577 (acid form, 100 mg) was dissolved in tetra- hydrofuran (20 mL). Water (3 ml) and 2N 40H (6 mL) were added and the mixture was stirred for 15 minutes, Vater (30 ml) was “Rw

77 9 yr Y. ry dded and the solution vas extracted tidee with diethyl etlry nae ) ae »3 (£0 ml each). The combined extracts were evaparstied sndey waenum iavan~ 1 freon. ried

T 19a diesnlved in Jdiavann and to dryness, The resi-ne uo le v - TA . to yield 100 mg of ARIST as the potassiom aali(n,n, 270-2720) -33 aro

BAD ORIGINAL d

Claims (2)

21°73 CLANS

1. Antibiotic ANO577 which has the Formula 1 4 Et NS 3 4 " CYA % = (¥ NG " Ma Hl H / I HO "\ Lor i 1 e sn con HOHLC N\ (0c, n HC + ~ ~ AN n+ 0s O ~0 or a pharmaceutically acceptable salt thereof.

2. A compound of Claim 1 which 4s the sodium salt of AROS77, «Y= BAD ORIGINAL 9