CA1200777A - A-21978c cyclic peptide derivatives and their production - Google Patents

Abstract

Abstract of the Invention A-21978C cyclic pep ides of the formula Wherein R is selected from the group consisting of hydrogen, an amino-protecting group, 8-methyldecanoyl, 10-methylundecanoyl, 10-methyldodecanoyl, the specific C10-alkanoyl group of A-21978C factor C0 and the specific C12-alkanoyl groups of A-21978C factors C4 and C5; R1 and R2 are, independently, hydrogen or an amino-protecting group, and salts thereof, are prepared by enzymatic deacylation of an antibiotic selected from A-21978C complex, A-21978C factors C0, C1, C2, C3, C4 and C5 and blocked A-21978C complex and factors C0, C1, C2, C3, C4 and C5, using an enzyme produced by the Actinoplanaceae, preferably by Actinoplanes utahensis.
These A-21978C cyclic peptides or a pharmaceutically-acceptable salt thereof are useful intermediates for preparing new semi-synthetic antibiotics.

Description

~2~3t~7~

X~5279 -1-CYCLIC PEPTIDE DERIVATIVES AND THEIR PRODUCTION
This invention relates to novel derivatives of cyclic peptides which possess antibiotic properties and to the method of producing these antibiotic deriv-atives by semisynthetic means.
There is a great need to develop new anti-biotics because of the great possibility and constant threat that antibiotic-specific resistant strains of 1~ pathoyenic microoryanisms will develop. In particular, pathogens within the gram positive genera Staphylococcus and Strept~cccc~s often are resistant to commonly used antibiotics such as penicillin and erythromycin;
see for example, W.O. Foye, Principles of Medicinal 1~ Chemistry, pp. 684-686 (1974).
In accordance wich the invention, new deri.v-a-tives of the A-21978C cyclic peptides and the pharma-ceutically-acceptable salts thereof, have been found to be ef~ective agents.
These new derivatives can be prepared by reacting the A-21978C nucleus or protected derivatives thereof, prepared by deacylating the appropriately blocked A-21978C complex or any of its appropriately blocked indlvidual factors C0, Cl, C2, C3, C~, or C5, with the desired acylating agent or an activated derivative thereof.

~1.2~ 7 In accordance with the present invention, there are provided A-21978C cyclic peptides of formula 1:
p ~IHR

H~/H~< H~ f ,~
o=O O=o\ f;H~
NH 1~02H

15 =~ \9 ~02C/~f~ f~l~ "~

~02C' in which R is selected from the group consisting of hydrogen, an amino protecting group, 8-methyldecanoyl, 10-methylundecanoyl, 10-methyldodecanoyl, the specific C10-alkanoyl group of A-21978C factor C0 and the specific C12-alkanoyl groups of A-21978C factors C4 and C5; R and R are, independently, hydrogen or an amino-protecting group; provided that, when R is other than hyar~gen or an amino-protecting group, at least one of Rl and R2 must be an amino-protecting group; or ,~

` ~L2~3777 pharmaceutically-accepta~le salts of these peptides. These peptides are useful as intermediates in the preparation of semi-synthetic antibacterial agent~.
The compounds of ormula 1 in which R is other than hydrogen or an amino-protecting group are blocked antibiotic A-21978C factors C0, Cl, C2, C3, C4 and C5. These blocked antibiotic compounds are useful intermediates to certain peptides of formula 1, i.e.
those in which R is hydrogen and at least one of Rl and R is an amino-protecting group.
The compound of formula 1 wherein R, Rl and R2 each represent hydrogen is the common cyclic peptide present in antibiotic A-21978C factors C0, Cl, C2, C3, C~ and C5, For con~enience, this compound will be called the A-21978C nucleus. This compound can be represented also by formula 2:
L-Asp D~Ala Gly '¦` D--Ser L-Asp ~ 3~1G
L-Orn ~ L-~yn Gly L~Thr L-Asp L-Asn L-Trp ~IH2 ~tl~ '7'~

X-5279 ~4~

in which 3MG represents L-threo-3-methylglutamic acid.
In another aspect, this inventio~ relates to a method of enzymatically deacylating an antibiotic selected from A-21978C complex, A-21978C factors C0, Cl, C2, C3, C4, and C5, blocked A-21978C complex, and blocked A-21978C factors C0, Cl, C2, C3, C4 and C5.
The naturally occurring A-21378C factors have a common cyclic peptide nucleus, but each has a different fatty acid group. The method of removing the fatty acid 10 groups from the A-21978C factors and blocked factors provided by -this invention comprises exposing the anti-biotic or blocked antibiotic, in an aqueous medium, to an enzyme produced by a microorganism of the family Actinoplanaceae until substantial deacylation is 15 accomplished.
A preferred method of this invention com-prises using an en~yme produced by the microorganism Actlnoplanes utahensis NRRL 12052 to cleave the fat-ty acid side chain. Deacylation is ordinarily accom-plished by adding the appropriate antlbiotic or blocked antibiotic to a culture of A. utahensis and permitting the culture to incubate until deacylation is accom-plished. The A-21978C cyclic peptide or blocked pep~
tide thereby obtained is separated from the fermenta-tion broth by known methods.
The A-21978C cyclic peptides of formula 1 are useful in that they can be reacylated to provide new antibiotic substances.

7~

The accompanyin~ infrared absorption spectra, run in KBr, represent the following:
Figure 1 - A-21978C nucleus (formula 1, R, R , R = H) Figure 2 - A-21978C Norn-t-BOC nucleus (formula 1, R and R = H; R
tert-butyloxycarbonyl) In this specification, the following abbreviations, most of which are commonly known in the art, are used: -Ala: alanine Asp: aspartic acid Asn: asparagine Gly: glycine Kyn: kynurenine Orn: ornithine Ser: serine Thr: threonine Trp: tryptophan t-BOC: tert-butoxycarbonvl Cbz: benzyloxycarbonyl DMF: dimethylformamide THF: tetrahydrofuran HPLC: high-performance liquid chromatography TLC: thin-layer chromatography . UV: ultraviolet ., ~

~-5279 -6 The A-21978C antibiotics are closely related, acidic peptide antibiotics and are described in U . S .
Patent No. 4,208,403, of R.L. Hamill and M.~. Hoehn, issued June 17, 1980. As drscribed in U.S. Patent No. 4,208,403, 5 ~he A-21978 anti}:)iotic complex contains a major com ponent, factor C, which is itself a complex of closely related factors. A-219 78 factor C, which is called the A-21978C complex, contains individual factors C0, Cl, C2 ~ C3, C4 and C5 Factors Cl ~ C2 and C3 are ma jor 10 factors; and factors C0, C4 and C5 are minor factors.
The structure of the A-21978C factors is shown in formula 3:
L--Asp ~AI a ~
1` ~S~r L--Asp ~
1` 3MG
L-Orn ~1,

2 0 '~ L~yn Gly O /
r~\ /
L--Thr L--Asp 2 5 L-Asn L--Trp NH
R' .,~

~1~~'~3~
.~ o ~7 ~-5279 7 in which 3MG represents L-threo-3 methylglutamic acid, and R represents a specific fatty acid moiety~ The specific R groups of the factors are as follows.
A~21978C FactorR Moiety Cl 8-methyldecanoyl C2 10-methylundecanoyl C3 10 methyldodecanoyl CO C10-alkanoyl*
c4 C12-alkanoyl*
c5 C12-alkanoyl*
*Identity not yet determined It is extremely difficult, when confronted with the problem of deacylating a peptide antibiotic to know whether an enzyme exists which can be used for this purpose. Finding such an enzyme is even more difficult when the substrate antibiotlc contains a cyclic peptide nucleus. Because enzymes have a high degree of specificity, differences in the peptide moiety and in the side chain of the substrate will affect the outcome of the deacylation attempt. In addition, many microorganisms make a large number of peptidases which attack different portions of the peptide moiety. This frequently leads to intractable mi~tures of products.
In each of the A~21978C antibio-tics (formula

3), the fatty acid side chain (RN) is at-tached at -the a-amino group of the tryptophan residue. We have discovered that the fatty acid side chain can be cleaved by an enzyme without affec~ing the chemical integrity of the xemainder of the A-21978C peptide.

~2~77~

~-5279 -8-The present in~ention also provides a novel process for obtaining A-21978C cyclic peptides having formula 1 IIJHR

1~ ~ T c~ ~

0 0~ ~H2 ~ H ~ /

~N~ IH 02H \~
0=~ 0=O H
~o ÇH3 H H~ H
HO~C/H~ R2 H
H02~
in which R is selected from the group consisting of hydrogen, an amino-protecting group, 8-methyldecanoyl, 10-methylundecanoyl, 10-methyldodecanoyl, the specific 25 C1~-alkanoyl group of A-21978C factor CO and the spe-cific C12-alkanoyl moieties of A-21978C factors C4 and C5; R and R are, independently, hydrogen or an amino-protecting group; provided that, when R is other than hydrogen or an amino protecting group, at least one of ~9~377~
~-5279 ~9 Rl and R2 must be an amino-protecting group; or to a pharmaceutically~acceptable salt of these compounds which comprlses:
(a) the protec~ion of one or more of the amino groups NHR, NHR and NHR of a compound of formula (1) as defined herelnabove in which R, R or R are hydrogen so as to prepare a c~mpound of said formula ~1) in which one or more of R, Rl and ~2 represent an amino~
protecting group, provided that one or more of R, Rl, and R2 were initially hydrogen, and/or (b) the enzymatic deacylation using an enzyme produced by a micxoorganism of the family Actinoplanaceae of a compound of ~ormula t.l) in which ~ is other than hydrogen or an amino pro~ecting group, so as to prepare a compound of ~O~Dula ( 1 ~ in which R is hydrogen, and, optionally, if so desired, removing any R or R amino protecting groups which may be present in the product of the reaction; and if desired, ~orming a pharmaceutically acceptable salt of said compound o~ formula (1~.
The cyclic peptides of formula 1 or their pharma ceutically-acceptable salts are useful in~ermediates in the preparation of new semi-synthetic antibacterial agents which are especially useful against gram-posi-tive microorganisms.

~2~3~3~'7'7 -9a-The term "amino~protecting group" refer~ to a recognized amino-protecting group which is compatible with the other functional groups in the A-21978C ~ole-cule. Preferably, amino-protecting groups are those which ca~ be readily removed subsequently. Examples of suitable protecting groups can be found in "Protective Groups in Organic Synthesi " by Theodora W. Greene, John Wiley and Sons, New York, 1981, Chapter 7. Espe-cially preferred amino~protecting groups are the t _ -butoxycarbonyl and benzyloxycarbonyl groups.
The cyclic peptides of formula 1 in which R
is hydrogen include the common cyclic peptide of A-21978C
~ 1~ 2~ C3, C4 and C5 (A~21978C nucleus) and blocked derivatives of A-21978C nucleus. A-21978C
nucleus, 1.e. the compound of formula 1 in which ~a_h of R, Rl and R2 represents hydrogen, is alternately de-scribed by formula 2.
A-21978C nucleus has the following charac-teristics:
Form: white amorphous solid which fluoresces under shoxt-wave UV
Empirical formula: ~62~83N1725 Molecular weight~ 1465 ~5 ~-5279 -10-Solubility: soluble in water Infrare~ absorption spectrum (KBr): shown in Figure 1 of the accompanying drawings; ab~orption maxima are observed at the fol~owing frequensies - 1 ~
3300 (broad), 3042 ~weak), 2909 (weak3~ 1655 (strong), 1530 (strong), 1451 (weak), 1399 (medium), 1~22 (medium), 1165 (weak), 1063 (weak) and 758 (medi-um to weak) 1~ U]traviolet (UV) absorption spectrum (meth-anol): UV maxima 223 nm (~ 41,482) and 260 nm (~ 8,687) Electrometric titration (66~ aqueous dimethyl-formamide): indicates the presence of four titratable groups with PKa valuec of about 5.2, 6.7, 8.5 and 11.1 (initial pH 6.12) A particularly useful cyclic peptide of formula 1 is the compound in which R and Rl represent hydrogen and R2 represents tert-butoxycarbonyl. This compound is designated "t-BOC nucleus" for convenience herein. A-21978C t-BOC nucleus has the following characteristics:
Form: white amorphous solid which f luor2sces under short-wave UV
Empirical formula: C67HglN17O27 Molecular weight: 1565 Solubility: soluble in water Infrared absorption spectrum (XBr): shown ir. Figure 2 of the accompan~yillg drawings; absorption maxima are observed at the following fre~uencies ~cm~l):

.~ ", 3345 Ibroad), 3065 (~eak), 2975 (weak), 2936 (weak), ~1710 (shoulder), 1660 (strong), 1530 (strong), 1452 (wealc), 1395 (medium), 13~8 (weak), 1341 (weak), 1250 (medium), 1228 (medium), 1166 (medium to weak), and 1063 (weak).
Vltraviolet absorption spectrum (90% ethanol):
UV maxima 220 nm (~42,000) and 260 nm (E10,600) High-performance liquid chromatography:
retention time = 6 min o~ 4.6- x 300-nm silica-gel 10 CI8 column, using H2O/CH3CN/CH3OH (80:15:5) solvent containing 0.2% NH40Ac at a flow xate of 2 ml/min with UV detection.
The blocked A-21978C factors of this invent-ion are those compounds of formula 1 in which at least 15 one of R and R is an amino~protecting group and R is selected from 8~methyldecanoyl, 10-methylundecanoyl, 10-methyldodecanoyl, the specific C10-alkanoyl group of A-21978C factor C0 and the specific C12-alkanoyl groups of A-21978C factors C4 and C5~
The A-21978C blocked factors and cyclic peptides of this invention are capable of forming salts which are also part of this invention. Such salts are useful, for example, Eor separating and purifying the compounds. Pharmaceutically-acceptable alkali-metal, 25 alkaline-earth-metal, amine and acid-addition salts are particularly useful. "Pharmaceutically-acceptable"
salts are salts which are useful in the chemotherapy of warm-blooded aminals.
For example, the A-21978C cyclic peptides of 30 formula 1 have four free carboxyl groups which can form l"b~,;i..

~a2~ '7'~

salts. Partial, mixed and complete salts of these carboxyl groups are contemplated therefore, as part of this invention. In preparing these salts, pH levels greater than 10 should be avoided due to the instability of the compounds at such levels~
Representative and suitable alkali~metal and alkaline-earth metal salts oE the A-21978C cyclic peptides oE formula 1 include the sodium, po-tassium, lithium, cesium, rubidium, barium, calcium and mag~
nesium salts. Suitable amine salts of the A-21978C
cyclic peptides include the ammonium and the primary, secondary, and tertiary Cl-C4-alkylammonium and hydroxy-C2-C4-alkylammonium salts. Illustrative amine salts include, among others, those formed by reaction of an A-21978C cyclic peptide with ammonium hydroxide, methyl-amine, sec-butylamine, isopropylamine, diethylamine, di-isopropylamine, ethanolamine, triethylamine, and 3-amino-1-propanol.
The alkali-metal and alkaline~earth-metal 20 cationic salts oi- the A-21978C cyclic peptides of ~ormula _ are prepared according to procedures commonly used for the preparation of cationic salts. For e~ample, the free acid form oi the ~-21978C cyclic peptide is dissolved in a suitable solvent such as warm methanol or ethanol; a solution containing the stoichio-metric quantity oE -the desired inorganic base in aqueous methanol then is addedA The sal-t thus formed can be isolated by routine methods, such as Eiltration or evaporation o-E the solvent.

~2~ '7 The salts formed with organic amines can be prepared in a similar manner, For example, the gaseous or liquid ami.ne can be added to a solution of an A-21978C cyclic peptide in a suitable solvent such as acetone; the solvent and excess amine can be removed by evaporation~
The A-21978C cyclic peptides of this in-vention also have free amino groups and can form therefore, acid addition salts which are also part of this invention. Representative and suitable`acid~
addition salts of the A-21978C cyclic peptides lnclude, among others, those salts formed by standard reaction with both organic and inorganic acids such as, for example, hydrochloric, sulfuric, phosphoric, acetic, 1~ succinic, citric, lactic, maleic, fumaric, palmitic, cholic, pamoic, mucic, D-glutamic, d-camphoric, glutaric, glycolic,.phthalic, tartaric, lauric, stearic, sali~
cy'ic, methanesulfonic, benzenesulfoni.c, sorbic, picric, benzoic, and cinnamic acids.
Preparation of the A-21978C Cyclic P ptides The A-21978C cyclic peptides of formula 1 wheren R is hydrogen are obtained by deacylating a peptide antibiotic selected from the group consisting of A-21978C factors C0, Cl, C2, C3, C4 and C5 a~d blocked A-21978C factors C0, Cl, C2, C3, C4 and C5.
I. Preparation of the Substrates _ A-21978C factors C0, Cl, C2, C3, 4 5 are prepared as described in U.S. Patent No. 4,208,403.
These factors are components of the A-21978C complex which is part of the A-21978 comple~.

y The A-21978 complex may be produced by cul-turing Streptomyces roseosporus NRRL 11379 (deposited .
August 29, 1978) under submerged aerobic fermentation condi-tions until a substantial level of antibiotic activity is produced. The A-21978 complex is separated by filtering the fermentation broth, lowering the pEI oc the filtrate to about pH 3, allowing the complex to precipitate, and separating the complex by filtration.
The separated complex may be purified further by ex-traction techniques. For isolation of the individualA-21978C complex and factors, chromatographic separations are required.
Blocked A-21978C complex and the blocked A-21978C factors C0, Cl, C2, C3, C4 a 5 invention (compounds of formula 1 wherein R is selected from the group consisting of 8-methyldecanoyl, 10~
methylundecanoyl, 10-methyldodecanoyl, and the C10-and C12-alkanoyl groups of factors C0, C4 and C5) are prepared using procedures for protecting amino groups in peptides. The protecting groups are selected from the various known amino~protecting groups such as, for example, ben~yloxycarbonyl, t~butoxycarbonyl, t-amyloxy-carbonyl, isobornyloxycarbonyl, aclamantyloxycarbonyl, o~nitrophenylthio, diphenylphosphinothioyl, chLoro~ or nitrobenzyloxycarbonyl Blocked A-21978C complex is especially advan-tageous as a substrate. It is prepared from the A-21978C
complex, thereby avoiding the separation steps required -to obtain the individual A-21978C factors; but, when it is deacylated, it gives a sinc~le product, i.e. the appropriately blocked nucleus.

77'~

II. The Deacylation Procedure A. Preparation of th~
1. The Producing Microorganisms The en~ymes which are useful for deacylation of A-21978C factors C0, Cl, C2, C3, 4 5 blocked factors C0, Cl, C2, C3, C4 5 duced by certain microorganisms of the family Actino-planaceae, preferably the microorganism Actinoplanes 10 utahensis NRRL 12052 (deposited October 9, 1979~.
Although a preferred method of cultivating A. utahensis NRRL 12052 to produce this enæyme is described in Example 1, those skilled in the art will recognize that other methods may be used.
The Actinoplanaceae are a family of micro-organisms of the order Actinomycetales. First de-scribed by Dr. John No Couch, this family was estab-lished in 1955 _J Eli~ha L~itchell Sci. Soc. 71, 148-155 (1955)]. The characteristics of the family and of many individual genera are found in "Bergey's Manual of Determinative Bacteriologyl', 8th ed., R. E. Buchanan and N. E. Gibbons, Eds., The Williams & Wilkins Co., Bal-timore, Md., 1974, pages 706-723. Ten genera have thus far been distinguished: I. Actinoplanes (the type genus and thus far the most common genus); II. Spiril-lospora; III. Streptosporangialm; IV. Amorphosporang-ium; V. Ampullariella; VI. Pilimelia; VII. Planomono-.
spora; VIII. Planobispora; IX. Dactylosporangium; and X. Kitasatoa.

77~
X~5279 Some of the species and varieties which havebeen isolated and characterized so far are: Actino~
planes philippinensis, Actinoplanes armeniacus, Actino planes utahensis, and Actinoplanes missouriensis;
Spirillospora albida; Streptosporiangium roseum, - _ ~
Streptospoxangium vulgare, Streptosporangium roseum =
var. hollandensis, Streptosporangium album, Strepto-sporangium viridialbum, Amorphosporangium auranticolor, Ampullariella regularis, Ampullariella campanulata, Ampullariella lobata, Ampullariella digitata, Pilimelia terevasa, Pilimelia anulata, Planomonospora paronto-.
spora, Planomonospora venezuelensis, Planobispora _ongispora, Planobispora rosea, _actylosporangium aurantiacum, and ~ thailandense.
The genus Actinoplanes is a preferred source ~
of the enzyme which is useful for this invention.
Within the genus Actinoplanes, the species Actinoplanes utahensis is an especially preferred source.
Cultures of other representative useful species are available to the public from the Northern Regional Research Center, Agricultural Research Culture Collection (NRRL), U.S. Depar-tment of Agriculture, 1815 North University St., Peoria, Illinois 61604, U.S.A., under the ~ollowing accession numbers:
Actinoplanes utahensis NRRL 12052 (deposited October 9, 1979) Actinoplanes missouriensis NRRL 12053 (deposited October 9, 1979) Ac-tinoplanes sp. N~RL 8122 (deposited Ocrober 17, 1975) Actinoplanes sp. NRRL 12065 (deposited November 5, 1979) Streptosporangium roseum var. hollandensis NRRL 12064 (deposited November 5, 197~) 7~7 A. utahensis WRRL 12052 was derived from a parent culture which was also deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852 (A. utahensis ATCC 14539).
The A. utahensis ATCC 14539 culture may also be used as a source of the enzyme.
A. missouriensis NRRL 12053 was derived from a culture which was also deposited with ATCC (A.
missouriensis ATCC 14538) and which is another source __ of the enzyme.
The effectiveness of any given strain of microorganism wi-thin the family Actinoplanaceae for carrying out the deacylation of this invention is determined by the following procedure. A suitable growth medium is inoculated with the microorganism.
The culture is incubated a-t about 30C. for two or three days on a rotary shaker. One of the substrate antibiotics is added then to the cul-ture maintaining the pH of the fermentation medium at about pH 7Ø The culture is monitored for activity using a Micrococcus luteus assay. This procedure is described in Sect. D.
Loss of antibiotic activity is an indication that the microorganism produces the requisite enzyme for de-acylation. This must be verified, however, using one of the following methods: 1) analysis by HPLC for presence of the intact nucleus; or 2) re-acylation with an appropriate side chain (e.g. lauroyl, n-decanoyl or n-dodecanoyl) to restore activity. Reduction in anti-biotic activity is difficult to distinguish when de-3 ~

acylating blocked A-21978C material because addition of the blocking group causes an 80-90% reduction in anti-biotic activity.
2. Conditions for Enzyme Production Production oF the enzyme occurs under con-ditions satisfactory for growth of the ctinoplanaceae, i.e., a temperature between about 25 and about 30C.
and a pH o~ between about 5.0 and abou-t d.0, with agitation and aeration. The culture medium should contain a) an assimilable carbon source such as su-crose, glucose, glycerol, or the like; b) a nitrogen source such as peptone, urea, ammonium sulfate, or the like; c) a phosphate source such as a soluble phosphate salt; and d) inorganic salts found generally to be effective in promoting the growth of microorganisms.
An effective amount of the enzyme is generally obtained in from about 40 to about 60 hours after the beginniny of the growth cycle and persists for some time after the effective growth has been reache~. The amount of enzyme produced varies from species to species of the organism and in response to different growth conditions.
As will be apparent to those in the field, th~ microorganisms which produce the enzyme, such as Actinoplanes utahensis NRRL 12052, are subject to variation. For e~ample, artificial variants and mutants of these strains may be obtalned by treatment with various known mutagens such as ultraviolet rays, X-rays, high-frequency waves, radioactive rays, and chemicals.
All natural and artificial variants and mutants which are obtained from -the Actinopla _~eae and which produce the enzyme may be used in this invC~ntion.

~v~

X-~279 -19-B. Deacylation Conditions The substrate used as the starting material preferably is added to the culture of Actinoplanaceae after the culture has been incubated for about 48 hours. The concentration of substrate in the conver-sion medium can vary widely. For maximum use of the enzyme and for substantially complete deacylation within a three-hour period, however, the concentration of substrate will generally range frorn about one to about three mg/ml. Lower concentrations can be used, but may not make maximum use of the enzyme; higher concentrations can be used also, but the substrate may not be completely deacylated unless the fermentation time is extended.
Conversion of the substrate antibiotic to A-21978C nucleus according to this invention proceeds best when the pH of the fermentation medium is main-tained in the range of Erom about 7O0 to about 7.2.
Below pH 7, deacylation proceeds slowly; as pH values move above pH 7.2, the nucleus which is formed is increasingly subject to alkaline hydrolysis. In stirred fermentoxs the pH may be controlled hy sensor con-trollers. Where this is impractical, such as in flask fermentors, pH may be controlled by adding 0.1 molar phosphate buffer to the medium prior to addition of the substrate.
After addition of the substrate, incubation of the culture should be continued for about 3-6 hours or longer. The purity of the substrate will affect the rate of deacylation. For example, a sub-X-5~79 -20-s~rate having a purity of greater than 50 percent is deacylated at a rate of about 2.5 mg/ml of antibiotic in 3 hours. When substrates of lower purity are used, the deacylation proceeds at a somewhat slower rate.
Multiple substrate feedings may be made. For example, 0.75 mg~ml of antibiotic may be fed at 24-hour intervals for at least five or more addit ons.
The deacylation can be carried out over a broad temperature range, e.g. from about 20 to about 45C. It is preferable, however, to carry out the deacylation at a temperature of about 30C. for optimum deacylation and stability of substrate and nucleus.
C. The Substrate It is preferable, but not essential, to use puriried antibiotic as the substrate. Because purified substrate is soluble in water ox in bufEer, it can be handled more conveniently. Moreover, with purified substrate the deacylation proceeds more rapidly.
Semipurified substrates containing as little as 15 percent of the starting antibiotic have been deacylated successfully.
The substrate antibiotics have antibacterial activityO Thus, althouyh the substrate materials (especially those of low purity) may harbor bacterial cells or spoxes which presumably could grow in the deacylation medium and affect th^e deacylation reaction or the stability of the starting antibiotic or the product nucleus, this has not been observed. It is not necessary, therefore, that the substrates be sterile, especially for short deacylation periods.

3~ 7 X-5279 -21~

D. Monitoring the Deacylation A-21978C factors C0, Cl, C2, C3, 4 5 are antibacterial agents which are especially active against Micrococcus luteus. For this reason an assay using M. luteus is prefexred for determininy quanti-ties of substrate present. The A-21978C nucleus which is formed is water soluble, but is biologically inactive.
Reduction in biological activity is, therefore, a quick, presumptive test for deacylation.
The amount of nucleus formed can be quanti-tated by HPLC analysis, using the system herein de-scribed.
E. Use of Resting Cells An alternate method of deacylation involves removing the Actinoplanaceae cells from the culture medium, resu~pending -the cells in a buffer solution, and carrying out the deacylation as described in Sect.
B. When this method is used, the enzymatically active mycelia can be reused. For example, _. utahensis NRRL
1205~ mycelia retain deacylase activity after storage for one month or longer under refrigeration (4-8 C.) or in the frozen state (-20 C.). A preferred buffer solution is 0.1 molar phosphate buffer.
~5 F~ Immobilized Enzymes .
Yet another method of carryiny out the deacylation is to immobilize the enzyme by methods known in the art. (See, Eor example, "Biomedical Applications of Immobilized Enzymes and Prokeins", Thomas Ming Swi Chang, Ed., Plemlm Pre~s, New York, ~-5279 -22-lg77; Vol. l.) The immobilized enzyme can then be us~d in a column (or other suitable type of reactor) to effect the deacylation.
In addition, the microorganism itself can be immobilized and used to cataly~e the deacyla~ion reaction.
Utility of the A-21978C Cyclic Peptides _ _ _ The A~21978C cyclic peptides and their salts are useful intenmediates in the preparation of semi-svnthetic antibacterial comPounds.
Chemotherapeutically useful compounds described in our co-pending Canadian Application Serial No. 428,101, filed May 13, 1983, have the general formula shown in formula 4:
~ Q NR3R
ll l =~ / /CONH2 r H~< C/\ ~a~ /N\ ~~J~f~

~1~=0 0=3'~ ~H2 ¦¦ H o~~ o ~' 2 5 /N--H NH ~02H 0\~ o 0=0' `-=0 H
HO~ R ~ R2 lir T

H ,~
H02C' S
.........

7~

in which R, Rl and R2 are, independently, hydrogen, C4-C14-alkyl, optionally substituted C2-C19-alkanoyl, C5-Cl~-alkenoyl or an amino-protecting group, R3, R4 and R are all hydrogen, or (i) R and R ; and/or (ii) R4 and R, and/or (iii) R5 and R2, taken toge-ther may represent a C4-C14 alkylidene group, provided that 1) at leas-t one of R, R or R must be other than hydrogen or an amino-protecting group, 2) at least one of Rl or R2 must be hydrogen or an amino-protecting group, 3) the R, R and R groups mus-t together contain at least four carbon atoms, and 4) when Rl and R2 are both selected from hydrogen or an amino-protecting yroup, R cannot be 8-me-thyldecanoyl, 10-methylundecanoyl, 10-methyldodecanoyl, the specific C10-alkanoyl group of A-21978C factor C0 or the specific C12-alkanoyl groups of A-21978C factors C4 and C5; or a pharmaceutically-acceptable salt thereof.
The term "C4-C14-alkylideny]." refers to a group o-f the formula \C= in which R3 and R4 are hydro-gen or an alkyl group of from 3 to 13 carbon atoms, ~rovided that one of R3 and R4 must be other -than hydrogen and further provided that the sum of the carbon atoms in R3 and R4 must be no greater than 13.
Those compounds in which one of R, Rl or R is C4-C14 alkylidenyl are known as Schiff's basesO
The term "C4-C14-alkvl" refers to a univalent saturated, straight- or branched-chain alkyl group containing from 4 to 14 carbon atoms. Those compounds in which one of R, R or R are C4-C14-alkyl are pre pared by reduction of the corresponding compounds where ~2~q~

~-5279 -~4-the R, Rl or R group is C4-C14 alkylidenyl and are referred to as "reduced Schiff's bases".
The terms "optionally substituted C2-C19-alkanoyl" and "C5-C19-alkenoyl" refer to acyl groups derived from carboxylic acids containing from 2 to 19 and 5 to 19 carbon atoms, respectively. 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 group or from one to three halo substituents selected from chlorine, bromide, 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 a recognized amino-protecting group as defined supra.
The following are preferred embodiments of the compounds of ormula 4:
(a) The compounds in which R is alkanoyl of o the formula CH3(CH2)n~C-, in which n is an integer from 3 to 17;
(b) The compounds in which R is alkanoyl of o the formula CH3(CH2)n-C-, in which n is 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14;
(c) The compounds in which R is alkanoyl of the formula CH3(CH2)nCH(CH2)m-C-, in which n and m are each, independently, an integer from ~ to 14, provided that ~2'l3`~

n + m must be no less than 1 and no greater than 15; and fur~hQr provided that, when n is 0, m cannot be 8 and, when n is 1, m cannot be 6 or 8;
(d) The compounds in which R is cis or trans alken~l of the formula O
CH3(CH2)nCH=CH(CH2)m-C-in which n and m are each, independently, an integer from O to 14, provided that n + m must be no less than 1 and no greater than 15;
(e) 'rhe compounds in which R is cls or trans alkenyl of the formula o CH 2 ~CH ( CH 2 ) nC
in which n is an integer from 4 to ].5;
(f) The compounds in whlch R is alkyl of the formula CH3(CH2)n~ and n is an integer ~rom 5 to 12; and (g) The compounds in which R is:
o CH3(CH2)5-C-~5 0 CH3(CH2)6-C-O
CH3(CH2)7-C-o 3~ CH3(CH2)8-C-~iu~

~-5279 -26-CH3(CH2)9-C-o H3(CH2)10 C
O
CH2=C~-(CH2~8-C-o H3(CM2)11 C
o 3( 2)12 o CH3-(CH2)3-CH=CH (CH2)7 CH3(CH2)13 C
O
3 2 ( 3) ( 2)6 CH3(CH2)11-CH3(CH2)10CH

CH3(CH2)6-CH3(C~I2)5~H= -CH3(CH2)9-CH3(CH2)8CH=

~%6~7~

X-5279 ~27~

Chemotherapeutically-useful compounds described in co-pending Canadian applications Nos. 428,100 and 428,103, of Manuel Debono, filed May 13, 1983, have the general formula 5:

~ ~

0=~ =0 H
/~ Ç~ p Hi 1~,~

H~

The derivatives of structure 5~described in Canadian Application No. 428,103 are those in which R is hydrogen, 8-methyldecanoYl, 10-methyldodecanoyl, _ ,~
10-methylundecanoyl, the specific C10-alkanoyl group of ~-21978Co or the specific C12-alkanoyl groups of A-21978C factors C4 and C5, an amino-protecting group, o an aminoacyl. q..oup o~ the formula -C-Q-NH2 in which Q is Cl-C16 alkylene or an N-alkanoylaminoacyl group of ~2'~ 7t~

X-5279 -~8 the formula -W~C~R2 in which:
W is a divalent aminoacyl radical of the formula:
o (a) -C-A-NH_ in which A is Cl-C10 alkylene or C5-C6 cyclo-alkylene;
o R3 "
(b) -C-CH-NH-in which R3 is hydroxymethyl, hydroxyethyl, mercaptomethyl, mercaptoethyl, methylthio-ethyl, 2-thienyl, 3-indole-methyl, phenyl, benzyl, or substituted phenyl or substituted benzyl in which the benæene ring thereof .is substituted with chloro, bromo, iodo, nitro, Cl-C3 alkyl, hydroxy, Cl-C3 alkoxy, C1-C3 alkylthio, carbamyl, or Cl-C3 alkylcarbamyl;

(C) ~ ~NH--2 0 ~>~X

in which X is hydrogen chloro, bromo, iodo, amino, nitro, Cl-C3 alkyl, hydroxy, Cl-C3 alkoxy, mercapto, Cl-C3 alkylthio, carbamyl, or Cl-C3 alkylcarbamyl;

(d) ~ NH-- ~-X-5279 ~29-in which X is chloro, bromo, iodo, amino, hydroxy, Cl-C3-alkyl or C~-C3-alkoxy;
llH

~ o~H--in which B is a divalent radical of the formula: (CH2)n~ and n is an in-teger from 1 to 3; -CH=CH-; -CH=CH-CH2-; or -C~I2NHC-;
R is Cl-C17 alkyl or C2-C17 alkenyl; and R is hydro-gen, an amino-protecting group, an aminoacyl group of o the Eormula -C-Q NH2 as herein defined, or an N-alkanoylaminoacyl group of -the formula ~W-C-R2 as herein defined; provided that, when R is other than aminoacyl or N-alkanoylaminoacyl, Rl must be aminoacyl or N-alkanoylaminoacyl; and, when Rl is an amino-protecting group, R must be aminoacyl or N-alkanoyl-aminoacyl; or a pharmaceutically acceptable salt thereof.
The terms "alkylene", "alkyl", "alkoxy", "alkylthio", and "alkenyl" comprehend both straight and branched hydrocarbon chains. "Alky.L" means a univalent ~3~

X-~279 -30~

saturated hydrocarbon radical. "Alkenyl" means a univalent unsaturated hydrocarbon radical containing one, two, or three double bonds, which may be oxiented in the is or trans configuration. "Alkylene" means a divalent saturated hydrocarbon radical. "Cycloal-kylene" means a divalent cyclic saturated hydrocarbon radical.
1 10 or Cl C16 alkylene radicals which are preEerred are:

-CH2-; -CH- in which R5 is C1-C4 alkyl (i.e., methyl, ethyl, _-propyl, l-propyl, n--butyl, t-butyl, l_butyl, or 1-methylpropyl); -(CH2)m in which m is an integer from 2 to 10; and CH3~(CH2)q~CH~(CH2)p~, in 1~ which p is an integer from 1 to 8 and q is an integer from 0 to 7, provided that p + q must be no greater than 8.
Illustrative Cl-C17 alkyl groups which are preferred are:
(a) CH3-;
(b) -(CH2)nCH3 in which n is an integer from 1 to 16; and C,H3 (c) -(CH2)rCH(CH2)sCH3 in which r and s are, in-dependently, an integer ~rom 0 to 14 provided that r + s can be no greater than 14.
Illustrative C2-C17 alkenyl radicals which are preferred are:
(a) -(CH2)t CH=CH-(CH2)u-CH3 in which t and u are, independently, an in'eger from 0 to 14 pro-vided that t + u can be no greater than 14~

(b) -(CH2)v CEI=CH~(CH2)y~CH=CH~(CH2)z-CH3 in which v and z are, independently, an integer from 0 to 11 and y is an integer from 1 to 12 provided that v + y + z can be no greater S than 12.
In particular, the following embod.iments of the Cl-C17 alky]. groups are preferred:

CH3(CH2)5-CH3(CEI2)6-CH3(CH2)8-CH3(CH2)10-CH3(CH2)12-H3~CH2)14 CH3(CH2)16-In particular, the following embodiments of the C2-C17 alkenyl groups are preferred:
c15-CH3(CH2)5CH=CH(CH2)7-trans-CH (CH ) CH=CH(CH ) -cis-cE~3(cH2)locH CH(CH2)4 trans-CH (CH ) CH=CEI(CH ) -cls CH3(C~I2)7CH=CH(CH2)7 trans-CH3(CH2)7CH=CH(CH2)7-Cls-CH3(CH2)5CH=CH(CH2)9 tralls~CH3(CH2)5CH=CH(C~I2)9-cis, cis-CH (CH ) CH=CHCE~2CH=CH(CH2)7-trans, trans-CH3(CH2)4CE~=CHCH2CH=CH(CH2)7-cis,cis,cis-CH CH CH=CHCH~CH=CHCH CH=CH-(CH2) -.
- - 3 2 ~ 2 7 ~2~ 7~

X-527~ -32-When '!W" is a dival~nt radical of the foxmula ~Xx it will be recognized by those skilled in the art that o the C- function and the -NH- function m~y be oriented on the ben7ene ring in the ortho, meta, or para con-figuration relative to each other. The substituent represented by X may be substituted at any available position of the benzene ring. Preferred embodiments O
are those in which X is hydrogen and the -C- and -NH-functions are oriented in the para configuration.
The terms "substituted phenyl" and "substi-tuted benzyl", as defined by R3 in formula 5, contem-plate substitution of a group at any of the availablepositions in the benzene ring~-i.e. the substituent may be in the ortho, meta, or para configuration. The term "Cl-C3 alkyl" as defined by R3 or X in formula 5 includes the methyl, ethyl, n-propyl, or i-propvl groups.
The derivatives of structure 5 described iII
Canadian Application No. 428,100 of M. Debono are those in which R is a substituted benzoyl group of the formula ~X
o X
in which X is hydrogen, chloro, bromo, iodo, nitro, C~-C3 alkyl, hydroxy, Cl-C3 alkoxy, or Cl-C3 alkylthio;
R is hydrogen or an amino-protecting group; and R is C8-Cl5 alkyl; or a pharmaceutically-acceptable salt thereof.
The substituted benzoyl group, the -C-function and the -OR function may be oriented on the benzene ring in the ortho, meta, or para position relative to each other. The para orientation for these groups is preferred. The substituent represented by X
may be substituted at any available position of the benzene ring not occupied by these two group~.
The term "alkyl" comprehends both straight and branched hydrocarbon chains.
Illus rative C8-C15 alkyl radicals which are preferred for R are:
(a) -(CH~)nCH3 in which n is an integer from 7 to 14; and ,CH3 (b) -(CH2)rCH(CH2)sCH3 in which r and s are, independently, an.intager from 0 to 12, provided that r + s can be no greater than 12 or no less than 5.

X-5279 -3~-The compounds of formulas 4 and 5 may be prepared by acylating a compound of formula 1 with the appropriate acyl side chain using methods conve}ltional 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 acid corresponding to the desired acyl side chain group.
The term "activated derivative" means a derivative which renders the carboxyl function of the acylating agent reactlve to coupling with a primary amino group to form the amide bond which links the acyl side chain to the nucleus. Suitable activated deriva-tives, their methods of preparation, and their use as acylating agents for a primary amine will be recognized by those skilled in the art. Preferred activated derivatives are: ta~ an acid halide (e.g. an acid chloride), (b) an acid anhydride (e.g. an alkoxyformic acid anhydride or aryloxyformic acid anhydride) or (c) an activated ester (e.g. a 2,4,5-trichlorophenyl ester). Other methods for activating the carboxyl function include reaction oE the carboxylic acid with a carbonyldiimide (e.g. N,N'-dicyclohexylcarbodiimide or N,N'-diisopropylcarbodiimide) to give a reactive inter-mediate which, because of instability is not isolated.
Such a reaction with the primary amine is carried out in situ.
A preferred metho~ for preparing the com-pounds of formulas 4 and 5 is by the active ester me-thod. The 2,4,5-trichlorophenyl ester of the desired acid is a preferred acylating agent. In this method, ~2~ 7 an excess amount of the active ester is reacted with a formula 1 compound at room temperature in a non-reactive org~lnic solvent such as DM~'. 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 i5 removed, and the residue is purified by a recognized method, such as by column chromatography. t-BOC groups can be removed by treat-ment 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 reversed-phase HPLC.^
The 2,4,5-trichlorophenyl esters of the corresponding acids can be prepared conveniently by treating the desired acid with 2,4,5-trichlorophenol in the presence of a coupling agent, such as N,N'-dicyclo-hexylcarbodiimide. Other methods suitable for pre-paring acid esters will be apparent to those skilled in the art.
The alkanoic and alkenoic acids used as starting materials for derivatives of Eormula 4 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 made conveniently 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-'7'~

trichlorophenol in the presence of N,N'~dicyclohexyl-carbodiimide. The 2,4,5-trichlorophenyl ester deriv-ative can be purified by column chromatography over silica gel.
The N-alkanoylamino acids or N-alkenoylamino acids used as startiny materials for derivatives of formula 5 are either known compounds or they can be made by acylating the appropriate amino acid with the desired alkanoyl or alkenoyl group using conventional methods. A preferred way of preparing the N-alkanoyl-amino acids is by treating the appropriate amino acid with an alkanoic acid chloride in pyridine. The alkanoic or alkenoic acids, the activated derivatives thereoE, and the amino acids used are either known compounds or they can be made by known methods or by modification of known methods apparent to those skilled in the art.
If a particular amino acid contains an acylable functional group other than the amino group, it will be understood by those skilled in the art that such a group must be protected prior to reaction of the amino acid with the reagent used to attach the N-alkanoyl or N-alkenoyl group. Suitable protecting groups can be any group known in ~the art to be useful for the protection of a side chain functional group in peptide synthesis. Such groups are well known, and the selection of a particular protecting group and its method of use will be readily known to one skilled in the art ~see, for example, "Protective Groups In Organic Chemistry", M. McOmie, Editor, Plenum Press, N.Y., 1973].

~. ~6:~r~7 7 7 X-5279 -~7-Certain amino acids used in the synthesis of these products may e~ist in optically active forms.
Both the natural configuration (L-configuration) and unnatural configuration (D-configuration) may be used as starting materials.
The substituted benzoic acids used as start-ing materials for some of the A-21978C derivatives, and the activated derivatives thereof are either known compounds or they can be made from known compounds by methods known in the art. The alkoxybenzoic acids can be prepared conveniently from an appropriate hydroxy-benzoic acid by reacting an appropriate alkyl halide with the disodium salt of the appropriate hydroxyben-zoic acid.
The hydroxybenzoic acids and substituted derivatives thereof used as starting materials in the processes described are either known compounds or can be prepared by conventional methods.
The derivatives prepared from the inter-mediates o this invention, i.e. the compounds offormulas 4 and 5, inhibit the growth of pathogenic bact-eria as evidenced by standard biological test procedures. The compounds are useful, therefore, for controlling the growth of bacteria on environmental surfaces (as an antiseptic) or in treating infections caused by bacteria. The antibacterial activity of the compounds has been demonstrated ln vitro in agar-plate disc-difusion tests and in agar-dilution tests and in vivo in tests in mlce infected with Staphylo-__ .
coccus aureus and Streptococcus pyogenes. ~he com-7~

pounds are particularly useful in treating infections caused by gram-positive organisms.
When an A-21978C cyclic peptide 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 A-21978C
compound is co~nonly administered together with a pharmaceutically-acceptable carrier or diluent. The dosage of A-21978C 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 appropri-ate dosage ranges and/or dosage units for administra-tion may be determined by considering the MIC and ED50 values and toxicity data provided together with factors such as pharmacokinetics, characteristics of the patient or host and the infecting microorganism.
In order to illustrate more fully the in-vention, the following non-limiting examples are provided.

Preparation of A-21978C Nucleus .
~. Fermentation of Actinoplanes utahensis . ~
A stock culture of Actinoplanes utahensis NRRL 12052 is prepared and maintained on an agar slant. The medium used to prepare the slant is se-lected from one of the following:

~Z~ 77~

X-5279 ~39-MEDIUM A
Ingredlent Amount Pre-cooked oatmeal 60.0 g Yeast 2.5 g K2HPO4 1.0 g Czapek's mineral stock* 5.0 ml Agar 25.0 g Deionized waterq.s. to 1 liter pH before autoclaving is about 5.9; adjust to pH 7.2 by addition of NaOH; after autoclaving, pH is about 6.7.
Czapek's mineral stock has the following composition:
Ingredient Amount . _ FeSO4 7H2O (dissolved in 2 ml conc HCl) 2 g KCl 100 g MgSO4-7H O 100 g Deionized waterq.s. to 1 liter ~Z00~7~

MEDIUM B
Ingredient Amount Potato dextrin 5.0 g Yeast e~tract 0.5 g Enzymatic hydrolysate of casein* 3.0 g Beef extract 0.5 g Glucose 12.5 g Corn starch 5.0 g Meat Peptone 5.0 g Blackstrap molasses2.5 g MgSO4.7H2o 0.25 g CaCO3 1.0 g Czapek's mineral stock 2.0 ml Agar 20.0 g Deionized water q.s. to 1 litre *"N-Z-Amine A," Humko Sheffield Chemical, Lyndhurst, N.J.
2Q The slant is inoculated with Actinoplanes utahensis NRRL 12052, and the inoculated slant is incubated at 30C for about 8 to 10 days. About ~
of the slant growth is used to inoculate 50 ml of a vegetative medium having the following composition:

1. Trademark ~2~ '7~7~

~-5279 -41-Ingredient Amount , Pre-cooked oatmeal 20.0 g Sucrose 20.0 g Yeast 2.5 g Distiller's Dried Grain*5.0 g K2HPO4 1.0 g Czapek's mineral stock5.0 ml Deionized water q.s. to 1 liter adjust to pH 7.4 with NaOH; after autoclaving, pH is about 6.8.
National Distillers Products Co., 99 Park Ave., New York, N.Y.
The inoculated vegetative med~um is incubated in a 250-ml wide-mouth Erlenmeyer flask at 30C for about 72 hours on a shaker rotating through an arc two inches in diameter at 250 RPM.
This incubated vegetative medium may be used directly to inoculate a second-stage vegetative medium~
Alternatively and preferably, it can be stored for later use by maintaining the culture in the vapor phase of liquid nitrogen. The culture is prepared for such storage in multiple small vials as follows: In each vial is placed 2 ml of incubated vegetative medium and 2 ml of a glycerol-lactose solution [see W. A. Dailey and C. E. Higgens, "Preservation and Storage of Micro-organisms in the Gas Phase of Liquid Nitrogen, Cryo-biol 10, 364-367 (1973) for details]. The prepared suspensions are stored in the vapor phase of liquid nitrogen.

7'7'7 X-5279 -~2-A stored suspension (1 ml) thus prepared is used to inoculate 50 ml of a first stage vegetative medium (having the composition earlier described). The inoculated first-stage vegetative medium is incubated as above-described.
In order to provide a larger volume of inoculum, 10 ml of the incubated first-staye vegetative medium is used to inoculate 400 ml of a second-stage vegetative medium having the same composition as the first-stage vegetative medium. The second-stage medium is incubated in a two-liter wide-mouth Erlen-meyer flask at 30C for ahout 48 hours on a shaker rotating through an arc two inches in diameter at 250 RPM.
Incubated second~stage vegetative medium (80 ml), prepared as above-described, is used to in-oculate 10 liters of sterile production medium selected from one of the following:
MEDIUM I
Ingredient Amount (g/L) Peanut meal 10.0 Soluble meat peptone 5.0 Sucrose 20.0 25 KH2PO~ 0.5 K2HPO4 1.2 MgSO4-7H2O 0.25 Tap water q.s. to 1 liter The pH of the medium is about 6~ after 30 sterilization by autoclaving at 121C for 45 minutes at about 16-18 psi.

~P~3~

X-5279 ~~3~

MEDIUM II
Ingredient Amount (g/L) Sucrose 30.0 Peptone 5.0 S K2HPO4 1.0 KCl 0.5 MgSO4~7H2O 0.5 FeSO ~7H2O 0.002 De.ioni~ed water q.s. to 1 liter Adjust to pH 7.0 with HCl; after autoclaving, pH is about 7Ø
MEDIUM III
Ingredient Amount (g/L) Glucose 20.0 NH4C1 3.0 2 o4 2.0 ZnC12 0.019 MgC12-6H2 0.304 FeCl 6H2O 0.062 MnC12 4H2O 0.035 CuC12-2~i2O 0.005 CaCO3 6.0 KH2PO4* 0.67 Tap water q.s. to 1 liter *Sterilized separately and added aseptically Final pH about 6.6.

'7 X~5279 _44_ The inocula~ed production medium is allowed to ferment in a 14-liter fermentation vessel at a temperature of about 30C for about 66 hours. The fermentation medium is stirred with conventional agitators at about 600 RPM and aerated with sterile air to maintain the dissolved oxygen level above 30% of air saturation at atmospheric pressure.
B. Deacylation of A-21978C
~ ~r =_ .. ___ _ . _... _._.__~_ 1 A fermentation of A. utahensis is carried out as described in Section A, using slant medium A and production medium I and incubating the production medium for about 67 hours. Crude A-21978C complex (100 g) is added to the -Eermentation medium.
Deacylation of the A-21978C complex is mon-itored by assay against Micrococcus luteus~ The fer-mentation is allowed to continue until deacylation is complete as indicated by disappearance of activity vs.
M. luteus, a period of about 24 hours.
This same procedure was used to determine whether other microorganisms would produce the desired A-21978C nucleus. In particular, Actinoplanes missouriensis NRRL 12053, Actinoplanes sp. NRRL 8122, .. .. _ _ _ _ .
Actinoplanes sp. NRRL 12065, and Streptosporangium _ 25 ros um var. hollandensis NRRL 12064, when used in place of Actinoplanes utahensis NRRL 12052 in the procedure above, were found to produce the desired nucleus. HPLC
comparisons using authentic samples o~ the nucleus obtained from this procedure using _. utahensis as the deacylating enzyme confirmed that these other micro-organisms produced the A-21978C nucleus.

7~

C. Isolation of A-21978 Nucleus .
Whole Eermentation broth (20 liters) obtain-ed as described in Section B was filtered with a filter aid (I'Hyflo Super-Cel'l* Johns Manville Corp.).
The mycelial cake was discarded. The filtrate thus obtained was passed through a column containing 1.5 liters of HP-20 resin (DIAION** High Porous Polymer, HP-Series Mitsubishi Chemical Industries Limited Tokyo Japan). The e:Efluent thus obtained was discarded.
The column was then washed with deionized water (10 L.) to remove residual filtered broth. This wash water was discarded. The column was then eluted with water:acetonitrile mixtures (10 L. each of 95:5 9:1 and 4:1) collecting 1 -liter fractions.
Elution was monitored by analytical HPLC
usi.n~ silica gel/C 18 and a solvent system of water:-methanol (3:1) containing 0.1% arnmonium acetate detecting the nucleus with a VV monito.r at 254 nm.
Fractions containing the nucleus were combined concentrated under vacuum to .remove the acetonitrile and freeze-dried to give 40.6 g of semi-purified A-21978C nucleus.
D. Purification of A-21978C_Nucleus Semi-purified A-21978C nucleus (15 g) obtained as described in Section C was dissolved in 75 ml of water:methanol:acetonitrile (82:10:8) containing 0.2% acetic acid and 0.8% pyridine. This solution was pumped onto a 4.7- x 192-cm column containing 3.33 L. of silica gel ("Quantum 1P~1"***)/Cl8.
The column was developed with the same solvent system.

* Trademark for a filter aid made from "Celite" (trade-mark) brand of diatomaceous earth specially processed to pro~ide a high flow rate.
** Trademark *** Trademark .
~, ., ~ .

~Z~ 7 ~-5279 --46-Fractions having a volume of 350 ml were collected.
Separation was mo~itored at 280 nm with a W monitor.
Fractions containing the nucleus were combined, con-centra~ed under vacuum to remove solvents and freeze~
dried to give 5.2 g o~ purified A-21978C nucleus.

Alternate Preparation of A-21978C_Nucleus A-21978C nucleus was prepared according to the method of Example 1 except ~or certain changes in Section R . The A. utahensis culture was incubated initially for about 48 hours; the substrate was semi-purified A-21978C complex (50 g); and incubation after addition of the substrate was about 16 hours. The broth filtrate was passed over a column containing 3.1 liters of HP-20 resin. The column was washed with 10 volumes of water and then was eluted with water:aceto-nitrile ~95:5). Elution was monitored as in Example 1.
After collecting 24 liters, the eluting solvent was changed to water:acetonitrile (9:1). Fractions con-taining the nucleus were eluted with this solvent.
These fractions were combined, concentrated under vacuum to remove acetonitrile, and freeze-dried to give 24.3 a of semi-purified A-21978C nucleus.
This semi-purified A-21978C nucleus (~4.3 g) was dissolved in water (400 ml). The solution ~as pumped onto a 4.7- x 192-cm steel colu~ cor.taining 3.33 liters of silica gel ~uantum LP-l)~C18 prepared in water:methanol:acetonitrile (8:1:1) containing 0.2 acetic acid and 0.8~ pyridine. The column was devel-*Trademark ~Z~7'7 X-5279 -47~

oped with the same solvent at a pressure of about 2000 psi, collecting 350 ml fractions~ Elution was monitored by UV at 280 nm. Fractions containing the nucleus were combined, concentrated under vacuum to remove solvents, and freeze~dried to give 14 ~ of highly purified A-21978C nucleus.

Preparation of Norn-t-BOC A-21978C Factors C2 and C3 A mixture of A-21978C factors C2 and C3 (10 g), prepared as described in U.S. patent 4,208,403, was dissolved in water (50 ml) wi-th sonication (200 mg/ml). The pH of the solution was adjusted from 4O05 to 9.5 with 5N NaOH (3.6 m]). 3i-tert-butyl dicarbon-ate (3.0 ml) was added, and -the reaction mixture was stirred at room ~empera-ture for 2 hours. The pH of the reaction was main-tained at 9.5 by manual addition of SN
NaOH (6.5 ml added in 2 hours).
The reaction was monitored periodically by TLC on silica gel, using CH3CN-H2O (7:3 and 8 2) sol-vent systems and detecting by UV.
After about 10 minutes the reaction solution became rapidly turbid, and base consumption increasedO
After 30 minutes, the rate of increase in turbidity and the rate of base consumption decreased, indicatiny that the reaction was complete. Nevertheless, the reaction was cortinued for an additional 90 minutes to insure completion. At the end or the two-hour reaction, the reaction material was lyophilized immediately to give 12.7 g of Norn-t BOC-A-21978 factors C2 and C3.

~2~777 Using similar procedures, two 10-g reactions and a 30-g reaction were run. In each of these the reaction time was only 40 minutes. The two 10-g experiments gave 11.9 and 12.1 g of product, respectively.
The 30-g reaction gave 35.4 g. of product.

Preparation of A-21978C No -t-BOC Nucleus A. Fermenta-tion of A. utahensis A fermentation of A. utahensis was carried out as described in Example 1, Section A, using slant medium A and production medium I and incubating the production medium for about 66 hours.
B- Deacylation of Norn-t-BOC Complex The A-21978C Norn-t-BOC complex (1185 g of crude substrate which contained about 176 g of A-21978C complex was added to the fermentation mediumO
Deacylation was carried out as described in Example 1.
Section B. Deacylation was complete, as indicated by HPLC, after about 24 hours.
C. Isolation of A-21978C Norn-t-BOC Nucleus ... .. .
Fermentation broth (100 L.), obtained as described in Section B, was filtered with a filter aid "(Hyflo Super-cel3" . The filtrate was passed over a column con-taining 7.5 L~ of HP-20 resin (DIAION) the column was washed wi-th water (38 L.). Flution was monitored by silica gel/C18 HPLC with UV detection at 254 nm. Some nucleus was eluted in the wash.

Subsequent elution o~ nucleus was carried out with water:
acetonitrile mixtures as follows: (95:5)-40 L.; (9:1)-40 L.; and (85:15)-100 L. Fractions containing the nucleus were combined, concentrated under vacuum to remove solvent, and freeze-dried to give 298.5 g of purified A-21978C Norn-t-BOC nucleus.
D. Purification of A-21978C Nor -t-BOC Nucleus Semi-purified A-21978C Norn-t-BOC nucleus ~30g), obtained as described in Section C, was dissolved in water:acetonitrile (9:1) containing 0.2% acetic acid and 0.8% pyridine (75 ml). This solution was applied to a

4.7 x 192-cm steel column containing 3.33 L. of silica gel "(Quantum LP-1)"/Cl8 equilibrated in the same solvent system. The column was developed under pressure with water:acetonitrile:methanol (80:15:5) containing 0.2%
acetic acid and 0.8% pyridine, collec-ting 350-ml fractions and detecting product by UV at 280 nm.
Fractions containing the product were combined, con-centrated under vacuum to remove solvent and ~reeze-dried to give 18.4 g of purified A-21978C Norn-t-BOC
nucleus.

Alternative Purification of A-21978C Norn-t-BOC Nucleus Semi-puri~ied A-21978C Norn-t-BOC nucleus (10.8 g), obtained as described in Example 4, ~ection ~
was dissolved in water and applied to a column contain-ing 80 ml of "Amberlite IRA~68" (acetate cycle). The column was washed with water and, at a flow rate of * Trademark for a "gel"-type weakly basic anion exchange resin having polyamine functionality.

~ ,7,~..

lZ~ 77~

5 ml/min, was eluted sequentially with 0.05 N acetic acid (1080 ml), 0~1 N acetic acid (840 ml), and 0.2 N
acetic acid (3120 ml), collecting 120-ml fractions.
The column was monitored with analytical HPLC over silica gel/C18, using a system of water:acetonitrile:
methanol (80:15:5) containiny 0.2~ ammonium acetate and detecting product with UV at 2S4 nm. Fractions contain~
ing the product were combined; the pH of the solution was adjusted to 5.8 with pyridine; the resulting solu-tion was concentrated under vacuum to a volume of about200 ml. Water was added to the concentrate, and the resulting solu-tion was reconcentrated to remove pyridine.
This concentrate was freeze-dried to give 3.46 g of purified A~21978C Norn~t-BOC nucleus.

The following procedure illustrates the preparation of the compounds of formula 4 by the "active ester" method. The speciEic compounds prepared by this procedure are the compounds of formula 4 wherein R i5 CH3(CH2)8CO- (n-decanoyl), R is hydrogen and R is t-BOC or hydrogen~
NTrp-~n-Decanoyl) A-21978C Nucleus __ A. Preparation of 2,4,5-Trichlorophenyl _-Decanoate _ _ A solution of decanoyl chloride (Pfaltz and Bauer, 5.6 mI) and 2,4,5-trichlorophenol (5.6 g) in diethyl ether (1 L) and pyridine (120 ml) is stirred for 4 hours. The reaction mixture is Eiltered and dried _ vacuo. The 2,4,5-trichlorophenvl n-decanoate ~oa~7~ , is purified on a silica-yel col~ (Woelm)~ using toluene as the eluent. Fractions are monitored by TLC, using short-wave UV for detection. Appropriate frac tions are pooled and dried in vacuo to give 10~4 g of 2,4,5-trichlorophenyl n-decanoate.
B. Acylation of N~rn t~BOC-A-21978C Nucleus with 2,4,5-Trichlorophenyl n-Decanoate A solution of Norn-t-BOC A-21978C nucleus (15.0 g) and 2,4,5-trichlorophenyl _-decanoate (15.0 g) in dry D~F (500 ml) is stirred under N2 at ambient temperature for 25 hours. The mixture is then 5 tirred at 60C. for 5 hours or until TLC shows reaction com pletion. The reaction mixture is concentrated in vacuo to about 200 ml and is stirred with 1.2 liters of Et2O/toluene (5:1). The product is separated by fil-tration, washed with Et2O, and dried under vacuum to give 15.05 g of the NTrp-(n-decanoyl)-Norn-t-Boc A-21978C nucleus intermediate (formula 4: R=_-decanoyl, Rl=H~R2=t-Boc)o C. Purification of NTrp-(_-Decanoyl)-Nor~-t-B
A-21978C Nucleus The NTrp-(n-^decanoyl)-Norn-t~Boc-A-2l~78c nucleus intermediate is purified in the following manner: The crude preparation is dissolved in about 50 ml of the eluting solvent sys~ Gm~ and this~is puri-ied by HPLC, using the Waters'~rep 500"system contain-ing a cartxidge packed with reversed;phase Cl~ silica-gel adsorbent. The system is eluted with H2O.MeOH:-*Trademark g~7i7~7 CH3CN(50:15:35) containing 0.2~ pyridine and 0.2% HOAc.
Fractions are monitored by UV at 280 nm. Appropriate fractions are combined and dried ln vacuo to give 8O56 g of purified NTrp~(_-decanoyl)-NO
A-21978C nucleus.
D. Removal of the Norn-t~BOC Group The t-BOC group is removed by stirring NTrp-(_-decanoyl)-NOrn~t-BOC A-21978C nucleus (1~47 g) in 15 ml of trifluoroacetic acid/1,2-ethanedithiol (10:1) at ambient temperature for 3 minutes. The reaction mixture is drled ln vacuo, and the residue is tri-turated with Et2O (50 ml). After a 20-ml Et2O wash, the triturate is dr~ied ln vacuo to give 2.59 g of crude NTrp-(n-decanoyl)-A-21978C nucleus (formula 4:
R=n-decanoyl; R and R =H).
E. Purification of NTrp-(--Decanoyl)-A-21978C Nucleus The crude NTrp-(n-decanoyl)-A-2l978c nucleus is purified by reversed-phase HPLC in the Eollowing manner: The sample (2.59 g), dissolved in 4.0 ml of H2O:MeOH:CH3CN:pyridine:HOAc (50:15:35:2:2), is in-jected into a 33- x l-inch stainless-steel column packed with LP-l/C18 adsorbent. The column is eluted with this same solvent system. Elution is performed at a pressure of 1200-1700 psi with a flow rate of 10-12 ml~min, using an LDC duplex pump (Milton-Roy). The erfluent is monitored by a UV detector (Isco Model UA-5, Instrum~nt Specialist Co., 4700 Superior Avenue, 30 Lincoln, Nebraska 68504) at 280 nm. Fractions (20-24 3L~ 0~ 7 77 ~-5279 -53-ml) are collected every two minutes. The desired fractions, as indicated by antimicrobial activity, are combined and dried ln vacuo to give 1.05 g of product.
This purification procedure was repeated with 4.35 g, 4.25 g, 2.14 g, 2.00 g and 1.75 g cxude start-ing derivative to give a total of 5.58 g of purified NTrp-(n~decanoyl) -A-21978C nucleus.

1 This example illustrates the preparation of compounds of formula 5. The specific compounds pre~
pared by this procedure are the compounds of formula 5 wherein R is N-(_-decanoyl)-L-phenylalanyl and R2 is t-sOC or hydrogen.
PreParatin of NTrp-[N-(_-Decanoyl)-L-phenylalanyl]
.. . ..
A-21978C Nucleus A. Preparation of N-(n~Decanoyl)-L-phenylalanyl 2,4,5-, . . . .. .
Trichlorophenolate A solution of N-(n-decanoyl)-L-phenylalanine (31.9 g, 0.1 mole) and 2,4,5-trichlorophenol (19.7 g, 0.1 mole~ in 1 liter of anhydrous ether was treated with N,N'-dicyclohexylcarbodiimide (20.6 g, 0~1 mole).
The reaction was stirred overnigh-t at room temperature.
The precipitated N,NI-dicyclohexylurea was removed by filtration and discarded. The filtrate was concen-trated under vacuum -to dryness. The residue obtained was tri-turated with ether, and the solids (residual cyclohexylurea) were removed by filtration. The fil '7~

X-527g -5~-trate was evaporated to dryness under reduced pressureO
The residu~ was crystallized from acetonitrile to give 36.9 g of crystalline N-(n-decanoyl)-L-phenylalanyl 2,4,5-trichlorophenolate, m.p. 122-124C.
B- Preparation of NTrp-[N~ Decanoyl)-L-phen N ~t-BOC-A 21978C Nucleus A solution of N-(n-decanoyl)-L-phenylalanyl 2,4,5-trichlorophenolate (10 g, 0.02 mole), ~orn-t-BOC-A-21978C nucleus (10 g~ 0.006 mole~ in anhydrous DMF
(1 L) was stirred at room temperature for 96 hours under an atmosphere of nitrogenO The solvent was removed by evaporation under reduced pressure. The residual material was stirred with a mixture oE diethyl lS ether (800 ml) and chloro~orm (200 ml) fcr 2 hours.
The product was separated by filtration to give a light brown powder (10O3 g). This material (9.9 g) was dissolved in methanol (200 ml) and purified by prepara-tive HPLC, using a "Prep LC/System 500" unit and a PrepPak-50d/C18 Co~umn as the stationary phase. The column was eluted isocratically, using a water:methanol-acetonitrile (2:1:2) solvent system and collecting 250-ml fractions at a rate of one fraction per minute.
The desired compound was eluted in the 9th through the 2?nd fractions.
Fractions were combined on the basis of TLC
[reversed phase silica gel/C18; developed with water:-methanol:acetonitrile (3:3:4); detected with Van Urk spray]. Combined fractions were examined by bioautog-raphy [silica ~el TLC acetonitrile:acetone:water *Trademark 7~7~

X-5279 -55_ 52:2:1) solvent system and Micrococcus luteus as the detecting organism] and were shown to consist of a single bioactive componentD This procedure gave 6.02 g of NTrp-[N~~_-decanoyl)-L-phenylalanyl]~Norn-t-Boc-A-21978C nucleus [compound of formula 5: R = N-(n-decanoyl-L-phenylalanyl); RI= t-BOC]~
C. Preparation of NTrp-~N-( -Decanoyl)-L-phenyl alanyl]-A~21978C Nucleus A flask (100 ml) was cooled to 5C. in an icebath. NTrp-[~-(--decanoyl)-L phenylalanyl]-NOrn-t-BOC A-21978C nucleus (6.02 g, 0.008 mole), prepared as described in Section B, and then anhydrous tri-fluoroacetic acid containing 2P~ anisole (50 ml) were added to the flask. The mixture, which went into solution in approximately two minutes, was stirred under an atmosphere of nitroyen for ten minutes. The solution was evaporated to dryness under reduced pres-sure at below 40~C. to give a gummy solid which was triturated twice with a diethyl ether:dichloromethane (4:1) solution (two 100-ml volumes). The solids were collected by filtration and washed with diethyl ether to give the TFA salt. This was dissolved in water (50 ml), and the pH of the solution was adjusted to 5.4 with pyridine. The solution was then lyophilized to yive 6.1 y of off-white lyophilizate.
The lyophil1~ate, dissolved in methanol (35 ml), was purified using a reverse-phase C18 silica-gel column (Waters Associates, Rrep 500), elutiny in 30 stepwise gradient with H2O:CH3OH:CH3CN containing 0.1%

~2~1~7~

pyridinium acetate at ratios of 3:1:2, 2:1:2 and 1:2:2 and collecting fractions having a volume of 250 ml~
The desired product was eluted during the 2:1:2 elution The fractions containing the product were lyophilized to give 2.23 g of cream-colored NTrp-[N-(n-deeanoyl) phenylalanyl]-A-21978C nueleus (eompound of formula 5:
R = M-(n-deeanoyl)-L-phenylalanyl; Rl= H).
The produet was evaluated by analytieal HPLC
[reversed-phase C18 siliea-gel eolumn, MeOH:CH3CN:H20:
il~ PyOAe (15:35:49:1) solvent and UV deteetion at 230 nm], by TLC [reversed-phase C18 silica-gel plates (Whatman), H~O:CH30H:CH3CN (3:3:4) solvent and Van Urk's spray and short-wave UV for deteetion] and by bioautography [silica-gel TLC (Merek), an H20:CH3CN:acetone (1:2:2) i5 solvent, and Mieroeoccus luteus as the deteeting organ-~sm] Eaeh of these methods demonstrated that the product was homogeneous. Substitution at the tryptophan ,~-terminus positlon was confirmed by 360 MHz PMR.
~mino-acid analysis confirmed the incorporation of one equivalent of L-phenylalanine into the product.
EX~IPLE 8 The following procedure illustrates the preparation of other eompounds of formula S. The ?5 speeifie eompounds prepared by this proeedure are the eompounds of formula 5 wherein R is _-(n-dodecyloxy)-benzo~fl ar.a F.l is t-sOC or hydrogen.

3C!

; ~ ' ~2~i7~7 X 5279 ~57~

A- Preparation of NTrp-~(n~Dodecyloxy)benzoyl-N
t-BOC-A-21978C Nucleus A solution of p-~n-dodecyloxy)benzoyl 2,4,5-trichlorophenolate (0.9 g, mole), A 21978C t-~OC nucleus (0.9 gm, mole) in 400 ml o anhydrous dimethylformamide was allowed to stir at room temperature for 120 hours under an atmosphere of nitrogen. The solvent was removed by evaporation under reduced pressure. The residual material was stirred with a mixture of diethyl ether (~00 ml) and chloroform (400 ml) for 2 hours.
The product was separated by filtration and dried to glve a light brown powder (0.962 g). A portion of this material (0.78 g) was dissolved in methanol (200 ml) and purified by preparative HPLC, using a "Prep LC/
System 500" unit (Waters Associates; Inc., Milford Mass.) and a ~rep Pak-500~C18 Column (Waters Associates) as a stationary phase. The column was operated iso-cratically, using a water:methanol:acetonitrile (2:1:2) solvent system and collectin~ 250-ml fractions (1 frac-tion/min.). The desixed compound was eluted in the 2nd to the 6th fractions.
Fractions were combined on the basis of TLC
[reverse phase/C18 silica gel, developed with water:
methanol:acetonitrile (3:3:4), detected with Van Urk spray]. Bioautography of the combined fractions, using silica gel TLC, an acetonitrile:acetone:water (2:2:1) solvent, and Staphylococcus aureus as the detecting organism, indicated that the product was a single bioactive component. This procedure gave 0.~21 g of N -p-(n-dodecyloxy)benzoyl-Norn-t-Boc -21978C
nucleus.

,. ~

~2~

X-5279 -58~

B. Preparation of NTrp-_-(n-dodecyloxy)benzoyl-A-2l978c ~ucleus __ N -p-(n Dodecyloxy)benzoyl NO~n 21978C nucleus (230 mg) was dissolved in 5 ml of tri-fluoroacetic acid containing 2% anisole and stlrred for 5 minutes at 0C. The solution was concentrated to an oil under vacuum, and the oil was triturated with Et2O
(100 ml). The solids were separated, air dried, and taken up in water (10 ml). The pH of this solution was adjusted from 3.25 to 7 by the addition of pyridine.
The resulting solution was lyophilized to give 179 mg of white amorphous NTrp-p-(-~dodecyloxy)benzoyl-~-21978C nucleus. This compound has an Rf value of about 0.78 on silica-gel TLC, using an acetonitrile:acetone:
water (2:2:1) solvent system and Van Urk spray for detection.

The antibacterial activity of the compounds of formulas 4 and 5 can be demonstrated n vitro in standard ayar-plate disc-difEusion tests and agar-dilution tests and in vivo in standard tests in mice which assess efectiveness against a systemic bacterial infection~ The results of the antibacterial testing of representative compounds of formulas 4 and 5 are set forth in Tables I, II, and III.
Table I gives -the results of the testing n vitro of the compounds of Examples 6-8 by agar-plate disc-diffusion methods~

U~7~7 Table II gives the results of the testing of the compounds o~ Examples 6-8 by standard agar-dilu-tion tests. In Table II activity is measured by the minimal inhibitory concentration (MIC), i.e. the lowest concentration at which the test compound inhibits growth of -the microorganism.
The results of in vivo tests to evaluate the effectiveness oE the derivatives against experimental bacterial infections in mice are given in Table III.
In these -tests two doses of test compound were adminis-tered subcutaneously or orally -to mice with illustra-tive infections, The activity observed was measured as an ED50 value [effective dose in mg/kg to protect fifty percent of the test animals: See Warren Wick, et al., J. Bacteriol. 81, 233-235 (19613]~
The toxicity of representative compounds of formulas 4 and 5 is set forth in Table IV.

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Claims (19)

1. A process for preparing an A-21978C
cyclic peptide of the formula (1):

in which R is selected from the group consisting of hydrogen, an amino-protecting group, 8-methyldecanoyl, 10-methylundecanoyl, 10-methyldodecanoyl, the specific C10-alkanoyl group of A-21978C factor C0 and the specific C12-alkanoyl groups of A-21978C factors C4 and C5; R1 and R2 are, independently, hydrogen or an amino-protecting group; provided that, when R is other than hydrogen or an amino-protecting group, at least one of R1 and R2 must be an amino-protecting group; or a pharmaceutically-acceptable salt thereof, which process comprises (a) the protection of one or more of the amino groups NHR, NHR1 and NHR2 of a compound of formula (1) as defined hereinabove in which R, R1 or R2 are hydrogen so as to prepare a compound of said formula (1) in which one or more of R, R1 and R2 represent an amino-protecting group, provided that one or more of R, R1, and R2 were initially hydrogen, and/or (b) the enzymatic deacylation using an enzyme produced by a microorganism of the family Actinoplanaceae of a compound of formula (1) in which R is other than hydrogen or an amino protecting group, so as to prepare a compound of formula (1) in which R is hydrogen, and, optionally, if so desired, removing any R1 or R2 amino protecting groups which may be present in the product of the reaction; and if desired, forming a pharmaceutically acceptable salt of said compound of formula (1).

2. A process according to claim 1 in which the method of deacylation comprises exposing the com-pound of formula (1) in an aqueous medium to said enzyme which deacylates and which is produced by a micro-organism of the family Actinoplanaceae until substan-tial deacylation is accomplished.

3. A process according to claim 2 in which the microorganism of the family Actinoplanaceae is a member of the genus Actinoplanes.

4. A process according to claim 3 in which the microorganism is Actinoplanes utahensis.

5. A process according to claim 3 in which the microorganism is A. utahensis NRRL 12052 or a mutant or variant thereof.

6. A process according to claim 3 in which the microorganism is Streptosporangium roseum var. hol-landensis NRRL 12064, or a mutant or variant thereof.

7. A process according to claim 3 in which the microorganism is Actinoplanes missouriensis NRRL
12053 or a mutant or variant thereof.

8. A process according to claim 3 in which the microorganism is Actinoplanes sp. NRRL 12065 or a mutant or variant thereof.

9. A process according to claim 3 in which the microorganism is Actinoplanes sp. NRRL 8122 or a mutant or variant thereof.

10. The process of claim 2 in which the enzyme is present in a culture of the producing Actinoplanaceae microorganism.

11. A process according to claim 1 in which R
is hydrogen or a pharmaceutically-acceptable salt thereof.

12. A process according to claim 11 in which R1 is hydrogen or a pharmaceutically-acceptable salt thereof.

13. A process according to claim 11 in which R2 is hydrogen or a pharmaceutically-acceptable salt thereof.

14. A process according to claim 11 in which R2 is an amino-protecting group, or a pharma-ceutically-acceptable salt thereof.

15. A process according to claims 11, 13 or 14 in which R1 is an amino-protecting group, or a pharmaceutically-acceptable salt thereof.

16. A compound of formula (1) as defined in claim 1, whenever prepared by the process according to any one of claims 1 to 3 or by an obvious chemical or biological equivalent thereof.

17. A compound of formula (1) as defined in claim 1 in which R is hydrogen or a pharmaceutically acceptable salt thereof, when prepared by the process of claim 11 or by an obvious chemical or biological equivalent thereof.

18. A compound of formula (1) as defined in claim 1 in which R and R1 are both hydrogen or a pharmaceutically acceptable salt thereof, when prepared by the process of claim 12 or by an obvious chemical equivalent thereof.

19. A compound of formula (1) as defined in claim 1 in which R and R2 are both hydrogen or a pharmaceutically acceptable salt thereof, when prepared by the process of claim 13 or an obvious chemical equivalent thereof.