Derivatives of A54145 cyclic peptides

SUMMARY OF THE INVENTION
This invention relates to derivatives of A54145 cyclic peptides, which can be represented by formula 1: ##STR3## wherein: R is C.sub.8 -C.sub.18 -alkyl; optionally substituted C.sub.8 -C.sub.18 -alkanoyl or C.sub.8 -C.sub.18 -alkenoyl;
(Lys-R.sup.1) represents --NH(CH.sub.2).sub.4 CH(NHR.sup.1)CO--;
(R,R.sup.2)-Trp represents a group of formula ##STR4## R.sup.1 is hydrogen or an amino-protecting group; and R.sup.2 is hydrogen; or
R and R.sup.2, taken together, represent a C.sub.8 -C.sub.18 -alkylidene group;
X is Ile or Val; and
Y is Glu or 3-MG;
provided that: 1) when R.sup.1 =H, X=Ile and Y=Glu or 3-MG, R cannot be 8-methylnonanoyl, 8-methyldecanoyl or n-decanoyl; 2) when R.sup.1 =H, X=Val and Y=3-MG, R cannot be 8-methyldecanoyl; 3) when R.sup.1 =H, X=Val and Y=Glu, R cannot be 8-methylnonanoyl; and 4) when R.sup.1 is an amino protecting group, R cannot be 8-methylnonanoyl, 8-methyldecanoyl or n-decanoyl; and salts of these derivatives.
The formula 1 compounds wherein R.sup.2 is hydrogen (1a compounds) are antibacterial agents and improve growth performance in animals such as poultry. The formula 1 compounds wherein R.sup.2 is other than hydrogen (1b compounds) are useful intermediates.
Feed compositions for improving growth performance in animals containing the 1a compounds and methods for increasing growth in animals with these compositions are also part of this invention.
DETAILED DESCRIPTION OF THE INVENTION
In this specification, the following abbreviations, most of which are commonly known in the art, are used:
______________________________________Abbreviation Term______________________________________Ala: AlanineAsn: Asparagine(HO) Asn: .beta.-Hydroxy-asparagineAsp: Aspartic acid(MeO) Asp: .beta.-Methoxy-aspartic acidGlu: Glutamic acidGly: GlycineIle: IsoleucineLys: LysineThr: ThreonineTrp: TryptophanSar: SarcosineVal: Valine3-MG: 3-Methylglutamic acidHPLC: High performance liquid chromatography.sup.1 H NMR: Proton nuclear magnetic resonanceTLC: Thin-layer chromatographyIR: InfraredUV: UltravioletFABMS: Fast-atom-bombardment mass spectrometry______________________________________
The abbreviation "R.sup.a -Trp" is used to designate a group of the following structure: ##STR5##
The abbreviation "R.sup.b -Trp" is used to designate a group of the following structure: ##STR6##
The A54145 cyclic peptide derivatives of this invention are shown in formula 1. The starting materials from which the formula 1 compounds are prepared, the A54145 antibiotics, are lipopeptides. Each contains a cyclic peptide unit with a fatty acid side chain. LaVerne D. Boeck, David S. Fukuda, Jon S. Mynderse, Marvin M. Hoehn, Ralph E. Kastner and Harold R. Papiska describe antibiotic A54145, comprising major components A and B: and minor components B, C, D, E, F and A.sub.1, and its production by Streptomyces fradiae strains NRRL 18158, NRRL 18159 and NRRL 18160 in their copending application entitled A54145 ANTIBIOTICS AND PROCESS FOR THEIR PRODUCTION, Ser. No. 07/179,773, now U.S. Pat. No. 4,994,270, filed this same day. The A54145 antibiotics inhibit the growth of Gram-positive bacteria and promote growth in animals.
The A54145 components are believed to have general structure 2 and specific structures 2a-2h
______________________________________ ##STR7## 2Structure Component X Y R.sup.a______________________________________2a A Ile Glu 8-methylnonanoyl2b B Ile 3-MG n-decanoyl2c C Val 3-MG 8-methyldecanoyl2d D Ile Glu "2e E Ile 3-MG "2f F Val Glu 8-methylnonanoyl2g A.sub.1 Ile Glu n-decanoyl2h B.sub.1 Ile 3-MG 8-methylnonanoyl______________________________________
In our copending application entitled "A54145 CYCLIC PEPTIDES", Ser. No. 07/179,928, filed herewith this same day, we describe our discovery that the fatty acid side chains of the A54145 antibiotics can be removed enzymatically to give the cyclic peptide unit ("nucleus"). For convenience, this cyclic peptide is called an A54145 nucleus. Thus far, we have obtained four unique A54145 nuclei. These nuclei have been designated the A54145A, A54145B, A54145C and A54145F nuclei.
The four A54145 nuclei appear to have the common structure 3 and individual structures 3a-3d:
______________________________________ ##STR8## 3Structure Nucleus X Y______________________________________3a A Ile Glu3b B Ile 3-MG3c C Val 3-MG3d F Val Glu______________________________________
The A54145 nuclei and blocked A54145 components and nuclei, which are called A54145 cyclic peptides, can be represented by formula 4: ##STR9## wherein: R.sup.b is selected from the group consisting of hydrogen, an amino-protecting group, 8-methylnonanoyl, 8-methyldecanoyl and n-decanoyl;
(Lys-R.sup.1) represents --NH(CH.sub.2).sub.4 CH(NHR.sup.1)CO--;
R.sup.1 is hydrogen or an amino-protecting group;
X is Ile or Val; and
Y is Glu or 3-MG;
The formula 4 compounds are useful intermediates to the formula 1 compounds of this invention.
The compounds of formula 4 wherein R.sup.b is other than hydrogen or an amino-protecting group and R.sup.1 is an amino-protecting group (4a compounds) are called "blocked" A54145 components.
A formula 4 compound wherein R.sup.b and R.sup.1 differ but are selected from hydrogen or an amino-protecting group (a formula 4b compound) is called a "blocked nucleus".
A formula 4 compound wherein R.sup.b and R.sup.1 are both hydrogen (a formula 4c compound) is called a "nucleus".
The present invention relates to novel compounds derived by acylating an A54145 nucleus (a 4c compound) or a blocked A54145 nucleus (a 4b compound). These novel compounds have the chemical structure depicted in formula 1.
The term "C.sub.8 -C.sub.18 -alkylidenyl" refers to a group of the formula ##STR10## wherein R.sup.3 and R.sup.4 are hydrogen or an alkyl group of from 7 to 17 carbon atoms, provided that one of R.sup.3 and R.sup.4 must be other than hydrogen and further provided that the sum of the carbon atoms in R.sup.3 and R.sup.4 must be no greater than 17. Those compounds wherein R and R.sup.2 represent C.sub.8 -C.sub.18 -alkylidenyl are known as Schiff's bases.
The term "C.sub.8 -C.sub.18 -alkyl" refers to a univalent saturated, straight- or branched-chain alkyl group containing from 8 to 18 carbon atoms. Those compounds wherein R is C.sub.8 -C.sub.18 -alkyl, referred to herein as "reduced Schiff's bases", are prepared by reduction of the corresponding compounds where R and R.sup.2 represent a C.sub.8 .sub.]C.sub.18 -alkylidenyl group.
The term "optionally substituted C.sub.8 -C.sub.18 -alkanoyl and C.sub.8 -C.sub.18 -alkenoyl" refer to acyl groups derived from carboxylic acids containing from 8 to 18 carbon atoms. When the R group is alkanoyl, the alkyl portion is a univalent, saturated, straight-chain or branched-chain hydrocarbon radical which can optionally bear one hydroxyl, carboxyl, or C.sub.1 -C.sub.8 -alkoxy group or from one to three halo substitutents selected from chlorine, bromine, and fluorine. When R is alkenoyl, the alkenyl portion is a univalent, unsaturated, straight-chain or branched-chain hydrocarbon radical containing not more than three double bonds. The double bond portion(s) of the unsaturated hydrocarbon chain may be either in the cis or trans configuration.
The term "amino-protecting group" refers to an art-recognized amino-protecting group which is compatible with the other functional groups in the A54145 molecule. Preferred amino-protecting groups are those which can be readily removed subsequently. Examples of suitable protecting groups can be found in "Protective Groups in Organic Synthesis" by Theodora W. Greene, John Wiley and Sons, N.Y. 1981, Chapter 7. Especially preferable amino-protecting groups are the tert-butoxycarbonyl (t-BOC) and benzyloxycarbonyl groups.
Subgeneric aspects of the invention include the following formula 1 compounds:
(a) The compounds wherein R is alkanoyl of the formula ##STR11## wherein n is an integer from 6 to 14, provided that, when X is Ile, n cannot be 8.
(b) The (a) compounds wherein n is 6 to 10;
(c) The compounds wherein R is alkanoyl of the formula ##STR12## wherein n and m are each, independently, an integer from 0 to 14, provided that 1) n+m must be no less than 4 and no greater than 14; and 2) m cannot be 6 when: a) n=0 and either (i) X=Ile or (ii) X=Val and Y=Glu; or b) n=1 and either (i) X=Ile or (ii) X=Val and Y=3-MG.
(d) The compounds wherein R is cis or trans alkenyl of the formula ##STR13## wherein n and m are each, independently, an integer from 0 to 14, provided that n+m must be no less than 4 and no greater than 14;
(e) The compounds wherein R is cis or trans alkenyl of the formula ##STR14## wherein n is an integer of from 5 to 15; (f) The compounds where R is alkyl of the formula CH.sub.3 (CH.sub.2).sub.n -- and n is an integer from 7 to 12; and
(g) The (a) compounds wherein R is: ##STR15## (h) the compounds where R and R.sub.2 represent:
CH.sub.3 (CH.sub.2).sub.10 CH.dbd.
CH.sub.3 (CH.sub.2).sub.12 CH.dbd.
CH.sub.3 (CH.sub.2).sub.8 CH.dbd.
Table I lists illustrative formula 1a compounds of this invention.
TABLE I______________________________________Formula 1a Derivatives.sup.aCompoundNo. X Y R R.sup.1______________________________________ 1 Ile Glu n-undecanoyl t-BOC 2 Ile Glu n-undecanoyl H 3 Ile Glu n-dodecanoyl t-BOC 4 Ile Glu n-dodecanoyl H 5 Ile Glu n-nonanoyl t-BOC 6 Ile Glu n-nonanoyl H 7 Ile Glu n-undec-10-enoyl t-BOC 8 Ile Glu n-undec-10-enoyl H 9 Val Glu n-decanoyl t-BOC10 Val Glu n-decanoyl H12 Val Glu 8-methyldecanoyl H14 Val 3-MG n-decanoyl H15 Ile 3-MG n-tetradecanoyl t-BOC16 Ile 3-MG n-tetradecanoyl H17 Val 3-MG n-octanoyl t-BOC18 Val 3-MG n-octanoyl H19 Ile Glu decyl H20 Ile 3-MG n-undec-10-enoyl t-BOC21 Val Glu 8-ethyldecanoyl H22 Val Glu 8-methoxy-decanoyl H23 Ile Glu 7-methyloctanoyl t-BOC24 Ile Glu 7-methyloctanoyl H25 Ile Glu n-(tetradecanoyl) t-BOC26 Ile Glu n-(tetradecanoyl) H27 Val 3-MG 8-methylnonanoyl H28 Ile 3-MG n-nonanoyl H29 Ile 3-MG n-undecanoyl H30 Ile 3-MG n-dodecanoyl H______________________________________ .sup.a R.sup.2 = H
The formula 1 compounds have both carboxyl and amino groups which can form salts such as alkali-metal, alkaline-earth-metal, amine and acid addition salts.
Partial, mixed and complete salts are, therefore, contemplated as part of this invention. Such salts are useful, for example, for separating and purifying the compounds.
Representative alkali-metal and alkaline-earth-metal salts of the formula 1 compounds include the sodium, potassium, lithium, cesium, rubidium, barium, calcium and magnesium salts.
The alkali-metal and alkaline-earth-metal cationic salts of the formula 1 compounds are prepared according to procedures commonly used for the preparation of cationic salts. For example, the free acid form of the compound is dissolved in a suitable solvent such as warm methanol or ethanol; a solution containing the stoichiometric quantity of the desired inorganic base in aqueous methanol is added to this solution. The salt thus formed can be isolated by routine methods, such as filtration or evaporation of the solvent.
Suitable amine salts of the formula 1 compounds include the ammonium and the primary, secondary, and tertiary C.sub.1 -C.sub.4 -alkylammonium and hydroxy-C.sub.2 -C.sub.4 -alkyl-ammonium salts. Illustrative amine salts include those formed by reaction of a formula 1 compound with ammonium hydroxide, methylamine, sec-butylamine, isopropylamine, diethylamine, di-isopropylamine, ethanolamine, triethylamine, 3-amino-1-propanol and the like.
The salts 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 compound in a suitable solvent such as acetone; the solvent and excess amine can be removed by evaporation.
Representative and suitable acid-addition salts of the formula 1 compounds include those salts formed by standard reaction with both organic and inorganic acids such as, for example, hydrochloric, sulfuric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, palmitic, cholic, pamoic, mucic, D-glutamic, d-camphoric, glutaric, glycolic, phthalic, tartaric, lauric, stearic, salicyclic, methanesulfonic, benzenesulfonic, sorbic, picric, benzoic, cinnamic and like acids.
Pharmaceutically acceptable salts are especially useful. "Pharmaceutically acceptable" salts are those in which the toxicity of the compound as a whole toward warm-blooded animals is not increased relative to the non-salt form. Pharmaceutically acceptable alkali-metal, alkaline-earth-metal and amine salts and acid-addition salts are particularly useful compounds of this invention.
Preparation of the A54145 Cyclic Peptide Derivatives
A. The Starting A54145 Components
The formula 1 compounds are prepared by starting with the A54145 components. These components are produced by an A54145-producing strain of Streptomyces fradiae under submerged aerobic conditions in a suitable culture medium. Cultures of three A54145-producing Streptomyces fradiae strains have been deposited and made part of the stock culture collection of the Midwest Area Northern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, Ill. 61604, from which they are available to the public under the accession numbers NRRL 18158, NRRL 18159 and NRRL 18160.
The culture medium used to grow the A54145-producing Streptomyces fradiae strains can be any one of a number of media. Preferred carbon sources are glucose, maltose, galactose, methyl oleate and peanut oil. Preferred nitrogen sources are soybean grits, soybean flour or an enzymatic hydrolysate of soybeans.
Among the nutrient inorganic salts which may advantageously be incorporated in the culture media are the customary soluble salts capable of yielding zinc, 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 substituents of the medium in amounts sufficient to meet the growth requirements of the organism.
Small amounts (i.e. 0.2 ml/L) of an antifoam agent such as polypropylene glycol may be added to large scale fermentation media if needed.
The A54145 antibiotics are produced by the Streptomyces fradiae strains when grown at temperatures between about 20.degree. and about 35.degree. C. A good temperature for A54145 production appears to be from about 25.degree. C. to about 29.degree. C.
As is customary in submerged aerobic culture processes, sterile air is blown into the vessel from the bottom while the medium is stirred with conventional turbine impellors. In a fully baffled 165-liter fermentor containing approximately 115 liters of broth, an aeration rate of 0.25 v/v/m with an agitation rate of 150-200 rpm is sufficient to maintain the level of dissolved oxygen at or above 30% of air saturation.
Production of antibiotic A54145 can be followed during the fermentation by testing samples of the broth for antibiotic activity against organisms known to be sensitive to the antibiotic. One assay organism useful in testing for antibiotic A54145 is Bacillus subtilis. The bioassay is conveniently performed by the agar-well plate test.
LaVerne D. Boeck has developed improvements in the process for preparing some of the A54145 components and some of the compounds of this invention which he describes in his copending application entitled "PROCESSES FOR PREPARING A54145 COMPOUNDS", Ser. No. 07/179,930, now U.S. Pat. No. 4,977,083, filed herewith this even date.
One process comprises feeding a C.sub.4 -C.sub.18 -alkanoic or alkenoic acid or alcohol, or an ester or salt thereof, to an A-54145-producing culture during its fermentation and recovering the A54145 components. This process provides significantly increased product yields of A54145 components.
In this process the alkyl portion of the alkanoic or alkenoic acid or alcohol (the substrate) used can be a straight or branched chain. The straight-chain acids or alcohols, or their esters or salts, are recommended because of availability and lower cost. An especially preferred substrate is n-decanoic acid and its esters and salts.
When using an alkanoic acid ester, the C.sub.1 -C.sub.4 -alkyl esters are preferred. In such an ester, the group may also be straight or branched.
Representative suitable salts of alkanoic or alkenoic acids which may be used in this process include those formed from alkali metals and alkaline-earth metals such as sodium, potassium, lithium, cesium, rubidium, barium, calcium and magnesium. Suitable amine salts include the amnonium and the primary, secondary and tertiary C.sub.1 -C.sub.4 -alkyl-ammonium and hydroxy-C.sub.2 -C.sub.4 -alkyl-ammonium salts.
For this process it is preferable to add the substrate to the fermentation in the form of a sterile solution. For example, n-decanoic acid is a solid at room temperature, whereas its ethyl ester is a liquid. Thus, the ethyl ester is preferred because the acid must be dissolved in a compatible liquid, such as oleic acid or methyl oleate, for efficient feeding. Oleic acid is a particularly useful solvent for this purpose, although other solvents such as ethanol, ethyl acetate and C.sub.1 -C.sub.4 esters of unsaturated fatty acids can be used. Those substrates which are suitably fluid at fermentation temperatures may be added directly and are, therefore, preferred.
The rate of addition of the substrate to the fermentation must be low enough to avoid producing a toxic effect on the fermentation, but high enough to increase the yield of the desired compound. Rates of addition of about 0.5 to about 4 mL of substrate per liter of fermentation broth per day can be used. A rate of from about 1.5 to about 3 mL of substrate per liter of fermentation broth per day is preferred.
The substrate is added to the growing A54154-producing culture during the production stage of the fermentation, beginning at from about 20 to about 26 hours and continuing until the fermentation is terminated. The substrate can be added by various methods. It is preferable, however, to add it by a method which best approaches a steady flow.
Another improved process for preparing A54145 compounds described in Boeck's application comprises feeding glucose at a rate from about 6 to about 9 grams/liter/day to an A54145-producing culture, starting from about 18 to about 24 hours after initiating the production stage and continuing throughout its fermentation. The improvement obtained by this process is illustrated in Table II, which compares the results obtained by standard methods with results obtained using this process.
TABLE II______________________________________Effect of Continuous GlucoseFeed on A54145 Biosynthesis A54145Glucose YieldLevel (%) Glucose Addition Method (mcg/mL)______________________________________4 Included at time of 520 medium make-up4 Continuous feed from 1370 day 1 to day 8.sup.a______________________________________ .sup.a Beginning 23 hours after initiating the production stage
As the results in Table II indicate, glucose feeding increases final A54145 yield by at least 150%.
In the continuous glucose feed process, the rate of addition of the glucose must he low enough to avoid toxic affects on the fermentation, but high enough to cause a significant increase in the yield of A54145 compound. A rate of about 6 to about 9 grams of glucose/liter of fermentation/day is recommended, but a rate of about 7.5 g/L/day is preferred for this process.
A third method for increasing product yields of A54145 components which Boeck describes comprises feeding an enzymatic soy digest to the fermentation at a rate of from about 2 to about 4 grams of soy digest/liter of fermentation broth/day to an A54145-producing culture, starting from about 90 to about 120 hours after initiating the production stage, and continuing throughout its fermentation.
Each of the Boeck processes can be carried out over a temperature range of from about 20.degree. to about 34.degree. C. Temperature affects the amount of total antibiotic produced and the type of nucleus and side chain produced. Thus, the temperature of the fermentation should be adjusted appropriately in order to obtain optimum yields of the desired product. Table III summarizes temperature effects on A54145 production which were observed in fermentation studies in which only the temperature was varied.
TABLE III______________________________________EFFECT OF TEMPERATURE ON A54145 NUCLEUSAND ACRYL-CHAIN BIOSYNTHESIS IN A STIRRED165-L BIOREACTOR Total AcylTemperature Antibiotic Nuclei (%) Chains (%).sup.a(.degree.C.) (mcg/mL) A B C F iC.sub.10 nC.sub.10 aC.sub.11______________________________________21 1015 60 31 -- 9 79 15 625 1582 62 30 -- 8 74 18 829 1623 39 51 3 6 64 21 1431 1341 32 59 2 6 59 23 1733 923 19 73 2 6 60 23 16______________________________________ .sup.a iC.sub.10 = 8methylnonanoyl nC.sub.10 = ndecanoyl aC.sub.11 = 8methyldecanoyl
B. The Cyclic Peptide Intermediates
The A54145 components or blocked components are deacylated by exposing the compound in an aqueous medium to an enzyme produced by a microorganism of the family Actinoplanaceae until substantial deacylation is accomplished. A preferred method comprises using an enzyme produced by the microorganism Actinoplanes utahensis NRRL 12052 to cleave the fatty acid side chain.
Deacylation is ordinarily accomplished by adding the appropriate A54145 component or blocked A54145 component to a growing culture of the A. utahensis strain and permitting the culture to incubate until deacylation is accomplished. The A54145 nucleus or blocked A54145 nucleus (formula 4 compound) is then separated from the fermentation broth by methods in the art.
C. The Acylation Step
The compounds of formula 1 wherein R is alkanoyl or alkenoyl are prepared by acylating a compound of formula 4b (which is blocked at the .epsilon.-amino group of lysine) with the desired alkanoyl or alkenoyl side chain, using methods conventional in the art for forming an amide bond. The acylation is accomplished, in general, by reacting the selected compound with an activated derivative of the alkanoic acid or alkenoic acid corresponding to the desired acyl side chain group (R). The term "activated derivative" means a derivative which renders the carboxyl function of the acylating agent reactive to coupling with the primary amino group to form the amide bond which links the acyl side chain to the nucelus. Suitable activated derivatives, their methods of preparation, and their methods of use as acylating agents for a primary amine will be recognized by those skilled in the art.
Preferred activated derivatives are: (a) an acid halide (e.g. an acid chloride), (b) an acid anhydride (e.g., an alkoxyformic acid anhydride) or (c) an activated ester (e.g. a 2,4,5,-trichlorophenyl ester). Other methods for activating the carboxyl function include reacting the carboxylic acid with a carbonyldiimide (e.g. N,N'-dicyclohexylcarbodiimide or N,N'-diisopropylcarbodiimide) to give a reactive intermediate which, because of instability, is not isolated, the reaction with the primary amine being carried out in situ.
Those skilled in the art will recognize that the compounds of formula 1 are prepared using selective acylation procedures and with the assistance of amino-protecting groups. For example, when a formula 4c compound (R and R.sup.1 are hydrogen) is the starting material, acylation can occur at both the .alpha.-amino group of tryptophan and the .epsilon.-amino group of lysine to give N.sub.Trp, N.sub.Lys -diacyl derivatives. To obtain derivatives monoacylated at the a-amino group of tryptophan, therefore, it is preferable to acylate a compound of formula 4b wherein the .epsilon.-amino group of lysine (the position) is blocked by an amino-protecting group. Such starting materials are preferably obtained by blocking the A54145 component at this position before it is deacylated.
Scheme I outlines the general procedure for preparing the formula 1 compounds. In this Scheme, the following symbols are used:
[*]=remainder of the A54145 component
N.sub.T =.alpha.-amino group of tryptophan
N.sub.L =.epsilon.-amino group of lysine
R=substituents as defined
R.sub.N =acyl group of natural factor
B=amino-protecting group
Acyl=an acylation step
Deacyl=a deacylation step
Block=acylation with an amino-protecting group
Deblock=removal of an amino-protecting group ##STR16##
D. The Schiff's Bases
The compounds of formula 1 in which a) R and R.sup.2 represent an alkylidenyl group (1b compounds; the Schiff's bases) or b) R is an alkyl group (the reduced Schiff's bases) can be prepared by known methods for preparing Schiff's bases and reducing such bases, respectively. Thus, the Schiff's bases are prepared by reacting (condensing) the primary amino group of tryptophan of the corresponding A54145 nucleus with an appropriate aldehyde or ketone in a suitable solvent. Reduction of the imine bond of the Schiff's base, to obtain the corresponding formula 1 compound in which R is alkyl can be accomplished by known selective reduction procedures. A preferred reducing agent for this reaction is sodium cyanoborohydride.
The Schiff's bases are useful as intermediates to the reduced Schiff's bases. When the Schiff's base is used as an intermediate, it is not necessary to isolate the intermediate prior to reducing it to form the reduced Schiff's base.
A preferred method for preparing a formula 1 compound in which R is alkanoyl or alkenoyl is by the active ester method. A compound of formula 4 wherein R.sup.b =H and R.sup.1 =t-BOC, i.e. an A54145 N.sub.Lys -(t-BOC)-nucleus or "t-BOC nucleus", is an especially preferred starting material in the preparation of formula 1 compounds. The 2,4,5-trichlorophenyl ester of the desired alkanoic or alkenoic acid is a preferred acylating agent. In this method, an excess amount of the active ester is reacted with the t-BOC nucleus at room temperature in a non-reactive organic solvent such as DMF, THF, diethyl ether or dichloromethane. The reaction time is not critical, although a time of about 6 to about 20 hours is preferred. At the conclusion of the reaction, the solvent is removed, and the residue is purified. A particularly useful purification method is reverse-phase HPLC, using LP-1/C.sub.18 as the stationary phase and a mixture of H.sub.2 O/CH.sub.3 OH/CH.sub.3 CN/pyridine/HOAc as the solvent system. The t-BOC group is removed by treatment with trifluoroacetic acid/anisole/triethylsilane or, preferably, trifluoroacetic acid/1,2-ethanedithiol for from about three to about five minutes at room temperature. After the solvent is removed, the residue can be purified by reverse-phase HPLC.
An alternative acylation method is a modified Schotten-Baumann procedure in which the unblocked nucleus is treated with the acid chloride of the desired alkanoic acid or alkenoic acid in a pyridine/water mixture. In this method, an excess of the acid chloride in a non-reactive organic solvent (such as acetone) is added slowly to a solution of the nucleus in 90% pyridine/10% water (by volume). The unreacted acid chloride is separated from the reaction product by extraction into an immiscible organic solvent (e.g., diethyl ether). Final purification can be accomplished by reverse-phase HPLC, as previously described.
The alkanoic and alkenoic acids used as starting materials for the acylation reaction, and the activated derivatives thereof (in particular, the acid chlorides and the 2,4,5-trichlorophenyl esters), are known compounds or can be prepared from known compounds by known methods. The 2,4,5-trichlorophenyl esters are conveniently made by treating the acid chloride of the alkanoic or alkenoic acid with 2,4,5-trichlorophenol in the presence of pyridine or by treating the free alkanoic or alkenoic acid with 2,4,5-trichlorophenol in the presence of N,N'-dicyclohexylcarbodiimide as a coupling agent. The 2,4,5-trichlorophenyl ester derivative can be purified by column chromatography over silica gel.
The formula 1a compounds inhibit the growth of pathogenic bacteria. For example, Table IV shows the inhibitory concentrations (MIC's) at which formula 1a compounds inhibit certain organisms. The MIC's in Table IV were determined by conventional agar-dilution assays.
TABLE IV__________________________________________________________________________Antibacterial Activity of Formula 1 Compounds MIC's of Compound.sup.bTest Organism 2 4 6 10 26__________________________________________________________________________Staphylococcus aureus X1.1 2 1 32 16 0.25Staphylococcus aureus V41 4 1 32 16 0.25Staphylococcus aureus X400 4 1 32 16 0.5Staphylococcus aureus S13E 4 2 32 16 0.25Staphylococcus epidermidis EPI1 2 1 16 16 0.5Staphylococcus epidermidis 222 2 1 16 16 0.5Streptococcus pyogenes C203 0.5 0.125 4 4 0.06Streptococcus pneumoniae Park 1 2 2 64 32 0.125Streptococcus sp. group D X66 16 4 128 128 2Streptococcus sp. group D 2041 32 8 7128 128 4__________________________________________________________________________ .sup.a MIC's in mcg/mL .sup.b Compound numbers from Table I
The in vitro activity of the A54145 antibiotics is stimulated by the presence of calcium ions. For example, the in vitro activity of A54145A against Staphylococcus aureus 209P and Streptococcus faecalis ATCC 29212 was stimulated about 10 to 100 times by the presence of Ca.sup.++ (50 mg/mL).
The formula 1a compounds have shown in vivo antimicrobial activity against experimentally-induced infections in laboratory animals. When two doses of test compound were administered to mice experimentally infected with Streptococcus pyogenes or Staphylococcus aureus, the activity observed was measured as an ED.sub.50 value [effective dose in mg/kg to protect 50% of the test animals: see Warren Wick, et al., J. Bacteriol. 81, 233-235 (1961)]. The ED.sub.50 values observed for illustrative compounds are given in Table V.
TABLE V______________________________________ED.sub.50 Values for Formula 1a Compounds.sup.avs. Streptococcus pyogenesCompound No..sup.b ED.sub.50.sup.c______________________________________2 1.654 1.156 10.610 10.8______________________________________ .sup.a Administered subcutaneously .sup.b Compound numbers from Table I .sup.c mg/kg .times. 2; doses given 1 and 4 hours postinfection
Table VI summarizes the relative toxicity of illustrative formula 1a compounds.
TABLE VI______________________________________Relative Toxicity ofFormula 1a CompoundsCompound No..sup.b Median Lethal Dose (mg/kg)______________________________________2 >5204 3216 >51010 >505______________________________________ .sup.a When administered IP in mice .sup.b Compound numbers from Table I
The formula 1a compounds improve growth performance in animals. The compounds are especially effective growth promoters in fowl, such as chickens and turkeys, but should also be effective in other animals such as swine.
The formula 1a compounds are typically effective in improving growth performance in animals when administered with feeds at a rate of from about 0.05 to about 100 grams of compared per ton of feed (0.055 to 110 ppm). A preferred rate is from about 0.05 to about 50 g/ton, and an especially preferred rate is from about 1 to about 20 g/ton.
The formula 1a compounds can be administered to animals orally or parenterally. The most practical way to administer the compound is by formulation into the feed supply. A variety of feeds, including the common dry feeds, liquid feeds, and pelleted feeds, may be used. Although the preferred method of administration is by mixing it with the animals' feed, it can also be administered in other ways, for example, tablets, drenches, boluses, or capsules. Each individual dosage unit should contain a quantity of compound directly related to the proper daily dose for the animal to be treated.
The methods of formulating drugs into animal feeds are well known. A preferred method is to make a concentrated drug premix which in turn is used to prepare medicated feeds. Typical premixes may contain from about 1 to about 200 grams of drug per pound of premix. Premixes may be either liquid or solid preparations.
The final formulation of feeds for animals or poultry will depend upon the amount of drug to be administered. The common methods of formulating, mixing, and pelleting feeds may be used to prepare feeds containing a formula 1a compound.
The compounds may be formulated for parenteral administration by methods recognized in the veterinary pharmaceutical art. Effective injectable compositions containing the compounds may be in either suspension or solution form. In the solution form, the compound is dissolved in a physiologically acceptable carrier. Such carriers comprise a suitable solvent, preservatives such as benzyl alcohol, if needed, and buffers. Useful solvents include, for example, water, alcohols, glycols, or inert oils such as vegetable oils or highly refined mineral oils.
Injectable suspension compositions are prepared using a nonsolvent for the compound with adjuvants, as a carrier. The nonsolvent can be, for example, a glycol such as polyethylene glycol.
Suitable physiologically acceptable adjuvants are necessary to keep the compound suspended in suspension compositions. The adjuvants may be chosen from among thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin, and the alginates. Many surfactants are also useful for suspending the compounds. Lecithin, alkylphenol polyethylene oxide adducts, naphthalenesulfonates, alkylbenzenesulfonates, and the polyoxyethylene sorbitan esters are useful suspending agents in liquid nonsolvents.
Many substances which affect the hydrophilicity, density, and surface tension of the liquid nonsolvent can assist in making injectable suspensions in individual cases. For example, silicone antifoams, glycols, sorbitol, and sugars can be useful suspending agents.
When an A54145 cyclic peptide derivative of this invention is used as an antibacterial agent, it may be administered either orally or parenterally. As will be appreciated by those skilled in the art, the A54145 compound is commonly administered together with a pharmaceutically acceptable carrier or diluent. The dosage of A54145 compound will depend upon a variety of considerations, such as, for example, the nature and severity of the particular infection to be treated. Those skilled in the art will recognize that appropriate dosage ranges and/or dosage units for administration may be determined by considering the MIC and ED.sub.50 values and toxicity data herein provided together with factors such as pharmacokinetics, the patient or host and the infecting microorganism.

The following non-limiting examples are provided to illustrate this invention. In these examples the following numbers will be used to represent specific solvent systems:
______________________________________No. System Ratio______________________________________1 Pyridine/HOAc/H.sub.2 O 1:1:982 Pyridine/HOAc/H.sub.2 O/CH.sub.3 CN 1:1:88:102a " 1:1:87:112b " 1:1:86:122c " 1:1:83:152d " 1:1:82:162e " 1:1:78:202f " 1:1:73:252g " 1:1:70.5:27.52h " 1:1:68:302i " 1:1:67:312j " 1:1:66:322k " 1:1:65:332m " 1:1:63:353a Pyridine/HOAc/H.sub.2 O/CH.sub.3 CN/MeOH 1:1:70:18:103b " 1:1:68:20:103c " 1:1:63:25:103d " 1:1:61:27:103e " 1:1:58:30:103f " 1:1:56:32:103g " 1:1:53:35:103h " 1:1:68:25:53i " 1:1:73:15:103j " 1:1:60.5:25:12.53k " 1:1:71:20:74 CH.sub.3 CN/H.sub.2 O 1:14a " 15:855 CH.sub.3 OH/H.sub.2 O 1:1______________________________________
Separation of the individual antibiotic A54145 components can be followed by TLC or HPLC. One convenient analytical HPLC system is:
Analytical HPLC System for A54145 Components
Column: 4.6-.times.250-mm Zorbax C8 (Dupont)
Mobile Phase: acetonitrile/water containing 0.2% tri ethylamine and adjusted to pH 3 with phosphoric acid (35:65)
Detection: UV at 223 nm
Flow Rate: 2 mL/min
A54145 components A-F have the following approximate retention times in this system:
______________________________________ A54145 Retention Factor Time (min)______________________________________ A 12.1 .sub. A.sub.1 13.1 B 14.9 .sub. B.sub.1 13.7 C 17.0 D 19.6 E 22.4 F 9.4______________________________________
Formation and purification of the A54145 nuclei can be monitored by analytical HPLC, using the following system:
Analytical HPLC System for A54145 Nuclei
Column: 4.6-.times.250 mm Zorbax ODS (Dupont, 5.mu.)
Detection: UV at 223 nm
Flow Rate: Usually 2 mL/min
Solvent System: CH.sub.3 CN/0.04M aq. NH.sub.4 OAc (9:91)
PREPARATION 1
Producing Antibiotic A54145 with Streptomyces fradiae A54145.1
A. Shake-flask Fermentation of A54145.1
The culture Streptomyces fradiae NRRL 18158, either as a lyophilized pellet or as a suspension maintained in liquid nitrogen, is used to inoculate 50 mL of a vegetative medium having the following composition:
______________________________________Vegetative Medium IIngredient Amount (g/L)______________________________________Glucose 15.0Potato dextrin 20.0Soybean grits 15.0Corn steep liquor 10.0Yeast extract 1.0CaCO.sub.3 5.0Tap water q.s. 1 liter(Adjust the pH of the medium from .about.6.1 to.about.6.5 with NaOH before sterilizing; post-sterilization pH______________________________________.about.7)
The inoculated first-stage medium is incubated in a 250-mL Erlenmeyer flask at 30.degree. C. for about 48 hours on a shaker orbiting in a two-inch (5.08 cm) circle at 250 rpm.
This incubated first-stage medium (1 mL) is used to inoculate 50 mL of a production medium having the following composition:
______________________________________Production Medium IIngredient Amount (g/L)______________________________________Glucose 45Soybean grits 35Blackstrap molasses 3CaCO.sub.3 2.5Tap water q.s. 1 liter(Presterilization pH .about.6.9; post-sterilization pH .about.6.8)______________________________________
The inoculated production medium is incubated in a 250-mL wide-mouth Erlenmeyer flask at 25.degree. C. for 6 to 7 days on a shaker orbiting in a two-inch circle at 250 rpm.
B. Tank Fermentation of A54145.1
In order to provide a larger volume of inoculum, 10 mL of incubated vegetative medium, prepared as described in Section A, is used to inoculate 400 mL of a second-stage growth medium having the same composition as that of the first-stage vegetative medium. This second-stage vegetative medium is incubated in a two-liter wide-mouth Erlenmeyer flask for about 24 hours at 30.degree. C. on a shaker orbiting in a two-inch circle at 250 rpm.
Incubated second-stage vegetative medium (800 mL) thus prepared is used to inoculate 115 liters of sterile production medium, prepared as described in Section A, except that 0.2 g/L of a silicone antifoam such as Sag-471 (Union Carbide) is added. The inoculated production medium is allowed to ferment in a 165-liter stirred fermentation tank for 6 to 7 days at a temperature of 25.degree. C. Low airflow (0.25 v/v/m) and low rpm (200-300) in the stirred vessel maintain a dissolved oxygen level above 30% of air saturation. The pH is not allowed to rise above 7.5.
PREPARATION 2
Producing Antibiotic A54145 with Streptomyces fradiae A54145.2
A. Shake-flask Fermentation of A54145.2
Shake-flask fermentation is carried out as in Preparation 1, Section A, with the following exceptions:
1) the culture is Streptomyces fradiae NRRL 18159;
2) the vegetative medium has the following composition:
______________________________________Vegetative Medium IIIngredient Amount (g/L)______________________________________Glucose 10Potato starch 30Soybean flour 20Defatted cottonseed flour 20CaCO.sub.3 2Tap water 1 liter______________________________________
3) the vegetative medium is incubated at 25.degree. C.; and
4) the production medium has the following composition:
______________________________________Production Medium IIIngredient Amount (g/L)______________________________________Glucose 25.0Soybean grits 18.75Blackstrap molasses 3.75Casein 1.25CaCO.sub.3 3.125Sodium acetate 8.0Tap water q.s. to 1 L(Pre-sterilization pH .about.6.9; post-sterilization pH______________________________________.about.6.8)
B. Tank Fermentation of A54145.2
Incubated vegetative medium prepared as described in Section A is used, and the procedures of Preparation 1, Section B, are followed with these following exceptions:
1) the amount of incubated vegetative medium used to inoculate the second-stage growth medium is 8 mL;
2) the amount of second-stage medium used to inoculate the production medium is 2 L;
3) the air flow is 0.125 v/v/m; and
4) the pH is allowed to rise above 7.5.
PREPARATION 3
Producing Antibiotic A54145 with Streptomyces fradiae A54145.3
The procedures of Preparation 2, Section B, are followed except that 1) the culture used is Streptomyces fradiae NRRL 18160, 2) dissolved oxygen is controlled at 40% of air saturation, 3) pH is controlled at 7.0 and 4) the production medium has the following composition:
______________________________________Production Medium IIIIngredient Amount (g/L)______________________________________Soybean flour 20.0Glucose 5.0Blackstrap molasses 2.5Fe(SO.sub.4).(NH.sub.4).sub.2 SO.sub.4.6H.sub.2 O 0.6Silicon defoamer 0.2Polypropylene glycol 0.1(M.W. 2000)Tap water q.s. to 1 liter______________________________________
PREPARATION 4
Isolating Antibiotic A54145
Procedure A
Whole fermentation broth from two 100-L tanks (217 L), prepared as described in Preparation 2, was filtered through a filter press with 3% filter aid (Hyflo Super-Cel, Manville Products, Lompoc CA). The filtrate (185 L) was adjusted to pH 6.4, using 5N HCl. Diaion HP-20 resin (20 L) was added to the filtrate. The initial effluent (85 L) and a water wash (60 L) were discarded. The resin was then eluted as follows:
______________________________________Eluate Solvent No. Amount______________________________________1 4a 40 L2 4 30 L3 4 30 L______________________________________
Eluate 1 was discarded.
Eluates 2 and 3 were combined and chromatographed on 2 L of IRA-68(OAc.sup.-) (2.5".times.32"). The initial effluent (60 L), a wash with Solvent No. 4 (10 L) and an eluate with 0.1N HOAc:CH.sub.3 CN (1:1, 10 L) were discarded. The column was then eluted with 14 L of 1.0N HOAc:CH.sub.3 CN (1:1). This fraction was concentrated under vacuum and lyophilized to give 101.1 g of antibiotic A54145.
Procedure B
Whole fermentation broth from a large tank (4600 L), prepared as described in Preparation 2, was adjusted to pH 6.5 with HCl and filtered through a filter press with the aid of 4% Celite 545 to give 4600 L of filtrate having a pH of 6.3.
The filtrate was absorbed batch-wise onto Diaion HP-20 resin (200 L), adjusted to pH 6.0 and maintained at this pH while stirring for 2 hours. The mixture was filtered, and the filtrate was discarded.
The saturated HP-20 resin was transferred to a small tank with a welded membrane. The resin was washed first with water (800 L), agitating for 35 minutes, and then with Solvent No. 4a (400 L), agitating for 3 minutes. These washes were discarded. The resin was then eluted twice with Solvent No. 4 (600 L), agitating for 35 minutes.
The eluates were combined (1200 L) and chromatographed on an IRA-68 resin column (100 L), equilibrated in Solvent No. 4 and washed with this solvent (500 L). The column was then eluted with CH.sub.3 CN:0.2N HOAc (1:1), discarding the first fractions (300 L) and combining, concentrating and lyophilizing subsequent fractions (750 L) to give 3.65 kg of antibiotic A54145.
PREPARATION 5
Separating A54145B, A54145C, A54145D and A54145E
Antibiotic A54145 (60 g), obtained as described in Preparation 4, was subjected to preparative HPLC using a Chromatospac 100, 4-L Quantum LP-1/C18 silica-gel column (3".times.39"). The antibiotic was dissolved in Solvent No. 1 and added to the column. Elution was monitored by UV at 280 nm.
Fractions were combined based on analytical HPLC as described supra, but detecting at 289 and 223 nm and collecting 500-mL fractions at a flow rate of 100 mL/min. The column was eluted as follows:
______________________________________ Solvent Fractions______________________________________ 1 1-8 3b 9-29 3c 30-73 3d 74-161 4 8-L strip______________________________________
On the basis of the analytical HPLC results, fractions 114-161 were combined to give a total of 8.5 g of antiobiotic A54145 enriched with components B, C, D and E. This material was rechromatographed on a Chromatospac column, repeating the previous conditions, but detecting at 233 nm and using the following solvents:
______________________________________ Solvent Fractions______________________________________ 1 1-8 3c 9-41 3f 42-60 3h 61-83 4 8-L strip______________________________________
From this column, fractions 76-78 gave 1.75 g of A54145B-enriched material, fractions 79-83gave 1.02 g of A54145C-enriched material, and the strip fraction gave 0.8 g of A54145D-enriched material.
PREPARATION 6
Separating A54145 Enriched with A54145A, A54145C and A54145F
A54145 (60 g), obtained as described in Preparation 4, Procedure B, was chromatographed as in Preparation 5, but using the following solvents:
______________________________________ Solvent Fractions______________________________________ 1 1-8 3c 9-102 4 103-122______________________________________
Fractions were combined on the basis of analytical HPLC to give 2.54 g of A54145F-enriched material, 5.1 g of A54145A-enriched material and 10.56 g of A54145C-enriched material.
PREPARATION 7
Isolating A54145A
A54145A-enriched material (1 g), obtained as described in Preparation 6, was purified, using the following preparative HPLC system: two 1".times.12" stainless steel columns packed with Zorbax ODS (12.mu.) in series.
Detection: UV at 280 nm
Flow Rate: 9 mL/minute
The material was dissolved in Solvent No. 1 for injection onto the column. The column was eluted as follows:
______________________________________ Solvent Fractions.sup.a______________________________________ 1 1-18 3a 19-145 4 146-165______________________________________ .sup.a Fraction volume = 18 mL
Fractions containing A54145A (fractions 86-96) were combined, concentrated under vacuum and lyophilized to give 212 mg of purified A54145A.
Characteristics of A54145A
Mol. Wt.: 1643
Mol. Formula: C.sub.72 H.sub.109 N.sub.17 O.sub.27
High Resolution FABMS(M+H): Found: 1644.7778, Calcd. for C.sub.72 H.sub.110 N.sub.17 O.sub.27 : 1644.7757
UV (EtOH) .lambda..sub.max : 219 nm (.epsilon. 35,000), 280 (.epsilon. 5,250), shoulder 288 (.epsilon. 4,600)
IR (KBr): essentially the same as that of A54145B, infra
Optical Rotation: [.alpha.].sub.589.sup.25.degree. C. No Rotation (CH.sub.3 OH). [a].sub.365.sup.25 C. -14.0.degree. (c 0.1, CH.sub.3 OH).
Amino-acid Analysis: Asp 973(2), Thr 441(1), Glu 1056(2), Gly 528(1), Ala 549(1), Ile 469(1), Trp 465(1)
PREPARATION 8
Isolating A54145B
The A54145B-enriched A54145 material obtained in Preparation 5 (500 mg) was chromatographed using the procedure of Preparation 7. The column was eluted as follows:
______________________________________Solvent No. Fractions.sup.a______________________________________1 1-163g 17-955 96-115______________________________________ .sup.a Fraction volume = 18 mL
Fractions containing A54145B (fractions 64-70) were combined, concentrated under vacuum and lyophilized to give 330 mg of purified A54145B.
Characteristics of A54145B
Mol. Wt.: 1657
Mol. Formula: C.sub.73 H.sub.111 N.sub.17 O.sub.27
High Resolution FABMS(M+H): Found: 1658.7954, Calcd. for C.sub.73 H.sub.112 N.sub.17 O.sub.27 : 1658.7914
UV (EtOH) .lambda..sub.max : 220 nm (.epsilon. 41,854), 281 (.epsilon. 5,613), 289 (.epsilon. 5,084)
IR (KBr): ranging from 3335 to 3313; 2930, 1660, 1531, 1407, 1255 cm.sup.-1
Optical Rotation: [.alpha.].sub.589.sup.25.degree. C. =-8.55.degree. (c 0.47, H.sub.2 O). [.alpha.].sub.365.sup.25.degree. C. =-36.32.degree. (c 0.47, H.sub.2 O).
Amino-acid Analysis: Asp 1039(2), Thr 466(1), Glu 564(1), Gly 528(1), Ala 525(1), Ile 491(1), Lys 514(1), Trp 491(1), 3-MG 512(1).
PREPARATION 9
Isolating A54145C
A54145C-enriched material (11.76 g), obtained as described in Preparations 5 and 6, was purified using the following preparative HPLC system:
Column: 2".times.60-cm stainless steel
Packing: Quantum LP-1/C18 silica gel (20 m.mu.)
Detection: UV at 280 nm
Flow Rate: 18 mL/min
The material was dissolved in pyridine/HOAc/H.sub.2 O (1:1:98, 37 mL) for application to the column. The column was eluted as follows:
______________________________________Solvent No. Fractions.sup.a______________________________________1 1-103e 11-1603h 161-5504 551-582______________________________________ .sup.a Fraction volume = 18 mL
Fractions containing A54145C were combined (fractions 320-331, 817.8 mg). This material (800 mg) was further purified by HPLC using a 1".times.20" stainless-steel column packed with Quantum LP-1/C18 (20 m.mu.) silica gel column, detecting as in Preparation 7, and applying the material in pyridine/HOAc/H.sub.2 O (1:1:98, 15 mL). The column was eluted at a flow rate of 8 ml/min as follows:
______________________________________Solvent No. Fractions.sup.a______________________________________1 1-183f 19-693g 70-1145 115-137______________________________________ .sup.a Fraction volume = 16 mL
Fractions containing A54145C (fractions 84-86 and 92-98) were combined, concentrated and lyophilized to give 350 mg of C-enriched material.
This process was repeated with some variation in the solvents used, i.e., varying the amount of CH.sub.3 CN in the solvent and sometimes eliminating methanol in the solvent mixture, to give an additional 27.6 mg of purified A54145C.
Characteristics of A54145C
Mol. wt.: 1657
Mol. Formula: C.sub.73 H.sub.111 N.sub.17 O.sub.27
High Resolution FABMS(M+H): Found: 1658.7905, Calcd. for C.sub.73 H.sub.112 N.sub.17 O.sub.27 : 1658.7914
UV (EtOH) .lambda..sub.max : 219 nm (.epsilon. 29,500), 281 (.epsilon. 4,200), 288 (.epsilon. 3,600)
IR (KBr): essentially the same as that of A54145B, supra;
Amino-acid Analysis: Asp 934(2), Thr 414(1), Glu 594(1), Gly 501(1), Ala 459(1), Val 359(1), Lys 451(1), 3-MG 487(1), Trp 308(1).
PREPARATION 10
Isolating A54145D
A54145D-enriched material (750 mg), obtained as described in Preparation 5 was purified using the preparative HPLC system described in Preparation 7, except that only one column was used. The material was applied to the column in 25 mL of solvent 1, and the column was eluted at a flow rate of 7.5 mL/min as follows:
______________________________________Solvent No. Fractions.sup.a______________________________________1 1-63g 7-892k 90-1014 102-115______________________________________ .sup.a Fraction volume = 15 mL
Fractions containing A54145D (19-22) were combined, concentrated and lyophilized to give 219 mg of material further enriched with A54145D.
This material was purified by a second HPLC column, using the same conditions except that 5% methanol was added to solvent 4 and solvent 2k was eliminated.
The fractions from this column containing A54145D (fractions 72-74) were combined, concentrated and lyophilized to give 70 mg of purified A54145D.
Characteristics of A54145D
Mol. Wt.: 1657
Mol. Formula: C.sub.73 H.sub.111 N.sub.17 O.sub.27
High Resolution FABMS(M+H): Found: 1658.7913, Calcd. for C.sub.73 H.sub.112 N.sub.17 O.sub.27 : 1658.7914
UV (EtOH) .lambda..sub.max : 219 nm (.epsilon. 37,500), 280 (.epsilon. 5,040), 289 (.epsilon. 4,500)
IR (KBr): essentially the same as that of A54145B, supra;
Amino-acid Analysis: Asp 1011(2), Thr 427(1), Glu 967(2), Gly 515(1), Ala 487(1), Ile 434(1), Lys 543(1), Trp 577(1)
PREPARATION 11
Isolating A54145E
A54145E-enriched material (1.0 g), obtained as described in Preparation 5, was purified using a preparative HPLC system as in Preparation 9, but using a 1".times.20". The material was applied in 15 mL of solvent 1, and the column was eluted at a flow rate of 9 mL/min as follows:
______________________________________Solvent No. Fractions.sup.a______________________________________1 1-192h 20-1182j 119-2154 216-225______________________________________ .sup.a Fraction volume = 18 mL
Fractions containing A54145E (fractions 147-160) were combined, concentrated and lyophilized to give 49.7 mg of material further enriched with A54145E.
This material was purified using two 9.4.times.250-mm Zorbax ODS (5.mu.) columns in series, detecting by UV at 280 nm. The material was applied to the column in 3 mL of solvent, and the column was eluted at a flow rate of 3.25 mL/min as follows:
______________________________________Solvent No. Fractions.sup.a______________________________________1 1-122i 13-1804 181-193______________________________________ .sup.a Fraction volume = 6.5 mL
Fractions containing A54145E (fractions 143-160) were combined, concentrated and lyophilized to give 16.07 mg of purified A54145E.
Characteristics of A54145E
Mol. Wt.: 1671
Mol Formula: C.sub.74 H.sub.113 N.sub.17 O.sub.27
High Resolution FABMS(M+H): Found: 1672.8065, Calcd. for C.sub.74 H.sub.114 N.sub.17 O.sub.27 : 1672.8069
UV (EtOH) .lambda..sub.max : 221 nm (.epsilon. 29,714), 278 (.epsilon. 4577), 289 (4044)
IR (KBr): essentially the same as that of A54145B, supra
Amino-acid Analysis: Asp 826(2), Thr 367(1), Glu 494(1), Gly 437(1), Ala 422(1), Ile 378(1), Lys 410(1), Trp 387(1), 3-MG 437(1)
PREPARATION 12
Isolating A54145F
A54145F-enriched material (800 mg), obtained as described in Preparation 6, was purified using an HPLC system as in Preparation 9, but with a 1".times.20" column. The material was applied to the column in 10 mL of solvent, and the column was eluted at a flow rate of 8 mL/min as follows:
______________________________________Solvent No. Fractions.sup.a______________________________________1 1-102f 11-602g 61-992k 100-1344 135-150______________________________________ .sup.a Fraction volume = 16 mL
Fractions containing A54145F (fractions 120-128) were combined, concentrated and lyophilized to give 366.2 mg of purified A54145F.
Characteristics of A54145F
Mol. Wt.: 1629
Mol Formula: C.sub.71 H.sub.107 N.sub.17 O.sub.27
High Resolution FABMS(M+H): Found: 1630.7634, Calcd. for C.sub.17 H.sub.108 N.sub.17 O.sub.27 : 1630.7601
UV (EtOH) .lambda..sub.max : 219 nm (.epsilon. 36,750), 280 (.epsilon. 5,100), 288 (.epsilon. 4,450)
IR (KBr): essentially the same as that of A54145B, supra
Optical Rotation: [.alpha.].sub.589.sup.25.degree. C. =-3.0.degree. (c 1.0, H.sub.2 O). [.alpha.].sub.365.sup.25.degree. C. =-6.0.degree. (c 1.0, H.sub.2 O).
Amino-acid Analysis: Asp 959(2), Thr 428(1), Glu 965(2), Gly 494(1), Ala 487(1), Val 363(1), Lys 492(1), Trp 452(1).
PREPARATION 13
Isolating A54145A.sub.1
Procedure A
A54145A.sub.1 -enriched material was obtained using the following procedure: Whole broth (103 L), prepared as described in Preparation 2, was treated as described in Preparation 4 Procedure A except that instead of the IRA-68(OAc.sup.-) column, the combined eluates were chromatographed over a 40-.times.780-mm BioRex 5 (Cl.sup.-) column, using gradient elution with a 0.1N-1.0N NaCl solvent system and collecting 100-mL fractions.
Fractions containing A54145 were combined and desalted over a 40-.times.400-mm HP-20 column, again collecting 100-mL fractions. Fractions containing A54145 were combined and lyophilized to give 12.08 g of antibiotic A54145.
A portion of this antibiotic A54145 (2 g) is subjected to preparative HPLC using a Waters PrepPak 500 (C18) column, using a linear gradient of water to H.sub.2 O/CH.sub.3 CN (1:1) containing 1% NH.sub.4 H.sub.2 PO.sub.4. Fractions containing A54145A.sub.1 are collected and desalted over an HP-20 column, eluting with Solvent 4.
This step is repeated twice, and the A.sub.1 -enriched material is combined (937 mg).
The A.sub.1 -enriched material is chromatographed over two 1".times.12" Zorbax ODS columns in series as described in Preparation 7. Fractions containing A54145A.sub.1 are eluted with Solvent 2j, combined, concentrated and lyophilized to give crude A54145A.sub.1 (109 mg).
This material is further purified by repeating this step to give more purified A54145A.sub.1 (69.29 mg).
The material is even further purified by repeating this procedure three times, using Solvents 3j, 3h and 3k, respectively. The product obtained is desalted over HP-20 to give purified A54145A.sub.1 (12.21 mg).
Procedure B
Whole fermentation broth (160 L) is prepared as described in Preparation 25. With this procedure, the fermentation volume increases with time; therefore, beginning at 138 hours, 10-L aliquots are removed at intervals and frozen. By harvest (287 hours), a total of 50 L is removed and frozen. The frozen broth is added back to the fermentation at harvest. The whole broth is filtered with a filter aid or separated using a centrifuge. A portion of the filtrate (55 L) is worked up using the procedure of Preparation 14. Fractions containing A54145A.sub.1 are eluted with solvent 4concentrated and freeze-dried. Following this procedure gave 111.3 g of A54145A.sub.1 -enriched material.
This material is chromagraphed over a 1".times.16" Zorbax C8 (12.mu.) column. The column is eluted with solvent 2h. Following this procedure gave 374 mg of further A54145A.sub.1 -enriched material, which contained approximately 46% A54145A.sub.1, 19% A54145B.sub.1, 14% A54145A, 13% A54145B and 8% of an unidentified material (HPLC analysis).
Preparative HPLC using appropriate solvents is carried out on the further purified material to obtain A54145A.sub.1 in pure form.
Characteristics of A54145A.sub.1
Mol. Wt.: 1643
Mol. Formula: C.sub.72 H.sub.109 N.sub.17 O.sub.27
High Resolution FABMS(M+H): Found: 1644.7691, Calcd. for C.sub.72 H.sub.110 N.sub.17 O.sub.27 : 1644.7757
UV (EtOH) .lambda..sub.max : 220 nm (.epsilon. 41,623), 281 (.epsilon., 5,750), 289 (.epsilon. 4,950)
Optical Rotation: [.alpha.].sub.589.sup.25.degree. C. -10.4.degree. (c 0.69, CH.sub.3 OH)
Amino-acid Analysis: Asp 1209(2), Thr 554(1), Glu 1209(2), Gly 636(1), Ala 617(1), Ile 576(1), Lys 604(1), Trp 514(1)
PREPARATION 14
Isolating A54145B.sub.1
Whole fermentation broth (100 L), prepared as described in Preparation 3, was worked up as described in Preparation 4, Procedure A, except that chromatography on IRA-68 was omitted. The material was eluted with solvent 4a, concentrated and freeze-dried to give 248.2 g of crude antibiotic A54145.
A portion of this material (60 g) was chromatographed on a 2".times.60-cm LP-l/Cl8 silica gel column.
Detection: UV at 254 and 280 mm.
Flow Rate: 25 mL/minute/fraction.
The column was eluted as follows:
______________________________________Solvent No. Fractions______________________________________1 1-1382f 139-4112h 412-5602m 561-9764 977-1000______________________________________
Fractions containing A54145B and A54145B.sub.1 were pooled as follows:
______________________________________Pool Fraction Weight (g)______________________________________1 951-1000 1.102 635-667 4.623 685-719 3.95______________________________________
The A54145B and A54145B.sub.1 -enriched fractions (Pools 2-3) were further purified over two 1".times.12" Amicon C18 columns.
Detection: UV at 280 mm.
Flow Rate: 20 mL/1.6 minute/fraction.
The columns were eluted with pyridine/HOAc.sub.2 /H.sub.2 O/CH.sub.3 CN (0.1/0.1/67.3/32.5). Fractions containing A54145B were combined to give 554 mg of A54145B, and fractions containing A54145B.sub.1 were combined to give 207 mg of purified A54145B.sub.1.
Other A54145B-enriched fractions (Pool 1) were also purified in this manner to give an additional 394.5 g of A54145B.
Characteristics of A54145B.sub.1
Mol. Wt.: 1657
Mol. Formula: C.sub.73 H.sub.111 N.sub.17 O.sub.27
High Resolution FABMS(M+H): Found: 1658.7911 Calcd. for C.sub.73 H.sub.112 N.sub.17 O.sub.27 : 1658.7914
UV (EtOH) .lambda..sub.max : 221 nm (.epsilon. 39,100), 282 (.epsilon., 5,500), 290 (.epsilon. 740)
IR (KBr): essentially the same as that of A54145B, supra.
Amino-acid Analysis: Asp 935(2), Thr 422(1), Glu 556(1), Gly 480(1), Ala 434(1), Ile 438(1), Lys 467(1), Trp 440(1), 3-MG 426(1);
PREPARATION 15
Preparing A54l45A Nucleus
A. Fermenting Actinoplanes utahensis
A stock culture of Actinoplanes utahensis NRRL 12052 is prepared and maintained under liquid nitrogen as described in U.S. Pat. No. 4,524,135 (cols. 22-23) or on slants, using the following medium:
Medium A
______________________________________Ingredient Amount______________________________________Oatmeal 20.0 gSucrose 20.0 gYeast (debittered dried brewer's) 2.5 gCorn distillers dried solubles* 5.0 gK.sub.2 HPO.sub.4 1.0 gCzapek's mineral stock** 5.0 mlDeionized water q.s. to 1 liter______________________________________*Nadrisol, National Distillers Products Co.,99 Park Ave., New York, NY**Czapek's mineral stock has the following composition:Ingredient AmountFeSO.sub.4 .multidot. 7H.sub.2 O (dissolved in 2 g2 mL conc HCl)KCl 100 gMgSO.sub.4 .multidot. 7H.sub.2 O 100 gDeionized water q.s. to 1 liter
For agar slants, 20.0 g agar is added to Medium A. Slants are incubated at 30.degree. for about 8 to 10 days.
Liquid nitrogen suspension (1 mL) is used to inoculate 200 mL of a vegetative medium (Medium A). The inoculated vegetative medium is incubated in a 1-L wide-mouth Erlenmeyer flask at 30.degree. C. for about 76 hours on a shaker rotating through an arc two inches in diameter at 250 RPM.
The incubated vegetative medium (500 mL) is used to inoculate 10 liters of sterile production medium having the following composition:
Medium B
______________________________________Ingredient Amount (g/L)______________________________________Peanut meal 10.0Soluble meat peptone 5.0Sucrose 20.0KH.sub.2 PO.sub.4 0.5K.sub.2 HPO.sub.4 1.2MgSO .multidot. 7H.sub.2 O 0.25Tap water q.s to 1 liter______________________________________ Post-sterilization pH is .about.6.9.
The inoculated production medium is allowed to ferment in a 14-liter fermentation tank at a temperature of about 30.degree. C. for about 68 hours. The fermentation medium is stirred with conventional agitators at about RPM and aerated with sterile air to maintain the dissolved oxygen level at or above 30% of air saturation at atmospheric pressure.
B. Deacylating A54145A
A fermentation of A. utahensis is carried out as described in Section A. After incubating the production medium for about 68 hours, A54145A (50 g) in water (300 mL) is added. The pH of the medium is adjusted to 7.2 with NaOH.
The fermentation is allowed to continue at 30.degree. C., stirring at 600 RPM to maintain the dissolved oxygen level at 30% or above, until deacylation is complete. Deacylation is measured by disappearance of activity vs. Micrococcus luteus (about 22 hrs).
C. Isolating A54145A and A54145F Nuclei
Whole fermentation broth (10 liters), obtained as described in Section B, was vacuum filtered with Hyflo SuperCel (2 L). The mycelial cake was washed with water (3 L) and then discarded. The original filtrate (9.5 L) and the mycelial wash (3 L) were combined. This solution was adjusted to pH 3.5 with HCl, mixed with 1.4 liters of HP-20 resin (Diaion High Porous Polymer, HP-Series, Mitsubishi Chemical Industries Limited, Tokyo, Japan), stirred for 1 hr and placed in a 6.5-.times.75-cm column. The first effluent (13 L) and a water wash (adjusted to pH 2 with HOAc) were discarded. The column was then eluted with CH.sub.3 CN:H.sub.2 O (1:9) (5 L). This eluate was concentrated under vacuum and lyophilized to give 14.25 g of crude nucleus.
The crude nucleus was subjected to preparative HPLC using the following system:
Column: 2".times.60-cm reverse phase silica gel (Quantum LP-1/C.sub.18)
Detection: UV at 280 nm
The crude nucleus (14 g) was dissolved in Solvent No. 1 and injected onto the column. The column was eluted at a flow rate of 12 mL/min. as follows:
______________________________________Solvent No. Fractions.sup.a______________________________________1 1-162 17-2694 270-330______________________________________ .sup.a Fraction volume: 24 mL
Fractions containing A54145F nucleus (fractions 84-106) were combined, concentrated and lyophilized to give 1.95 g of impure A54145F nucleus, and fractions containing A54145A nucleus (fractions 150-250) were combined, concentrated and lyophilized to give 4.8 g of impure A54145A nucleus.
D. Purifying A54145A Nucleus
Impure A54145A nucleus (1.9 g), obtained as described in Section C, was purified by HPLC as in Section C, but using two 1".times.12" silica gel (Zorbax ODS, 12.mu.) columns in series.
The nucleus was applied to the column in solvent 1. The column was eluted at a flow rate of 8 mL/min as follows:
______________________________________Solvent No. Fractions.sup.a______________________________________1 1-252 26-932a 94-1774 178-200______________________________________ .sup.a Fraction volume: 16 mL
Fractions containing A54145A nucleus (fractions 111-117) were combined, concentrated and lyophilized to give 457 mg of A54145A nucleus.
A54145A nucleus has the following characteristics:
Mol. wt. (FABMS): 1489
pKa (H.sub.2 O): 3.2, 4.3, 4.9, 5.6, 9.5
UV (EtOH): 217 nm (.epsilon. .about.50,000), 279 (.epsilon. 4,100), 288 (.epsilon. 3,600)
IR (KBr): 3324, 3317, 3313, 3069, 1668, 1543, 1407, 1253, 1214 and 1200 cm.sup.-1
Optical Resolution: .alpha..sub.589D.sup.25.degree. C. =-4.93.degree. (c, H.sub.2 O). [.alpha.].sub.365.sup.25.degree. C. =-23.65.degree. (c, H.sub.2 O).
Analysis, Found: C, 45.36; H, 5.66; N, 12.74
Amino-Acid Analysis: Asp 722(2), Thr 321(1), Glu 734(2), Gly 368(1), Ala 362(1), Ile 339(1), Lys 362(1), Trp 286(1), Sar 377(1).
PREPARATION 16
Preparing A54145B Nucleus
A. Deacylating A54145B
A culture of Actinoplanes utahensis NRRL 12052, prepared in medium A, incubated for eight days at 30.degree. C. and stored at 4.degree. C., was used to inoculate 50 mL of a vegetative medium (medium A) in a 250-mL Erlenmeyer flask. The vegetative medium was incubated at 30.degree. C. on a rotary shaker at 250 RPM for 48 hours.
Incubated vegetative medium (5 mL) was used to inoculate 100 mL of sterile production medium (medium B) in a 500-mL Erlenmeyer flask. The production medium was incubated at 30.degree. C. on a rotary shaker at 250 RPM for 120 hours.
Semi-pure antibiotic A54145B (100 mg) in water (2 mL) was added to the production flask. The culture was then incubated at 30.degree. C. on rotary shakers at 250 RPM for another 20 hours to achieve complete deacylation.
B. Isolating A54145B Nucleus
Whole fermentation broth (100 mL at pH 6.8), prepared as described in Section A, was vacuum filtered. The mycelial cake was washed with water (20 mL), and the water wash was combined with the filtrate. This solution was adjusted to pH 4.5 with HCl and added to an HP-20 resin column (38 mL). The initial efluent (112 mL) and a water wash (adjusted to pH 3 with HCl, 111 mL) were discarded. The column was then eluted with CH.sub.3 CN:H.sub.2 O (3:1, 100 mL). This eluent was concentrated under vacuum and lyophilized to give 169 mg of crude A54145B nucleus.
C. Purifying A54145B Nucleus
The crude nucleus (169 mg) was dissolved in solvent No. 1 and injected on a preparative HPLC column, using the following system:
Column: Two 9.4-.times.250-mm Zorbax ODS (5.mu.) columns in series
Detection: UV at 280 nm
Flow Rate: 2 mL/min
The column was eluted a follows:
______________________________________Solvent No. Fractions.sup.a______________________________________1 1-202b 21-1532d 154-1683h 169-1864 187-200______________________________________ .sup.a Fraction volume: 4 mL
Fractions containing A54145B nucleus (#122-150) were combined, concentrated and lyophilized to give 39 mg of A54145B nucleus.
A54145B nucleus has the following characteristics:
Mol. wt. (FABMS): 1503
UV (EtOH): 218 nm (.epsilon. 31,193), 365 (.epsilon. 472), 279 (.epsilon. 5,308), 288 (.epsilon. 4,615)
IR (KBr): 3346, 3338, 3327, 3324, 3319, 3315, 3309, 1669, 1665 and 1540 cm.sup.-1
Analysis, Found: C, 41.20; H, 5.58; N, 12.33
Amino-Acid Analysis: Asp 1009(2), Thr 442(1), Glu 575(1), Gly 571(1), Ala 513(1), Ile 480(1), Lys 503(1), Trp 489(1), 3-MG 557(1).
PREPARATION 17
Preparing A54145C Nucleus
A. Deacylating A54145C
A production culture of Actinoplanes utahensis NRRL 12052 was obtained as described in Preparation 5, but using two 50-mL Erlenmeyer flasks each containing 10 mL of medium. Impure A54145C (10 mg) in water (0.2 mL) was added to each flask.
B. Isolating A54145C Nucleus
The culture was harvested and worked up as described in Preparation 16, using a 3-mL water wash and combining it with the filtrate. The wash/filtrate solution was adjusted to pH 4.5 with HCl and chromatographed over HP-20 resin (7 mL). The initial effluent (14 mL) and a water wash (adjusted to pH 3 with HCl, 10 mL) were discarded. The column was then eluted with CH.sub.3 CN:H.sub.2 O (3:1, 12 mL). This eluate was concentrated and lyophilized to give 35.6 mg of crude A54145C nucleus.
C. Purifying A54145C
The crude nucleus (35 mg) was dissolved in solvent No. 1 (3 mL) and injected onto a preparative HPLC column, using the system of Example 2.
The column was eluted as follows:
______________________________________Solvent No. Fractions.sup.a______________________________________1 1-22 23-662c 67-852e 86-1052h 106-1244 125-136______________________________________ .sup.a Fraction volume: 6 mL
Fractions containing A54145C nucleus (#75-81) were combined, concentrated and lyophilized to give 5.5 mg of A54145C nucleus.
A54145C nucleus has a molecular weight (FABMS) of 1489.
PREPARATION 18
Preparing N.sub.Lys -(t-BOC)-A54145
Crude A54145 (25 g) was dissolved in water (200 mL adjusted to pH 3.7). This solution was adjusted to pH 9.1 with 5N NaOH and stirred at ambient temperature. Di-tert-butyl dicarbonate (32 mL) and tert-butanol (50 mL) were added, and the solution was stirred for 41/2 hours. The solution was then lyophilized. The residue thus obtained was dissolved in water (400 mL) and extracted 3 times with equal volumes of dichloromethane. The aqueous phase was then concentrated and lyophilized to give 39 g of N.sub.Lys -(t-BOC)-A54145.
The reaction was monitored by analytical HPLC using the following system:
Column: 4.6-.times.250-mm Zorbax ODS (C.sub.18, 5.mu.)
Detection: UV at 289 and 223 nm
Flow Rate: 2 mL/min
Solvent: CH.sub.3 CN:MeOH:0.04M aq.NH.sub.4 OAc (25:12.5:62.5)
This reaction was repeated except that the pH was maintained at 8.5-9.0 with NaOH and only 7 mL of di-tert-butyl dicarbonate was added. Under these conditions, the reaction took place in only 80 minutes.
PREPARATION 19
Preparing N.sub.Lys -(t-BOC)-A54145 Nucleus
The procedure of Preparation 15, Section A, was repeated except that the vegetative medium had the following composition:
Medium C
______________________________________Ingredient Amount (g/L)______________________________________Sucrose 20Soybean flour 10K.sub.2 HPO.sub.4 1.2KH.sub.2 PO.sub.4 0.5MgSO .multidot. 7.sub.2 O 0.25Defoamer* 0.3Tap water q.s. to 1 LAdjust pH to 6.8 if necessary______________________________________ *Sag 471 (Union Carbide)
The first-stage medium had a volume of 800 mL 400 mL/2-L flask) and was incubated at 30.degree. C. on a rotary shaker for 72 hr.
The second stage medium (Medium C) had a volume of 950 L. The fermentation was carried out in a 350-gal. fermentor at 30.degree. C., stirring at 155 RPM with an air flow of 28 cfm for 72 hours.
The second-stage medium was used to inoculate 810 L of production medium (Medium B) in a 350-gal. fermentor. This fermentation was carried out at 30.degree. C., stirring at 130 RPM with an air flow of 10 cmf for 43 hours.
A portion of this production medium (15 L) was transferred to a 68-L fermentor and N.sub.Lys -(t-BOC)-A54145 (prepared from 50 g of A54145 as described in Preparation 16 and dissolved in water) was added. The fermentation was continued at 30.degree. C. until deacylation was complete as determined by HPLC to give N.sub.Lys -(t-BOC)-A54145 nucleus.
PREPARATION 20
Purifying N.sub.Lys -(t-BOC)-A54145 Nucleus
Whole fermentation broth (18 L), obtained as described in Preparation 19, was vacuum filtered with 2-3 L of filter aid (Hyflo Super-Cel). The mycelial cake was rinsed with water (2 L) and filtered. The water wash was combined with the original filtrate (18 L). The mycelial cake was extracted with methanol (4 L) and again filtered. The methanol extract was concentrated to remove the methanol, and the aqueous solution remaining was lyophilized. The lyophilized material was reconstituted in water (500 mL) and combined with the filtrate and water wash to give a total volume of 20.5 L.
This solution was adjusted to pH 4.7 with HCl mixed with HP-20 resin (2.4 L), stirred for 2 hours and then placed in a 2".times.60" column. The initial effluent (22 L) and the initial water wash (adjusted to pH 3.5 with HOAc, 14.5 L) were discarded. The column was then eluted with CH.sub.3 CN/H.sub.2 O in the following ratios and amounts: 5:95 (4 L); 1:9 (4 L); 15:85 (2 L); 3:7 (8 L). The 3:7 eluate was concentrated and lyophilized to give 23.0 g of purified N.sub.Lys -(t-BOC)-A54145 nucleus.
PREPARATION 21
Separation of N.sub.Lys -(t-BOC)-A54145F Nucleus and N.sub.Lys -(t-BOC)-A54145F Nucleus
N.sub.Lys -(t-BOC)-A54145 nucleus (23 g), prepared as described in Preparation 20, was subjected to preparative HPLC, using a Chromatospac 100 silica-gel column (4 L, Quantum LP-1/C18). The material was dissolved in Solvent 1 (200 mL) and added to the column. Elution was monitored by UV at 280 nm. The column was eluted at a flow rate of 60 mL/min as follows:
______________________________________Solvent No. Fractions.sup.a______________________________________1 1-5 3i 6-704 71-73______________________________________ .sup.a Fraction volume: 480 mL
Fractions were combined based on analytical HPLC, using the following system:
Column 4.6-.times.250-mm Zorbax ODS (5.mu.)
Detection: UV at 223 nm
Solvent: CH.sub.3 CN/MeOH/0.04 M aq. NH.sub.4 OAc (12.5:5:82.5)
Flow rate: 2 mL/min
From this column, fractions 28-31 gave 1.8 g of N.sub.Lys -(t-BOC)-A54145F nucleus, and fractions 39-42 gave 2.6 g of N.sub.Lys -(t-BOC)-A54145A nucleus.
N.sub.Lys -(t-BOC)-A54145F nucleus is deblocked using standard procedures to give A54145F nucleus.
A54145F nucleus has the following characteristics:
Mol. Wt. (FABMS): 1475
UV (EtOH): 218 nm (.epsilon. 30,672), 278 (.epsilon. 4,547), 289 (.epsilon. 3,883)
IR (KBr): 3367, 3355, 3345, 3340, 3387, 1667 and 1542 cm.sup.-1
Analysis, Found: C, 37.87; H, 4.88; N, 11.29
Amino-Acid Analysis: Asp 962 (2), Thr 415 (1), Glu 974 (2), Gly 510 (1), Ala 486 (1), Val 459 (1), Lys 488 (1), Trp 393 (1).
PREPARATION 22
Preparation of Synthetic A54145B
A. Acylation
N.sub.Lys -(t-BOC)-A54145B nucleus (930 mg, 0.58 mmoles) was dissolved in dimethylformamide (DMF) (anhyd.), and O-(n-decanoyl)-hydroxybenzotriazole active ester (335 mg, 1.16 mmoles) was added. The reaction mixture was stirred at room temperature under nitrogen for 3 hours when silica gel TLC, using a CH.sub.3 CN/H.sub.2 O/HOAc (70:25:5) solvent system, indicated no starting material remained. The volatile solvent was removed under vacuum to give a near-white tacky residue. The residue was triturated with diethyl ether/CHCl.sub.3 (2:1) (3.times.200 mL) and worked up to give 1.1 g of N.sub.Lys -(t-BOC)-N.sub.Trp -(n-decanoyl)-A54145B nucleus as a cream-white powder.
B. Deblocking
This product was added to a prechilled solution of TFA containing 10% anisole, and the solution was stirred for .about.35 min. under nitrogen. Volatile solvents were removed under vacuum to give the product as an off-white residue. This residue was triturated with iethyl ether/CHCl.sub.3 (3:1) (3.times.50 mL). The solids (TFA-salt) were collected by filtration and dissolved in H.sub.2 O (50 mL). The pH of this solution was adjusted to .about.6.3 with several drops of pyridine and filtered. The filtrate was lyophilized to give 940 mg of N.sub.Trp -(n-decanoyl)-A54145B nucleus.
PREPARATION 23
Effect of Lipid Precursors, Media and Feeding Enzymatic Soy Digest on A54145 Production
A54145 fermentations were carried out as in Preparation 3, but using the following three production media, with and without lipid feeding:
______________________________________Ingredient Amount (g/L)______________________________________Glucose 25.0Soybean grits 15.0Blackstrap molasses 3.0Acid-hydrolyzed casein 1.0CaCO.sub.3 2.5Tap water q.s. 1 liter______________________________________ (Pre-sterilization pH.about. 7.0; poststerilization pH.about. 7.1)
Medium B
______________________________________Ingredient Amount (g/L)______________________________________Soybean flour 20.0Glucose 5.0Blackstrap molasses 2.5Fe(SO.sub.4).(NH.sub.4).sub.2 SO.sub.4.6H.sub.2 O 0.6Silicon defoamer 0.2Polypropylene glycol 0.1(M.W. 2000)Potato dextrin 30.0Tap water q.s. to 1 liter______________________________________
Medium C
______________________________________Ingredient Amount (g/L)______________________________________Soybean flour 20.0Glucose 5.0Blackstrap molasses 2.5Fe(SO.sub.4).(NH.sub.4).sub.2 SO.sub.4.6H.sub.2 O 0.6Silicon defoamer 0.2Polypropylene glycol 0.1(M.W. 2000)Tap water q.s. to 1 liter______________________________________
Medium D
Medium C with an enzymatic-soy-digest (Hy Soy, Sheffield Products, Norwich New York) feeding.
Table VII summarizes the results of these studies.
TABLE VII______________________________________EFFECT OF LIPID PRECURSORS AND MEDIA ONYIELDS AND FACTOR SIDE CHAINS OF A54145IN A 165-L BIOREACTOR TotalLipid Antibiotic Factor Side Chains (%).sup.bMedium Precursor.sup.a (mcg/mL) iC.sub.10 nC.sub.10 aC.sub.11______________________________________A -- 97 68 20 12A nC.sub.10 179 20 79 1B -- 570 70 17 13B nC.sub.10 1046 7 91 2C -- 1100 76 14 10C nC.sub.10 /C.sub.18 :.sub.1 2316 19 74 7D nC.sub.10 /C.sub.18 :.sub.1 3570 21 71 8______________________________________ .sup.a nC.sub.10 = ethyl caprate nC.sub.10 /C.sub.18 :.sub.1 = ndecanoic acid in methyl oleate (1:1) .sup.b iC.sub.10 = 8methylnonanoyl nC.sub.10 = ndecanoyl aC.sub.11 = 8methyldecanoyl
PREPARATION 24
Effect of Amino-Acid Enrichment on A54145 Production
A54145 fermentations were carried out as in Preparation 2, Section A, but using the culture used in Preparation 3 and the following production medium:
______________________________________Ingredient Amount (g/L)______________________________________Glucose 30.00Soybean flour 25.0Blackstrap molasses 5.0CaCO.sub.3 4.0Fe(SO.sub.4).(NH.sub.4).sub.2 SO.sub.4.6H.sub.2 O 0.6Tap water q.s. 1 liter______________________________________
Different amino acids were added to study their effects on the A54145 nuclei and acyl side chains produced. Table VIII summarizes the results of these studies.
TABLE VIII______________________________________EFFECT OF AMINO ACID ENRICHMENT ON BIO-SYNTHESIS OF A54145 NUCLEI AND ACYL CHAINSIN SHAKEN FLASKS TotalAmino Level Antibiotic Nuclei.sup.a Acyl Chains.sup.a,bAcid (M) (%) A B C F iC.sub.10 nC.sub.10 aC.sub.11______________________________________ -- 100.sup.c 50 39 1 10 65 18 17L-Val .03 32 32 20 2 54 98 0 2L-Leu .02 56 42 40 2 16 76 7 17L-Ile .04 73 48 47 2 0 16 18 66L-Glu .02 85 49 39 1 11 63 20 16L-Asp .005 134 56 34 1 10 64 19 17L-Tyr .02 59 12 76 1 11 67 18 15______________________________________ .sup.a Percent of total produced .sup.b iC.sub.10 = 8methylnonanoyl nC.sub.10 = ndecanoyl aC.sub.11 = 8methyldecanoyl .sup.c 550 mcg/mL
Table IX summarizes the results of a similar study of the effect L-tyrosine has on A54145 production. Thus study was made in a 115-liter fermentation run.
TABLE IX______________________________________EFFECT OF L-TYROSINE ENRICHMENT ON BIO-SYNTHESIS OF A54145 NUCLEI IN A BIOREACTOR(115 L OF MEDIUM) TotalAmino Level Antibiotic Nuclei.sup.a Acyl Chains.sup.a,bAcid (M) (%) A B C F iC.sub.10 nC.sub.10 aC.sub.11______________________________________-- -- .sup. 100.sup.c 19 78 1 2 19 74 7L-Tyr 0.01 102 10 87 1 1 28 67 5______________________________________ .sup.a Percent of total produced .sup.b iC.sub.10 = 8methylnonanoyl nC.sub.10 = ndecanoyl aC.sub.11 = 8methyldecanoyl .sup.c 3200 mcg/mL
Comparing the results in Tables VIII and IX shows that the scale of the fermentation affects the amount of a) total antibiotic produced, b) nuclei produced and c) acyl side chains produced. Adding L-tyrosine decreased total antibiotic production in the shaken-flask fermentation, but did not adversely affect production in the tank fermentation. The unsupplemented shaken-flask fermentation produced more A nucleus and more iC.sub.10 side chain product, whereas the unsupplemented tank fermentation produced more B nucleus and more nC.sub.10 side chain. L-tyrosine increased the percentage of B-nucleus produced in both shaken flasks and tanks, but the effect was more pronounced in flasks.
Adding L-valine or L-leucine increased the percentage of F nucleus produced and the percentage of iC.sub.10 side chain product. This effect was more pronounced with L-valine.
Adding L-isoleucine increased the percentage of both B nucleus and aC.sub.11 side chain produced.
PREPARATION 25
An A54145 fermentation was carried out as described in Preparation 3 except that the following production medium was used:
______________________________________Ingredient Amount (g/L)______________________________________Soybean flour 30.0Blackstrap molasses 5.0Glucose 3.0Fe(SO.sub.4).(NH.sub.4).sub.2 SO.sub.4.6H.sub.2 O 0.6Deionized water q.s. 1 liter______________________________________
Antifoam agents were added, and the pH was adjusted from .about.6.2 to .about.7.2 with 5N NaOH.
Beginning about 23 hours after the fermentation was initiated, glucose was fed to the fermentation at a rate of approximately 6.5 g/L/day. Beginning at about 25 hours after the fermentation was initiated, a sterile solution consisting of decanoic acid and oleic acid (1:1, v/v) was fed to the fermentation at a rate of approximately 6.0 mL/L/day.
At about 117 hours after the fermentation was initiated, a feeding of enzymatic soy digest was initiated and continued at a rate of about 3.0 g/L/day.
The yield of A54145 from the fermentation after about 280 hours was 3969 mcg/mL. This yield is substantially greater than the yield of about 500 mcg/mL ordinarily obtained using similar conditions, but without the glucose, enzymatic soy digest and decanoic acid feeds used in this fermentation.
PREPARATION 26
Another series of fermentations was carried out using the procedures of Preparation 23 with Medium C, but adding different C.sub.4 -C.sub.18 -alkanoic acids and esters to enhance A54145 production. The results of these studies are shown in Table X.
TABLE X__________________________________________________________________________EFFECT OF LIPID PRECURSORS ON BIOSYNTHESISOF A54145 SIDE CHAINS IN A 165-L BIOREACTOR Total Known Side Chains RQ.sup.a A54145 Percent of Total New Analogs.sup.b,cPrecursor Calcd..sup.d Found.sup.e (%) iC.sub.10 nC.sub.10 aC.sub.11 C.sub.6 A C.sub.6 B C.sub.8 A C.sub.8 B C.sub.9 A C.sub.9 B__________________________________________________________________________None 1.0 1.0 100.sup.f 76 14 10Acetate 1.0 1.0 104 73 15 11Propionate 0.88 0.96 28 69 22 8Butyrate 0.8 0.93 49 28 58 15Hexanoate 0.75 0.83 56 2 2 -- 67 29Caprylate 0.73 0.8 84 17 9 5 33 36Nonanoate 0.72 0.85 95 -- -- -- 75 25Caprate 0.71 0.86 184 17 91 2Undecanoate 0.76 0.9 136 11 3 26 17 16Undecylenate 0.71 0.87 153 27 56 2Laurate 0.71 0.9 154 43 54 3Tridecanoate.sup.g 0.7 0.76 64 40 19 5 12 28Myristate 0.7 0.81 207 10 85 5Oleate 0.7 0.9 142 49 48 3Decyl Alcohol 0.67 0.86 157 22 75 3__________________________________________________________________________ .sup.a Respiration Quotient .sup.b Abbreviations as follows: "C.sub.6 A" = A nucleus with a C.sub.6 side chain .sup.c Undecanoate and tridecanoate precursors each produced two additional unknown factors [amounts: 13 and 14% (undecanoate) and 7 and 8 (tridecanoate) .sup.d For metabolism as sole carbon source .sup.e Represents glucose metabolism or cometabolism with glucose .sup.f 1100 mcg/mL .sup.g In 50% methyl oleate
EXAMPLES 1-2
Preparation of N.sub.Lys -(t-BOC)-N.sub.Trp -(n-Undecanoyl)-A54145A Nucleus (Compound 1) and N.sub.Trp -(n-Undecanoyl)-A54145A Nucleus (Compound 2)
A. n-Undecanoyl Trichlorophenyl Ester
Undecanoic acid (7.5 g) was added to a solution of 2, 4, 5-trichlorophenol (8.6 g) in tetrahydrofuran (THF) (175 mL, anhyd.). This mixture was chilled in an ice bath and stirred under nitrogen for 10 minutes. The reaction mixture was removed from the ice bath, and N,N-dicyclohexylcarbodiimide (DCC) (9.0 g) was added.
The reaction mixture was stirred overnight under nitrogen at room temperature and then was concentrated to a volume of about 100 mL under vacuum. The concentrate was filtered to remove precipitated material, and the filtrate was evaporated to dryness.
The residue obtained was dissolved in diethyl ether and hexane. This solution was concentrated under vacuum and filtered to remove the red-pink precipitate which formed. The filtrate was evaporated under vacuum. The white precipitate which formed was collected by filtration and dried to give 5.27 g of n-undecanoyl "active ester".
B. Acylation of N.sub.Lys -(t-BOC)-A54145A Nucleus
N.sub.Lys -(t-BOC)-A54145A nucleus (500 mg) was added to anhydrous DMF (15 mL) in a 50-mL round bottom flask. After the solution was purged with nitrogen, the undecanoyl "active" ester (300 mg) and hydroxybenzotriazole (HBT) (8 mg) were added. The reaction mixture was stirred under nitrogen at room temperature for 21 hours and then concentrated under vacuum to a low volume (about 1/10th). Diethyl ether (30 mL) was added, and the precipitate which formed was separated by sonicating the suspension and filtering. The precipitate was washed twice with diethyl ether (30 mL each) and dried under vacuum to give 425 mg of N.sub.Lys -(t-BOC)-N.sub.Trp -(n-undecanoyl)-A54145A nucleus (Compound 1).
C. Deblocking
The t-BOC derivative obtained in Section B was dissolved in TFA (5 mL) containing anisole (0.5 mL) and stirred at room temperature for one hour under nitrogen. The solution was concentrated under vacuum to a low volume. A precipitate was formed by adding CH.sub.2 Cl.sub.2 /diethyl ether (2:1, 30 mL). The precipitate was separated by filtration and washed twice with CH.sub.2 Cl.sub.2 /diethyl ether (2:1, 30 mL each) and dried. The residue was dissolved in water (10 mL). The pH of the solution (1.72) was adjusted to 6.2 with pyridine, and the solution was lyophilized to give 645.5 mg of product.
D. Purification by Preparative HPLC
The product obtained in Section C was purified by preparative HPLC, using the following conditions:
Column: 1-.times.12-in. Zorbax ODS (Dupont, 12.mu.)
Detection: UV at 280 nm
Flow Rate: 9 mL/min
The material, dissolved in solvent 1 (5 mL), was injected onto the column. The column was eluted at a flow rate of 9 mL/min, collecting 18-mL fractions, as follows:
______________________________________ Solvent Fractions______________________________________ 1 1-10 3g 11-63 4 64-80______________________________________
Fractions were monitored by analytical HPLC, using the following conditions:
Column: 4.6-.times.250-mm Zorbax ODS (Dupont, 5.mu.)
Detection: UV at 223 nm
Flow Rate: 2 mL/min
Solvent: CH.sub.3 CN:MeOH:0.04 M aq. NH.sub.4 OAc (25:12.5:62.5)
Fractions containing the product were combined, concentrated and lyophilized to give 241 mg of N.sub.Trp -(n-undecanoyl)-A54145A nucleus (compound 2).
FABMS (M+1): 1658
UV (EtOH): 280 nm (.epsilon. 4,752) and 220 nm (.epsilon. 34,413).
EXAMPLES 3-4
Preparation of N.sub.Lys -(t-BOC)-N.sub.Trp -(n-Dodecanoyl)-A54145A Nucleus (Compound 3) and N.sub.Trp -(n-Dodecanoyl)-A54145A Nucleus (Compound 4)
A. Preparation of n-Dodecanoyl Trichlorophenyl Ester
n-Dodecanoyl trichlorophenyl ester was prepared by heating n-dodecanoyl chloride (2.5 g) and 2,4,5-trichlorophenol (1.75 g) in diethyl ether (40 mL). The reaction mixture was stirred under nitrogen at room temperature until materials were dissolved. Pyridine (2 mL) was added, and the mixture was stirred under nitrogen at room temperature for 21 hours.
The reaction mixture was filtered to remove the precipitate which formed. The precipitate was washed with diethyl ether, and the diethyl ether wash was combined with the filtrate and concentrated under vacuum to give crude product.
This product was purified over a 2.5-.times.30-cm silica-gel column (Woelm), eluting the n-dodecanoyl "active" ester with toluene. The fractions containing the ester were combined and concentrated to dryness to give 3.0 g of the "active" ester.
B. Acylation of N.sub.Lys -(t-BOC)-A54145A Nucleus
The active ester prepared in Section A was used to acylate N.sub.Lys -(t-BOC)-A54145A nucleus, using a procedure like that described in Examples 1-2, Section B, to give 438 mg of N.sub.Lys -(t-BOC)-N.sub.Trp -(n-dodecanoyl)-A54145A nucleus (Compound 3).
C. Deblocking
The material obtained in Section B was
deblocked using the procedure of Examples 1-2, Section C, to give 630 mg of product.
The product was purified by preparative HPLC using the procedure of Examples 1-2, Section D, with the following eluting solvents:
______________________________________ Solvent Fractions______________________________________ 1 1-13 3g 14-86 4 87-105______________________________________
The column was monitored by analytical HPLC as in Examples 1-2 except that the solvent system used was CH.sub.3 CN:0.04 M aq. NH.sub.4 OAc (35:65). Fractions containing the product (#42-49) were combined, concentrated and lyophilized to give 225 mg of N.sub.Trp -(n-dodecanoyl)-A54145A nucleus(Compound 4).
FABMS (M+1): 1671
UV (EtOH): 280 nm (.epsilon. 5,056) and 219 nm (.epsilon. 36,055).
EXAMPLES 5-6
Preparation of N.sub.Lys -(t-BOC)-N.sub.Trp -(n-Nonanoyl)-A54145A Nucleus (Compound 5) and N.sub.Trp -(n-Nonanoyl)-A54154A Nucleus (Compound 6)
A. n-Nonanoyl Trichlorophenyl Ester
The procedure of Examples 1-2, Section A, was followed, except using 2.5 g (2.7 mL) of nonanoic acid, 2.9 g of 2,4,5-trichlorophenol, 55 mL of anhydrous THF and 3.0 g of DCC to give approximately 3 g of n-nonanoyl "active" ester.
B. Acylation of N.sub.Lys -(t-BOC)-A54145A Nucleus
The active ester from Section A was used to acylate N.sub.Lys -(t-BOC)-A54145A nucleus, using a procedure analogous to that of Examples 1-2, Section B, to give 437.1 mg of N.sub.Lys -(t-BOC)-N.sub.Trp -(n-nonanoyl)-A54145A nucleus.
C. Deblocking
The product obtained in Section B was deblocked, using a procedure like that of Examples 1-2, Section C, to give 605 mg of product.
The product was purified by preparative HPLC as described in Examples 1-2, but using the following solvents:
______________________________________ Solvent Fractions______________________________________ 1 1-37 3c 38-132 4 133-146______________________________________
The column was monitored by analytical HPLC as an Example 1, except that the solvent system was Ch.sub.3 CN:MeOH:0.04 M aq. NH.sub.4 OAc (22.5:7.5:70). Fractions containing the product (#91-110) were combined, concentrated and lyophilized to give 178 mg of N.sub.Trp -(n-nonanoyl)-A54145A nucleus.
FABMS (M+1): 1630
UV (EtOH): 279 nm (.epsilon. 5,459) and 220 nm (.epsilon. 39,124).
EXAMPLES 7-8
Preparation of N.sub.Lys -(t-BOC)-N.sub.Trp -(n-Decanoyl)-A-54145F Nucleus (Compound 9) and N.sub.Trp -(n-Decanoyl)-A54145F Nucleus (Compound 10)
The n-decanoyl trichlorophenyl ester was prepared as described in Examples 1-2, Section A. This material was used to acylate N.sub.Lys -(t-BOC)-A54145F nucleus, using a procedure analogous to that of Examples 1-2, Section B, to give about 400 mg of N.sub.Lys -(t-BOC)-N.sub.trp -(n-decanoyl)-A54145F nucleus (Compound 9).
This material was deblocked using a procedure like that of Examples 1-2, Section C, to give 527.05 mg of product.
The product was purified using preparative HPLC as described in Examples 1-2, Section D, except that fractions of 16 mL were collected and the flow rate as 8 mL/min. The following solvents were used:
______________________________________ Solvent Fractions______________________________________ 1 1-12 3d 13-89 4 90-100______________________________________
The column was monitored by analytical HPLC as in Examples 1-2, Section D, except that the solvent ratio was 25:5:70. Fractions containing the product (#55-67) were combined to give 160.37 mg of N.sub.Trp -(n-decanoyl)-A54145F nucleus (Compound 10).
EXAMPLES 9-10
Preparation of N.sub.Lys -(t-BOC)-N.sub.Trp -(n-Tetradecanoyl)-A54145A Nucleus (Compound 25) and N.sub.Trp -(n-Tetradecanoyl)-A54145 Nucleus (Compound 26)
The general procedure described in Preparation 22 was followed, but using N.sub.Lys -(t-BOC)-A54145A nucleus (550 mg), O-(n-tetradecanoyl) active ester (400 mg) and DMF (50 mL) to give 630 mg of Compound 25:
FABMS (P+1+Na): 1822.
Deblocking Compound 25 as in Examples 1-2, Procedure C, gave 550 mg of Compound 26:
FABMS (P+1): 1700.
EXAMPLE 11
A54145-Improved Broiler Starter Ration
The following recipe provides a balanced broiler starter ration adapted to feed chicks for improved weight gains.
______________________________________Ingredient Percent Lbs/Ton______________________________________Ground Yellow Corn 55.99 1119.8Animal - Vegetable Fat 3.13 62.6Soybean Meal (48%) 32.37 647.4Fish Meal 2.50 50.0Feather Meal - Hydr. 2.50 50.0Dicalcium Phosphate 1.66 33.2Ground Limestone 0.77 15.4Salt 0.30 6.0Vitamin Premix.sup.1 0.50 10.0Trace Mineral Premix.sup.2 0.10 2.0Methionine Hyd. Anal. 0.13 2.6A54145 Premix.sup.3 0.05 1.0Total 100.00 2000.0______________________________________ .sup.1 Vitamin premix provides 3000 IU of vitamin A, 900 ICU of vitamin D.sub.3, 40 mg of vitamin E, 0.7 mg of vitamin K, 1000 mg of choline, 70 mg of niacin, 4 mg of pantothenic acid, 4 mg of riboflavin, 100 mcg of vitamin B.sub.12, 100 mcg of biotin and 125 mg of ethoxyquin per kg of complete feed. .sup.2 Trace mineral premix provides 75 mg of manganese, 50 mg of zinc, 2 mg of iron, 1 mg of iodine and 0.1 mg of selenium per kg of complete feed .sup.3 Premix containing 1-20 g of formula 1a compound per pound premix provides a corresponding 1-20 g of A54145 compound/ton of finished feed.
This diet is usually fed to chicks from day 1 until a day between 17 and 28.
EXAMPLE 12
A54145-Improved Broiler Finisher Ration
The following recipe provides a balanced broiler finisher ration adapted to feed chicks for improved weight gains:
______________________________________Ingredient Percent Lbs/Ton______________________________________Ground Yellow Corn 66.35 1327.0Animal - Vegetable Fat 1.53 30.6Corn Glut. Meal (60%) 4.00 80.0Soybean Meal (48%) 19.19 383.8Fish Meal 2.50 50.0Feather Meal - Hydr. 2.50 50.0Dicalcium Phosphate 1.71 34.2Ground Limestone 0.83 16.6Salt 0.30 6.0Vitamin Premix.sup.1 0.50 10.0Trace Mineral Premix.sup.2 0.10 2.0Methionine Hyd. Anal. 0.15 3.0Lysine HCl 0.29 5.8A54145 Premix.sup.3 0.05 1.0Total 100.00 2000.0______________________________________ .sup.1 Vitamin premix provides 3000 IU of vitamin A, 900 ICU of vitamin D.sub.3, 40 mg of vitamin E, 0.7 mg of vitamin K, 1000 mg of choline, 70 mg of niacin, 4 mg of pantothenic acid, 4 mg of riboflavin, 100 mcg of vitamin B.sub.12, 100 mcg of biotin and 125 mg of ethoxyquin per kg of complete feed. .sup.2 Trace mineral premix provides 75 mg of manganese, 50 mg of zinc, 2 mg of iron, 1 mg of iodine and 0.1 mg of selenium per kg of complete feed .sup.3 Premix containing 1-20 g of formula 1a compound per pound of premi provides a corresponding 1-20 g of A54145 compound/ton of finished feed.

Number	Name	Date
RE31396	Hamill et al.	Sep 1983
RE32333	Hamill et al.	Jan 1987
RE32455	Hamill et al.	Jul 1987
4024245	Hoehn et al.	May 1977
4208403	Hamill et al.	Jun 1980
4287120	Abbott et al.	Sep 1981
4320052	Abbott et al.	Mar 1982
4320053	Abbott et al.	Mar 1982
4320054	Abbott et al.	Mar 1982
4322338	Abbott et al.	Mar 1982
4331594	Hamill et al.	May 1982
4482487	Abbott et al.	Nov 1984
4524135	Abbott et al.	Jun 1985
4537717	Abbott et al.	Aug 1985
4638047	Szelke et al.	Jan 1987

Derivatives of A54145 cyclic peptides

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