DD210257A5 - PROCESS FOR PREPARING A-21978C CYCLOPEPTIDE DERIVATIVES - Google Patents

Abstract

A process for the preparation of A-21978C cyclopeptide derivatives having the formula I resulting from the formula sheet, wherein R, R is high 1 and R is highly independent of each other 2 hydrogen, C is deep 4-C deep 14-alkyl, optionally substituted Ctief2-C deep 19-alkanoyl , Ctief5-Ctief 19-alkenoyl or an amino-protecting group, R 3, R 4 and R 2 together represent a C 4-C 14 -alkynylidene group, with the proviso that (I) at least one of the substituents R, Rhoch1 or a (2) at least one of R 1 or R 2 must represent hydrogen or an amino protecting group, (3) the substituents R, R contain high 1 and R 2 together contain at least 4 carbon atoms and (4) if R 1 and Rhoch 2 are selected from hydrogen or an amino-protecting group, then the substituent R is not 8-methyldecanoyl, 10-methyldecanoyl, 10-methyldodecanoyl, the special C 1 deep 0 alkanoyl group of the factor C deep 0 of A-21978 C or the special C deep 12-alkanoyl groups of the factors C deep 4 and C may be deep 5 of A-21978 C, or of pharmaceutically acceptable salts thereof by per se known reaction of a A-21978 C-Cyclopeptide nucleus of formula I wherein the substituents R, R are high 1, R is high 2, R is high 3, R is high 4 and R is high 5 but optionally hydrogen or protecting groups, or the pharmaceutically acceptable salts thereof with corresponding acylating agents or carbonyl derivatives with selective reduction of an alkylidene group and removal of any protecting groups.

Description

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

The great likelihood and constant danger of developing antibiotic-specific resistant strains of pathogenic microorganisms makes the development of new antibiotics a great need. Especially pathogens within the gram-positive species of Staphylococcus

30 and Streptococcus are often resistant to commonly used antibiotics, such as penicillin and erythromycin, and in this connection reference is made, for example, to W.O. Foye, Principles of Medicinal Chemistry ,. Pages 634 to 686 (1974).

The object of the invention is therefore the creation of new and particularly effective antibiotics.

1 Presentation of the essence of the invention :

This object is achieved according to the invention by novel A-21978C cyclopeptide derivatives and pharmaceutically acceptable salts thereof, which have been found to be effective antibiotic agents.

These novel derivatives can be prepared by reacting the nucleus of A-21978C obtained by deacylation of the appropriately blocked complex of A-21978C or any of its suitably blocked single factors Cp, C ,, C-, C-, C ₄ or C _n . available, with the desired acylating agent or an acylated derivative thereof or, in the case of the alkylated derivatives, by formation of the corresponding Schiff base and subsequent reduction of the imine bond of this base.

The A-219 7 8C cyclopeptide derivatives according to the invention have the formula I.

HOsC,

> H ₃

rv-a

^π ° HM

. * - J

, N-H

0 = »

, _π3

ι ^

0 H

HOaCT ^

0 N

!! I

* , ο

⁹ ^

, 0

9 ϊ

QJ

19th

wherein R, R ^ and R ~ are independently hydrogen, C, -. C, _d alkyl, optionally substituted C - C, _Q -A.lkanoyl / C _r -C, _Q alkenoyl or an amino, R , R "and R ° are all hydrogen or (i) R and

- - 2 2

R "and / or (ii) R * and R and / or (iii) R and R may together represent a C, -C, ^ -alkylidene group, with the proviso that (i) at least one of the substituents R, R" or R is other than hydrogen or an amino protecting group, (2) at least one of the sub-

l ~ 2

(R) or R is hydrogen or an amino protecting group, (3) the substituents R, R and R ~ together must contain at least 4 carbon atoms

1 2

and (4) if R and R are selected from hydrogen or an amino-protecting group, then the substituent R does not represent 8-imidodecanoyl, 10-methylundecanoyl, 10-methyldodecanoyl, the particular C, "- alkanovel group of the factor C _n of A-21978C or the special C ₁ alkanoyl

U J. Z.

groups of the factors C ₁ and C _r of A-21978C, or are pharmaceutically acceptable salts of these peptides, and these compounds are either valuable semisynthetic antibacterial agents or intermediates for the preparation of such agents.

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

Ala Alanin

Asp aspartic acid

30 Gly glycine

Kyn Kynurenin

Om ornithine

Ser serine

Thr 'threonine

35 Trp tryptophan

t-30C t-3utoxycarbonyi

Cbz 'benzyloxycarbonyl

DMF dimethylformamide

1 THF 'tetrahydrofuran

HPLC High Performance Liquid Chromatography

ι NMR. ^ H nuclear magnetic resonance

TLC thin layer chromatography 5 UV ultraviolet

The antibiotics A-21978C are closely related acid peptides. antibiotics as described in JS-PS 4,208,403, which is hereby incorporated. As is apparent from US-PS 4,208,43, the antibiotic complex A-21978 eir.e contains predominant component, namely the factor C, which in turn is itself a complex of closely related. Factors. Factor C of A-21978, designated Kc-plex A-21978C ', contains the single factors.

C _n , C ₁ , C-, C-, C, and C-. The factors C ₁ , C and C,

U 1 2 J 4 3 12 J

predominant factors, while the factors C ^, C ^ and Z. are subordinate factors. The structure of the factors of A-219 73C is shown by the following formula II:

20 ^l ' ^{As p}

-t

D-rtla.

f C-Ser L-Asp -1 /

t 3MG

25 L-Om ^

\ L-Kvn

^G 'y

L-hr

30. L-ksp

L-Asp

t L-Trp

NH 35

_p M

II

- J ₁ J

wherein 3MG is L-threo-3-methylglutamic acid and R

means a specific fatty acid residue. The specific radicals R "of the individual factors have the meanings given below

 5

Factors of A-21978C residue R ^N

C, 8-methyl decanoyl

C ₉ 10-methyl undecanoyl

10 C ₃ 10-methyldodecanoyl

C ₀ C _1Q alkanoyl *

C ₄ C-, "-alkanoyl *

C _r C-, alkanoyl *

* The exact nature of these remains has not yet been determined.

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 string chain of the substrate therefore influence the result of the attempt at deacylation. A number of microorganisms also form a large number of peptidases that attack different parts of the peptide residue. This often leads to difficult to handle product mixtures.

The enzyme, by the use of which the deacylation of the fatty acid side chain of the Cu-amino group of tryptophan can be achieved, by a microorganism from the family of Actinoplanaceae, preferably by the micro-organism Actinoplanss utahensis NRRL 12052 or a variant thereof, are formed. To effect such deacylation, an antibiotic selected from the complex of A-21978C, the factors

C, C "and

-S

from A-21978C, the blocked

Complex of A-21978C or the blocked factors C

C ₁ , C _n , C-, C and C- of A-21978C, to a culture of the microorganism. The indications blocked factors of A-21978C and blocked complex of A-21978C refer to either single factors of A-21978C or factor mixtures (complex) in which the amino group of ornithine and / or kynurenine is blocked with an amino protecting group. Preferably, the amino protecting group is attached to the o-amino

> 0 nogroup. of ornithine at the respective single factor or the mixture of factors arranged. The culture is allowed to act on the substrate until the deacylation is practically completed. The corresponding A-21978C cyclopeptide thus obtained is passed through the fermentation broth

^ 5 using known methods separated.

The cyclopeptides obtained by this enzymatic deacylation have the following formula III

Hoa

MHR '

..y..y Γ 9

] wherein R 'and R are independently hydrogen or an amino protecting group, or are salts thereof.

Removal of the acyl residue from the complex of A-21978C or of the single factors C-, C-, C-, C-, C _d and C- of A-21978C results in the compound of formula III wherein R and R ' each hydrogen. The deacylated molecule is the common cyclopeptide present in each of the factors of antibiotic A-21978C. For the sake of simplicity, this compound is referred to as the core of A-21978C. This compound can also be represented by the following formula IV.

L-Asp

15 Giy

D-Ala | ^y

T C-Ser

L-Asp I

T 3MG

L-Om I

20 \ L-kyn

GIy /

L-Thr

L-Asp

25 ^f

^{/ 3} . L-Asp

f L-Trp

Mha

IV 30

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

The compounds of formula III wherein R or R 'are other than hydrogen are prepared by deacylating the appropriately blocked factors C ₀ , C ₁ , C ₂ , C ₃ , C ₄ and C ₅ of antibiotic A-21978C. These compounds are referred to herein for convenience as blocked nuclei of A-21978C. This bluff

] kiertsn compounds are suitable as intermediates for the preparation of certain peptides of the formula I, namely those compounds in which at least one of the sub-

1 2

R and R represents an amino protecting group.

Of course, the core of A-21978C or the blocked cores of A-21978C can be obtained either in the form of free amines or acid addition salts. Any suitable acid addition salt may be used for this purpose, but preference is given to pharmaceutically acceptable acid addition salts. Pharmaceutically acceptable salts are understood as meaning those salts which are useful for the chemotherapy of warm-blooded animals.

The process for preparing the core of A-21978C of the formula III is described in detail using various microorganisms in the copending application filed on the same date as the present application with the internal reference X-5279. The preferred deacylation system, Actionoplanes utahensis MRRL 12052,. is available from the Agricultural Research Culture Collection (NRRL), Northern Regional Research Center, U.S. Pat. Department of Agriculture, 1815, North University St., Peoria, Ill. 61604, V.St.A. deposited and from there under the number NRRL 12052 freely available. Later Production Example 1 shows the production of the nucleus of λ-21.978C by fermentation using the complex of A-2197 8C as substrate and Actinoplanes utahensis NRRL 12052 as microorganism.

Other Actinoplanaceae cultures that can be used to produce the core of A-21978C of Formula III have also been deposited with the Northern Regional Research Laboratory and are subject to the following comments.

35 reservation numbers freely available:

Actinoplanes utahensis NRRL 12052

Actinoplanes missouriensis NRRL 12053

1 Actinoplanes sp. NRRL 8122

Actinoplanes sp. NRRL 12065

Streptosporangium roseum var. Hollandensis NRRL 12064

The efficacy of a particular strain of a microorganism of the Actinoplanaceae family to carry out the deacylation is determined by the following procedure. For this purpose, a suitable growth medium is inoculated with the microorganism. The culture is incubated for 2 or 3 days at about 28 C on a ³ Rotationsschuttier. The culture is then mixed with one of the substrate antibiotics and the pH of the fermentation medium is maintained at about 6.5. The culture is monitored for activity by examination with Micrococcus luteus. 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.

The present invention relates to novel compounds formed by acylating a nucleus of A-219 78C (compound of formula III), a factor of A-2T9 78C, or a blocked factor of A-21978C, whereby compounds having the cephem structure of the

30 following formula I arise.

15

, CH3

HO ₂ C ,,

- 10 -

I II

ο ":

, 0

in which R, R ¹ and R independently of one another are hydrogen, C ¹ -C ₄ , _4- alkyl, optionally substituted C ₁ -C ₄ , g-alkanoyl, C ₁ -C 4 -alkenoyl or an amino-protecting group,

45 R "and R are all hydrogen or

R ³ and R ⁴

4 5 2 ·

and / or (ii) R "and R and / or (iii) R and R 'may together represent a C, -C, alkylidene group, provided that (1) at least one of the substituents R, R or R (2) at least one of substituents R or R is hydrogen or an amino-protecting group,

1

(3) the substituents R, R and R together must contain at least 4 carbon atoms

1 2

and (4) if R and R are selected from hydrogen or an amino protecting group, then the substituent R is not for 8-methyldecanoyl, 10-methylundecanoyl, IQ-methyldodecanoyl, the particular C, "alkanoyl group of factor C ₀ of A-21978C or the specific C 1-4 alkanoyl groups of factors C ₄ and C ₅ of A-21978C, or the salts thereof.

The term C.-C, .alpha.-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 one

3 4

the substituent R and R "must have a meaning other than hydrogen 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 da ~ nen one of the substituents R, R 'or R is C ₄ -C ^ ₁ is alkylidenyl are known as Schiff bases.

The term C ₁ -C ₄ alkyl, refers to a monovalent saturated straight or branched chain alkyl group of 4 to 14 carbon atoms. The compounds in which

1 ?

, one of the substituents' R, R or R "is C ^ -C, ~ _d alkyl, are prepared by reducing the corresponding

The compounds in which the group R, R or R is C " -C-, ○ -alkyiidenyl and are referred to herein as reduced Schiff's bases.

The statements optionally substituted C ₀ -C, _Q alkanoyl and C ₁ -C 8 q alkenoyl refer to acyl groups derived from carboxylic acids containing from 2 to 19 and from 5 to 19 carbon atoms, respectively. According to the group R alkanoyi, the alkyl moiety is a monovalent saturated straight or branched chain hydrocarbon radical optionally containing a hydroxyl, carboxyl or C ₁ -C 12 alkoxy group or 1 to 3 halogen substituents selected from chlorine, bromine or fluorine can carry. 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

τ 2

'have either the ice or the trans configuration.

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 easily be eliminated after acylation of the respective compound. Examples of suitable protecting groups are given in Protective Groups in Organic Synthesis by Theodora W. Green, John Wiley and Sons, New York, 1981, Chapter 7. Particularly preferred amino-protecting groups are t-butoxycarbonyl and benzoyloxycarbony1.

As subclasses, the following compound groups from the general formula I are preferred according to the invention:

(a) the compounds wherein R is alkanoyl of the formula 'O

I!

CH-, (CH ₀ ) -C- and η stands for an integer of 3, j 2 η

20 to 17 stands;

(b) the compounds wherein R is alkancyl of the formula

t!

CH, (CH-) -C- and η is 5 to 14; ,

(c) the compounds wherein R is alkanoyl of the formula

CH O

CH-, (CH-) CH (CH _n ) -C- and the indices η and m 3 2 η 2 m

each independently represents an integer from 0 to 14, with the proviso that η + m may not be less than 1 nor greater than 15, and with the further proviso that if η is 0 then m can not be 8 and, if η is 1, m can not be 6 or 8; 35

(d) the compounds wherein R is cis- or trans-alkenyl of formula O.

CH ₃ (CH ₂ ) _n CH = CH (CH ₂ ) -C-, and η and m are each

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

(e) the compounds wherein R is cis- or trans-alkenyl of formula 0

Il

CH = CH (CR -) - C- and η is an integer from 4 to 15;

10 (f) the compounds wherein R is alkyl of formula

CH ₀ (CH _n ) - and η is an integer from 5 to 12 j Z η ^Ό

is;

(g) the compounds wherein R has the following meanings: O

CH ₃ (CH ₂ J ₅ -C-

O CH

3 ^v 2 '6

Cn, (CH ₉ ) -, - C-

CH, (CH_) _Q -CO

Il

Il

1)

CH ₂ = CH- (CH ₂ J ₈ -C 30 O

O CH ₃ (CH ₂ ) ₁₂ -C-

O ³⁵ CH ₃ - (CH _{2) 3} -CH = CH- (CH _{2) 7} -C

O CH ₃ (CH ₂ ) ₁₃ -C-

CH ₁ CK ₀ CK (CP ') - (CH ₀ ).-C

CH ₃ (CH ₂ J ₆ -

CH ₃ (CK ₂₎ CH = 10

CH ₃ (CH ₂ J ₉ -

CK ₃ (CH ₂ J ₈ CH =

The compounds of formula I 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.

The compounds of the formula I have, for example, 5 free carboxyl groups which 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 compounds of formula I include the sodium, potassium, lithium, cesium, rubidium, barium, calcium and magnesium salts. Suitable amine salts of compounds of formula I include the ammonium salts as well.

3-5 the primary, secondary and tertiary C ₁ -C 4 alkyl ammonium and hydroxy C ₀ -C 4 alkylammonium salts. Exemplary amine salts are those obtained by reacting a compound of the formula I with ammonium hydroxide, methylamine, s-butylamine, isopropyl

amine, diethylamine, diisopropylamine, cyclohexylamine, ethanolamine, triethylamine or 3-amino-l-propanol formed compounds.

The cationic alkali metal and alkaline earth metal salts of compounds of formula I are prepared using procedures common to the formation of cationic salts. For this purpose, for example, the free acid form of a compound of the formula I is dissolved in a suitable solvent, such as warm methanol or ethanol, and then such solution is treated with a solution containing a stoichiometric amount of the desired inorganic base in aqueous methanol. The salt formed in this way can be isolated by the use of conventional methods, for example by filtration or evaporation of the solvent. A convenient method for the preparation of salts consists in the use of ion exchange resins.

Similarly, the salts of organic amines can be formed. For this purpose, for example, the respective gaseous or liquid amine to a solution of a compound of formula I in a suitable solvent, such as ethanol, and the solvent and the

25 Excess amine then remove by evaporation.

The compounds of the invention also have free amino groups and can therefore form acid addition salts, which are also part of the invention. Examples of suitable acid addition salts of compounds of formula I 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, muconic, D-glutamic, d-camphoric, glutaric, glycolic, phthalic, tartaric, lauric, stearic, salycilic, methanesulfonic, benzenesulfonic, sorbic

- 16 1 crinsäure, benzoic acid or cinnamic acid.

The compounds of formula I wherein R, R or R are alkanoyl, alkenoyl or an amino-protecting group are prepared by acylating a factor of A-21978C, a blocked factor of A-21978C, a compound of formula III or a corresponding compound of Formula III, which is blocked at the <χ-amino group of tryptophan, with the desired alkanoyl '] 0 or alkenoyl side chain using methods that are conventional for forming an amide bond. This acylation is generally carried out by reacting the particular compound with an activated derivative of the alkanecarboxylic acid or alkenecarboxylic acid, which is the

1 ^

corresponds to the desired side chain group (R, R or R),

  implements. 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 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 chloride, (b) acid anhydrides,

25 such as alkoxyformic anhydride

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. The reaction with the primary amine is carried out directly in the reaction mixture in such a case.

It is obvious to the person skilled in the art that the compounds of the formula I are obtained by the use of selective acylation methods and the use of amino-protecting groups.

let set. If, for example, a compound of the formula III in which R 'and R are hydrogen are used as starting material, then an acylation may occur both at the c6-amino group of tryptophan and at the 6-amino group of ornithine, so that M , N _n -

i rp - urn

Diacyl derivatives are formed. The preparation of derivatives which are monoacylated at the cc-amino group of tryptophan is therefore preferably carried out by acylation of a compound of the formula III in which the δ-amino-

0 group of ornithine (namely the position R) is blocked by an amino protecting group. Such starting materials are preferably prepared by blocking Factor A-21978C at this position prior to its deacylation. The aromatic amino group of kynurenine is the least reactive group of the three free amino groups in core form A-21978C. Sine acylation of kynurenine therefore requires suitable blocking of the amino groups of tryptophan and ornithine, while acylation on the substituent R or R does not usually require blocking of the amino group of kynurenine.

From the reaction schemes x, il and III general procedures for the preparation of compounds of formula I, wherein one of the substituents R, R or R is alkanoyl, alkenoyl or an amino protecting group. In these reaction schemes, the symbols contained have the following meanings:

30 / * 7 = remainder from A-21978C

N = ^σ - amino group of tryptophan

N _n = δ-amino group of ornithine

N "= Aromatic aminoarupoe of kynurenine

12 "

R, R, R = indicated substituents

35 R = acyl group of the natural factor

B, B = amino protecting groups

Acyl = acylation level

Deacyl = deacylation stage

-IS-

] Block = acylation with an amino protecting group deblock = removal of an amino protecting group

In Reaction Scheme 1, the N- monoacyl derivatives of A-21978C are represented by the general formula III, while the N, N -diacyl derivatives of A-21978C correspond to the general formula IV. The N, N -dialyl derivatives in which the N_ acyl group is that of the natural factor A-21978C correspond to the general formula 10. VIII.

ul

to Cn

K) O

LTi

Reaction note I

Preparation of N, _n- monoacyl and N _1n , N-diacyl-A-21978C derivatives

_ Trp ^ ! ££ IilQ £ DI

acyl

K a

0 \%

DeacyI

/ V ¹¹ Vi

DeacyI

HJI II K a

A ¹¹ ,

-) [*] - N ₀ HQ

VI

NI ₁ J

XIX ^

Acyl

1 * 1

IV

Deb lock

VII

In Reaction Scheme II, the N _T , N -diacyl derivatives of ft-21978C are represented by the general formula X.

The N, _,, "N _T, -Diacylderivate wherein the N acyl group

i rp i \ yn irp

which is the natural factor A-21978C, have the general formula XII. The compounds of the general formulas V, VI and VII are likewise described in Reaction Scheme I.

LO O

ro

UI

Reaction note IT

Preparation of N _1n , N ₁ , diacyl A 21978C derivatives

TrgKyn

acyl

A ^NR N

/ ^N T ^1IR N [*] M ₀ HE

., ^X N _K IIR ¹ 11

Deblock

A ^HR N

\ l

Acyl

N HR

Deblock

N _K IIR ¹

VI

VII

- 22 -

In Reaction Scheme III, the N _Q -Monoacylderi- corresponding derivatives of A-21978C of the formula XVIII, while

the N-monoacid derivatives of A-21978C of the general j

Formula XV and the N, N _T -diacyl derivatives of A-21978C are represented by the general formula XX. The compounds of general formula VI are also described in Schemes I and II.

OJ O

N)

K)

ln

ln

Reaction Slide III

Preparation of NL monoacyl, N ₁ . -Monoacyl- and l \ L, N _r -Diacyl- Om ^{J J J} Ky η Orn Kyn ^J

MB

Λ-21978C derivatives

, NJIB ¹ Selective NJ-IB ¹

XIX

acyl

N ₁ -

\ | ..HR XX K 1

XIII

I N JIB

NJ

N _n HB

XVII

/ ^N T ^H

XVIII

XIV

XV

The compounds of the formula I ₇ in which one or more amino groups are substituted by an alkylidene group (Schiff bases) or an alkyl group (reduced Schiff bases) can be prepared by processes known per se for the preparation of Schiff bases and reduction of such bases be formed. The Schiff bases are therefore prepared by reaction (condensation) of the primary amino group of tryptophan, ornithine or kynurenine of A-21978C with an appropriate aldehyde or ketone in a suitable solvent. A reduction of the imine bond of the Schiff base to give the corresponding compound of the formula I,

1 2

wherein R, R or R is alkyl can be achieved by known selective reduction methods. A preferred reducing agent for this reaction is sodium IS cyanoborohydride.

Under the conditions used in an in vitro experiment, Schiff bases show no antibacterial activity, possibly due to their instability in the test medium. However, they are useful as intermediates for the preparation of the reduced Schiff bases. When using Schiff base as an intermediate, one need not isolate this intermediate prior to reduction to form the reduced Schiff base.

A preferred process for the preparation of the compounds of the formula I in which one of the substituents R, R or R is alkanoyl or alkenoyl is the active ester method. The compound of formula III wherein R 'is H and R is t -30C, namely

A-21978C N_t-BOC core or the t-BOC core is a beurn

particularly preferred starting material for the preparation of compounds of formula Γ. The 2,4,5-Trichlorpheny1- ester of the desired alkane or Aikencarbonsäure is preferred as the acylating agent. In this method, an excess amount of the active ester having the t-BOC core is reacted at room temperature in a non-reactive organic solvent such as DMF, THF, diethyl ether.

"Ο ς"

ether or dichloromethane. The reaction time is not critical, although a reaction time of about 6 to about 20 hours is preferred. After completion of the reaction, the solvent is removed and the residue is purified. A particularly suitable purification method is reverse-phase HPLC using LP-1 / C _n "as a stationary phase and a mixture of KO / CH 2 OH / CH 2 CN / pyridine / HOAc as the solvent system. The t-BOC group It is removed by treatment with trifluoroacetic acid / anisole / triethylsilane or preferably with trifluoroacetic acid / 1,2-ethanedithiol over a period of about 3 to about 5 minutes at room temperature. After removal of the solvent, the residue is purified by reverse phase HPLC.

Another method of acylation is a modified Schotten-Baumann method in which the unblocked core is treated with the acid chloride of the desired alkanecarboxylic acid or alkene carboxylic acid in a mixture of pyridine and water. In this process, an excess of the acid chloride in a non-reactive organic solvent, such as acetone, is slowly added to a solution of the core in a mixture of 90% pyridine and 10% water (volume). The unreacted acid chloride is separated from the reaction product by extraction into an insoluble organic solvent such as diethyl ether. Final purification is by reverse phase HPLC in the manner already described.

The alkane and alkene carboxylic acids used as starting materials for the acylation reaction and the activated derivatives thereof, in particular the acid chlorides and the 2,4,5-trichlorophenyl esters, are either known compounds or can be prepared from known compounds in a manner known per se. The 2,4,5-trichlorophenyl esters are best prepared by treatment of the acid chloride of the alkanecarboxylic acid or

Alkenecarboxylic acid with 2,4,5-trichlorophenol in the presence of pyridine or by treatment of the free alkanecarboxylic acid or alkene carboxylic acid with 2,4,5-trichlorophenol in the presence of N, N'-dicyclohexyl'carbodiimide as coupling agent.

The 2,4,5-trichlorophenyl ester derivative can be purified by column chromatography over silica gel.

The A-21978C cyclopeptides of the present invention can be administered as antibacterial agents either orally or parenterally. The compound A-21978C is usually administered naturally along with a pharmaceutically acceptable carrier or diluent. The dose of A-21978C compound depends on. a number of considerations, such as the nature and severity of the particular infection to be treated. The appropriate for administration dosage ranges or dosage units can be with the basis of the MIC-values and ED_ _n values and toxicity data in compound

_ / U

determine other factors by the skilled person, such as the pharmacokinetics, the characteristics of the patient or host; and the responsible for the infection "microorganism.

embodiments

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

Production Example 1 30

Preparation of the Core "of A-21978C

A. Fermentation of Actinoplanes utahensi s

Make a stock culture of Actinoplanes utahensis NRRL 12052 and keep it on an agar slope. To make the bevel use one of the following media:

Medium A constituents amount

Pre-cooked oatmeal 60.0 g

Yeast 2.5 g

K ₂ HPO ₄ · .1, Og

Czapek Mineral Blend *. · 5.0 ml

Agar 25.0 g

Deionized water q.s. to 1 liter

The pH before autoclaving is about 5.9. It is adjusted to pH 7.2 by addition of NaOH. After autoclaving, the pH is about 6.7. 15

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

FeSO ₄ -7H ₉ O (dissolved in 2 ml conc. HCl) 2 g KCl 100 g

MgSO ₄ -7H ₂ O: 1 00 g

Deionized water q.s. to 1 liter

- 2 8 _ Medium B

Ingredients . amount

Potato dextrin · 5.0 g

Yeast extract 0.5 g

Enzyme hydrolyzate of casein * 3.0 g

Beef extract 0.5 g

Glucose. 12.5 g

Cornstarch 5, 0 g

Meat peptone. 5.0 g

Cane molasses 2.5 g

MgSO ₄ -7H ₂ O 0.25 g

CaCO ₃ . 1.0g.

Czapek mineral blend. 2.0 ml

Agar 20.0g

Deionized water q.s. to 1 liter

* NZ-Amine A, Humko Sheffield Chemical, Lyndhurst-NJ, ²⁰ 'V.St.A.

The slant is inoculated with Actino.planes utahensis TiRRL 12052 incubating the inoculated slant approximately 8 to 10 days at 30 ⁰ C. Using about half of the

^A ~> oblique growth is then inoculated 50 ml of a vegetative medium with the following composition:

Constituents ' quantity

^ou Pre-cooked oatmeal 20,0 g

Sucrose 20.0 g

Yeast 2.5 g

Dried grain veal * 5.0 g

K HPO 1,0g -ώ *

Czapek mineral mix 5.0 ml

Deionized water q.s. to 1 liter

The whole is adjusted to pH 7.4 with NaOH, the pH after autoclaving being about 6.8.

* National Distillers Products Co., 99 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 hours at 30 ^° C. on a rotary shaker moving in a 5 cm diameter bow at 250 rpm.

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 keeping the culture in a vapor phase of liquid nitrogen. For such storage, the culture is filled into a number of small vials in the following manner: 2 ml of incubated vegetative medium and 2 ml of a glycerol-lacto.se solution (Cryobiol 10, 364-367 (1973) are added to each vial )). The suspensions thus formed are liquid in a vapor phase

Kept nitrogen. 25

Then, using a stored suspension (1 ml) prepared in this way, 50 ml of a first-stage vegetative medium having the above-described composition is inoculated. The inoculated first stage vege- ^ou tative medium is then incubated in the manner described above.

To inoculate a larger volume of inoculum, inoculate 400 ml of a second stage vegetative medium,

which has the same composition as the first stage vegetative medium, with 10 ml of the incubated first stage vegetative medium. The second stage medium is placed in a 2 liter Erlenrneyer wide-mouth flask.

] Butt about 48 hours at ⁰ .30 C on a rotary shaker, which is moved under a sheet of 5 cm x diameter with 250 U / min.

The incubated second stage vegetative medium (80 ml) prepared in the manner described above is then inoculated with 10 1 of one of the following production media:

Medium I

ingredients

Peanut flour 15 Soluble meat peptone sucrose KH ₂ PO ₄

K ₂ HPO ₄

MgSO ₄ -7H ₂ O 20 tap water

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. ·.

Medium II

Ingredients . Quantity (g / l)

,

Sucrose. 30.0

Peptone - 5.0

K ₂ HPO ₄ 1/0

KCl 0.5

amount ig / i) 10.0 5.0 20.0 0.5 1.2 0.25 to 1: liter

MgSO ₄ -7H ₂ O - 0.5

FeSO ₄ -7H ₂ O 0.002

Deionized water q.s. to 1 liter

The whole is adjusted with HCl to a pH of 7.0, wherein the pH after autoclaving is about 7.0.

Medium III

Ingredients Quantity (g / l)

10 glucose 20.0

NH ₄ Cl '3.0

Na ₂ SO ₄ . 2.0

ZnCl ₂ . 0.019

MgCl ₂ "6H ₂ O '0.304

15 FeCl -, - 6H-0 0.062

MnCl ₂ -4H ₂ O 0.035

CuCl ₂ '2H ₂ O 0.005

CaCO ₃ . 6.0 KH ^ PO ₄ * 0.67

Tap water q.s. to 1 liter

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

, '' '

It is allowed to the inoculated production medium in a 14 1 fermentation vessel comprehensive about 66 hours at a temperature of about 0 ⁰ C 3 ferment. The fermentation medium is added by means of conventional stirrers

^ou so vented at a rate of about 60 0 U / min and with sterile air so that the dissolved amount of oxygen is maintained above 30% of air saturation at atmospheric pressure.

^Β · Degmentation of A-21978C

Actinoplanes utahensis is fermented using the method and use of medium A for the slope described in section A and the production medium I, incubating the production medium for about 67 hours. The fermentation medium is treated with crude complex of A-21978C (100 g), the. according to US Pat. No. 4,208,403.

The deacylation of the complex of A-21978C is carried out by

Surveillance against Micrococcus luteus monitored. The fermentation is allowed to continue until complete deacylation, which manifests itself in a disappearance of activity against the micrococcus luteus, which is the case after a period of about 24 hours.

C. Isolation of the core of A-21978C

The total 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 l HP-20 resin (DIAION Highly Porous Polymer, HP series, Mitsubishi Chemical Industries Limited, Tokyo, Japan). The resulting Säulenabs tr om is discarded. The column is then filtered to remove ^residual: broth washed with deionized water {10 1). The resulting wash water is also discarded. The column is then distilled with mixtures of water and acetonitrile (in each case 10 l in ratios of 95: 5, 9: 1 and 4: 1),

35 collecting fractions of 1 liter each,

The elution is monitored by paper chromatography using a solvent system of n-butanol, pyridine, acetic acid and water {15: 10: 3: 12} and detection of the compounds by UV fluorescence. In this system, the factors of A-21978C have an R "value of about 0.56, while the core of A-21978C has a fU value of about 0.32. The product can also be further purified 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 25: 4 Monitor 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.

20 D. Purification of the core of A-21978C

Dissolve the semi-purified core obtained in Section C above from A-21978C (15 g) in 75 ml of a mixture of water, methanol and acetonitrile (82: 10: 8) containing 0.2% acetic acid and 0.8% pyridine contains. The solution is pumped into a 4.7 χ 192 cm column, the. 3.33 1 silica gel Cη (Quantum LP-1, Quantum Industries, 341 Kaplan Drive, Fairfield, NJ, 07006, V, St.A.). never pillar will be the same

^ Solvent system developed. Fractions of 350 ml each are collected. Separation is monitored at 280 nm with a UV monitor. The fractions containing the core are pooled under vacuum to remove the solution.

^OJ concentrated and freeze-dried, resulting in 5.2 g of purified core of A-2197 8C.

1 E. Properties of the core of A-21978C

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

(a) Shape: White amorphous solid which fluoresces under short-wave UV light

(b) Empirical formula: C ^ ₀ H ₀₀ N, _r 0 ~ _r

QZ OZ IO / D

10 (c) Molecular weight: 1466

{d) Solubility: Soluble in water

(e) The infrared absorption spectrum (KBr) shows "! 5 absorption maxima at the following frequencies (cm ~ ' ^! ):

3300 (broad), 3042 (weak), 2909 (weak), 1655 (strong), 1530 (strong), 1451 (weak), 1399 (middle), 1222 (middle), 1165 (weak),

1063 (weak) and 758 (medium to weak). 20

{f) The UV absorption spectrum in methanol shows

Maxima at 223 nm (£ = 41482) and 260 nm: (S = 8687).

2-> (g) The electrometric titration in 66%

Aqueous dimethylformamide gives the presence of four titratable groups with pK.

Values of about 5.2, 6.7, 8.5 and 11.1 (initial pH = 6.12).

-jō "

1 Production Example 2

Another method of making the core of A-21978C

The core of A-21978C is prepared according to the procedure described in Preparation Example 1, except for certain changes 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, elution is continued using a solvent mixture of water and acetone (9: 1). The fractions containing the core are eluted with this solvent mixture. The resulting fractions were combined, concentrated under vacuum to remove acetonitrile, and lyophilized to give 24.3 g of the 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 x 192 cm steel column containing 3.33 liters of silica gel (Quantum LP-1 / C, J, which is contained in a

IO - -

Mixture of water, methanol, and acetonitrile (8: 1: 1) ^ prepared containing 0.2% acetic acid and 0.8%

Contains pyridine. The column is developed with the same solvent mixture at a pressure of about 136 bar, collecting fractions of 350 .ml each. The elution is monitored by UV light at 280 nm ~~ '~'. The fractions containing the core were combined, concentrated under vacuum to remove the solvent and freeze-dried to give about 14 g of highly purified A-21978C nucleus passes.

1 Production Example 3

Preparation of Factors C ~ and C-, of N -t-BOC-

A-21978C \

A mixture of factors C_ and C- 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 4.0 to 5.5 by the addition of 10 5 N NaOH (3.6 mL). Di-t-butyldicarbonate (3.0 ml) is added and the reaction mixture is stirred 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 (7: 3 and 8: 2) and detection by UV light.

After about 10 minutes, the reaction solution becomes rapidly cloudy and the consumption of base increases. After about 3 0 minutes, the speed of the

^ ³ Increase in turbidity and consumption of base, indicating completion of the reaction. Regardless, the response is to ensure its termination

, but continued for another 90 minutes. At the end of the two hour reaction time, the reaction material is immediately lyophilized, yielding 12.7 g of C ₂ and C factors of N -t-BOC-A-21978.

Using similar procedures one also carries out two reactions with 10 g of material and a reaction with 30 g

Material through. In each of these reactions, the reaction time is only 40 minutes. The two experiments with 10 g of material lead to 11.9 g and 12.1 g of product. The reaction with 30'g material gives 35.4 g product.

1 production example 4

Preparation of the core of λ-2197 8C-N-t-BCC

_Orn

A fermentation of Actinoplanes utahensis

Actinoplanas utahensis are fermented according to the procedure described in Preparation Example 1 under Section A.

Method using the inclined medium A and 10

of the production medium I _7, whereby the production

Medium incubated for about 6 6 hours.

B. Deacylation of the complex of N-t-BOC-A-219-78C

The complex of A-219.7 8C-N. -t-BOC (1185'σ to raw

Orn.

Substrate containing about 176 g complex of A-21978C) is added to the fermentation medium. The deacylation is carried out as described in Preparation Example 1 in Section B. The deacylation is completed by HPLC after about 24 hours.

C. Isolation of the Kerr of A-21973C-N-t-BOC

The fermentation broth (100 L) obtained according to Section 3 is filtered using a filter aid (HyfIo. Super-Cel). The filtrate is passed through a column containing 7.5 L of HP-20 resin (DIAlON) and the column is washed with water (38 L). Elution is monitored by silica gel Cg-HPLC with 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 (35:15). The fractions containing the core are combined, concentrated under vacuum to remove the solvent and lyophilized to gelate to 298.5 g semi-purified core of A-2197 8C-N-t-BOC.

Orn.

D. Purification of the core of A-21973C-N-t-BOC

The semi-purified core of A-2197 8C-N-t-BOC (30 g) obtained in Section C 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 1 silica gel (Quantum L -1 / C ₁₀ )

Ί ö

has been 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 0.3% pyridine, collecting fractions of 350 ml each and detecting the detection of the Product by UV light at 280 nni performs. The fractions containing the product are combined, eingeengt'und freeze-dried under vacuum to remove the solvent, so as to obtain 18.4 g of purified A-21978C nucleus of _N-Q t-BOC.

, The core of A-21978C-N _Q -t-30C has the following properties;

(a) Shape: White amorphous solid which fluoresces under short-wave UV light.

(b) Empirical formula: 0, -, ENT

(c). Molecular weight: 1566

(d) Solubility: Soluble in water

(e) The infrared absorption spectrum (KBr) shows

Absorption maxima at the following frequencies (cm -1 '): 3345 (broad), 3065 (weak), 2975 (weak), 2936 (weak), -1710 (shoulder), 1660 (strong), 1530 (strong), 1452 (weak ), 1395 (medium), ·

1368 (weak), 1341 (weak), 1250 (medium), 1228 (medium), 1166 (medium to weak) and 1 063 (weak).

(f) The UV absorption spectrum in 90% ethanol

shows maxima at 220 nm (£ = 42,000) and 260 nm

(ε = 10600).

(g) HPLC residence time: 6 minutes on a 4.6 x 300 mm column filled with silica gel C "using a mixed solvent of H ₂ O, CH ₃ CN and CH ₃ OH. (80: 15: 5) containing 0.2% NH.OAc at a flow rate of 2 ml / min. With detection by UV light.

Production Example 5

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

The solution according to Preparation Example 4, section

  C, obtained half-purified core of A-2-T9 7 8C-N-t-

Om

op, BOC (10.8 g) in water and the solution is applied to a column containing 80 ml of Amberlite IRA-6 8 (Rohm and Haas, Philadelphia, PA, V-St.A., Acetatecycle). The column is washed with water and taken at a flow. speed of 5 ml / min in turn first with

0.05n acetic acid (1080mL), then eluted with 0.1N acetic acid (840mL) and finally with 0.2N acetic acid (3120mL), collecting fractions of 120mL each. The column is purified by analytical HPLC over silica.

oxide gel-C g using a solvent system of water, acetonitrile and methanol (80: 15: 5) containing 0.2% ammonium acetate and detecting the product by UV light at 254 nm. 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 added with water and the resulting solution is reconcentrated to remove pyridine. This concentrate is freeze-dried, yielding 3.46 g of purified A-21978C-N-t-BOC core.

15 Examples 1 to 16

The preparation of various alkanoyl and alkencyl derivatives by acylation of compounds of the formula III, namely the preparation of compounds of the formula I, is evident from the following Table I. The derivatives indicated in Table I are either used according to the modified Schotten-Bauman reaction using an acid chloride as the acylating agent (Method A) or by the active ester method using the 2,4,5-trichlorophenyl ester as the acylating agent (Method B). manufactured. The general procedures for carrying out the acylation reactions according to Method A or Method B are described below.

Method A (modified Schotten-Baumann reaction)

This method consists of reacting a nucleus of formula III with the alkanecarboxylic acid chloride or alkene carboxylic acid chloride corresponding to the desired acyl side chain.

A compound of the formula III (1.95 to 2.16 g,

1.33 to 1.47 mmol) or a compound of the formula II

(409 mg, 0.2 5 mmol) in 20 0 ml of a mixture of pyridine and water (9: 1). The acylating agent (solution of up to 20 mmol excess of acyl chloride in 15 ml acetone) is added dropwise over a period of 1 to 3 hours and the reaction mixture is stirred at ambient temperature for a further 2 to 3 hours. The reaction mixture is then concentrated to remove the acetone. The remaining aqueous layer is diluted with water to a volume of about 2 00 ml. The pH of this solution is adjusted to pH 3.0 to 3.5 with glacial acetic acid. The solution is washed eight times with equal volumes of diethyl ether and then lyophilized.

5 The crude acyl derivative is purified by reverse phase HPLC as follows: The sample is sprayed as a solution in water or in the eluent system (about 4 to 6.5 ml) onto a 1.6 x 85 cm stainless steel column, which is filled with LP, / C, _o as a carrier. The pillar

1 oo

is eluted with a solvent system of HO: MeOH: CH, CN: pyridine: HOAc. The elution is carried out at a pressure of about 103 to 138 bar and a flow rate of about 10 to 12 ml / min using an LDC duplex pump (Milton-Roy). The effluent is monitored by UV detec tion using an ISCO UA-5 detector at 280 nm. The desired fractions are combined and evaporated to dryness under vacuum to give the desired alkanoyne derivative. The purified product is analyzed by TLC using reverse phase plates (Whatman KC, ο) ^{and <} 3 of a solvent system of H ₂ O: MeCH: CH ₃ GT: pyridine: HOAc (45: 15: 40: 2: 2). The plates are observed to detect the product under UV light. The products are further analyzed by UV analysis (extinction coefficients at 220 nm and 260 nm) and by amino acid analysis. The purity is determined by analytical reverse phase HPLC (C, _O -Microbondapak

- 1 O -

by Waters Co.) using a solvent system of H-O: MeOH: CH, CN: pyridine: HOAc and

Monitoring the eluate with UV light at 28 0 nm determined.

Method B (method over the active 2,4,5-trichxorphenyl-

ester) 5

A solution of N -t-BOC A-21978C core (1.0 g, 0.64 mmol), A-21978C core (0.5 to 1.0 g, 0.34 to 0.68 mmol) or a-21978C (9 46 mg, 0.5 8 mmol) and a 3.5 molar excess of active Trichlorphenylacylester is dissolved in DMF (100 ml) and about 6 to about 20 hours at a temperature of about room temperature up to about 50 ⁰ C stirred. The reaction mixture is concentrated under vacuum to an oil. The oil is treated with 50 ml of a mixture of diethyl ether and toluene (1: 1) and washed with diethyl ether. The monoacylated and diacylated A-21978C core derivatives, the acylated A-21978C-, and the acylated t-BOC A-21978C core are as described. cleaned in the method A described above.

The acylated t-BOC-A-21978C nucleus is prepared using 50 mg / ml of a Gemisches- of trifluoroacetic acid, anisole 'and triethylsilane (10: -1: 1) at about -1O ⁰ C to ⁰ ° C over a period deblocked from 3 to 5 minutes. The reaction mixture is concentrated under vacuum to an oil, which is treated twice with 20 ml of diethyl ether. The crude acyl product is purified and analyzed by reverse phase HPLC as described above for Method A.

The individual compounds of these Examples prepared in the above manner are shown in the following Table I to VI with reference to the following Formula I.

NR ³ R ¹

HOaC, - 9

? ΐ

Y /

ii

HOA (Z ,,

 1-13

 Ι

HOSC

H

H 9!

0 = <

β- ♦ -

H H-N

i!

2QMA11933 * 091322

TABLE I

Preparation of N-monoacyl derivatives of A-21978C cyclopeptides

Example no.

10 11 12 13 14 15 16

Connection R

a)

CH ₃ (CU ₂ ) ₅ C0-CH ₃ (CH ₂ ) ₆ C0-

C0

CH ₃ (CH ₂ ) _? C0-CH (CH _{2) 7} CO-CH ₃ (CII,) ₈ C0-

CH

(CH ₂ ) ₁₀ C0-

CO

co-

(CiI ₂ )

₂ )

CH ₃ (ClLj) ₁₃ CO

(CH ₂ )

CO

CII ₉ = CU- (CIl ₂ ) _β C0-CH ₃ (CH ₀ ) ₃ CH "C

_y C0-

CH CH = CH (CIl ₂ ) ₇ C0-

Production method Weight of Ke (mg) B 1028 A 1950 B 10 00 A 2000 B 1000 A 216 0 B 10 0 0 A 10 0 0 ß 10 0 0 B 1000 B 1000 B 1000 B 1000 B 1000 B 1000 B 1000

Weight of acylating agent (mg)

800

3421 350 882 400 5 95 288 4 0 6 800

812 900 31 74 4 37

400

00

Reaction time (h). Intermediate HPLC eluent Prod u [IPLC El uie- _c. mngsmittel 35 20 55:15:30 50:15 30 3 5 0:15:35 55:15 64 24 50: 15:35 115: 15 .35 2 No cleaning 50:15 35 20 No cleaning 50:15 : 40 5 4 5:15 .40 22 No cleaning .45: 15 40 2 45:15 45 24 10:15:75 4 0:15 L ^e) ₂₂ d) 50:15:35 1: 0: 1 35 20 50:15:35 5 0:15 55 ^e) 22 ^d) No cleaning 35:10 18 ^d) No cleaning 2: 0: 1 40 20 2 0:15:65 45:15 45 48 No cleaning 40:15 50 28 3 5: 1 5: 5 0 * 35:15

a) el)

R '

R - H;

b)

Acyl-N-t-BOC; ° Elution medium from HO: C1I., O11: C1LCN (V: V: V) with a ^Tr P Om 'fiphall- (1.?% Pvri di η and 0. '>%. HCtAr.

Content of 0.2% pyridine and 0.2% HOAc

G ¹ "

Reaction temperature maintained at 5 ⁰ C; ¹ "containing 1% pyridine and 1% HOAc

TA BELLE I I

Properties of the N-monoacyl derivatives of A-21978C cyclopeptides

R R ¹  ) M " < P r (j u Li k t UV : i c, 260niu Amax CH ₃ (CH ₉ ) ₅ C0- H H 0.85 Anal. IIPL.C c. 22Onii Amax 11,400 Verbin CH ₃ (CH ₂ ) ₆ C0- 11 11 0.80 NiUel ^Uny3 " ^b 48,100 10,500 dung CH ₃ (CH ₉ ) ₇ CO- H H 0.75 60:15:25 46,900 11,000 1 CII ₃ (ClI ₂ J ₈ CO- H 11 0.70 60:15:25 46,000 10,000 2 CH ₃ (ClI.,) YCO- H Il 0.65 45:15:40 46,500 10,400 3 C ₃ (CH ₂ ) ₁₀ CO- H H 12:59 45:15:40 46,200 9,500 4 CIL ₁ (CIl ..) .., CO- H H 12:53 45: L5: 4UE 44,000 9,800 5 ClI ₃ (CIl ₂ ) j ₉ C0- 11 II 12:42 45:15:40 48,500 9.170 6 CIL ₁ (CIl ₂ ). _n C0- H 11 12:34 45:15:40 48.447 9.751 7 CH ₃ (CIl ₂ ) ^ CO- H H 12:25 50: 0: 49 ° 50.172 12,000 8th CII ₂ = CIl- (ClI ₂ ) _β C0- H H 0.70 1: 0: 1 ° ^ 54,000 9,200 9 CH (CH ₉ ) ClU-Cn (CII ₉ ) CO- Il H 12:54 39: 0: 60 ° 44,000 9, 600 10 CIl CH CHClClI (Ml * (Ml) 45:15:40 45,000 11 CH ₉ CH = Cll (CH ₂ ) CO-, H Il> 12:50 45: 15:40 11,600 12 50,000 13 45: 15: 40 °

The R values are determined by reverse phase silica gel TLC (Whatman KC with fluorescent indicator) and eluting with HLChCH-OI-IrCt-LCN eluant (45:15:40) containing 0.2 % pyridine and 0.2 Contains % HOAc. ^

Elution agent from HO: CH ₃ QH: CILCN (V: V: V) containing 0.2 % pyridine and 0.2 % HOAc. Containing 1% pyridine un

TABLE III

Preparation of N "-N -Diacy.l-A-2197.8C Derivatives

at CH ₃ CH ₂ CH R (CH 9> game CH ₃ CIl ₂ CH (CII 2> No. CHClI ₂ Cl (CH ₃ ) (ClI 2> 17 CIl ₃ (CIl 9) (CH ₃ ) 18 CH ₃ (CH 2> (CH ₃ ) 19 ClI ₃ (CH 2> 6 ^C0 ~ 20 CIl (ClI 2> ₉ co- 21 CH ₃ CH ₂ 9 ^C ° - (CH 2> 22 Cbz ii ^co - 2 3 CH (CH ₃ ) 2A 25

.CO-, CO

, CO

R ²

CH ₃ CO CH CO

CH ₃ (CH ₉ ) ₆ C0-CH.J (CH ₂ ) ₉ C0-CH ₃ (CH ₂ ) ₉ C0-CH ₃ (CH ₂ ) _n C0-

t-LiOC

₁₀

CO

manufacturing

development method

B)

e)

Weight of starting material (mg)

409

  946

500

500

1000

1000

1000

1000 weight acylation agent (mg)

2208

139 62

530

292 1119 1066.9

123

Reaction time (h) HPLC elution. medium ^a] 1.5 45:15:40 17 95:30:75 20 45:15:40 18 ^C) 35:25:40 24 18 ^C) 18 ^C) 45:15:40 2: 0: 3 ^d) 2: 0: 3 ^d) 20

Wt 1 product j = * cn (Mg) I 273 365 201 60 158 366 245.9

550

Eluent of fI ₉ O: CILOH: CiLCN (V: V: V), 0.2 %. Pyridine and 0.2% HOAc

Succinic anhydride, 90% pyridine

The reaction temperature is kept at 5 ° C

With a salary. 1% pyridine and 1% HOAc

(t-BOC) _? / H ₉ O

properties of

TABLE IV • 'r' ^N orn ~ ^DiaCYI ~ ^A ~ ^21978C ~ ^Derivatives

product

Connection No.

CIl ₃ CH ₂ CH (CH ₃ ) (CIl ₂ ) ₆ C0-

15 ClI CII ₉ ClI (CIl ₃ ) (ClI ₉ ) ₆ C0-

16 CII ₃ (ClI ₉ ) ₆ C0-: i7 ClL (ClL) .. CO-

Cn ₃ CH ₉ ClI (CIl ₃ ) (CH ₉ ) ₆ C0 -Cbz

H CH CO-

H HOCO (CH ₂ ) ₂ C0-

11 CIL ₁ (ClI ₉ ), CO-3 ~ η

II CH ₃ (CIl ₂ ) _(J C0-

H Cn ₃ (CII ₉ ) _n C0-

H C-ßOC

π CH ₃ (CH ₉ ) _LO co-

0.69 0.75 0.67 0.31

Anal. HPLC elution; medium

45 45 45 30 45 45

15:40 15:40 15: 4Ü ^C 15:55 L5: 40 ^e

15:40

NU NU

22Onm e, 260nm c aniax

46,000 47,000 45,000 48,200

49,000 NU NU

10,400

11,000

9,000

8,000

9,000

NU NU

The R _r values are determined by reversed-phase silica gel TLC (Whatman KC fluorescence indicator) and using an eluent of H ₂ OrCH ₃ OH

With. -CH ₃ CN

, (45:15:40), containing 0.2% pyridine and 0.2 % HOAc, contains ^D eluent from H ₂ O ₁ CH ₃ OIIiCH ₃ CN (V: V: V) containing 0.2% pyridine and 0 , 2% HOAc contains .jWith 1 % pyridine content and 1 % HOAc NU = Not tested

TABLE V

Preparation of an N _m , N ₁ , -Diacyl derivative '

Example no. R, R ¹ R ² Manufacture of products Weight of starting material (g) Weight of acylation agent (g) Real-time (h) t-BOC-Diacyl Intermediate - HPCL Elution Ether ^a Produc HPCL EIuation, medium t Gewi-clit (mg) 26 CH ₃ (CH ₂ ) g CO H B 15 15 30 50:15:35 50:15:35 211

See example 28 above

Eluent of H ₂ OtCH ₃ OHrCl ₃ CN (VtVtV) containing 0.2% pyridine and 0.2% HOAc

TABLE VI

Properties of the N, N -diacyl derivative

Anal. I] PICU V

\ RR ¹ R ² R _F ^a Elution _{bf 22Q r 2GQ}

no. f medium λ max Λ max

21 ClUCIL) ₀ CO-CH ₀ (CIL) ₀ CO-H 0.66 61: 15: 23: 1 4 1800 11500

The Rp values are determined by reversed-phase silica gel TLC (Whatman KC,., With fluorescent indicator) and using an eluent of H ₂ OiCl-I ₃ OIl: CH ₃ CN '[45: 1 5:40], ie 0.2%. Pyridine and 0.2% HOAc

^b LI ₂ O eluent: ClI ₃ OH: CH ₃ CN: NH ₄ OAc containing 0.2 % pyridine and 0.2% HOAc

- 50 1 Example 27

Ν-, --- (n-decanoyl) -A-21978C core (compound 4) Irp

The following procedure shows the large-scale production of compounds by the active ester method.

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 purified over a silica gel filled column (WoeIm) using toluene as the eluent. The fractions are monitored by TLC, using short-wave UV light for detection. The appropriate fractions are pooled and dried under vacuum, yielding 10.4 g of 2,4,5-trichlorophenyl n-decanoate.

B. Acylation of the N 1 -t-BOC-A-21978C core with 2,4,5-trichlorophenyl n-decanoate.

A solution from N _Or . _n -t-BOC-A-219 7 8C-core (15.0 g) and 2, 4, 5-trifluorophenyl n-decanoate (15.0 g) in dry DtMF (500 ml) under nitrogen atmosphere for 25 hours stirred at ambient temperature. The mixture is then

° 0 6O ⁰ C for 5 hours or until a TLC shows the completion of the reaction. 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, yielding to 15.05 g des.N _Tr - (n-decanoyl) -N _Orn -t-BOC-A-21978C nucleus intermediate passes (formula I with R = n-decanoyl, R ¹ = H, R ² = t-BOC).

C. Purification of the N _Tr - _O ~ (n-decanoyl) -N ^^ - t-BOC-A-21 978C core

The N, _r - (n-decanoyl) -N-t-BOC-A ^^ TSC core intermediate is purified in the following manner. The crude product is dissolved in about 50 ml of the eluent system and then purified by HPLC using the Waters Prep / 500 system containing a cartridge packed with reversed-phase C "silica gel as the adsorbent. The system is purified with H ₉ OrMeOHrCH 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 combined and dried under vacuum to give 8.5 g of purified N- (n-decanoyl) -N _n

1rp urn

15 t-3OC-A-21978C core.

D. Cleavage of N ₀ ^. t-BOC group

The t-BOC group is removed by stirring the N (n-Decancyl) -N _OT _ _n -t-BOC-A-21978C nucleus (1.47 g) in 15 ml of a mixture of trifluoroacetic acid and 1,2- Ethanedithiol (10: 1) at ambient temperature over a period of 3 minutes. The reaction mixture is dried under vacuum and the residue is treated with diethyl ether (50 ml). The treated product is washed with 20 ml of diethyl ether and dried under vacuum to give 2.59 g of crude N- (n-decanovl) -A-21978C core

^ rp ^ _{1 2}

(Formula I with R = n-decanoyl, R ~ and R = H), 30 E. Purification of the N ^ - (n-decanoyl) -A-21978C core

The crude Ν _φ - (n-decanoyl) -A-21978C core is purified by reverse phase HPLC in the following manner. The sample (2.59 g) is injected in the form of a solution in 4.0 ml of H ₉ O: MeOH ': CH ₃ CN: pyridine: HOAc (50: 15: 35: 2: 2) to a 2.5 χ 85 cm stainless steel column, the

filled with LP-l / c, "adsorbent. The column is filled with the 1 o

same solvent system eiuiert. The elution is added

] at a pressure of 83 to 117 bar at a flow rate of 10 to 12 ml / rnin and using a LDC Dupiexpumpe (Milton Roy) performed. The eluate is monitored by a UV detector (ISCO model UA-5, Instrument Specialist Co., 4700 Superior Avenue, Lincoln, NB 68504, V.St.A.) at 2,800 nm. Every 2 minutes, fractions (20 to 2 4 ml) are collected. The fractions desired as a result of antimicrobial efficacy are combined and dried under vacuum to give 1.0 g of product.

This purification procedure is repeated using 4.3 g , 4.25 g, 2.14 g, 2.00 g and 1.7 g of crude starting derivative to give a total of 5.58 g of purified N ₁ → ( n-decanoyl) -A-21978C core.

Example 28

N _mT . - (n-decanoyl) -N ^ - (n-decanoyl) -A-219 / 8C (Compound 21) The Ν _φ - (n-decanoyl) -N - (n-decanoyl) -A-21973C core

-L-Lp -. rs. yi ^

(Formula I with R and R = n-decanoyl, R = H) is a minor reaction product in the preparation of N, -. - (n-decanovinyl) derivative of Example 27.

= Trp

This core is isolated by reverse phase KPLC purification as described in Section E. The desired fractions are combined and dried under vacuum to give 211 mg of crude product. The compound is purified by analytical HPLC (C ₁ "microbonda

pak, Waters Co.) using RO : MeOH:. CH ₃ CM: NH _d OH: HOAc (6: 23: 15: 0.5: 0.5) as the eluent system (32 replicates, 5 00 μg per sample injected), yielding 4.4 mg of N ₁ - , ^ - (n-decanoyl) -N "- (n-decanoyl) -A-21978-C nucleus, as indicated by 360 MHz proton NMR. 35

- 53 1 Example 29

N _m -Cbz-N 1 -Laurovl A 21978C core (Compound 20) prpurn

This method requires a protection of the oC-amino group of tryptophan in the N _n -t-BOC-A-21978C nucleus with a Cbz group with subsequent cleavage of the t-BOC group and acylating (at Deroc-amino group of the ornithine) with the Trichlcrphenyllauroylester.

A. Preparation of the N _m -Cbz N ^ t-3OC-A 21978C nucleus

irp around

The N _n -t-BOC-A-21978C nucleus (1.0 g) is dissolved in DMF (150 ml) and the solution heated to 50 ⁰ C. Add first Ν, Ο-bistrimethylsilylacetamide (0.55 ml) and then benzylpentachlorophenyl carbonate (257 mg). After stirring for 20 hours, the reaction mixture is concentrated to a volume of about 20 to 25 ml. Water (100 ml) is added and the pH of this solution is adjusted to 6.0 with 1N sodium hydroxide solution. The mixture is washed with diethyl ether (6 times with 200 ml each time) and then lyophilized.

The crude derivative is purified by reverse phase HPLC as follows. The sample is dissolved in Forin in a solution in about 6 ml of a mixture of ~0: MeOK: CH., CN: pyridine: HOAc (55: 15: 30: 2: 2) in a 1.7 x 85 cm stainless steel column Sprayed steel with LP-l / C ·! "Carrier is filled. The desired fractions are located by UV absorption (280 nm) and their antimicrobial efficacy. These fractions are then combined and lyophilized, yielding 951 mg

the N _m -CbZ-N ₀ -t-3OC-A-21978C core derivative., Trp Orn ^ ^

35 B. Cleavage of the t-BOC group

To cleave the t-30C group, the intermediate product is dissolved in an amount of 50 mg / ml in a mixture of trichlorethylene

fluoroacetic acid, anisole and triethylsilane (10: 1: 1) and left at -1O ⁰ C for 3 minutes. The solution is concentrated to an oil and the oil treated twice with 2 5 ml each of diethyl ether, giving 5 20 mg crude

5 N "-Cbz-A-21978C core. Trp ^ ^

C. Acylation of the N -Cbz-A-2197 8C core

The N -Cbz-A-21978C Ksrn is acylated by the acylation method via the active trichlorophenyl ester. To this is added N -Cbz-A-21978C (520 mg) to a solution of 1-hydroxy-citizotriazole (7 mg) and active lauroyltrichlorophenyl ester (123 mg) in pyridine (150 ml). After 20 hours of stirring at 6O ⁰ C, the reaction mixture is concentrated to a residue by treating it with diethyl ether (three times with each 25 ml) to N "-Cbz-N, -

Tró Orn

Lauroyl-A-21978C core (550 mg).

Example 30 20

Production of ship bases and reduced ship bases

Several reactions are carried out in the following way. Dissolve A-21978C-Kem (40 mg) in water (2 ml). The pH of the solution is adjusted from 4 to 9 with 1N sodium hydroxide solution. Aliquots (0.5 ml) of this solution are then mixed in each case with a solution of one of the following aldehydes (5 μl) in methanol (4 ml):

1) heptaldehyde

2) octylaldehyde

3) Decyl aldehyde ³⁵ 4) Undecyl aldehyde

The reaction mixtures are stirred overnight at room temperature. The resulting ship bases are being used

] treatment with NaBEuCN (2.5 mg per reaction) for 5 minutes at room temperature.

The reactions are assayed by TLC using silica gel, using a solvent system of CH-. CN and H-O (7: 3), and testing for Staphylococcus aureus. Schiff's bases are not active in this trial against Staphylococcus aureus, possibly due to their instability under the assay conditions. In each reduction reaction, two factors are formed. These compounds are active against Staphylococcus aureus and show similar mobility in the TLC system, proving that the following compounds of formula I have been formed

15 are where each R is hydrogen:

V J

Manufacture of Ship Bases and Reduced

Schiff bases

product

Implementation of A-21978C with

heptaldehyde

octyl

decyl

undecylaldehyde

CII ₃ (CH _{2) 5} CH ₃ CII (CII _{2) 6} -cli ₃ (CII _{0) 5} CH CH ₃ (CH ₂ J ₆ -CIl ₃ (CH _{2) 6} CH CH ₃ (CH _{2) 7} -CIl ₃ (CH ₂ ) g CH CH ₃ (CIl ₂ ) ₇ -CH

CH ₁ (CIl ₂ ) _a CH = Cll ₃ (CH ₂ ) ₉ -CH ₃ (CH ₂ ) ₉ C 11 = CH ₃ (CII ₂ ) ₁₀ -

II CH ₃ (CH ₂ ) ₅ CH

II

CIl ₃ (CII ₂ ) ₇ -HH

HH CH ₃ (CII ₂ J ₉ CII

Active against Staphylococcus aureus

el

No

Yes

No

Yes

No

Yes

No

Yes

no yes no

Yes

no Yes

no Yes

The lack of activity of the ship bases in this attempt to be unstable under the experimental conditions.

one episode

- 57 1 Example 31

Preparation of the Ship Base N-Lauraldehyde and the Schiff Base N, N-Dilauraldehyde and the Reduced Schiff Bases Thereof (Compounds 22 and 23)

A-21978C core (1 g) is dissolved in water (50 ml) and the solution is treated with a solution of dodecyl aldehyde (500 μΐ) in methanol (200 ml). The reaction mixture is stirred overnight at room temperature. The reaction mixture is then reduced by addition of NaBH.CN (291 mg) for 50 minutes. The reaction mixture is filtered under vacuum using Whatman No. 1 paper to remove particulate matter. The supernatant liquid is concentrated to an aqueous solution, the lyophilization of which leads to 1.256 g of product. This product is analyzed by analytical HPLC and purified by preparative reverse phase HPLC in proportions. Each portion (350 mg) is dissolved in 50% aqueous methanol (6 ml) with sonication and heating to dissolve the material. The solution is then applied to a 1.5 × 8 cm-cm column filled with LP, -C-, _fl and which is treated with a mixture of CH-, OH: CH ₃ CN: H ₃ O (25:40: 35) containing 0.2% pyridine and 0.2% acetic acid, eluted at a flow rate of about 7.5 ml / min. The elution is monitored by UV light at 280 nm. The fractions containing the desired products are combined, yielding a total of 104 mg of M- (n-dodecyl) -A-21978C-

Kern (Compound 22) and 19.2 mg Ν _φ - (n-dodecyl) -N_ (n-dodecyl) -A-21978C core (Compound 23).

Example 32

The antibacterial activity of the compounds of the formula I can be demonstrated in vitro. The results obtained in the study of representative compounds of the formula I with respect to their antibacterial activity using the disc diffusion test on agar plates

These are shown in the following table VII. In this Table VII the efficacy is measured by the size (diameter in mm) of the observed zone in which a growth of the microorganism is determined by the respective

5. sought compound is inhibited.

TABLE VII

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

on agar plates

Connection no.

1 2 3 4 5 6 7 8 9

10 11. 12 13

cu ₃ (cn ₂ ) ₅ co-

k (ClL), CO-3 2 6

CH CH ₃

C0-CO-

CH ₃ (CIl ₂ ) ₀ CO-

cii ₃ (cii ₂ ) _lo co

CH ₃ (CH ₉ ) ₁₁ CO-

^C11 3 ^(CH 2 ⁾ 14 ^CO ~ CII ₂ = CII- (CH ₂ ) ₈ CO-

CH (CIl ₂ ) CH = Cll (CH ₂ ) CO-ClI CH ₂ Ch

R ² Size of Heniinzone (mm) Bacillus H Staphylococcus Bacillus Micrococcus sUbL11 is 1 H aure.us tiubL.il is luteus ATCC 6633 R ¹ H ATCC 6738P ATCC 6633 ATCC 9341 16 H H 17 15 21 20 H H 23 18 23 27 H H 20 16 18 29 II H 24 20 22 21 H H 19 19 21 32 H II 25 20 19 29 H H 21 17 19 28 H H 22 21 21 24 H II 20 19 19 23 II 19 17 17 25 H 21 17 20 30 11 2 2 19 20

13

14

TA BELLE VII (continued)

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

on agar plates

Great of the Ue mm ζ one (now)

Micrococcus Bacillus

Ve rb in- CH ₃ R (CH ₉ ) , CO-0 1 R ² , CO-6 aureus subtllis luteus is subtle, dimg no. ClL CH ₂ CH (CH ₃ ) (CH _? ) , .co- R ¹ CH CO- qCO- ATCC 6738P ATCC 6633 ATCC 9341 ATCC 6633 14 CH ₃ CH ₂ CH (CH ₃ ) Il 110CO (CH _{0 n} co- 21 18 20 25 15 CH ₃ (CH ₂ ) ₆ co- H CH ₃ (CH ₂ ) 21 18 19 25 16 CH ₃ (CH ₂ ) _(J CO- H CH ₃ (CH ₂ ) ! 0 ^C ° - 11 , Sp ° 10 17 17 ClI ₃ (CH ₂ ) _1X CO- (CH ₂ ) , CO-D H CH ₃ (CH ₂ ) 17 13 14 20 18 Cbz CH ₂ CH (CH ₃ ) H t-BOC 12 sp - 13 19 11 CH (CH) 14 11 10 22 20 Il 15 10 17 23

The respective compounds are suspended in water at a concentration of 1 mg / ml. Into the suspension is dipped a 7 mm slice / which is then placed on the agar surface. It is incubated for 24 to 48 hours at 25 to 37 ° C.

Grown on minimal nutrient agar

track

] The d- in the investigation _{= J} _- en antimicrobial activity of representative compounds of formula 1 using the conventional agar dilution test results obtained are shown in the following Table VIII. In this Table VIII, the activity is measured by the minimum inhibitory concentration (MIC value), namely the lowest concentration of the compound which inhibits the growth of the microorganism.

^ y

TABLE VIII

Antibiotic activity of A-21978C cyclopeptides

MIC values of the compounds investigated Experimental organisms

1 8th 16 2 ^d 3 4 ^e 5 ^d 6 ^e 7 8th 9 10 11 I 8th 8 2 4 2 1,0.5 12:25 0.5,0.5 0125 0.5,1 1 1 0.5 cn NJ I 8th 8th 4 2 1,0.5 12:25. 0.5,1 0125 0.5,2 1 1 0.5 8th 128 8th 4 2.1 0.5 1.1 0.5 1.2 2 ') L. 0.5 128 4 2 1,0.5 0.5 0.5,0.5 12:25 1.2 1 1 12:25 8th 4  2.1 0.5 1.1 0.5 1.4 2 2 1 4 1 2 0.25 1.0.5 0.25, 0.125 0.5 0.125 1.1 0.125, 0.25 0.5 0.25 2.2 0.5.2 4 1 2 1 1 0.5 8th 2 1,0.5 12:25 0.25, 0.25 12:25 0.03, 0.125 12:06 12:06 12:25 64 32 16 ', 16 2 2.4 12:03 0.5,2 2 2 1 16 8th 2.2 0.5 0.5,0.5 32 0.125,> 128 12:25 12:25 0.5

Staphylococcus aureus Xl.1 Staphylococcus aureus V41 Staphylococcus aureus X400 Staphylococcus aureus SI3E Staphylococcus epidermidis EPIII Staphylococcus epidermidis ΕΡΪ2 Streptococcus pyogenes C2O3

Streptococcus pneumoniae park

Streptococcus group I) X66 Streptococcus group 9960

^a MIC values in pg / ml. The numbers of the respective compounds are from Tables II, IV and VI and Example

Penicillin-resistant strain d

^c Methicilin-resistant strain Mean of three experiments Two trials

TABLE VIII (continued)

Ask for Organisms

Staphylococcus aureus Xl.1 Staphylococcus aureus V41 Staphylococcus aureus X400 Staphylococcus aureus S13E Staphylococcus epidermidis EPIII Staphylococcus epidermidls El ^J 12 Streptococcus pyogenes C2O3 Streptococcus pneuinoniae Park Streptococcus Cruppe D X66 Streptococcus group 9960

13

MIC values of the compounds tested

16

18

20

21

22

1 > 128 4.4 4.4 8th 1 8th 8th 1 0.5 8th σ \ UJ I 1 > 128 4.4 8.8 16 4 64 16 4 1 8th 2 > 128 8.8 4.0 8th 4 > 128 32 32 1 16 1 > 128 8.4 4.8 8th 1 > 128 8th 16 0.5 8th 2 > 128 8.8 8.8 16 0 > 128 32 2 1 8th 2 > 128 0.4 8.8 16 8th > 128 32 2 1 8th 0.5 0.5 > 128> 128 1.1 2.2 1.1 2.4 4 4 0.25 0.125 1 0.5 4 0.125 0.5 0.5 1 4 8 2 > 128 64.32 64.64 > 128 8th > 128 128 > 128 4 32 1 > 128 32.8 32.32 64 8th > 128 64 128 1 8th

The A-21978C cyclopeptides of formula I also exhibit antimicrobial activity in vivo against experimentally induced bacterial infections. If mice with the infections shown are given subcutaneously or orally two doses each of the compound to be tested, a corresponding activity can be observed which is measured in the form of the ED- _Q values (effective dose in mg / kg, which provides protection of 5% of the test animals) (J. Bacteriol 81, 233 to 235 (1961)). The ED - ^ values obtained for typical A-219TSC compounds of the formula I are shown in Table IX below.

TABLE IX In vivo potency of A-21'JVSC cyclopeptides

ED _5Q values ^a

Connection no.

Compound of formula I

₂ ) ₅ C0-

ClL (ClL), CO-J Zb

CII

C0

CH ₃ (CH ₂ ) ₈ C0-

cn ₃ (cii ₂ ) _g co-

CH ₃ (Cn ₂ ) _1L CO-Cll ₃ (CH ₂ ) ₁₂ C0-) _{] 3} C0-CH ₃ (Cll ₂ ) _U CO-Cll = CH- (ClL) ₀ CO-CH ₃ (CH ₂ ) CH = CH (CH ₂ ) CO-

CiL ₁ CILCH (CIL) (CIL), CO-J / j Ib

CIL ₁ CILCII (CIl) (CIL), CO- Sl J Ib

Stapliylococcus aureus

JL

H II II II II II II II 11

H H II II H H II H H H II H

S ii bleu tan

II CH CO-II HOCO-

2.65

3.75,1.4 0.5 0.28 0.37 0.44 0.54

1.17,3.08 8.3

0.93 3.28 1.3

27.6, CO-Strepto coccus p yogenes

Subcutaneous Orally 1.49,5.l ^b NU ^C <2.2,0.65,1.9 ^d > 200 0.14,0.243 92.117 12:03 66 12:13 138 12:05 69 0046 45 O.98, O.20, <12:54 <50 <200 <0.5,0.18 > 200 12:18 > 200 0068 138 0134 75 2.2,1.9 163> 2OO 1.4 > 200

₃ (CH ₂ ) _g CO

H CH (CH) CO- ^J > 35

11 Il

1.25 2.85 1.1,0.85

> 200> 200

'Mg / kg; χ 2; Two attempts; ^c Not tested; Three attempts

Some A-21978C cyclopeptides of formula I have also been tested for their toxicity, and the. Test results obtained from this are shown in Table X below.

TABLE X

Toxicity of A-2ly7BC cyclopeptides

/ herd no. Formula I R R ¹ R ² LD ₁ values (rag (kg) on the mouse 1 CII ₃ (CH ₂ J ₅ CO- H H > 600 2 CH ₃ (ClI ₂ J ₆ CO- H II > 600 3 ClI ₃ (CH ₀ J ₇ CO- II Il > 600 4 CIl ₃ (CH ₂ J ₈ CO- 11 ii > 300 5 CH ₃ (CII ₂ J ₉ CO- H H > 600 6 CH ₃ (CH ₂ J ₁₀ CO- H IL 144,265 ^b 7 CH ₃ (CH ₂ ) _u C0- ii H 112.5 8th CH ₃ (CH ₂ J ₁₂ CO-: H IL 62.5, <150 9 CIi ₃ (CH ₂ J ₁₃ CO- II M 56.25 10 · CH ₃ (CH ₂ J ₁₄ CO- H H 50 11 ClL - CH- (CILJ _o C0- Z Za ii H > 500 12 CIU (CIU) -, CH = CH (ClU) -, CO- 3 2 3 Zl H H 450 14 CH ₃ CH ₂ Cll (CH ₃ ) (CIU) ₆ CO- H CH ₃ CO- > 600 _; 600 15 CH ₃ CH ₂ Cll (CIi ₃ ) (CH ₂ J ₆ CO- H HOC 0 (CH) CO 2 2 450.600 16 CH ₃ (CH ₂ J ₆ CO- Il CH ₃ (CH ₂ ) ₆ C0- > 600 17 CII ₃ (CH ₂ ) ₉ C0- H CH ₃ JCH ₂ J ₉ CO- 94 22 CH ₃ (CH ₀ J ₁₁ - H- ii > 300

Intravenous administration

Two attempts

Claims (7)

1. A process for the preparation of A-21978C cyclopeptide derivatives of the formula I. HO2 KOs in which R, R and R independently of one another are hydrogen, C ₄ -C 12 -alkyl, optionally substituted C ₅ -C 10, _Q- alkanoyl, C-C, q-alkenoy.l or an amino-protecting group, r3, R 1 and R ^ are all hydrogen or (i) R and R 'and / or (ii) R and R and / or (iii) R ³ and R "together form a C.-C, may represent alkylidene group with dei proviso in that (1) at least one of the substituents R, R ^ 2 orV'-R must be other than hydrogen or an amino protecting group, (2) at least one of the sub- \ '2
R or R is hydrogen or an amino-protecting 1), (3) the substituents R, R and R together must contain at least 4 carbon atoms 1 2
and (4) if R and R are selected from hydrogen or an amino protecting group, then the substituent R then not for 8-methyldecanoyl, 10-methylundecanoyl, 10-methyldodecanoyl, the specific C, »alkanoyl group of Fak C _Q of A-21978C or the particular C alkanoyl groups of factors C and C, - of A-21978C, or of pharmaceutically acceptable salts thereof with organic or inorganic acids, thereby 5, characterized in that one has an A-21978C-Cyclopeptidkern the formula IU NHR ' ΗΟξ Hoa III or a suitably protected derivative thereof, wherein R ' 0
and R independently of one another denote hydrogen or an amino-protecting group, or a pharmaceutically acceptable salt thereof with an organic or inorganic acid, (a) with an acylating agent of the formula V R - COOH or an activated derivative thereof, wherein R is optionally substituted C.-C. η-alkyl or C.-C ₁ "alkenyl, or 22AuRiQ. A -i j κ- .. ~ 1 (b) with a carbonyl derivative of the formula VI R ⁷ - C - R ⁸ (VI) Il 7 Q wherein the group RRC = for C. -C ₁ . Alkylidene, ° converts and- (C) a C ₄ -C ₁₄ -Alkylidenprodukt obtained according to the above method (b) optionally converted by reduction into a compound of formula I wherein R, R or R is a C ₄ -C ₁ , alkyl group and ¹ ^ (d) from the product obtained according to one of the above reactions (a), (b) or (c), if desired, cleaving off any protective groups present and (e) optionally the compounds of formula I with organic or inorganic acids in their pharmaceutical , harmless salts transferred. 2. The method according to item 1, characterized in that R is alkanoyl of the formula 20 ^CH 3 ^(CH 2 ⁾ n "^" and η is an integer from 3 to 17. 3. The method according to item 1, characterized in that R is alkanoyl of the formula 25 CH ₃ "O CH ₀ (CH ₀ ) - CH (CH _n ) -C-3 Z η 2 m and η 'and m each independently represent an integer of 0 to 14, with the proviso that η + m may not be less than 1 nor more than 15, and with the further proviso that if η is O , m can not be 8, and if η is 1, m can not be 6 or 8. 4. The method according to item 1, characterized ge.kenn characterized in that R is cis or trans-alkenyl of the formula CH ₃ (CH ₉ J _n CH = CH (CH ₂ ) _m - C - _ "7 1 _ ι Χ 1, wherein η and m each independently represent an integer of 0 to 14, with the proviso that η + m may not be less than 1 nor more than 15. 5 5. The method according to item 1, characterized ge indicates that R is eis or trans-alkenyl of the formula CH ₀ - CH (CH _n) C 2 Zn wherein η is an integer of 2 to 16. 6. The method according to item 1, characterized in that R is C _d -C, _d -Alkyliden 15 stands. 7. Method according to item 6, characterized ge indicates that R is dodecylidenyl. 20 3 · Method according to point 1, thereby ke η η indicates that R is C ₄ -C ₁₄ -alkyl. 9. The method according to item 8, characterized in that R is dodecyl. 25