DD210285A5 - PROCESS FOR PREPARING A-21978C CYCLOPEPTIDE DERIVATIVES - Google Patents

Abstract

A process for the preparation of A-21978C cyclopeptide derivatives having the formula I formula wherein R is hydrogen, an amino-protecting group, 8-methyl decanoyl, 10-methyl undecanoyl, 10-methyl dodecanoyl, the special C low 10 alkanoyl group of factor C low O of A -21978C or the special C 12-alkanoyl groups of the factors C deep 4 and C deep 5 of A-21978C and R hoch1 and R 2 represent hydrogen or an amino protecting group independently, with the proviso that at least one of the substituents R is 1 and R 2 must be an amino-protecting group if R is other than hydrogen or an amino-protecting group, or pharmaceutically-acceptable salts thereof, by protection of one or more of the amino groups NHR, NHR-1 and NHR-2 to form a compound of the formula I in which one or more of the substituents R, R is 1 and R 2 is an amino-protecting group, with the proviso that one or more a plurality of the substituents R, R are highl and Rhigh 2 are initially hydrogen, and / or by deacylating a compound of formula I, wherein R has a meaning other than hydrogen or an amino protecting group, to form a compound of formula I wherein R is hydrogen and, optionally, removing any amino protecting groups R "1" or R "tall" 2 present in the reaction product, preferably using a microorganism of the Actinoplanaceae family for deacylation, especially microorganisms Actinoplanes utahensis NRRL 12052, Streptosporgangium roseum var. hollandensis NRRL 12064, Actinoplanes missouriensis NRRL 12053, Actinoplanes sp. NRRL 2065 or Actinoplanes sp. NRRL 8122 or mutants or variants thereof are used.

Description

Process for the preparation of A-21978C cyclopeptide derivatives

Field of application of the invention:

The invention relates to novel derivatives of cyclopeptides having antibiotic properties, and to the process for preparing these antibiotically active derivatives by semisynthetic route.

Characteristic of the known technical solutions and object of the invention:

The great likelihood and constant risk of the formation of antibiotic-specific resistant strains of pathogenic microorganisms makes the development of new antibiotics a major requirement. In particular, pathogens within the Gram-positive species of Staphylococcus and Streptococcus are often resistant to commonly used antibiotics such as penicillin and erythromycin in this context, reference is made, for example, to WO Foye, Principles of iMedicinal. Chemistry, pages 684 to 686 (1974). The object of the invention is therefore the creation of new and particularly effective antibiotics.

Explanation of the essence of the invention :

This object is achieved according to the invention by new

A-21978C cyclopeptide derivatives and pharmaceutically acceptable salts thereof which have been found to be effective antibiotic agents.

These new derivatives can be prepared by reacting the nucleus of A-21978C or slotted derivatives thereof, which nucleus is prepared by deacylation of the appropriately blocked complex of A-21978C or any of its suitably blocked single factors C-, C-, C ~, C, , C. or C ₁ . is obtainable with the desired acylating agent or an activated derivative thereof.

The A-21978C cyclopeptide derivatives according to the invention have the formula I.

Hoa

aC

> = O

Π3

 rf

Hoi

H3C '«

> IH

> = O

H H i

A /

r ϊ

, cosh

ΤΛ A>

1 r

β- R-

wherein R is hydrogen, an amino-protecting group, 8-methyldecanoyl, 10-methylundecanoyl, 10-methyldodecanoyl , the specific C, "- alkanoyl group of the factor C _n of A-21978C or the particular C," - alkanoyl groups of the factors C.

12 and C_ of A-21978C and R and R independently represent hydrogen or an amxno protecting group, with the proviso that at least one of the substituents R

2 and R must be an amino protecting group, if R is other than hydrogen or an amino. or are pharmaceutically acceptable salts of these peptides, and these compounds are valuable intermediates for the preparation of semisynthetic antibacterial agents.

10,

The compounds of the formula I in which R has a meaning other than hydrogen or an amino-protecting group are blocked factors Cq, C ,, C-, C ₃ , C and C _{5 of} the antibiotic A-21978C. These blocked antibiotic compounds are useful as intermediates for the preparation of certain peptides of formula I, namely those compounds in which R is hydrogen and at least one of the sub-protecting group means.

The compound of formula I, wherein R, R and R are each hydrogen, is the common cyclopeptide present in C ', C-, C-, C ,, C ₄ and C_ factors of antibiotic A-21978C is. For simplicity, this compound will be referred to as the core of A-21978C. It can also be represented by the following formula II:

1 2 at least one of the substituents R and R is an amino

Gi

D-Al 3 A L-A s o T 'L-O r r, L-Th r ΐ D-Ser ι 3MG i 0 \ L-ky η G I y

LA-Sp 15 ^! ,

N H 2

(II)

wherein 3MG is L-threo-3-methylglutamic acid.

The invention also includes a method for the enzymatic

the deacylation of the complex of A-21978C, the factors C _Q , C-,, C ₂ , C ₃ , C and C ₅ of A-21978C, of the blocked complex of A-21978C and the blocked

Factors C _Q , C ₁ ZC ₃ , C ₃ , C ₄ and C ₅ of A-21978C. The naturally occurring factors of A-21978C. Have one common nucleotide of cyclopeptide, but each have a different one. Fatty acid group on. The process of the present invention for removing the fatty acid groups from the factors of A-21978C consists of allowing the antibiotic or blocked antibiotic to act in an aqueous medium on an enzyme formed by a microorganism of the family Actinoplanaceae until the deacylation becomes practical finished.

A preferred embodiment of the method according to the invention consists in the use of an enzyme formed by the microorganism Actinoplanes utahensis NRRL 12052.

] zyms for cleavage of the fatty acid side chain. To effect such deacylation, the respective antibiotic or blocked antibiotic is added to a culture of Actinoplanes utahensis and the culture is allowed to incubate until the deacylation is complete. The resulting A - 21978C cyclopeptide or blocked peptide is separated from the fermentation broth using methods known per se.

The A-21978C cyclopeptides of the formula I are valuable products, since they can be converted by reacylation into new anti-biotic substances.

The infrared absorption [] spectra resulting from the drawing, which are recorded in KBr, show:

Fig. 1: A-21978C core (formula I: R, R ¹ , R ² = H)

Fig. 2: A-21978C N _n -t-BOC core (Formula I: R and R ¹ = ~ Orn

20 H, A ~ = t-butoxycarbonyl)

In the present description, the following abbreviations are used, which are mostly conventional designations:

Ala: alanine

Asp: aspartic acid

Gly: glycine

Kyn: Kynurenin

30 Orn: ornithine

Ser: serine ·:

Thr: threonine

Trp: tryptophan

t-BOC: t-butoxycarbonyl

35 Cbz: benzyloxycarbonyl

DMF: dimethylformamide

THF: tetrahydrofuran

HPLC: high performance liquid chromatography

1 TLC: thin layer chromatography UV: ultraviolet

Antibiotics A-21978C are closely related acid peptide antibiotics and are described in U.S. Patent 4,208,403, which is hereby incorporated by reference. As is known from US-PS 4,208,403. the antibiotic complex A-21978 contains a predominant component, namely factor C, which itself is a complex of closely related factors. Factor C of A-21978, referred to as complex A-21978C, contains the single factors

^C 0 ^'C l' ^C 2 ^'C V ^C 4 ^{and C} 5'^'D i ^{e factors c} i' C_ and C ₃ are predominant factors, while the factors C _{Q /} C, and C ₁ - represent minor factors. The structure of the factors of A-21978C is shown by the following formula III:

L-As ρ Gly -A I 3 .D-Ser 3MG i t L-A s ρ ί L-Om L-Ky π " / \ GIv

  L-T'nr

L ^ Asp f

30. ^L T ^P

    , L-Tr-p

35. -

Where 3MG is L-threo-3-methylglutamic acid and R is a particular fatty acid residue. The special ones

Residues R of the individual factors have the meanings given below:

Factors of A-21978C Rest R ^N 5 ^C l 8-methyldecanoyl ^C 2 10-methylundecanoyl ^C 3 10 Methyldodecanoyl S C, Q alkanoyl * U ^C 4 C ₁₂ alkanoyl * ^C 5 C, _2- alkanoyl *

* The exact nature of these remains has not been determined to date 15

When faced with the problem of deacylating a peptide antibiotic, it is extremely difficult to know if there is an enzyme that can be used for this purpose. The discovery of such an enzyme is

even more difficult if the antibiotic substrate contains a cyclopeptide nucleus. Enzymes have a high degree of specificity, and differences in the peptide moiety and in the side chain of the substrate therefore influence the result of the attempt at deacylation. A

In addition, a series of microorganisms forms a large number of peptidases which attack different parts of the peptide residue. This often leads to unmanageable product mixtures.

For each of the A-21978C antibiotics (Formula III), the

N fatty acid side chain (R) bound to the cX-amino group of the tryptophan residue. According to the invention, this fatty acid side chain can be cleaved off by an enzyme without thereby impairing the chemical integrity of the remainder of the A-21978C protein.

Peptides is impaired.

The invention also relates to a novel process for the preparation of A-21978C cyclopeptide derivatives

1 of the formula I

Q NHR ¹

I)

wherein R is hydrogen, an amino-protecting group, 8-methyldecanoyl, 10-methylundecanoyl, 10-methyldodecanoyl, the particular C, Q-alkanoyl group of the factor C _Q of A-21978C or

the specific C ₁ ^ alkanoyl groups of factors C and C _r

1 2 ⁴ ^

from A-21978C and R and R independently represent hydrogen or an amino protecting group provided that when R has a meaning other than hydrogen or an amino protecting group, at least one of the substituents R and R must be an amino protecting group and pharmaceutically acceptable Salts of these compounds

35 gen.

The cyclopeptides of the formula I or their pharmaceutically acceptable salts are suitable as intermediates for

] Preparation of new antimicrobial antibacterial agents which are particularly effective against gram-positive microorganisms.

The term amino-protecting group refers to the usual amino-protecting groups that are compatible with the other functional groups in the molecule of A-21978C. Preference is given to those amino protecting groups, which can then easily be split off again. Examples of suitable protecting groups are shown in "Protective Groups in Organic Synthesis" by Theodora W. Greene, John Wiley and Sons, New. York 1981, Chapter 7. Particularly preferred amino protecting groups are t-butoxycarbonyl and benzyloxycarbonyl. _

The cyclopeptides of formula 1 wherein R is hydrogen include the factors C _n , C _1, C _1, C ₁ , C ₄ and C ₁ . the cyclopeptide A-21978C (A-21978C core) and the blocked derivatives of the A-21978C core. The A-21978C nucleus, namely the compound of formula I wherein each of the substituents R, R and R is hydrogen is also designated by formula II.

The core of A-21978C has the following characteristics: 25

Form: white amorphous solid that fluoresces under short-wave UV light

Empirical formula: C, -_ H _o _N ,, 0_,

oz ο Z Io Zo

Molecular weight: 1466 Solubility: Soluble in water

Infrared absorption spectrum (KBr): according to FIG. 1 of the drawing with absorption maxima at the following frequencies (cm):

3300 (broad), 3042 (weak), 2909 (weak), 1655 (strong), 1530 (strong), 1451 (weak),

1399 (medium), 1222 (medium), 1165 (weak), 10 63 (weak) and 758 (medium to weak).

-ιοί ultraviolet absorption spectrum (methanol): UV-magenta

xima at 223 ran (£ = 41482) and 260 nm (t = 8687).

Electrometric titration (66% aqueous dimethylformamide): presence of four titration ba-

groups with pK values of about 5.2, 6.7,

8.5 and 11.1 (initial pH = 6.1.2).

A particularly useful cyclopeptide of formula I is the compound wherein R and R are each hydrogen

and R is t-butoxycarbonyl. This compound is also referred to as t-BOC core for the sake of simplicity. The A-21978C t-BOC core has the following characteristics:

Form: white amorphous solid that fluoresces under short-wave UV light.

Empirical formula: C, _, H _n , N, 0 ~ _o Molecular weight: 1566

Solubility: Soluble in water. Infrared absorption spectrum (KBr): according to FIG

Drawing with absorption maxima in the following

- 1 frequencies (cm):.

3345 (broad), 3065 (weak), 2975 (weak), 2936 (weak), about 1710 (shoulder), 1660 (strong), 1530 (strong), 1452 (weak), 1395

(medium), 13 68 (weak), 1341 (weak), 1250 (medium), 1228 (medium), 1166 (medium to weak) and 1063 (weak). Ultraviolet absorption spectrum (90% ethanol):

UV maxima at 220 nm it = 42000) and 260 nm

 (£ = 10600).

High Performance Liquid Chromatography: retention time = 6 minutes on a 4.6 χ 30 0 nun measuring and silica gel C- filled column using a solvent

from H ₂ O / CH ₃ CN / CH, OH (80: 15: 5) containing 0.2% ammonium acetate and using a flow rate of 2 ml / min

- 11 1 UV detection.

The blocked A-21978C factors of the invention are the compounds of formula I wherein at least one of the substituents R and R is an amino-protecting group and R is selected from 8-methyl decanoyl, 10-methyl undecanoyl, 10-methyldodecanoyl, the specific C-, " Alkanoyl group of the factor C _n of A-21978C and the specific C, _2- alkanoyl groups of the factors C ₄ and C ₁ . from A-21978C.

The blocked factors of A-21978C and cyclopeptides of the invention are capable of forming salts which are also part of this invention. Such salts are suitable for example for the separation and purification of the compounds. Pharmaceutically acceptable alkali metal, alkaline earth metal, amine and acid addition salts are particularly useful. Pharmaceutically acceptable salts are understood to mean those salts which are suitable for the chemotherapy of warm-blooded animals.

For example, the A-21978C cyclopeptides of Formula I have five free carboxyl groups that can form salts. The invention therefore includes partial salts, mixed salts and complete salts of these carboxyl groups. In preparing these salts, pH ranges above 10 should be avoided as the compounds are unstable at such pH levels.

Examples of suitable alkali metal and alkaline earth metal salts of the A-21978C cyclopeptides of Formula I include the sodium, potassium, lithium, cesium, rubidium, barium, calcium and magnesium salts. Suitable amine salts of the A-21978C cyclopeptides include the ammonium salts. ze as well as the primary, secondary and tertiary C -, - C ₄ -alkylammonium and hydroxy-C ^ -C, -alky! ammonium salts. Examples of amine salts include those obtained by reacting an A-21978C cyclopeptide with ammonium hydroxide, methylamine, s-butylamine, isopropylamine, diethylamine, di-methylamine.

  - 12 -

iso-propylamine, ethanolamine, triethylamine and 3-amino-l-propanol formed compounds.

The cationic alkali metal and alkaline earth metal salts of the A-21978C cyclopeptides of Formula I are prepared using procedures common to the formation of cationic salts. For this purpose, for example, the free acid form of the A-21978C cyclopeptide is dissolved in a suitable solvent, such as warm methanol or

TO ethanol, and then such solution is added to a solution containing a stoichiometric amount of the desired inorganic base in aqueous methanol. The salt formed in this way can be isolated by conventional methods, such as by filtering off or evaporating the

15 solvent.

Similarly, the salts of organic amines can be formed. For this purpose, for example, the respective gaseous or liquid amine may be added to a solution of an A-21978C cyclopeptide in a suitable solvent, such as ethanol, and the solvent and excess amine then removed by evaporation.

The A-21978C cyclopeptides according to the invention also have, also free amino groups and can therefore form acid addition salts, which are also part of the invention. Examples of suitable acid addition salts of the A-21978C cyclopeptides include the salts formed by conventional reaction with both organic and inorganic acids, such as hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, succinic acid , citric acid, lactic acid, maleic acid , Fumaric, palmitic, cholic, pamoic, mucic, D-glutamic, d-camphoric, glutaric, glycolic, phthalic, tartaric, lauric, stearic, salicylic, methanesulfonic, benzenesulfonic, sorbic, picric, benzoic and cinnamic acids.

- 13] Preparation of the A-21978C Cyclopeptides

The A-21978C cyclopeptides of formula I wherein R is hydrogen are accessible by deacylation of a peptide antibiotic selected from the group of factors C _Q , C ,, C ₂ , CU, C, and C ₅ of A-21978C and the blocked factors C ", C ,, C, C, C, and C of A-21978C.

10 I · Production of the substrates

The factors C ", C ,, C ~, C ₃ , C ₄ and C ₅ of A-21978C are prepared as described in U.S. Patent 4,208,403. These factors are components of the A-21978C complex, which in turn is part of the A-21978 complex.

The A-21978 complex can be prepared by cultivating Streptomyces roseosporus NRRL 11379 (deposited on August 29, 1978) under submerged aerobic fermentation conditions until a substantial amount of antibiotic activity is formed. The A-21978 complex is separated by filtration of the fermentation broth. Lowering the pH of the filtrate to about pH 3, precipitating the complex and separating the complex by filtration. The separated complex can be further purified by extraction techniques. For the isolation of the single A-21978C complex and its individual factors, chromatographic separation methods are required.

The blocked A-21978C complex and the blocked factors C _Q , Cw C ₂ , C ₃ , C, and C ₅ of A-21978C according to the invention (compounds of the formula I wherein R is selected from 8-imidodecanoyl, 10 Methylundecanoyl, 10-methyldodecanoyl and the C, C and C 1-4 alkanoyl groups of the C ", C and C" factors are prepared using procedures common to the protection of amino groups on peptides. Suitable protecting groups for this purpose can be prepared from the various known amino protecting groups

and examples of these are benzyloxycarbonyl, t-butoxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, adamantyloxycarbonyl, o-nitrophenylthio, diphenylphosphinothioyl, chloro or nitrobenzyloxycarbonyl.

5 ".

The blocked A-21978C complex is a particular substrate,

suitable. It is prepared from the A-21978C complex so that one can do without the separation steps required to form the individual factors of A-21978C. However, the deacylation of this complex results in a single product, namely the appropriately blocked nucleus.

II. Deacylation Procedure 15 A. Preparation of Enzyme I _{: The} Production Microorganisms

The microorganisms capable of deacylating the factors C _Q , C ₁ , C ₂ , C ₃ , C ₄ and C ₅ of A-21978C and the blocked factors C _Q , C-, C, C ₃ , C ₄ and C from A-21978C can be produced by certain microorganisms of the family Actinoplanaceae, preferably by the microorganism Actinoplanes utahensis NRRL 12052 (deposited October 9, 1979). A preferred method for cultivating Actinoplanes utahensis NRRL 12052 to produce this enzyme is described in Example 1 below. Instead, however, other methods may be used, all of which are within the ordinary skill of the art.

The Actinoplanaceae are a family of microorganisms from the order Actinomycetales. After a first description of Dr. med. This family was then founded by John N. Couch in 1955 (J. Elisha Mitchell, Soc., 71, 148-155- (1955).) The properties of this family, and of several individual genera thereof, can be found in "Bergey's Manual of Determinative Bacteriology" 8 Edition, RE

Buchanan and N.E. Gibbons, The Williams & Wilkins Co., Baltimore, Md., 1974, pages 706 to 723. So far, the following ten different genera have been identified:

I. Actinoplanes (the type genus and thus the most widespread genus),

II. Spirillospora,

III. Streptosporangium,

IV. Amorphosporangxum, 10 V. Ampullariella,

VI. Pilimelia,

VII. Planomonospora,

VIII. Planobispora,

IX. Dactylosporangium and 15X · Kitasatoa.

Some species and varieties that have hitherto been isolated and characterized are Actinoplanes philippinensis, Actinoplanes armeniacus, Actinoplanes utahensis and Actinoplanes missouriensis; Spirillospora albida; Streptosporangium roseum, vulgar Streptosporangium, Streptosporangium roseum var. Hollandensis, Streptosporangium album, Streptosporangium viridialbum, Amorphosporangium auranticolor, Ampullariella regularis, Ampullariella campanulata, Ampullariella lobata, Ampullariella digitata, Pilimelia terevasa, Pilimelia anulata, Planomonospora parontospora, Planomonospora venezuelensis, Planobispora longispora, Planobispora rosea , Dactylosporangium aurantiacum and Dactylosporangium thailandense.

The genus Actinoplanes is: a preferred source for the enzyme required according to the invention. Within the genus Actinoplanes, the species Actinoplanes utahensis is a particularly preferred enzyme source.

Cultures of other representative useful species are available from the Northern Regional Research Center, Agricultural Research Culture Collection (NRRL), US Department of Agriculture.

culture, 1815 North University St., Peoria, Ill. 61604, V.St.A. without restriction on their availability deposited and from there under the following reference numbers freely available:

Actinoplanes utahensis. NRRL 12052 (deposited on

9 October 1979 Actinoplanes missouriensis NRRL 12053 (deposited on

October 9, 1979) Actinoplanes sp. NRRL 8122 (deposited on

October 17, 1975) Actinoplanes sp. NRRL 12065 (deposited on

November 5, 1979)

, Streptosporangium roseum NRRL 12064 (deposited, at the var. Hollandensis November 5, 1979)

Actinoplanes utahensis NRRL 12052 is derived from a stock culture deposited at the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, V.St.A., without limitation of its availability · and from there the. Reference number ATCC 14539 is freely available. The culture 'Actinoplanes utahensis ATCC 14539 can also be used as a source of the enzyme.

Actinoplanes missouriensis NRRL 12053 is derived from a culture which is also deposited with the American Type Culture Collection (ATCC) without restriction on its availability and can be obtained therefrom under the reference number ATCC 1453.8 ·. This culture Actinoplanes missouriensis ATCC 14538 is another source of the enzyme .: '

The activity of a specific strain of a microorganism of the family Actinoplanaceae for carrying out the deacylation according to the invention is determined by the following method. A suitable growth medium is inoculated with the microorganism. The culture is applied zv / egg or three days at about 30 ⁰ C on a rotary

.- 17-

Broth then incubate the culture with one of the substrate antibiotics and keeps the pH of the fermentation medium to about pH 7.0. Culture is monitored by activity with Micrococcus luteus. This procedure will be described later in Section D. A loss of antibiotic activity is an indication that the microorganism forms the enzyme suitable for deacylation. However, this must be verified using one of the following methods: (1) analysis by HPLC for the presence of the intact nucleus or (2) reacylation with a suitable side chain such as lauroyl, n-decanoyl or n-dodecanoyl to restore activity. A decrease in antibiotic activity is difficult to detect when acylating a blocked A-21978C material, since the introduction of the blocking group results in an 80-90% reduction in antibiotic activity.

20 2_ ₁ _Conditions_for_EnzYmproduction

The formation of the enzyme takes place under conditions in which Actinoplanaceae growth is possible, namely at a temperature between about 25 and about 30 ° C. and a pH between about 5.0 and about 8.0 with thorough mixing and The culture medium is intended to contain (a) an assimilable carbon source such as sucrose, glucose, glycerin and the like, (b) a nitrogen source such as peptone, urea, ammonium sulfate and the like, (c) a phosphate source such as a soluble phosphate salt, and ( d) inorganic salts which have generally been shown to be effective in promoting the growth of microorganisms An effective amount of enzyme generally results within about 40 to about 60 hours after the start of the growth cycle and lasts for some time after effective growth is achieved The amount of enzyme produced varies from one species of organism to another and is

- 18. 1 depending on the different growing conditions.

The microorganism that forms the enzyme, such as Actinoplanes utahensis NRRL 12052, is naturally susceptible to variation. Artificial variants and mutants of these strains can be obtained, for example, by treatment with various known mutagenic. Agents, such as ultraviolet radiation ,. X-radiation, high frequency waves, radioactive radiation and chemicals. All natural and artificial variants and. Mutants available from Actinoplanaceae and. form the enzyme can be used according to the invention.

15 B. Deactivation Conditions

The substrate used as the starting material is preferably added to the culture of Actinoplanaceae after this culture has been incubated for about 48 hours. The concentration of the substrate in the conversion medium can vary within wide limits. However, with maximum utilization of the enzyme and virtually complete deacylation within a period of 3 hours, the concentration of substrate is generally between about 1 and about 3 mg / ml. Lower concentrations can be used, but can not give maximum utilization of the enzyme. · It is also possible to work with higher concentrations, but the substrate can not be completely deacylated if the fermentation

30. time is not extended.

Conversion of the substrate antibiotic to the A-21978C core of the present invention proceeds best at a pH of the fermentation medium ranging from about 7.0 to about 7.2. At a pH below 7, deacylation occurs. only slowly. At pH values above pH 7.2, however, the resulting nucleus is exposed to increasingly strong alkaline hydrolysis. In Rührfermentern can be

adjust the pH with appropriate sensors - If this is eliminated for practical reasons, such as in piston fermenters, then the pH can be adjusted by adding the medium before adding the substrate

5 mixed with a 0.1 molar phosphate buffer.

After the addition of the substrate, incubation of the culture should be continued for about 3 to 6 hours or more. The purity of the substrate affects the rate of deacylation. For example, a substrate having a purity above 5% is deacylated at a rate of about 2.5 mg / ml of antibiotic in 3 hours. When substrates with lower purity are used, the deacylation proceeds more slowly.

It is also possible to use a multiple addition to substrate. For example, it is possible to add amounts of 0.75 mg / ml each of antibiotics at intervals of 24 hours in the form of at least five or more additions.

The deacylation can be carried out over a wide temperature range, for example from about '20 to about 45 ⁰ C. For the sake of optimum deacylation and stability of substrate und.des core, however, a Deacylierungstemperatur of about 30 ⁰ C is preferred.

C. The substrate

Preferably, but not necessarily, a purified antibiotic is used as the substrate. A purified substrate is soluble in water or a buffer and therefore easier to handle. Furthermore, the deacylation proceeds more rapidly with a purified substrate. Even semi-purified substrates containing only about 15% of the starting antibiotic ^OJ have been successfully deacylated.

The substrate antibiotics have antibacterial activity. The

Suhstrat materials, particularly those of low purity, while capable of harboring bacterial cells or spores that could grow in the deacylating medium and affect the deacylation reaction or the stability of the parent antibiotic or nucleus formed, have not been observed. It is therefore not necessary for the substrates to be sterile, especially not for short deacylation times

 10

D. Monitoring Deacylation

The factors C _Q , C ,, C, C ₃ , C and C ₅ of A-21978C are antibacterial agents which are particularly effective against Micrococcus luteus. To determine the amounts of substrate present, therefore, an experiment using Micrococcus luteus is preferred. The formed A-21978C core is soluble in water but biologically inactive. A decrease in biological activity is therefore a rapid and presumptive test for deacylation.

The amount of nucleated core can be quantified by HPLC analysis using the system described herein.

E. Use of dormant cells

Another method of deacylation is removal of Actinoplanaceae cells from the culture medium, resuspension of the cells in a buffer solution, and deacylation in the manner described in Section B above. By using this method one can get the enzymatically active mycelium. reuse. For example, the mycelium of Actinoplanes utahensis NRRL 12052 retains a deacylase activity after storage of 1 month or longer under refrigeration (4 to 8 ⁰ G) or in the frozen state (-20 ⁰ C)

at. A preferred buffer solution is a 0.1 molar phosphate buffer.

F. Immobilized enzymes

Another method of performing the deacylation is to immobilize the enzyme using known methods. Reference is made, for example, to "Biomedical Applications of Immobilized Enzymes and Proteins", Thomas Ming Swi Chang, Ed., Plenum Press, New York (1977), Volume 1. Using such an immobilized enzyme can then be the desired deacylation, for example, in a Reach column or other suitable reactor.

In addition, one can also immobilize the microorganism itself and use it to catalyze the deacylation reaction.

Usefulness of the A-21978C cvclopeptides

The A-21978C cyclopeptides and their salts are valuable intermediates for the preparation of semisynthetic antibacterial compounds.

Chemotherapeutically useful compounds are described in copending application Serial No. X-5163 filed the same day, and these compounds have the following general formula IV:

S \ h 6o ₂ h il Il i

Il i

¹⁵ I Ϊ } I

HOzC

IV

1 2 wherein R, R and R are independently hydrogen, C.-C, alkyl., Optionally substituted C ₂ ^- C, g-alkanoyl, Cc ^- mean C-, Q-alkenoyl or an amino protecting group, R ^3, R ⁴ and R ^{5 are} all hydrogen or (i) R and R and / or (ii) R and R and / or (iii) R and R together may represent a C ₄ -C,, alkylidene group, with In that (1) at least one of the substituents R, R or R has a meaning other than hydrogen or an amino-protecting group, (2) at least one of the sub-

1 2 substituents R or R must represent hydrogen or an amino protecting group, (3) the substituents R, R and R.

must contain at least 4 carbon atoms together. and (4) if R and R are selected from hydrogen or an amino-protecting group, the substituent R then-not for 8-methyldecanoyl, 10-methylundecanoyl, 10-methyldodecanoyl, the specific C, _Q alkanoyl group of the factor Cq of A- 21978C or the particular C, ₂ alkanoyl groups of factors C _a and C ₅ of A-21978C, or are salts of these compounds.

The term C ₄ -C-, _4- alkylidenyl refers to a group of the formula

R ³

Wherein R and R are hydrogen or an alkyl group having 3 to 13 carbon atoms, with the proviso that. a ·

3 4 '

the substituent R and R must have a meaning other than hydrogen.muß and the further proviso that the sum

3 4

the carbon atoms in the substituents R and R may not be greater than 13. Those compounds in which one of the substituents R, R or R is C ₄ -C ₁₄ -alkylidenyl are referred to as Schiff bases.

The term C ₄ -C 1 -alkyl refers to a monovalent saturated straight-chain or branched-chain alkyl group having 4 to 14 carbon atoms. The compounds wherein one of the substituents R, R or R is C ₄ -C 10 alkyl are prepared by reduction of the corresponding compounds wherein the group R, R or R is C 1 -C ₄ -alkylidenyl and are referred to herein as reduced ship bases.

The statements optionally substituted C - C, g - alkanoyl and C ₅ -C, Q alkenoyl refer to acyl groups derived from carboxylic acids containing 2 to 19 and 5 to 19 carbon atoms, respectively. When the group R is alkanoyl, then the alkyl part is a monovalent saturated straight or branched chain hydrocarbon radical which may optionally carry one hydroxyl group or 1 to 3 halogen substituents selected from chlorine, bromine or fluorine. When R is alkenoyl, then the alkenyl moiety is a monovalent unsaturated straight chain or branched chain hydrocarbon moiety containing no more than three double bonds. The double bonds of the unsaturated hydrocarbon chain can

- 24 have either the cis or the trans configuration.

The term amino protecting group refers to the usual amino protecting groups of the type already mentioned above.

5 ... '

Subclasses include the following connection groups

Formula IV preferred: -

, Q- (a) The compounds wherein R is alkanoyl of the formula

11

CH- (CH-) -C- and η is an integer from 3 to 17;

12 (b) the compounds wherein R is alkanoyl of the formula

  .-O

Il

CH _{3 is} (CH ₂ ) _n -C- and η is 5 to 14;

(c) the compounds wherein R is alkanoyl of the formula 20 ^CH -5 O

I ^J Il

CH- (CH-) CH (CH-) -C- and the indices η and m j 2 η 2 m

each independently represent an integer from 0 to 14,: with the proviso that η + m is not less than 1 and

should not be greater than 15, and with the further proviso that, if η is 0, m is not 8

and, if η is 1, m can not be 6 or 8;

(d) the compounds wherein R is eis or trans-alkenyl of the formula. 0

CH (CH,). CH = CH (CH-) -C- and η and m je-3 2 η. 2m ^y .

because independently are an integer from 0 to 14, with the proviso that η + m must not be less than 1 nor greater than 15;

35. , -

(e) the compounds wherein R is cis- or trans-alkenyl

- 25 of formula 0

Il

CH = CH (CH-, J-C- and η is an integer from 4 to 15;

(f) the compounds wherein R is alkyl of formula

CH ₃ (CH ₂ ) - and η is an integer from 5 to 12;

(g) the compounds in which R has the following meanings:

?

CH ₃ (CH ₂ ) ₅ -C-

CH ₃ (CH

CH ₃ (CH ₂ ) ₇ -C-

CH ₃ (CH ₂ ) g CO

CH ₃ (CH ₂ J ₁₀ -C-

O CH ₀ = CH- (CH-) ₀ -C-

?

CH ₃ (CH ₂ J ₁₁ -C-

O CH ₃ (CH ₂ J ₁₂ -C-

CH ₃ - (CH ₂ J ₃ -CH = CH- (CH ₂ J ₇ -C-

O CH ₃ (CH ₂ J ₁₃ -C-

CH, CH-CH (CH,) - (CH ₀₎ -C- 35 · ^{32 3}

CH ₃ (CH ₂ J ₁₁ -

(CH ₂ ) ₁₀ CH =

10

CH ₃ (CH ₂ ) ₆ -CH ₃ (CH ₂ J ₅ CH =

CH ₃ (CH ₂ ) ₉ -

CH ₃ (CH ₂ )

15

The chemotherapeutically useful compounds described in the co-pending applications filed on the same day as the present application with the internal references .X-5108 and X-5205 have the following general formula V:

HO2

20 25

¹ HOa

35

ν.

In the derivatives described in the above-mentioned Parallelanmeildung with the internal application number X-5205 the

Structure V are those compounds wherein R is hydrogen, 8-methyldecanoyl, 10-methyldodecanoyl, 10-methylundecanoyl, the particular C, "- alkanoyl group of the C _n factor of A-21978C, or the particular C, -alkanoyl groups of Factors C and C, from A-21978C, an amino protecting group, an aminoacyl group of the formula

O - C - Q - NH ₂

wherein Q is C, -C ,, - alkylene, or an N-alkanoyl

aminoacyl group of the formula

Il

₁₅ -WCR ²

where W is one of the following divalent aminoacyl radicals:

O 20 (a) -C-A-NH-,

where A is C -C. »-Alkylene or C ₅ -C -cycloalkyleh.,

OR ³

(b) -C-CH-NH-,

wherein R is hydroxymethyl, hydroxyethyl ₇ mercaptomethyl, mercaptoethyl, methylthioethyl, 2-thienyl, 3-IndolinethyI, phenyl, benzyl or substituted phenyl or substituted benzyl, wherein the benzene ring by chlorine, bromine, iodine, nitro, C ₁ -C ₃ -Alkyl, hydroxy, C ₁ -C ₃ -alkoxy, C ₃ -C ₃ -alkyl.

thio, carbamyl or C ..- C alkylcarbamyl is substituted,

(C)

35 ^.

wherein X is hydrogen, chlorine, bromine, iodine, amino,. Nitro, C 1 -C 4 -alkyl, hydroxy, C 1 -C -alkoxy, mercapto, C 1 -C -alkylthio, carbamyl or C 1 -C 4 -alkylcarbamyl,

or Γ

wherein X is chlorine, bromine, iodine, amino, hydroxy, C.-C -.- alkyl or C.-C ^ -alkoxy,

15. 20

or [j; or

25 (f) 0

wherein B is a bivalent radical of the formulas

'- / wherein η is a number from 1 to 3,

₀ )

2. η

-CH = CH-, -CH = CH-CH-- or O

  ·. -CH-NHC- means and.

2 in which R is C ₁ -C -alkyl or C ₁ -C 4 -alkenyl,

and R is hydrogen, an amino protecting group, an aminoacyl

group of the formula -C-Q-NH- of the type indicated above or

an N-alkanoylaminoacyl group of the formula

"2

c -WCR of the above indicated kind means

with the proviso that R must be aminoacyl or N-alkanoylaminoacyl if R has a meaning other than aminoacyl or N-alkanoylaminoacyl, and further that R must be aminoacyl or N-alkanoylaminoacyl if R is an amino-protecting group or the pharmaceutically acceptable salts hereof.

The terms alkylene, alkyl,] alkoxy, alkylthio and alkenyl as used herein mean both straight-chain and branched-chain hydrocarbon radicals. By alkyl is meant a monovalent saturated hydrocarbon radical. Alkenyl means a monovalent unsaturated hydrocarbon radical containing one, two or three double bonds, each oriented in the cis or trans configuration. Alkylene means a divalent saturated hydrocarbon radical. Cycloalkylene means a divalent cyclic saturated hydrocarbon radical. 25

Examples of C -C _Q - or C -C _g -alkylene radicals, which are preferred, are as follows:

R ⁵

~ ^CE 2 ~ ' ~ ^CH ~' ^{in which r5 is} C -C -alkyl, such as methyl, ethyl, n-propyl, isopropyl, η-butyl, t-butyl, isobutyl or 1-methylpropyl, - (CH-) - in which m is a number from 2 to 10 and CH _- (CH_) -CH- (CH.) -, where ρ is a

J 2 q ι 2 p

Number is from 1 to 8 and q is a number from 0 to 7, 35

with the proviso that the sum of ρ + q is not greater than

8 may be.

Examples of C 1 -C 4 -alkyl groups which are preferred are the following:

(b) - (CH-) CH, wherein η is a number from 1 to 16, and

ί ^Η 3

(c) - (CH ₀ ) <-> H (CH ₀ ) CH, wherein r and s independently represent a number from 0 to 14, but the sum of r + s may not be greater than 14.

Examples of C ₀ -C alkenyl groups which are preferred are the following:

(a) - (CH ₀ K-CH = CH- (CH ₀₎ -CH, wherein t and u independently represent a number from 0 to 14, with the proviso that the sum of t + u is not greater than 14 " may,

(b) - (CH ₀₎ -CH = CH- (CH) -CH = CH- (CH ₀₎ -CH ,, wherein ν and ζ independently a number from 0 to 11 mean

on and y stands for a number from 1 to 12, with the proviso that the sum of ν + y + ζ may not be greater than 12.

The following C ₁ -C. _7- Alkyl groups are particularly preferred: ²⁵

CH ₃ (CH ₂ ) ₆ 30

35 CH ₃ (CH ₂ ) ₁₆ -

The following C ₀ -C, _ alkenyl groups are particularly preferred

CiS-CH ₃ (CH ₂₎ CH = CH (CH ₂₎ -. trans -CH ₃ (CH ₂ J ₅ CH = CH (CH ₂ J

31 CIS-CH ₃ (CH ₂ J ₁₀ CH = CH (CH ₂ ) ₄ -

trans -CH ₃ (CH ₂ ) _1Q CH = CH CiS-CH ₃ (CH ₂ ) ₇ CH = CH trans-CH ₃ (CH ₂ ) _? CH = CH CiS-CH ₃ (CH ₂ ) ₅ CH = CH

, trans -CH ₃ (CH ₂ ) ₅ CH = CH (CH ₂ ) _g

CXS, CiS-CH ₃ (CH ₂ ) ₄ CH = CHCH ₂ CH = CH (CH ₂ ) _? - ¹⁰ trans, trans -CH ₃ (CH ₂ ) ₄ CH = CHCH ₂ CH = CH (CH ₃ ) _? -

cis, cis, CIS-CH ₃ CH ₂ CH = CHCH ₂ CK = CHCH ₂ CH = CH- (CH ₂ ) ₇ -.

If the divalent aminoacyl radical W is a divalent radical of the formula

-NH-

Il

then, of course, the functions -C- and -NH- on the benzene ring may be oriented in ortho, meta or para configuration to one another. The substituent X may be present in any available position of the benzene ring. Preference is given to those compounds in which X is hydrogen, and the functions

O -C-. and -NH- are oriented in the para configuration.

Substituted phenyl and substituted benzyl as defined by the substituent R in formula V is taken to mean a substituent on any of the available positions in the benzene ring such that

25, this substituent in ortho, meta or para

Configuration can be located. The term C ₁ -C 4 -alkyl corresponding to the substituents R or X in the formula V includes methyl, ethyl, n-propyl or isopropyl.

The derivatives of structure V described in copending application Serial No. X-5108 filed on the same date as the present application are those wherein R is a substituted benzoyl group of the following formula:

ίο \ χ ·

in which X is hydrogen, chlorine, bromine, iodine, nitro, C 1 -C 4 -alkyl,

2 is hydroxy, C ₁ -C ₃ -alkoxy or C ₁ -C 4 -alkylthio and R is C ₁ -C 4 -alkyl, and R 'is hydrogen or an amino-protecting group, or pharmaceutically acceptable salts thereof.

In the substituted benzoyl group, the functions

"2

-C and -OR in ortho, meta or para position to each other. The para orientation is preferred for these groups. The substituent X may be present at any available position of the benzene ring which is not occupied by these two groups.

The term alkyl means both straight-chain and branched hydrocarbon chains.

Examples of C _g -C, ^ -alkyl radicals which are preferred as substituent R are the following:

.. (a) - (CH ₂ ) CH, wherein η is an integer from 7 to 14,

, and. CH

(b) - (CH ₉₎ CH (CH ₉₎ CH ,, wherein r and s are each independently an integer from 0 to 12, wherein the sum of r + s but not more than 12 or not smaller than 5 may be.

'-Year

The compounds of formulas IV and V can be prepared by acylating a compound of formula I with the appropriate acyl side chain using methods conventional in forming an amide bond. These. Acylation is generally carried out by reacting the particular compound with an activated derivative of the acid corresponding to the desired acyl side chain group.

By an activated derivative is meant a derivative which renders the carboxyl function of the acylating agent reactive for coupling with the primary amino group to form the amide linkage linking the acyl side chain to the nucleus. Suitable activated derivatives, processes for their preparation and methods for their use as acylating agents for a primary amine are known to those skilled in the art. Preferred activated derivatives are (a) acid halides such as acid chlorides, (b) acid anhydrides such as alkoxy-formic anhydrides or aryloxy-formic anhydrides, or (c) activated esters such as 2,4,5-trichlorophenyl ester. Other methods for activating carboxyl function include reacting the carboxylic acid with a carbonyldiimide such as, N, N''-dicyclohexylcarbodiimide or N, N'-diisopropylcarbodiimide to form a reactive intermediate which is not isolated due to its instability. Such a reaction with the primary amine is carried out directly in the reaction mixture.

A preferred process for the preparation of the compounds of the formulas IV and V is the active ester method. For this purpose, the 2,4,5-trichlorophenyl ester of the desired acid is used in particular as the acylating agent. In this method, an excess amount of the active ester is reacted with a compound of formula I at room temperature in a non-reactive organic solvent such as DMF. The implementation time is not critical, although a reaction

tion time of about 6 to about 20 hours is preferred. After completion of the reaction, the solvent is removed and the residue is purified by a suitable method, for example, by column chromatography. The t-BOC groups may be removed by treatment with a mixture of trifluoroacetic acid, anisole and triethylsilane or, preferably, a mixture of trifluoroacetic acid and 1,2-ethanedithiol over a period of about 3 to about 5 minutes at room temperature. After Entfer ^nen of the solvent can clean the residue, for example, by reverse phase HPLC.

The 2,4,5-trichlorophenyl esters of the corresponding acids are most easily prepared by treating the desired acid with 2,4,5-trichlorophenol in the presence of a coupling agent such as N, N'-dicyclohexylcarbodiimide. Other methods suitable for the production of acid esters are within the skill of the art.

The alkanecarboxylic acids and alkenecarboxylic acids used as starting materials for the preparation of derivatives of the formula I and the activated derivatives thereof, in particular the acid chlorides and the 2,4,5-trichlorophenyl esters, are either known compounds or can be known from known compounds. Be made way. The 2,4,5-trichlorophenyl esters are best prepared by treating the acid chloride of the respective alkanecarboxylic acid or alkene carboxylic acid with 2,4,5-trichlorophenol in the presence of pyridine or by treating the free alkanecarboxylic acid or alkene carboxylic acid with 2,4,5-trichlorophenol in Presence of N, N'-dicyclohexylcarbodiimide. For example, the 2,4,5-trichlorophenyl ester derivative can be purified by column chromatography on silica gel.

Die.N-alkanoylamino acids or N-alkenoylamino acids used as starting materials for derivatives of the formula V are either known compounds or can

by acylation of the respective amino acid, with the desired alkanoyl group or alkenoyl group, using methods known per se. A preferred way to prepare the N-alkanoylamino acids is to treat the respective amino acid with an alkanecarboxylic acid chloride in pyridine. The alkanecarboxylic acids or alkene carboxylic acids, the activated derivatives thereof and the amino acids are in turn either known compounds or can be prepared by known methods or modifications of known methods in a conventional manner.

Of course, if a particular amino acid contains an acylatable functional group other than the amino group, then such a group must be protected before reacting the amino acid with the reagent that employs the N-alkanoyl group or N-alkenoyl group becomes. Suitable protective groups for this purpose are all known groups which are already used for protecting a functional side chain group in peptide syntheses. Such groups are well known and the selection of a particular protecting group and method of use is within the skill of the art. For example, reference is made to "Protective Groups In Organic Chemistry", M. McOmie, Plenum Press, N.Y. (1973).

Certain amino acids used to synthesize these products can exist in optically active forms. As starting materials, both the natural configuration (L configuration) and the unnatural configuration (D configuration) can be used.

The substituted benzoic acids used as starting materials for some derivatives of A-21978C and the activated derivatives thereof are either known compounds or can be prepared from known compounds

be prepared per se known methods. The alkoxybenzoic acids are most easily prepared from a corresponding hydroxybenzoic acid by reacting a suitable alkyl halide with the disodium salt

5 of the respective hydroxybenzoic acid.

The hydroxybenzoic acids used as starting materials in the described processes and substituted derivatives thereof are also either known compounds or can be prepared using conventional methods.

The derivatives prepared from the intermediates of the invention, namely the compounds of formulas IV and V, inhibit the growth of pathogenic bacteria, as can be demonstrated by conventional biological assay methods. These compounds are therefore suitable for controlling the growth of bacteria on corresponding surfaces, for example as antiseptics, or for treating bacteria-induced infections. The antibacterial activity of these compounds has been determined in vitro using disc diffusion assays on agar plates and agar dilution assays as well as in vivo by experiments in mice infected with Staphylococcus aureus and Streptococcus pyogenes. Above all, the compounds are suitable for the treatment of infections caused by gram-positive organisms,

When an A-21978C cyclopeptide of the invention is used as the antibacterial agent, it can be administered either orally or parenterally. The administration of a compound of A-21978C is usually self-evident. Together with a pharmaceutically acceptable carrier or diluent. The dose, the particular compound of A-21978C is dependent on a number of considerations, such as the nature and strength of the infection to be treated. The for a

Administration of suitable dosage ranges and dosage units can be readily determined by one skilled in the art based on MIC and ED.sub.x values as well as toxicity data in conjunction with other factors such as pharmacokinetics, characteristics of the patient or host, and the microorganism responsible for the infection.

embodiments

The invention will be further explained by way of examples, which are not to be construed as limiting.

15 Example 1

Preparation of the core of A-2197 8C

A. Fermentation of Actinoplar.es utahensis

; . Preserve a stock culture of Actinoplanes utahensis.

NRRL 12052 and keeps it on an agar slope. To make the slope, use one of the following media: 25

Medium A ingredients amount Pre-cooked oatmeal 60.0 g yeast 2.5 g K ₂ HPO ₄ 1/0 g. Czapek Mineral Blend * 5.0 ml agar 25.0 g Deionized water σ. s. to .1 liters

The pH before autoclaving is about 5.9. It is adjusted to pH 7.2 by addition of NaOH. To

- 38 ..] the autoclaving, the pH is about 6.7. ,

* The Czapek mineral blend has the following composition; Ingredients Quantity

5 FeSO ₄ -7H ₂ O (dissolved in 2 ml conc. HCl) 2 g KCl 100 g

MgSO ₄ -7H ₂ O 100 g

Deionized water q.s. to 1 liter

Medium B

Ingredients Quantity.

J5 potato dextrin ( yeast extract enzyme hydrolyzate from casein * beef extract glucose corn starch meat peptone cane molasses. 'MgSO. * 7H-0

CaCO ₃ Czapek mineral blend

agar

Deionized water q.s.

* NZ-Amine A, Humko Sheffield Chemical, Lyndhurst, NJ, ³⁰ V. St.A.

Which slant is inoculated with Actinoplanes utahensis NRRL 12052 incubating the inoculated slant approximately 8 to 10 days at 30 ⁰ C. Using about half of the

      - '

Incidence growth is then inokuliert 50 ml of a vegetative medium having the following composition:

5.0 G G 0.5 G ml 3.0 G G 0.5 G to 1 liter 12.5 G 5.0 G 5.0 G 2.5 G 0.25 g 1.0 2.0 20.0

- 39] Ingredients Amount

Pre-cooked oatmeal 20,0 g

Sucrose 20.0 g

5 yeast 2.5 g

Dried grain veal * 5.0 g

K ₂ HPO ₄ 1.0 g

Czapek mineral mix 5.0 ml deionized water q.s. to 1 liter

The whole is adjusted with NaOH to pH 7.4, wherein the

pH after autoclaving is about 6.8.

* National Distillers Products Co., 9 9 Park Ave., New York, NY, V.St.A.

The inoculated vegetative medium is incubated in a 250 ml Erlenmeyer wide-mouth flask for about 7 2 hours at 3 0 ⁰ C on a rotary shaker, which moves in a sheet with a diameter of 5 cm at 25 0 U / min.

The incubated vegetative medium can be used directly to inoculate a second stage vegetative medium. Optionally and preferably, this vegetative medium is preserved until later use by maintaining the culture in a vapor phase of liquid nitrogen. For such storage, the culture is filled in a number of smaller ways in the following manner

In each vial, add 2 ml of incubated vegetative medium and 2 ml of a glycerol-lactose solution (Cryobiol 10, 364-367 (1973)). The suspensions thus formed are liquid in a vapor phase

Kept nitrogen

 35

Using a stored suspension prepared in this manner (1 ml), then inoculate 50 ml of a first stage vegetative medium containing the above

] has described composition. The inoculated first stage vegetative medium is then incubated in the manner described above.

To form a larger volume of inoculum, inoculate 400 ml of a second stage vegetative medium having the same composition as the first stage vegetative medium with 10 ml of the incubated first stage vegetative medium. The medium of the second stage TQ is incubated in a 2 1 Erlenmeyer wide-necked flask for about 48 hours at 3O ⁰ C.On a rotary shaker, which is moved under a sheet of 5 cm diameter with 250 U / min.

The incubated second stage vegetative medium (80 ml) prepared in the manner described above is then inoculated with 10 μl of one of the following sterile production media:

Medium I

Ingredients Quantity (g / 1)

Peanut flour 10.0

Soluble meat peptone. · 5.0

Sucrose 20.0

KH ₂ PO ₄ 0.5

K ₂ HPO ₄ 1,2

MgSO ₄ -7H ₂ O 0.25

Tap water q.s. to 1 liter

This medium has a pH of about 6.9 after sterilization by autoclaving at 121 ^° C. for 45 minutes under a pressure of about 1.1 to 1.3 bar.

- 41 Medium II

Ingredients Quantity (g / l)

5 sucrose 30.0

Peptone 5.0

K ₂ HPO ₄ 1.0

KCl 0.5

MgSO ₄ -7H 0.5

in ¹ FeS0.-7H_0 0.002

Deionized water q.s. to 1 liter

The whole is adjusted to pH 7.0 with HCl, the pH after autoclaving being about 7.0.

Medium III Quantity (g / l) ingredients 20.0 glucose 3.0 NH ₄ Cl 2.0 Na ₂ SO ₄ 0.019 ZnCl ₂ 0.304 MgCl- * 6H-0 0.062 FeCl ₃ -6H ₂ O 0,035 MnCl ₂ -4H ₂ O '0.005 CuCl ₂ -2H ₂ O 6.0. CaCO ₃ 0.67 KH ₂ PO ₄ * q.s. to 1 liter tap water

* Sterilized separately and added under aseptic conditions. At the end, the pH is around 6.6.

It is allowed to ferment the inoculated production medium in a 14 1 fermentation vessel for about 66 hours at a temperature of about 30 ° C. The fermentation medium is stirred by means of conventional stirrers at a rate of about 60 ° C / min and aerated with sterile air so that the dissolved amount of oxygen is maintained above 30% of atmospheric saturation air saturation.

in B- deacylation of A-21978C

Actinoplanes utahensis is fermented using the method described in section A. and using the medium A for the slope and the production medium I, incubating the production medium for about 67 hours. Raw A-21978C complex (100 g) is added to the fermentation medium.

The deacylation of the complex of A-21978C is monitored by examination against Micrococcus luteus. The fermentation is allowed to continue until complete deacylation, which manifests itself by a disappearance of activity against Micrococcus luteus, which after a period of about 24 hours Hours the case is.

Using the same procedure, it is also determined if another microorganism produces the desired core of A-21978C. The desired nucleus is mainly represented by Actinoplanes missouriensis NRRL 12053, Actinoplanes sp. NRRL 8122, Actinoplanes sp.

NRRL 12065 and Streptosporangium roseum var. Hollandensis NRRL 12064, as demonstrated by the use of these microorganisms instead of Actinoplanes utahensis NRRL 12052 in the above method. By HP.LC comparison using authentic samples of the nucleus obtained by this method using Actinoplanes utahensis as the deacylating enzyme, it is confirmed that these other microorganisms also form the nucleus of A-21978C. -

- 43 1 ^C * Insulation of the core of A-2197 8C

The whole fermentation broth (20 L) obtained in the manner described in Section B is filtered using a filter aid {Hyflo Super-Cel, Johns Manville Corp.). The mycelium cake is discarded. The resulting filtrate is passed through a column containing 1.5 liters of HP-20 resin (DIAION Highly Porous Polymer, HP series, Mitsubishi Chemical Industries Limited, Tokyo, Japan). The resulting column effluent is discarded. The column is then washed with deionized water (10 L) to remove any residual filtered broth. The resulting wash water is also discarded. Then, the column is eluted with mixtures of water and acetonitrile (each "10 1 in ratios of 95: 5, 9: 1 and 4: 1), wherein one captures fractions of 1 1 each.

The elution is monitored by analytical HPLC using silica gel C ^ and a solvent system of water and methanol (3: 1) containing 0.1% ammonium acetate and detection of the core with a UV monitor at 254 nm. The fractions containing the core are combined, concentrated under vacuum to remove the acetonitrile, and lyophilized to give 40.6 g of the semi-purified core of A-21978C.

D. Purification of the core of A-21978C

Dissolve the semi-purified core of A-21978C (15 g) obtained in Section C above in 75 ml of a mixture of water, methanol and acetonitrile (82: 10: 8) containing 0.2% acetic acid and 0.8% pyridine. The solution is pumped into a 4.7 x 192 cm column containing 3.33 liters of silica gel-C.jg (Quantum LP-D.) The column is developed using the same solvent system

collected with a volume of 350 ml each. Separation is monitored at 280 nm with a UV monitor. The fractions containing the core are pooled, concentrated under vacuum to remove the solvents and lyophilized to give 5.2 g of purified A-21978.C core.

Be. ispiel-2 Another method of making the core of A-21978C

The core of A-21978C will be after the im

Example 1 except certain changes made in Section B. The culture of Actinoplanes utahensis is first incubated for about 48 hours. The substrate is a semi-purified complex of A-21978C (50 g). After addition of the substrate is incubated for about 16 hours. The filtered broth is passed through a column containing 3.1 l HP-20 resin.

The column is washed with 10 volumes of water and then eluted with a mixture of water and acetonitrile (95: 5). The elution is monitored as in Preparation Example 1. After collecting 24 L of eluate, continue using a solvent mixture

^AJ eluted from water and acetonitrile (9: 1). The fractions containing the core are eluted with this solvent mixture. The resulting fractions are combined, concentrated under vacuum to remove acetonitrile and lyophilized to give 24.3 g

semi-purified core of A-21978C.

This semi-purified core of A-21978C \ (24.3 g) is dissolved in water (400 ml). The solution is pumped into a 4.7 × 192 cm steel column containing 3.33 liters of SiCl.

ciumdioxidgel-C. g (Quantum LP-1). which is prepared in a mixture of water, methanol and acetonitrile (8: 1: 1) containing 0.2% acetic acid and 0.8% pyridine. The column is filled with the same solution

developed mediumgenisch at a pressure of about 136 bar, collecting fractions of 350 ml each. Elution is by UV light at 280 nm. supervised. The fractions containing the core are combined, concentrated under vacuum to remove the solvents and lyophilized to give 14 g of highly purified core of A-21978C.

Example 3

Preparation of Factors C and C of N -t-BOC- A-21978C -

A mixture of the 1'5 factors C_ and C ₃ of A-21978C (10 g) prepared according to US Pat. No. 4,208,403 is dissolved under sonication (200 mg / ml) in water (50 ml). The pH of the solution is adjusted from 4.05 to 9.5 by addition of 5N NaOH (3.6 mL). Di-t-butyldicarbonate (3.0 ml) is added and the reaction mixture is stirred.

mixed for 2 hours at room temperature. The pH of the reaction mixture is maintained at 9.5 by the manual addition of 5 N NaOH (6.5 mL is added over 2 hours).

The reaction is monitored periodically by TLC over silica gel using a solvent system of CH ₃ CN and H 2 O (UV light.

from CH ₃ CN and HO (7: 3 and 8: 2) and detection by

After about 10 minutes, the reaction solution becomes cloudy

quickly and increases the consumption of base. After about 30 minutes, the rate of increase in cloudiness and in the consumption of base decreases, indicating "the completion of the reaction. Regardless, however, the reaction to ensure that it ends will continue for another 90 minutes. At the end of the two hour reaction time, the reaction material is immediately lyophilized. which gives you 12.7 g of the factors

C- and C- of N -t-BOC-A-219 78 arrives. 2 3 Orn.

Using similar procedures one also carries out two reactions with 10 g of material and one reaction with 30 g of material. In each of these reactions, the reaction time is only 40 minutes. The two experiments with 10 g of material lead to 10.9 g and 12.1 g of product. The reaction with 30 g of material gives 35.4 g of product.

  B e i s ρ "i e 1 4

Preparation of the core of A-21978C-N. -t-BOC

Orn

A. Fermentation of Actinoplanes utahensis

Actinoplanes utahensis is fermented according to that described in Example 1 under Section A.

Method using the inclined medium A and the production medium I, wherein the production medium is incubated for about 66 hours. ;

B. deacylation of the complex of N _Q -t-BOC-A-21978C ~~~

The complex of A-21978C-N. -t-BOC {1185 g of crude

orn

Substrate containing about 176 g complex of A-21978C) is added to the fermentation medium. The deacylation

becomes like in example 1 in section B

described carried out. The deacylation is due

a.HPLC ended after about 24 hours.

'C. Isolation of the core of A-21978C-N-t-BOC

35; ] ^Orn

The fermentation broth (100 L) obtained according to Section B is filtered using a filter aid (Hyflo.

] Super Cel). The filtrate is passed through a column containing 7.5 liters of HP-20 resin (DIAION) and the column is washed with water (38 L). The elution is carried out by silica gel-C. "HPLC monitored under UV detection at 254 nm. With the washing liquid, a certain amount of core is eluted. The subsequent actual elution of the core is carried out using the following mixtures of water and acetonitrile:

♦.

40 1 (95: 5), 40 1 (9: 1) and 100 1 (85:15). The fractions containing the core are combined, concentrated under vacuum to remove the solvent, and lyophilized to give 298.5 g of the semi-purified core of A-21978C-N 1 -t-BOC.

D. Purification of the core of A-21978C-N-t-BOC

The after. Section C obtained semi-purified core of A-21978C-N-t-BOC (30 g) is dissolved in a mixture of water and acetonitrile (9: 1) containing 0.2% acetic acid and 0.8% pyridine (75 ml ). The solution is placed on a 4.7 x 192 cm steel column containing 3.33 liters of silica gel (Quantum LP-1) equilibrated with the same solvent system. The column is developed under pressure with a solvent mixture of water, acetonitrile and methanol (80: 15: 5) containing 0.2% acetic acid and 0.8% pyridine, collecting fractions of 350 ml each

^ou and the detection of the product by UV light at 280 nm. The fractions containing the product are combined, concentrated under vacuum to remove the solvent and lyophilized to give 18.4 g of the purified core of A-21 t-BOC.

- 48 1 'Example 5

Other purification of the core of A-21978C-N -t-BOC

5 Dissolve the according to Example 4, section

C, obtained semi-purified core of A-21978C-NL · t-BOC (10.8 g) in water and the solution placed on a column containing 80 ml Amberlit IRA-68 (Rohm and Haas, Philadelphia, PA, V St.A., acetate cycle). The column is washed with water and, at a flow rate of 5 ml / min in turn first with 0.05N acetic acid (1080 ml), then with 0.1N acetic acid (840 ml) x and finally with 0.2N acetic acid (3120 ml), collecting fractions of 120 ml each.

The column is monitored by analytical HPLC over silica gel CQ using a solvent system of water, acetonitrile and methanol (80: 15: 5) containing 0.2% ammonium acetate and detection of the product by UV light at 254 nm

    is made. The fractions containing the product are combined. The pH of this solution is adjusted to 5.8 with pyridine. The solution is then concentrated under vacuum to a volume of about 200 ml.

The concentrate is mixed with water and the

concentrated again to remove pyridine. This concentrate is freeze-dried to give 3.

to 3.46 g of purified core of A-21978C-N -t-BOC

- 49 1 Example 6

The following procedure shows the preparation of compounds of formula IV by the active ester method. In particular, hereinafter compounds of formula IV are prepared wherein R is CH- (CH ₀ KCO- (n-decanoyl), R Wa substance.

1 2

R is hydrogen and R is for t-BOC or water

10N, _Tr - (n-decanoyl) A-21978C core

A. Preparation of 2,4,5-trichlorophenyl n-decanoate

A solution of decanoyl chloride (5.6 mL) and 2,4,5-trichlorophenol (5.6 g) in diethyl ether (1 liter) and pyridine (120 mL) is stirred for 4 hours. The reaction mixture is filtered and dried under vacuum. The 2,4,5-trichlorophenyl n-decanoate is dissolved in a column filled with silica gel (Woelm) using toluene gel.

Purified luol as eluent. The fractions are monitored by TLC using short wavelength UV light for detection. The appropriate fractions are pooled and dried under vacuum to give

10.4 g of 2,4,5-Trichlorphenvl-n-decanoate passes. 25

B. Acylation of N -t-BOC-A-21978C core with 2, 4,5 -trichlorophenyl-n-decanoate _:

A solution of N -t-BOC-A-21978C core (15.0 g) and JU

2,4,5-Trichlorophenyl n-decanoate (15.0 g) in dry DMF (500 ml) is stirred under nitrogen at ambient temperature for 25 hours. The mixture is then stirred for 5 hours at 60 ⁰ C until the completion of the reaction shows in TLC. The reaction mixture is concentrated under vacuum to about 200 ml and stirred with 1.2 1 of a mixture of diethyl ether and toluene (5: 1). The product is separated by filtration, washed with diethyl

ether and dried under vacuum to give 15.05 g of the N- (n-decanoyl) -N _Orn -t-BOC-A-21978C core intermediate (Formula IV: R = n-decanoyl, R = H,

R ² = t-BOC).

C. Purification of ISL · (n-decanoyl) -N _Q -t-BOC-A-21978C core

The N- (n-decanoyl) -N-t-BOC-A-21978C core intermediate is purified in the following manner. The crude preparation is dissolved in about 50 ml of the solvent system used for elution and then purified by HPLC using the Waters Prep 500 system containing a cartridge packed with reverse phase C, Q silica gel as the adsorbent. The system is named Ή O:

MeOH: CH ₃ CN (50:15:35) containing 0.2% pyridine and 0.2% HOAc. The fractions are monitored by UV light at 280 nm. The appropriate fractions are pooled and dried under vacuum to give 20.5 g of purified N- (n-decanoyl) -N-t-BOC-A-21 9 78C core. -.

D. Removal of the N ₀ -t-BOC group

to remove the t-BOC group, a solution of

ISL - (n-decanoyl), - N (1.47 g) urn _n -t-BOC-A-21978C nucleus in Xo-M

15 ml of a mixture of trifluoroacetic acid and 1,2-ethanedithiol (10: 1) for 3 minutes at ambient temperature. The reaction mixture is dried under vacuum and the residue is treated with diethyl ether (50 ml). The treated product, after washing with 20 ml of diethyl ether, is re-dried under vacuum to give 2.59 g of crude N '- (n-decanoyl) -A-21978C nucleus (Formula IV: R = n-).

    P 1 '2 decanoyl, R and R = H).

- 51 1 E. Purification of N - (n-decanoyl) -A-21978C core

The crude N- (n-decanoyl) -A-21978C core is purified by reverse phase HPLC in the following manner. The sample (2.59 g) is added as a solution in 4.0 ml of a mixture of H ₂ O: MeOH: CH ₃ CN: .pyridine. HOAc (50: 15: 35: 2: 2) was injected into a 2.5 χ 83 cm stainless steel column equipped with LP-l / C, _o as adsorber.

1 O

is filled. The column is eluted with the same solvent system. The elution is carried out at a pressure of 83 to 117 bar and at a flow rate of 10 to 12 ml / min using an LDC duplex pump (Milton Roy). The column effluent is monitored at 280 nm with a UV detector (Isco Model UA-5, Instrument Specialist Co., 4700 Superior Avenue, Lincoln, Nebraska 68504). Every 2 minutes, fractions (20 to 24 ml) are collected. The desired fractions, which show by antimicrobial activity, are combined and dried under vacuum, where

by passing to 1.05 g of product.

This purification procedure is repeated using 4.35 g, 4.25 g, 2.14 g, 2.00 g and 1.75 g crude starting derivative to give a total of 5.58 g.

N _Tr - (n-decanoyl) -A-21978C core. Example 7

This example shows the preparation of compounds of formula V. Following this procedure compounds of formula V are prepared wherein R is N- (n-decanoyl) -L-phenylalanyl and R is t-BOC or hydrogen.

Preparation of N _m - / N- (n-decanoyl) -L-phenylalanyl

irp -

A- 21978C-Ke rn _;

A. Preparation of N- (n-decanoyl) -L-phenylalanyl-2,4,5-5 '. Trichlorphenolat

A solution of N- (n-decanoyl) -L-phenylalanine (31.9 g, 0.1 mol) and 2,4,5-trichlorophenol (19.7 g, 0.1 mol) in 1 liter of anhydrous ether with N, N'-dicyclohexylcarbodiimide (20.6 g, 0.1 mol). The reaction mixture is stirred overnight at room temperature. The precipitated N, N ¹ -dicyclohexylurea is filtered off and discarded. The filtrate is concentrated to dryness under vacuum. The resulting residue is treated with ether and the solids (residual cyclohexylurea) are removed by filtration. The filtrate is evaporated to dryness under reduced pressure. The residue is recrystallized from acetonitrile to give (n-decanoyl) -L-phenylalanyl trichlorphenolat-2,4,5-crystalline reaches to 36.9 g N, melting at 124 ⁰ C 122'bis.

B. Preparation of N - / N- (n-decanoyl) -L-o-phenylalanyl

irp -

N _n -t-BOC-A-21978C core

Grn

A solution of N- (n-decanoyl) -L-phenylalanyl-2,4,5-trichlorophenolate (10 g, 0.02 MoIO and N _Orn -t-BOC-A-21978C core (10 g, 0.006 mol) in anhydrous DMF (1 liter) is stirred under nitrogen atmosphere for 96 hours at room temperature, the solvent is removed by evaporation under reduced pressure and the residual material is stirred for 2 hours with a mixture of diethyl ether (800 ml of I and chloroform (200 ml) The product is separated by filtration to give a light brown powder (10.3 g). This material (9.9 g) is dissolved in methanol (200 ml) and purified by preparative HPLC using a Prep LC / System 500 unit and a PrepPak-500 / C, _O column as a stationary phase

IO

Column is isocratic using a solvent

eluted from water, methanol and acetonitrile (2: 1: 2) and collecting 250 ml fractions at a rate of one fraction per minute. The desired compound is in fractions 9 to 22

5 eluted.

The fractions are combined on the basis of TLC (reverse phase silica gel / C, development with a mixture of water, methanol and acetonitrile (3: 3: 4), detection with Van Urk spray). The pooled fractions are assayed by bioautography (TLC with silica gel using a solvent system of acetonitrile, acetone and water (2: 2: 1) and use of Micrococcus luteus as a detection organism), indicating that they consist of a single bioactive component. This procedure yields 6.0 g of N 2 - / N- (n-decanoyl) -L-phenylalanyl / -N _n -t-BOC-A-21978C nucleus (compound of formula V: R = N- (n- de-

canoyl-L-phenylalanyl), R = t-BOC

C. Preparation of N- / N- (n-decanoyl) -L-phenylalanyl / -A-21978C core

A flask (100 ml) is cooled to 5 C in an ice bath.

The flask is first made with N- / N- (n-decanoyl) -L-phenylalanyl-7-N _Orn -t-BOC-A-21978C core (6.02 g, 0.008 mol) prepared according to section B above ^ and then treated with anhydrous trifluoroacetic acid containing 2% anisole (50 ml). The mixture, which goes into solution within about 2 minutes, is stirred for 10 minutes under a nitrogen atmosphere. The solution is evaporated under reduced pressure at a temperature of less than 4O ⁰ C to dryness so as to obtain a gummy solid, which was washed twice with a solution of diethyl ether and dichloromethane (4: .1) treated (100 ml twice). Dia solids are collected by filtration and washed with diethyl ether to give the TFA salt. This salt is dissolved in water

(50 ml) and adjust the pH of the solution to 5.4 with pyridine. The solution is then lyophilized to give 6.1 g of whitish lyophilisate.

The lyophilizate is dissolved in methanol (35 ml) and purified by means of a reverse phase C, _O silica gel column (Waters Associates, Prep 500), graded gradient with a mixed solvent of H "0: CH 2 OH: CH -CN containing 0.1% pyridinium acetate, eluted under ratios of 3: 1: 2.2: 1: 2 and 1: 2: 2, collecting fractions of 250 ml each. The desired product is eluted during the 2: 1: 2 elution. The product containing fractions are lyophilized to give

] 5 to 2.23 g of cream N- / N- (n-decanoyl) -L-phenylalanyl7-A-21978C nucleus (compound of formula V: R = N- (n-decanoyl) -L-ph'enylalanyl, R = H).

For evaluation, the product is analyzed by analytical HPLC (reverse-phase C, silica gel column, solvent).

^A V Io

Mixture of MeOH: .CH-CN: H_O: PyOAc (15:

35: 49: 1), UV detection at 230 nm), by TLC (reversed-phase C, silica gel plates (Whatman), solvent mixture of HO: CH-OH: CH CN (3: 3: 4), Detection by spraying with Van Urk spray and by short-wave UV light) and by bioautography (TLC with silica gel (Merck), solvent mixture of HO: CH ₃ CN: acetone (1: 2: 2), Micrococcus luteus detection organism) .Each of these Method shows that the product is homogeneous. Substitution at the N-terminal position of tryptophan is confirmed by 360 MHz PMR. An amino acid analysis confirms the introduction of one equivalent of L-phenylalanine into the product. ,

Example 8.

The following example shows the production of other

Compounds of the formula V. According to this method, those compounds of formula V are prepared, wherein R is p- (η-dodecyloxy material.

p- (n-dodecyloxy) benzoyl and R t-BOC or water

A. Preparation of N -p- (n-dodecyloxy) benzoyl-Nt-BOC-A-219 78C core

A solution of p- (n-dodecyloxy) benzoyl-2,4,5-trichlorophenolate (0.9 g, mol), A-21978C t-BOC nucleus (0.9 g, mol) in 400 ml of anhydrous dimethylformamide is stirred for 120 hours under nitrogen atmosphere at room temperature. Then the solvent is removed by evaporation under reduced pressure. The residual material is stirred for 2 hours with a mixture of diethyl ether (400 ml) and chloroform (400 ml). The product is separated by filtration and dried to give a light brown powder (0.962 g). A portion of this material (0.78 g) is dissolved in methanol (200 ml) and purified by preparative HPLC using a Prep LC / System 500 unit (Waters Associates, Inc. MiIford, Mass.) And a Prep Pak-500 / C, _O column (Waters Associates) as

i o

stationary phase cleaned. The column is operated isocratically using a solvent system of water, methanol and acetonitrile (2: 1: 2) and collecting 250 ml fractions (one fraction per minute). The desired compound is eluted in fractions 2 to 6.

The fractions are combined on the basis of TLC (reverse phase / C, silica gel, development with a

i o

Mixture of water, methanol and acetonitrile (3: 3: 4), detection by spraying with Van ürk spray). Bioautography of the pooled fractions using TLC over silica gel, a solvent system of acetonitrile, acetone and water (2: 2: 1) and using Staphylococcus aureus as the detection organism revealed that the product was prepared from a single biogenic gel.

active component. This procedure gives 0.421 g of N-p- (n-dodecyloxy) benzoyl-N _Q -t-BOC-A-21978C core.

B. Preparation of N -p- (n-dodecyloxy) benzoyl-A-21978C-

Core · ' .

Dissolve N _Tr -p- (n-dodecyloxy) benzoyl-N _0RN -t-BOC-A-21978C nucleus (230 mg) in 5 ml of trifluoroacetic acid containing 2% anisole, and the solution is stirred for 5 minutes at 0 ⁰ C. The solution is concentrated under vacuum to an oil and the oil is treated with diethyl ether (100 ml). The solids are separated, air dried and taken up in water (10 ml). The pH of this solution is adjusted from 3.25 to 7 by the addition of pyridine. The resulting solution is lyophilized to give 179 mg of white amorphous N_ -p- (n-dodecyloxy) benzoyl-A-21978C core. This compound has an R ^ value of about 0.78 on silica gel TLC using a solvent system of acetonitrile, acetone and water (2: 2: 1) and detection by spraying with Van Urk spray. ,

Example 9

The antibacterial activity of the compounds of formulas IV and V can be demonstrated in vitro by disk diffusion tests on agar plates and by agar dilution tests as well as in vivo by conventional tests in mice demonstrating efficacy against a systemic bacterial infection. The results obtained in the study of typical compounds of the formulas IV and V on their antibacterial activity are shown in the following Tables I, II and III. '35 ·. 'Ζ'. ...

Table I shows the experimental results in the in vitro study of the compounds of Examples 6 to 8 using the disk diffusion test on agar-platinum

- 57 1 th.

Table II shows the results obtained in the study of the compounds of Examples 6 to 8 using the usual agar dilution test. In this Table II, the activity is measured by the minimum inhibitory concentration (MIC values), namely the lowest concentration of the compound which inhibits the growth of the microorganism.

The in vivo tests to determine the effectiveness.

The results obtained for derivatives against experimentally induced bacterial infections in mice are shown in Table III. In these experiments, mice are administered subcutaneously or orally with the infections shown two doses of the compound to be tested. The observed efficacy is measured in terms of ED '', - values (effective dose in mg / kg giving protection to 50% of the experimental animals) (J. Bacteriol 81,233 bis

20 23 5 (1961)).

The toxicity of typical compounds of formulas IV and V is shown in Table IV.

TABELIiE.-I '

Antibacterial activity of compounds of the formula I according to the disc diffusion test

on agar plates

connection At game no. For mel No N substituent S size the zones of inhibition (mm) Mierococcus luteus ATCC 9341 Bacillus subtilis, ATCC 6633 6 7 8 IV V V CH ₃ (CH ₂ ) ₈ C0- (L) CT ₃ (CH ₂ ) gCONHCH (CH ₂ G ₆ H ₅ ) CO-CH ₃ (CH ₂ ) ₁₁ O- ^ CO Staphylococcus aureus ATCC 6738P Bacillus subtilis ATCC 6633 22 24 18 29 31 20 24 30 0 40 2 2 13

^a The respective compounds are in water in a concentration of. 1 mg / ml is suspended. A 7 mm slice is dipped into the suspension, which is then placed on top of the agar.

area lays. It is incubated for 24 to 48 hours at 25 to 37 ° C. Grown on minimal nutrient agar

TABLE II Antibacterial activity after the agar dilution test

test organisms

MTC values of the compounds investigated '6 ^d 7 ^e 8

Staphylococcus aureus Xl.1 Staphylococcus aureus V41 Staphylococcus aureus X400 Staphylococcus aureus S13E Staphylococcus epidermidis EPIII Staphylococcus epidermidis EPI2 Streptococcus pyogenes C203 Streptococcus pneumoniae Park I Streptococcus group D X66 Streptococcus group 9960

1, 0.5 0.5 1, 0.5 0.5 2, 1 1 1, 0.5 0.5 2, 1 1 1/. 0.5 0.5 0 25, 0.125 0.25 1, 0.5 0.5 16 , 16 8th 2, 2 2

1 1

0.2 0.015

MIC values in μg / ml, number of the compound = example number

Penicillin-resistant strain

Methicillin-resistant strain Two experiments

Average of three trials

TABLE III

In Vivo Efficacy of A-21978C Cyclopeptides

For mel No connection ED values ^a Streptococcus pyogenes subcutaneously orally At game no. IV N _m substituent Trp Staphylococcus aureus subcutaneously 0.0 3 66 6 V CH ₃ (CH ₂ ) ₈ C0- 0.28 0.39> 200 7 V (L) CH ₂ (CH ₀ ) _o C0NHCH (CH "C, H ₁ -) C0- 0.7, 0.98 ^b 0.31> 200 8th CH ₃ (CH ₂ J ₁₁ O - <^ ^Γ ^> - CO , 3.35

mg / kg x 2 Two experiments

TABLE IV

Toxicity of A-21978C Cyclopeptides

connection

For- play Rnel No. no.

N _ -Substituent LD »values (mg / kg) in the mouse

IV

CH-, (CH ₀ ) "CO-

V (L) CH (CH ₂ J ₈ CONHCH (CH ₂ C ₆ H) CO-> 300 600

8V CH ₃ (CH ₂

- / Λ-ΓΠ-

CO 67.5

Intravenously administered

Claims (13)

Erfindunqsansprüche: 1. Process for the preparation of A-21978C-cyclopeptide derivatives of the formula CI-) , ü l \ S HO (Z wherein R is hydrogen, an amino-protecting group, 8-methyldecanoyl, 10-methylundecanoyl, 10-methyldodecanoyl, the specific C, "- alkanoyl group of factor C _n of A-21978C or the specific C ₁ _ alkanoyl groups of factors C and C ^ 1 "of A-21978C and R and R independently represent hydrogen or an amino-protecting group, provided that at least one of the substituents R and R must be an amino-protecting group if R has a meaning other than hydrogen or an amino-protecting group, or of pharmaceutically acceptable salts thereof, thereby κ e η η ζ i c h η e t there 3 man .a) 'one or more of the amino groups NHR, NHR' and 2 NHR protects and thus a connection of the For- mel (I), in which one or more substituents 1 2
R, R and R represent an amino-protecting group, with the proviso that one or more of the substituents 1 2 tents R, R and R are originally hydrogen, 5 and / or (b) deacylating a compound of the formula (I) in which R has a meaning other than hydrogen or an amino group and thereby preparing a compound of the formula (I) in which R is hydrogen and optionally, if desired, in the reaction product. 1 2 available amino protecting groups R or R removed. 2. The method according to item 1, characterized in that for the deacylation to the respective compound of formula (I) in an aqueous medium as long as a microorganism from the family Actinoplanaceae formed deacylating enzyme is allowed to act until the deacylation is practically completed. , 3. The method according to item 2, characterized in that one uses as a microorganism from the family Actinoplanaceae a representative of the genus Actinoplanes. 4. The method according to item 3, characterized in that one uses as a microorganism Actinoplanes utahensis. 30 5. Method according to point 3, characterized ge indicates that as a microorganism. Actinoplanes utahensis NRRL 12052 or a mutant or variant thereof. 35 6. Method according to point 3, characterized ge indicates that Streptosporangium roseum var. hollandensis NRRL 12064 or a mutant or variant thereof is used as the microorganism. 7. The method according to item 3, characterized in that one uses as a microorganism Actinoplanes missouriensis NRRL 1205 3 or a mutant or variant thereof. 8. The method according to item 3, characterized in that as a microorganism Actinoplanes sp. NRRL 12065 or a mutant or variant thereof. 9. The method according to item 3, -dadurch characterized in that as a microorganism. Actinoplanes sp. NRRL 8122 or a mutant or variant thereof. 10. The method according to item 2, 3, 4, 5, 6, 7, -8 or 9, characterized in that one works with an enzyme which is present in a culture of the producing microorganism of Actinoplanaceae 20 is. 11. The method according to any one of items 1 to 10, characterized in that compounds wherein R is hydrogen, or a pharmaceutically 25 harmless salt thereof manufactures. 12. The method according to item 11, characterized in that one produces compounds in which R is hydrogen, or a pharmaceutically acceptable salt thereof. 13. The method according to item 11 or 12, characterized in that compounds, wherein 2
R is hydrogen, or a 35 salt from it. R is hydrogen, or a pharmaceutically acceptable 14. Method according to item 11 or 12, characterized in that compounds in which - 65 - R is an amino-protecting group or a pharmaceutically acceptable salt thereof. 15. Method according to item 11, 13 or 14, d a - characterized in that compounds wherein R is an amino-protecting group or a pharmaceutically acceptable salt thereof are prepared.