CS257766B2 - Method of cyclic peptide's a-21978c derivative preparation - Google Patents

Description

The present invention relates to novel A-21978C cyclic peptide derivatives and pharmaceutically acceptable salts thereof which are potent antibiotics. The novel derivatives of the invention are prepared by reacting the A-21978C nucleus prepared by deacylating the appropriately protected A-21978C complex or any of its protected Factors Co, Cl, C2, C8, C4 or Cs with the desired acylating agent or activated derivative thereof or of the appropriate Schiff base and subsequent reduction of the imine bond of the base.

The present invention relates to A-21978C cyclic peptides of formula I

where

R ¹ , R ¹ and R ² are each independently hydrogen, C 2 -C 19 alikanoyl optionally substituted with carboxyl, C 5 -C 19 alkenoyl or an amino protecting group selected from Icarbobenzyloxy or tert-butoxycarbonyl,

R ³ , R ⁴ and R ⁵ are hydrogen atoms, wherein il. at least one of R, R ¹ or R ² must be other than hydrogen or an amino protecting group as defined above,

2. at least one of R ¹ or R ² must be a hydrogen atom or an amino protecting group as defined above;

The third of R, R ¹ and R ² groups together must contain at least four carbon atoms and

4. when both R ¹ and R ² are hydrogen or an amino protecting group as defined above, R cannot be 8-methyldecanoyl, 10-methylundecanoyl, 10-methyldodecanoyl, a specific C 10 alkanoyl group A-21978C or a specific alkanoyl group having And the pharmaceutically acceptable salts of these peptides useful as semisynthetic antibacterial agents or as intermediates in the preparation of such agents.

The following abbreviations are commonly used in the literature:

Ala: alanine

Asp: aspartic acid.

Asn: asparaigin Gly: glycine Kyn: kynurenin Orn: ornithine Ser: serine Thr: threonine Trp: tryptophan t-BOC: tert-butoxyicarbonyl Cbz: benzyloxycarbonyl DMF: dimethylformamide THF: tetrahydrofuran NMR: nuclear magnetic resonance

UV: ultraviolet radiation.

A-21978C antibiotics are closely related acidic peptide antibiotics described in U.S. Patent No. 4,208,403, which is incorporated herein by reference, as described in U.S. Patent No. 4,208,403. C, which is itself a complex of closely related factors. The A-21978 factor C, which is called the A-21978C complex, contains the individual factors Co, Cl, C2, C, C4, and Cs. The factors Ci, C2 and Сз are the main factors and the factors Co, Ci and Cs are minor factors. The structure of A-21978C factors is evident from Formula II

257768

NH

AND

RN (II) where

3MG is L-threo-3-methylglutamic acid, and

R ^N is part of a specific fatty acid molecule.

Specific R ^N groups of factors are

dující: A-21978C factor R ^N part of the molecule Cl 8-methyldecanoyl C2 Сз 10-Methyllundecanoyl 10-methyldodecanoyl What Cto-alkanoyl * C4 C12-alkanoyl * C5 C12-alkanoyl *

Identity has not yet been established.

When performing deacylation of peptide antibiotics, it is very difficult to decide which enzyme is useful for this purpose. Finding such an enzyme is even more difficult if the substrate comprises a cyclic peptide core. Since the enzymes have a high degree of specificity, the difference in the peptide portion and in the side chain of the substrate affects the yield of deacylation. In addition, many microorganisms produce large amounts of peptidases that attack various portions of the peptide portion of the molecule. This often leads to unprocessable product mixtures.

The enzyme used for performing deacylation of the side chain of the fatty acid α-amino group of tryptophan is produced by a microorganism of the genus Actinoplanaceae, preferably a microorganism of Actinoplanes utahensis NRRL 12052 or a variety thereof. In performing deacylation, the antibiotic is selected from the group consisting of A-21978C complex, A-21978C factors Co, Cl, C2, Ciz, Ci and Cs blocked by A-21978C complex and blocked A-21978C factors by Co, Ci, Сг, Сз, Ci and Cs. Cs, added to the culture of the microorganism.

The terms "blocked A-21978C factors" and "blocked A-21978C complex" refer to individual A-21978C factors or a mixture of factors (complex) wherein the aminosulinipine of ornithine and / or kynurenine is protected by an amino protecting group.

Preferably, the amino protecting group is located 11a of the δ-amino group of ornithine of individual factors or a mixture of factors. The culture is allowed to incubate with the substrate until the deacylation is complete. The corresponding A-21978C cyclic peptide thus obtained is separated from the fermentation medium by methods known in the literature.

The cyclic peptides obtained by these enzymatic deacylations are represented by Formula III

R 1 and R 0 are each independently hydrogen or an amino protecting group and salts thereof.

Removal of the acyl moiety from the A-21978C individual factors of Co, C 1, C 2, C 2, C 4 and C 5 provides a compound of formula III wherein R ‘and R ° are hydrogen atoms. The deacylated molecule is a common cyclic peptide present in each of the A-21978C factors. For simplicity, this compound is called the A-21978C core. This compound may be represented by Formula IV

o-Ata F Gly 1 L-Áp D-Ser T L — Qru 3MG 1 ΐ 1 Gíy □ / of Wh-r 1 ’L — Ast> r L “ASii

t

L —Trp

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

Compounds of formula III wherein R 1 or R 0 are other than hydrogen are prepared by deacylating the respective blocked A-21978C antibiotic factors Co, Ci, C2, Coz, C4 and C5. For simplicity, these compounds are called blocked A-21978C nuclei. These blocked compounds are useful as intermediates for the preparation of certain peptides of Formula I, i.e., compounds wherein at least one of R ¹ and R ² is an amino protecting group.

It is known to those skilled in the art that the A-21978C core and the blocked A-21978C core may be prepared either in the form of free amines or acid salts. While any suitable acid salt may be prepared, pharmaceutically acceptable salts are preferred. Pharmaceutically acceptable salts are those useful in the chemotherapy of warm-blooded animals.

NRRL 12052 is available to the public from the Agricultural Research Culture Collection (NRRL), Norhern Regional Research Center, US Department of Agriculture, 1815 North University St., Peoria, Illinoifs 61604, USA, under NRRL 12052.

Preparation 1 discloses herein the preparation of the A-21978C core by fermentation using A-21978C (complex as substrate and Actinoplanes utahensis NRRL 12052 as microorganism).

Other Actinoplanaceae cultures useful for preparing the A-21978C core of Formula III are available to the public from the Northern Regional Research Laboratory under the numbers:

257769

Actinoplanes utahensis

Actinoplanes missouriensis

Actinoplanes sp.

Actinoplanes sp.

Streiptosporangium roseumj var. hollandensis

NRRL 12052

NRRL 12053

NRRL 8122

NRRL 12065

NRRL 12064

The efficacy of any of the Actinoplanaceae microorganism strains for deacylating the present invention is determined by the following procedure. A suitable growth medium is inoculated with a microorganism. The culture is incubated at about 28 ^and C for two days or three days on a rotary shaker. One of the antibiotic substrates is then added to the culture and the culture is maintained at a pH of about 6.5. Culture was evaluated for activity using Micrococcus Juteus. Loss of antibiotic activity is an indicator that the microorganism produces the desired enzyme for deacylation. However, this shall be verified by using one of the following methods:

1. by high pressure liquid chromatography analysis for the presence of an antibiotic nucleus or b

2. Reacylation with the appropriate side chain (eg, lauroyl, n-decanoyl or n-do is located on the ω-amino group of ornithine decanoyl) to restore activity.

The present invention provides a process for the preparation of compounds of formula (I)

(!)

Where

R ¹ , R ¹ and R ² are independently hydrogen, C 2 -C 19 alkanoyl optionally substituted with carboxyl, C 5 -C 19 alkenoyl or an amino protecting group selected from carbobenzyloxy or tert-butoxycarbonyl, wherein:

1. at least one of R, R ¹ or R ² must be other than hydrogen or an amino protecting group as defined above;

2. at least one of R ¹ or R ² must be a hydrogen atom or an amino protecting group as defined above;

The third of R, R ¹ and R ² groups together must contain at least four carbon atoms and

4. when both R ¹ and R ² are hydrogen or amino protecting groups as defined above, R cannot be 8-methyldecanoyl, 10-methylundecanoyl, 10-methyldodecanoyl, 10-carbon specific alkanoyl group A-21978C or specific alkanoyl groups having 12 carbon atoms of A-21978C factors Cd and Cs and pharmaceutically acceptable salts of these peptides, characterized in that the core of the cyclic peptide A-21978C of formula III

or an appropriately protected derivative thereof, wherein R 1 and R 0 are independently hydrogen or an amino protecting group as defined above, or a corresponding pharmaceutically acceptable salt thereof, is reacted with an acylating agent of formula V

R ⁶ —COCH (V) or an activated derivative thereof, wherein R ^B is C 1 -C 18 alkyl, optionally substituted with carboxyl, or C 4 -C 18 alkenyl, and optionally removes any protecting group that may be present in the of the above reaction.

The term "C 4 -C 14 alkyl" refers to monovalent saturated, unbranched or branched alkyl groups containing 4 to 14 carbon atoms. These compounds wherein one of R, R ¹ or R ² are C 4 -C 14 alkyl are prepared by reduction of the corresponding compounds wherein R, R ¹ or R ² is C 4 -C 14 alkylidenyl and are referred to herein as " reduced Schiff bases ".

The terms "optionally substituted C 2 -C 19 alkanoyl" and "C 5 -C 19 alkenoyl" refer to acyl groups derived from C 2 -C 19 and C 5 -C 19 carboxylic acids. When R is alkanoyl, the alkyl moiety is a monovalent saturated, unbranched or branched hydrocarbon radical which may optionally bear as a substituent hydroxyl, 1-carboxyl, alkoxy of 1 to 3 carbon atoms or one to 3 halogen atoms selected from the group consisting of chloro, bromo or fluorine.

When R is alkenyl, then the alkenyl portion of the molecule is a monovalent unsaturated, unbranched or branched hydrocarbon radical containing no more than three double bonds. The double bonds of the unsaturated hydrocarbon chains may be either cis or trans.

The term "amino protecting group" refers to an amino protecting group that is compatible with other functional groups in the A-21978C molecule. Preferred amino protecting groups are those that are readily cleaved from subsequent acylated compounds.

Examples of suitable protecting groups can be found in "Protective Groups in Organic Synthesis" by Theodore W. Greene, John Wiley and Sons, New York, 1981, Chapter 7. Particularly preferred amino protecting groups are tert-butoxycarbonyl and benzyloxycarbonyl.

In its preferred embodiments, the present invention relates to the following compounds of Formula I:

a) Compounds wherein R is an alkanoyl of formula

O

II

CH 5 (CH 2) n -c- and n is an integer from 3 to 17,.

(b) Compounds wherein R is alkanoyl of formula

О

СНз (СН2) „- С— а

n is an integer from 5 to 14,

c) Compounds wherein R is alkanoyl of the general formula

CH 3 (CH 2) _n CH (CH 2) _m —C-

I II

СНз o a

m and m are independently integers from 0 to 14, where n + m must be less than 1 and not more than 15, and if n is 0, m must not be 8, and if n is 1, m must not be 6 or 8,

d) Compounds wherein R is cis or transalkenyl of the general formula

O

II

CH 3 (CH 2 J 11 CH = CH (CH 2) c - a

n and m are independently integers from 0 to 14, with n + m not less than 1 and not more than 15,

e) Compounds wherein R is cis or trans alkenyl of the general formula

O

II.

СП2 ^ СИ (СН2) '- c a

n. is an integer from 4 to 15,

f) Compounds in which R is alkyl of the formula S (N 2) _p - and _n is an integer from 5 to 12; and

g) Compounds wherein R is

O

II

СИз (СН2) 5 — C— o

II

CH3 (CH2) 6-C10

II

CH 3 (CH 2) 7 -C 10

II

CH 3 (CH 2) 8 -C 10

CH 3 (CH 2) 9 -C 10

II

СНз (СН2) 10 — C — o

II

CH2 = CH- (CH2) 8-Co

II

СНз (СН2) 11 — C — o

II

CH5 (CH2) 12-C10

II

СНз— (CH2) 3 —CH = CH— (CH2) 7 — c— o

II

CH3 (CH2) 13-C10

II

CH3CH2CH (СNз) - (CH2) 6 — c—

Cil5 (Cil2) U-Cl13 (CH2) 10CH = CH3 (CH2) 6-CH3 (CH2) 5CH = СНЗ (СН2) 9— СНз (СН2) 8СН =

The compounds of formula I are capable of forming salts which are also included in the present invention. These salts are useful, for example, for separating and purifying compounds. Pharmaceutically acceptable alkali metal, alkaline earth metal, amine and acid addition salts are particularly useful.

For example, the compounds of formula I have four free carboxyl groups, which may form salts. Partial, mixed or complete salts of these carboxyl groups are included in the present invention. In the preparation of these salts, a pH greater than 10 should be avoided because of the instability of the compounds at the indicated values.

Representative and suitable alkali metal and alkaline earth metal salts of the compounds of formula I include:

sodium, potassium, lithium, cesium, rubidium, barium, calcium and magnesium salts.

Suitable ammonium salts of the compounds of formula I are ammonium and primary, secondary and tertiary C 1 -C 4 alkyl ammonium salts and C 2 -C 4 hydroxyalkylammonium salts. Examples of ammonium salts include, but are not limited to, those formed by the reaction of a compound of Formula I with ammonium hydroxide, methylamine, sec-butylamine, isopropylamine, diethylamine, di-isopropylamine, cyclohexylamine, ethanolamine, triethylamine and 3-amino-1-propanol.

The alkali metal and alkaline earth metal cation salts of the compounds of formula (I) are prepared by processes commonly used to prepare cationic salts. For example, the free acid form of the compound of formula I is dissolved in a suitable solvent such as hot methanol or ethanoan, followed by the addition of a solution containing a stoichiometric amount of the desired inorganic base in aqueous methanol. The salt thus formed can be isolated by conventional methods such as filtration or solvent evaporation. A suitable method for preparing salts is the use of ion exchange resins.

Salts formed with organic amines can be prepared in a similar manner. For example, gaseous or liquid amines may be added to a solution of the compound of formula I in a suitable solvent such as ethanol, solvent and excess amines are removed by evaporation.

The compounds of the invention also have free amino groups and can therefore also form acid addition salts, which also form part of the present invention. Representative and suitable addition salts of the compounds of formula (I) include, but are not limited to, those 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 acid, palmitic acid, cholic acid, pamoic acid, mucic acid, D-glutamic acid, D-camphoric acid, glutaric acid, glycolic acid, phthalic acid, tartaric acid, lauric acid, stearic acid, salicylic acid, acid methanesulfonic acid, benzenesulfonic acid, sorbic acid, picric acid, benzoic acid and cinnamic acid.

Compounds of formula I wherein R, R ¹ or R ² are alkanoyl, alkenoyl and / or an amino protecting group are prepared by acylating A-21978C factor, blocked A-21978C factor, a compound of formula III or a corresponding compound of formula III that is blocked at α- an amino group of tryptophan, the desired alkanoyl or alkenoyl side chain using methods conventional in the art for forming an amide bond. The acylation is conveniently carried out by reacting a selective compound with an activated alkanoic acid or alkenoic acid derivative corresponding to the desired side chain acyl group (R, R ¹ or R ² ). The term "activated derivative" refers to a derivative that causes the carboxyl functionality of an acylating agent to be reactive for coupling a primary amino group to an amide bond that attaches the acyl side chain to the core. Suitable activated derivatives, methods for their preparation and methods of using acylating agents for the primary amine are known to those skilled in the art. Preferred activated derivatives are a) an acid halide (eg an acid chloride), b) an acid anhydride (eg an alkoxy formic anhydride), or c) an activated ester (eg a 2,4,5-trichlorophenyl ester). Other methods for activating a carboxyl function include reacting the carboxylic acid with carbonyldiimide (e.g., N, N'-dicyclohexylcarbodiimide or N, N'-diisopropylcarbonyldiimide) to form a reactive intermediate which is not isolated due to instability (instability). in this case, it performs in network.

It is known to those skilled in the art that compounds of formula I are prepared by selective acylation procedures, with the assistance of amino protecting groups. For example, if a compound of formula III, wherein R 1 and R 0 are hydrogen atoms, is used as the starting material, acylation may occur at the α-amino group of tryptophan and the α-amino group of ornithine to form the Nr _rp , N _Orn- diacyl derivatives. For the preparation of monoacylated derivatives on the .alpha.-amino group of tryptophan, the compound of formula III is preferably acylated, wherein the .alpha.-amino group of ornithine (R0 position) is blocked by an amino protecting group. These starting materials are preferably prepared by blocking A-21978C factor at this position prior to deacylation. The aromatic-aminoskuipin of cynurein is reactive in at least three

2577G6 of the free amino groups of the A-21978C core. Acylation of kynurenine involves suitably blocking the amino groups of tryptophan and ornithine, but acylation at the R and R ^{1 positions} normally does not involve blocking the amino group of kynurenine.

Schemes I, II and III show general procedures for the preparation of compounds of formula I wherein one of R, R ¹ or R ² is alkanoyl, alkenoyl or an amino protecting group. The following abbreviations are used in the diagrams:

(x] = residue A-2197BC

N _r = α-amino of tryptophan

N _o = α-amino group of ornithine N _K = aromatic amino group of kynurenine

R ¹ , R ¹ , R ² = substituents as defined

R _n acyl group of natural factor

V, B ¹ = amino-protecting groups Acyl = acylation step

Deacyl-deacylation step Block = acylation with an amino-protecting group

Deblock = removal of an amino protecting group.

In Scheme I, A-21978C monτιρ-monoacyl derivatives are represented by the general formula III and Nt _1p , N-diacyl derivatives A-21978C are represented by the general formula IV. Ni-rp, οο, η-diacyl derivatives, where the N _Trp- acyl group is a group of natural A-21978C factor is represented by the general formula VIII.

Scheme I: Preparation of N _rrp- monoacyl and Nrr _P ,

Nom-diacyl-A21978C derivatives

In Scheme II, Ντ, ρ, N _Kyn -diacylderivatives A-21978C are represented by the general formula X. Ty N _T rp, N _Kyn -diacylderivatives, where N _Trp- acylcipridine is a group of natural A-21978C factor are represented by general formula XII. Compounds of formulas V, VI and VII are also described in Scheme I.

Scheme II: Preparation of N- _Ir- N- _Kyi -Diacyl-A-21978C derivatives

X

In Scheme III rnanoacylderiváty Nou-A-21978C are represented by formula XVIII NK -monoacylderiváty _yl-21978C are represented by the formula XV and _Om N, N _K Y _H -di®cylderlváty of A-21978C are represented by general Formula XX. Compounds of formula (VI) are also described in Schemes I and II.

Scheme III: Preparation of N-N _- monoacyl, N- _Kyil- monoacyl and N- _Om , N-n-diacyl A-21978C derivatives

> X v.-

Compounds of formula I wherein one or more amino groups are substituted with an alkylidenyl group (Schiff base) or an alkyl group (reduced Schiff base) can be prepared by known methods used to prepare Schifl bases and reduce these bases. , or.nithine or kynurenine A-21978C with the appropriate aldehyde or ketone in a suitable solvent Reduction of the imine bond of the Schiff base to give the corresponding compound of formula I wherein R, R ¹ or R ² is alkyl can be carried out by known selective reduction procedures. the reagent for this reaction is sodium cyanoborohydride.

Under the conditions of in vitro assays, Schiff's bases do not show antibacterial activity likely due to instability in the assay medium. However, they are useful as intermediates for the preparation of reduced Schiff bases. If Schiff bases are used as intermediates, they need not be isolated prior to reduction to reduced Schiff bases.

A preferred method for preparing compounds of formula I wherein one of R, R ¹ or R ² is alkanoyl or alkenoyl is the active ester method. A compound of formula III wherein R 1 H and R 0 = t-BOC, i.e., A-21978C N _{O 1} r -t-BOC core or "t BOC core" is a particularly preferred starting material in the preparation of compounds of formula I. 2,4 The 5-trichlorophenyl ester of the desired alkanoic or alikenoic acid is a preferred acylating agent. In this method, an excess amount of active ester is reacted with the t-BOC core at room temperature in a non-reactive organic solvent such as dimethylformamide, tetrahydrofuran, diethyl ether or dichloromethane. The reaction time is not critical, although a period of from 6 to about 20 hours is preferred. At the end of the reaction, the solvent was removed and the residue was purified. A particularly preferred purification method is reverse phase high pressure liquid chromatography using LP-1 / C18 as the stationary phase and a mixture of water, methanol, acetonitrile, pyridine and acetic acid as the solvent system. The t-BOC group is removed by reaction with a mixture of trifluoroacetic acid, anisole and triethylsilane, or preferably a mixture of trifluoroacetic acid and 1,2-ethanedithiol for 3-5 minutes at room temperature. After removal of the solvent, the residue was purified by reverse-phase high-pressure liquid chromatography.

An alternative acylation method is a modified Schotten-Baumann method in which an unprotected nucleus is reacted with the acid chloride of the desired alkanoic acid or alkenic acid in a mixture of pyridine and water. In this method, excess acid chloride in a non-reactive organic solvent (such as acetone) is slowly added to the core solution in a mixture of 90% pyridine and 10% water (v / v). The unreacted acid chloride is separated from the reaction product by extraction into a water-immiscible organic solvent (e.g. diethyl ether). The final purification is preferably carried out by high pressure liquid chromatography as described above.

The alkanoic and alkenoic acids used as starting materials for the acylations and their activated derivatives (especially acid chlorides α 2,4,54ггс111о ^ епу1е81егу) are known compounds or can be prepared from known compounds by known methods. The 2,4,5-trichlorophenyl esters are preferably prepared by reacting an alkanoic or alkenoic acid chloride with 2,4,5-trichlorophenol in the presence of pyridine or by reacting a free alkanoic or alkenic acid with 2,4,5-trichlorophenol in the presence of N, .beta.-dicyclohexylcarbodiimide as coupling agent.

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

When the A-21978C cyclic peptide of the invention is used as an antibacterial agent, it can be administered either orally or parenterally. As is known in the art, the A-21978C compound is conveniently administered together with a pharmaceutically acceptable carrier or diluent. The doses of the A-21878C compound depend on various factors, such as the type and severity of the infection being treated. It will also be appreciated by those skilled in the art that the appropriate dosage range and / or dosage units can be administered with respect to MCT and ED50 values and toxicities data, and with factors such as pharmacokinelics, patient or host characteristics and the microorganism causing the infection.

The following examples are given to illustrate the invention in full, but none of them are disclosed. ^! way.

Preparation 1

Preparation of A-21978C core

A. Fermentation of Actinoplanes utahensis

A stock culture of Actinoplanes utahensis NRRL 12052 is prepared and maintained on sloping agar. The medium used for the preparation of the inclined culture is selected from the following media :.

Medium A

Ingredient Amounts of pre-cooked oat flour 60.0g yeast 2.5g

K2HPO4 1.0g

Czapek's mineral solution * 5.0g agar 25.0g deionized water to 1 liter pH before autoclave sterilization is about 5.9, adjusted to 11a pH 7.2, addition of NaOH, after autoclave sterilization the pH is about 6.7.

* Czapek's mineral solution has the following composition:

Component Quantity

FeSOi. H2O (dissolved in 2 ml conc. HClj 2 g KC1 100 g MgSOd. 7 H2O 100 g deionized water up to .1 liter Medium В Component Amount potato dextrin 5.0 g yeast extract 0.5 g enzymatic hydrolyzate from casein * 3,0 g beef extract 0.5 g glucose 12,5 g cornstarch 5.0 g peptone from meat 5.0 g final molasses 2.5 g MgSOi. 7 H2O 0.25 g СаСОз 1.0 g Czapek's mineral solution 2.0 ml agar 20,0 g deionized water up to 1 liter 'N-Z-amine by A. Humko Sheffield Chemical, Lyndhurst, N.J. Inclined culture is inoculated Actinoplanes utahensis NRRL 12052 and inoculated culture Incubate at 30 ° C for about 8 to 15 ° C 10 days. About half of the grown culture is used for vaccination with 50 ml of vegetative medium · Composition: Component Amount pre-cooked oat flour 20,0 g sucrose 20,0 g yeast 2.5 g distilled granules residues * 5.0 g K2I-IPO3 1.0 g Czapek's mineral solution 5.0 ml deionized water up to 1 liter

The pH is adjusted with NaOH to pH 7.4, after sterilization in an autoclave, the pH is about 6.8.

* National Destillers Products Co., 99 Park Ave., New York, Ν. Y.

The inoculated vegetative medium is incubated in a 250 ml wide-necked Erlenmeyer flask at 30 ° C for about 72 hours on a rotary shaker with a 5 cm shift at 250 rpm.

This incubated vegetative medium can be used directly to inoculate the second stage vegetative medium. Alternatively and preferably, it may be stored for further use by keeping the culture in the liquid nitrogen gas phase. The culture is prepared for such storage in a plurality of small vials as follows.

2 ml of incubated vegetative medium and 2 ml of glycerollactose solution are placed in each vial (see WA Dailey and S. E. Higgens, "Preservation and Storage of MH Croorganism in the Gas Phase of Liquid Nitrogen, Cryobiol 10, 364-367 (1973)"). ). The prepared suspensions are stored in the gaseous phase of liquid nitrogen.

The stored suspension (1 ml) is used to inoculate 50 ml of the first stage of the vegetative medium (composition above). This medium is incubated as described above.

To prepare a larger seed volume, 10 ml of the incubated vegetative medium from the first stage were used to inoculate 400 ml of the second stage vegetative medium of the same composition as the vegetative medium of the first stage. The second stage medium was incubated in a 2 L wide-necked Erlenmeyer flask at 30 ° C for about 48 hours on a rotary shaker with a 5 cm shift at 250 rpm.

The inoculated second stage vegetative medium (80 ml) prepared as described above was used to inoculate 10 liters of sterile production medium selected from the following media:

Medium I

Component Amount (g / 1) peanut flour soluble peptone from meat sucrose KH2PO4 K2HPO4 MgSCh.7 H2O drinking water 10.0 5.0 20.0 0.5 1,2 Add 0,25 to 1 liter The pH of the medium after sterilization in an autoclave at 121 ° C and 0.11 to 0.12 MPa for 45 minutes is about 6.9. Medium II Component Quantity (g / l) sucrose peptone K2HPO4 KC1 MgSCH. 7 H2O FeSO1.7 H2O deionized water 30.0 5.0 1.0 0.5 0.5 Add 0,002 to 1 liter

the pH is adjusted to 7.0 by addition of HCl and after sterilization in an autoclave the pH is about 7.0.

5 7 7 6 N

Medium III

Component Amount (g / 1) glucose 20.0 NH4Cl 3.0 NazSCM 2,0 ZnC12 0.019 MgCl 2. 6 H2O 0,304 FeC13.6 H2O 0,062 MnC12.4 H2O 0,035 CuC12.2 H2O 0.005 СаСОз 6.0 KH2PO4 * 0.67 drinking water top up to 1 liter

Sterilization is performed separately and added aseptically to a final pH of about 6.6.

The inoculated production medium is ferunent in a 14 liter fermentation tank at a temperature of about 30-2 for about 66 hours. The fermentation medium is agitated with a conventional stirrer at about 600 rpm and aerated with sterile air to maintain dissolved oxygen at a boiling level above 30% air saturation at atmospheric pressure.

B. Deacylation A-21978C

Fermentation of A. utahensis is carried out as described in paragraph A using the sloping medium A and the production medium I and incubating the production medium for about 67 hours. Crude A-21978C complex (100 g) was added to the fermentation broth.

Deacylation of the A-21978C complex is monitored by a Micrococcus luteus assay. Fermentation is carried out until the deacylation, which is indicated by the disappearance of activity on M. luteus, is complete for about 24 hours.

C. Insulation of A-21978C Core

The entire fermentation medium (20 liters) obtained as described in section V is filtered using filter material (Hyflo Super-Cel, Johns Manville Corp.). The mycelium filter cake is discarded. The filtrate thus obtained was passed through a column containing 1.5 liters of HP-20 resin (DIAION High Porous Polymer, HP-Series, Mitsubishi Chemical Industries Limited, Tokyo, Japan). The eluate obtained in this manner is discarded.

The column was then washed with deionized water (10 L) and the residual medium was removed. This water is also poured out. The column was then eluted with water-acetonitrile (10 L of 95: 5, 9: 1 and 4: 1) and the 1 L fractions collected.

Elution is monitored by paper chromatography using the n-butanol: pyridine: acetic acid: water solvent system (15: 10: 3: 12) and detection of compounds by UV fluorescence. In this system they have

A-21978C factors Rf value about 0 _? 56 a

The A-21978C core has an Rf value of about 0.32. The product was also monitored by analytical high pressure liquid chromatography using Cib silica gel and a water: methanol mixture (3: 1) containing 0.1% ammonium acetate using UV detection at 254 mm.

The core-containing fractions were pooled, concentrated in vacuo to remove acetonitrile and freeze-dried to give 40.6 g of semi-pure A-21978C core.

D. Cleaning the A-21978C Core

The semi-pure A-21978C core (15 g) obtained as described in paragraph C was dissolved in 75 mL of water: methanol: acetonitrile (82: 10: 8) containing 0.2% acetic acid and 0.8% pyridine. This solution was pumped onto a 4.7 X 192 cm column containing 3.33 liters of silica gel (Quantum LP-1 / Cie). The column is developed with the same solvent system.

Collect 350 ml fractions. The separation is monitored at 280 nm and a UV detector. The core-containing fractions were combined, concentrated in vacuo and freeze-dried to yield 5.2 g of pure A-21978C core.

E. Characteristics of A-21978C core

The A-2197BC core has the following characteristics:

(a) Form:

white amorphous solid fluorescing in the shortwave UV region

(b) Empirical formula:

C62H83N17O25

(c) Molecular weight:

465

(d) Solubility: soluble in water

(e) The infrared spectrum (KBr) shows absorption maxima at the following lengths [om ^-1 ]:

300 (wide)

042 (weak),

909 (weak),

655 (strong)

530 (strong),

1451 (weak),

399 (Wednesday),

222 (medium),

165 (weak),

063 (weak) a

758 (medium to weak).

(f) The UV absorption spectrum in methanol shows maximums at:

223 nm (ε 41.482) a

260 nm (ε 8.687).

g) Electrometric titration in 66% aqueous dimethylformamide indicated the presence of four titrating groups pK _and values of about 5.2, 6.7, 8.5 and 11.1 (initial pH 6.12).

2577В6

Preparation 2

Alternative preparation of A-21978C core

The A-21978C core was prepared according to the method of Example 1, except for certain changes in paragraph B. The culture of A. utahensis was first incubated for about 48 hours, the substrate was semi-purified A-21978C colmplex (50 g) and incubation time after substrate addition was about 16 hours. The filtrate was passed through a column containing 3.1 liters of 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 was monitored as described in Preparation 1. After collecting 24 liters, the eluted solvent was changed to a 9: 1 mixture of water and acetonitrile. Fractions containing the core are eluted with this solvent. These fractions were combined, concentrated in vacuo to remove acetonitrile, and freeze-dried to give 24.3 g of semi-pure A-21978C core.

The semi-purified A-21978C core (24.3 g) was dissolved in water (400 mL). The solution is pumped into a stainless steel column (4.7 X 192 cm) containing 3.33 liters of silica gel (Quantum LP-1 / С1) prepared in a water-methanol-acetonitrile (8: 1: 1) mixture containing 0.2% acetic acid and 0.8% pyridine. Develop the column with the same solvent at a pressure of about 14 MPa. Fractions containing 350 ml were collected. The elution from the monitor is UV radiation at 280 nm. The fractions containing the core were combined, concentrated in vacuo and the solvent removed. Freeze-drying gave 14 g of a highly pure A-21978C core.

P rin g 3

Preparation of N, -, - t-BOC A-21978C Factors C2 and S

A mixture of A-21978C Factors C2 and C8 (10 g), prepared according to the method of US Patent No. 4,208,403, is dissolved in water (50 ml) with sonication (200 mg / ml). The pH of the solution was adjusted from 4.05 to 9.5 by addition of 5N NaOH solution (3.6 mL). Di-tert-butyl dicarbonate (3.0 µl) was added and the reaction mixture was stirred at room temperature for 2 hours. The pH of the reaction mixture was maintained at 9.5 (6.5 ml added over 2 hours) by addition of 5N NaOI-I solution.

The reaction was monitored periodically by thin layer chromatography on silica gel using CH 3 CN: H 2 O (7: 3 and 8: 2) and detection by ultraviolet light.

After about 10 minutes, the reaction solution became cloudy and the consumption of the base increased. After 30 minutes, the rate increased, which translates into turbidity and rate of base consumption, indicating that the reaction is complete. Nevertheless, the reaction is continued for a further 90 minutes to ensure complete progress. At the end of the two-hour reaction, the reaction material was immediately lyophilized to give 12.7 g of t-BOC A-21978 factors C2 and S.

Using similar procedures, two 10g reactions and 30g reactions were performed. For each of these reactions, the reaction time was only 40 minutes. Two 10g experiments yielded 11.9 and 12.1 g of product. Reaction of 30 g gave 35.4 g of product.

Preparation 4

Preparation of A-21978C Norn-t-BOC Core

A. Fermentation of A. utahensis

Fermentation of A. utahensis was carried out as described in Preparation 1, Section A using Inclined Medium A and Production Medium I. Incubation of the production medium was about 66 hours.

B. Deacylation of the Norn-t-BOC complex

Nom-t-BOC complex (1185 g of crude substrate containing about 176 g of A-21978C complex) is added to the fermentation medium. The deacylation is carried out as described in Preparation 1, paragraph B. The deacylation is complete by HPLC after about 24 hours.

C. Isolation of A-21978C Nom-t-BOC core

The fermentation medium (100 L) obtained by the procedure of paragraph V is filtered using filter clay (Hyflo Super-Cel). The filtrate was passed through a column containing 7.5 L of HP-20 resin (DIAION), and the column was washed with water (38 L). The elution was monitored by high pressure liquid chromatography on silica gel Cie detected by ultraviolet radiation at 254 nm. A portion of the core elutes upon washing. Subsequent elution of the core is performed with a mixture of water and acetonitrile (95: 5), 40 liters, (9: 1) 40 liters and (85: 15) 100 liters. The core-containing fractions were combined, concentrated in vacuo to remove the solvent, freeze-dried to give 298.5 g of semi-pure A-21978'CN _Oin- t-BOC core.

D. Cleaning A-21978C N _O1 nt-B0C Core

Semi-clean A-21978C N ₀ ,. _{The n-} t-BOC core, (30 g) obtained according to Part C, was dissolved in a mixture of water and acetonitrile (9: 1) containing 0.2% acetic acid and 0.8% pyridine (75 mL). This solution is applied to a stainless steel column 4.7 x 192 cm containing 3.33 L of Quantum LP-1 / Cis silica gel equilibrated in the same solvent system. The column is developed under pressure with a mixture of water, acetonitrile and methanol (80: 15: 5) containing 0.2% acetic acid and 0.8% pyridine. 350 ml fractions were collected and the product was detected by UV light at 280 nm. Product containing fractions were combined, concentrated in vacuo to remove solvent, and freeze-dried to give 18.4 g of purified A-21978C Nom-t-BOC core.

The A-21978C t-BOC core has the following characteristics:

(a) Form:

white amorphous solid fluorescing in the shortwave UV region

(b) Empirical formula:

C7SH91N97O27

(c) Molecular weight:

1565

(d) Solubility: soluble in water

(e) The infrared absorption spectrum (KBr) shall show absorption maxima at the following lengths (cm ^-1 ):

345 (wide),

065 (weak),

975 (weak),

936 (weak), ~ 1,710 (inflex),

660 (strong)

530 (strong),

452 (weak),

395 (medium)

368 (weak),

341 (weak),

250 (medium)

228 (medium),

1166 (medium to weak) a

063 (weak).

(f) The UV absorption spectrum in 90% ethanol / ethanol shows maximums at:

220 nm (s 42,000) a

260 nm (ε 10,600)

g) High pressure liquid chromatography retention time = 6 min on a 4.6 X 330 mm silica gel C18 column using: H 2 O / CH 3 CN / CH 3 OH (80: 15: 5) solvents containing 0.2% NH 4 EtOAc at 2 mL / min and UV detection .

5

Alternative purification A-21978C N _O111 -t-BOC core

The semi-purified A-21978C N _Om -t-B0C core (10.8 g) prepared according to Preparation 4, paragraph C was dissolved in water and applied to a column containing 80 ml of Amberlite IRA-68 (in an acetate cycle). The column was washed with water and eluted at 5 ml / ml with 0.05 N acetic acid (1080 ml), 0.1 N acetic acid (840 ml) and 0.2 N acetic acid (3120 ml) and collected with 120 ml fractions.

The eluate was monitored by analytical high pressure liquid chromatography on silica gel / Cie using water-acetonitrile-methanol (80: 15: 5) containing 0.2% ammonium acetate and detected by ultraviolet radiation at 254 nm. The product containing fractions were combined, adjusted to pH 5.8 with pyridine, and concentrated under vacuum to about 200 mL. Water was added to the concentrate and the solution was concentrated again and pyridine was removed. This concentrate was freeze-dried to give 3.46 g of pure A-21978C N _0.11- t-BOC core.

Examples 1 to 16

The preparation of various alkanoyl and alkenoyl derivatives by acylation of compounds of formula III, representative preparation of compounds of formula I, is summarized in Table I below. The derivatives in Table I are prepared either by a modified Schotten-Bauman reaction using an acid chloride as an acylating agent (Method A) or by an active ester method using 2,4,5-trichlorophenyl ester as an acylating agent (Method B). General procedures for carrying out acylation reactions by Method A or Method V are given below.

Method A (Modified Schotten-Bauman reaction)

This method involves reacting the core of formula III with an alkanoic or alkenic acid chloride corresponding to the desired acyl side chain.

The compound of formula III (1.95 to 2.16 g, 1.33 to 1.47 mmol) or the compound of formula II (409 mg, 0.25 mmol) was dissolved in 200 mL of a 9: 1 mixture of pyridine and water. The acylating reagent (18-20 mmol excess of acyl chloride dissolved in 15 mL of acetone) was added dropwise over 1 to 3 hours and the reaction mixture was stirred at room temperature for a further 2 to 3 hours. The reaction mixture was evaporated from acetone by evaporation and the remaining aqueous phase was diluted with water to a volume of about 200 ml. The pH of this solution was adjusted to pH 3.0 to 3.5 with glacial acetic acid. This solution was washed eight times with equal volumes of diethyl ether and then lyophilized. .

The crude acylated derivative is purified by reverse phase high pressure liquid chromatography as follows:

A sample dissolved in water or in a mixture of eluting solvents (about 4 to 6.5 ml) is loaded onto a 74 x 1.6 cm stainless steel column packed with LPi / Cie support. The column is eluted with a mixture consisting of water, methanol, acetonitrile, pyridine and acetic acid. The eluate is performed at a pressure of about 10.3 to 13.8 MPa at a rate of about 10 to 12 ml / min using an LDC duplex pump (Mllton-Royj. The eluate is monitored by a UV detector, using an ISCO-UA-5 detector at 280 nm. .

The desired fractions were combined and evaporated in vacuo to dryness to give the desired alkanoyl derivative. The purified product was analyzed using reverse layer thin layer chromatography (Whatman KCie) and a mixture of water, methanol, acetonitrile, pyridine, acetic acid (45: 15: 40: 2: 2). The plates are observed for UV detection. The product is also analyzed by UV absorption (extinction coefficients at 220 nm and 260 nm) and amino acid analysis. Purity was determined by analytical high-pressure reverse phase liquid chromatography (Cis Microbondapack, Waters Co.) with a solvent mixture of water, methanol, acetonitrile, pyridine, acetic acid and UV monitoring at 280 nm.

Method В (active 2,4,5-trichlorophenyl ester)

Norn-t-BOC solution of 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 (946 mg, 0.58 mmol) and a 3.5 molar excess of trichlorophenylacyl active ester were dissolved in dimethylformamide (100 mL) and stirred at room temperature to 50 ° C for 6 to 20 hours. The reaction mixture was concentrated in vacuo. The oil obtained is triturated with 50 ml of a 1: 1 mixture of ether and toluene and washed with ether. Monoacylated and diacylated A-21978C core derivatives, acylated A-21978C1 and acylated t-BOC A-21978C core are purified as described in Method A.

The acylated t-BOC-A-21978C core was deblocked using a 50 mg / ml mixture of trifluoroacetic acid, anisole and triethylsilane (10: 1: 1) at a temperature of about -10 ° C to about 0 ° C for 3-5 minutes. The reaction mixture was concentrated in vacuo and the resulting oil was triturated with two 20 mL volumes of ether. The crude acylated product is purified by reverse-phase high-pressure liquid chromatography and analyzed as described in Method A.

The specific compounds in the examples are summarized in Tables I to X and are compounds of formula I.

(AND)

Preparation of N _Trp- monoacyl derivatives of A-21978C cyclic peptides

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Characteristics of N _Trp- monoacyl derivatives of A-21978C cyclic peptides

Slouče- RR ¹ R ² R f UV ^and HPLC analysis Nina no. Eluate _^B; . _max 220 nm 260 nm

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2S7766

Preparation of N _Tr p'N- _O- diacyl A-21978C derivatives

Example RR ¹ R ² Method Starting Acylation Reaction HPLC Product

No. preparation material reagent elution time ² (mg)

what 00 Ю r — 4 O 00 WHAT HIM CD l> WHAT CD WHAT ID what PEOPLE CN 00 CN T — 1 oo CN in

N ДО CL >> CL O OjS <D CB О д Д CJ LO CN О Λ n лч

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Characteristics of N _Trp , N _O m-diacyl A-21978C derivatives> У

ю (those three <rH

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Table V

Honors o

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2S7766

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Example 27

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

The following procedure illustrates the preparation of the compounds in a large active ester method.

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

A solution of decanoyl chloride (Pfaltz and Bauer, 5.6 ml) and 2,4,5-trichlorophenol (5.6 g) in diethyl ether (1 liter) and pyridine (120 ml) was stirred for 4 hours. The reaction mixture was filtered and dried in vacuo. The 2,4,5-trichlorophenyl n-decanoate is purified on a silica gel column (Woelm) using toluene as the eluent. Fractions were monitored by thin layer chromatography using short wave UV detection. Appropriate fractions were combined and dried under vacuum. 10.4 g of 2,4,5-trichlorophenyl n-decanoate are obtained.

B. Acylation of N _{i) m} -t-BOC-A-21978C nucleus A '2,4,5-trichlorophenyl n-dekanvátem

A solution of Norn-t-BOC A-21978C core (15.0 g) and 2,4,5-trichlorophenyl n-decanoate (15.0 g) in anhydrous diimethylformamide (500 mL) was stirred at room temperature under nitrogen for 25 h. . The mixture was then stirred at 60 ° C for 5 hours until the reaction was complete by thin layer chromatography. The reaction mixture was concentrated in vacuo to a volume of about 200 mL and stirred with 1.2 L of Et 2 O / toluene (5: 1). The product was filtered off, washed with ether, dried under vacuum to give 15.05 g of N _Trp - (n-kanoyl) -N- _Orn- t-BOC A-21978C nucleus, intermediate (Formula I; R = n-decanoyl, R1 ⁾ = H, R ² = t-BOC).

C. Purification of N _Trp - (n-decanoyl) _-Nolll -t-B0C A-21978C Core

The N _Ttp - (n-decanoyl) -N _Orn -t-BOC-A-21978C core is purified as follows: The crude product is dissolved in 50 ml of a mixture of solvents used for elution and purified by high pressure liquid chromatography using a Waters / Prep 500 system packed with C18 reverse phase silica gel adsorbent. The system was eluted with water, methanol, acetonitrile (50:15:35) containing 0.2% pyridine and 0.2% acetic acid. The fractions are monitored in UV light at 280 nm. Appropriate fractions were combined and dried under vacuum. 8.56 g of purified N _Trp - (n-decanoyl) -N _Ori t-BOC A 21978C nucleus were obtained.

D. Removal of the _N-RN t-B0C t-BOC group is removed by stirring the N _Trp - (n-decanoyl) alkohol-t-BOC-21978C nucleus (1.47 g) in 15 mL of trifluoroacetic acid and 1 Of 2-ethanedithiol (10: 1) for 3 hours at room temperature. The reaction mixture was dried in vacuo and the residue was triturated with ether (50 mL). After washing with 20 ml of ether, the product is dried under vacuum to give 2.59 g of crude N _T1 β- (n-decanoyl) -A-21978C core (Formula I: R = in-decanoyl; R ¹ and R ² = Н] .

E. Purification of N _Tip - (n-decanoyl) -A-21978C nucleus

Crude N _T rp- (n-decanoyl) -A-21978C nuclei

The crude N _Tlp - (n-decanoyl) -A-21978C core was purified by reverse phase high pressure liquid chromatography as follows: A sample (2.59 g) was dissolved in 4.0 mL of a mixture of water, methanol, acetonitrile, pyridine and acetic acid. (50: 15: 35:: 2: 2) was applied to a 74 XX 2.54 cm steel column packed with LP-1 / Cie adsorbent. The column is eluted with the same solvent system. Elution is carried out at a pressure of 8.3 to 11.8 MPa at a rate of 10 to 12 ml / min using an LDC duplex pump (Milton-Royl). The eluate was monitored by a UV detector (Isco Model UA-5, Instrument Specialist Co., 4700 Superior Avenue, Lincoln, NB 68504) at 280 nm. Fractions (20-24 ml) were collected every two minutes. The desired fractions by antimicrobial activity were combined and dried under vacuum to give 1.05 g of product.

This purification procedure is repeated with 4.35 g,

4.25 g, 2.14 g, 2.00 g, and 1.75 g of crude starting derivative to give a total of 5.58 g of pure N _Tip - (n-decanoyl) -A-21978C core.

Example 28

N _TrP - (n-decanoyl) -N _Kyl - (n-decanoyl) -A-21970C (compound 21)

N _Tr p- (n-decanoyl) -N _Ky n- (n-decanoyl) -A-21978C core (Formula I: R and R ¹ = n-decanoyl; R ² = H) is a minor reaction product in the preparation of N _Trp - (n-decanoyl) derivative according to Example 27. It is isolated during purification by reverse-phase high-pressure liquid chromatography as described in paragraph (E). The desired fractions were combined and dried in vacuo. 211 mg of crude product are obtained. The compound was purified by analytical high pressure liquid chromatography (C18 Microbondapak Waters Co.) using water, methanol, acetonitrile, ammonia and acetic acid [6: 23: 15: 0.5: 0.5] as the eluent (32 reps for 500 / zg). in each injected sample). 4.4 mg of N _Trp - (n-decanoyl) -NI; _? n- ( ⁿ 'decanoyl) -A-21978C nuclei, identified by a NMR spectrum measured at 360 MHz.

Example 29

N _T1P -Cbz-Noin-lauroyl A-21978C nuclei (compound 20)

This method involves protecting "groups -amino257766 tryptophan nrt _O N-BOC-A-21978C nucleus A Cbz group, and then removing the t-BOC group and acylating (at the ornithine alpha-amino) lauroyl-trichlorophenyl ester.

A. Preparation of N ₁ -p-Cbz-N ₁ O ₍₁ -t-BOC-A-21978C nucleus)

The N- _III- t-BOC-A-21978C core (1.0 g) was dissolved in dimethylformamide (150 mL) and heated to 60 ° C. N, O-bis-trimethylsilylacetamide (0.55 mL) was added and Benzyl ipentachlorophenylcarboinate (257 mg) was added, after stirring for 20 hours, the reaction mixture was concentrated to a volume of about 20-25 ml, water (100 ml) was added and the pH of the solution was adjusted to 6.0 with 1 N NaOH. (6 X 200 ml volume) and then lyophilized.

The crude derivative is purified by reverse-phase high performance liquid chromatography as follows:

The sample was dissolved in about 6 mL of water, methanol, acetonitrile, pyridine and acetic acid (55: 15: 30: 2: 2) and injected onto a 74 X 1.25 cm stainless steel column packed with LP-1 / C18 support. Desired fractions are monitored by UV absorption (280 nm) and antimicrobial activity. These fractions are then combined and lyophilized. 951 mg of N- _Trp -Cbz-Nom-t-BOC A-21978C core derivative are obtained.

B. Removal of the t-BOC group The t-BOC group was removed by dissolving the intermediate in a 50 mg / ml mixture of trifluoroacetic acid, anisole and triethylsilane (10: 1: 1) at -10 ^and C for 3 minutes. The reaction mixture was concentrated to an oil which was triturated with 12 X 25 ml ethyl ether. 520 mg of crude N ₍₁₁ -Cbz-A-21978C core) are obtained,

C. Acylation of _{N] IP-Cbz-A-21978C} nucleus

The N 7 _ip -Cbz-A-21978C core is acylated by the acylation-activated trichlorophenyl ester method. Add N _Tip -Cbz-A-21978C (520 mg) to a solution of 1-hydroxybenzotriazole (7 mg) and active lauroyltrichlorophenyl ester (123 mg) in pyridine (150 µl). After stirring at 60 ° C for 20 h, the reaction mixture was concentrated to a residue which was triturated with ethyl ether (3 x 25 mL volumes). The NTrp-Cbz- _Nnn lauroyl-A-21978C core (550 milligrams) was obtained.

Example 30

Preparation of Schiff bases and reduced Schiff bases

Some reactions were performed as follows: A-21978C core (40 mg) was dissolved in water (2 ml). The pH is adjusted to 4-9 by adding 1N NaOH. Aliquots (0.5 ml) of this solution are then mixed with each of the following aldehydes (5 μΐ):

1. Heptaldehyde

2. octylaldehyde

3. De-cylaldehyde

4. undecylaldehyde in methanol (4 mL). The reactions were stirred at room temperature overnight. The resulting Schiff bases were reduced with N-aBH 3 CN (2.5 mg per reaction) for 5 minutes at room temperature.

The reaction mixture was evaluated by thin layer silica gel chromatography using a 7: 3 mixture of acetonitrile and water and a Staphylococcus aureus assay. Schiff bases are not on

S. aureus active in this test due to their instability under test conditions. In each reduction reaction two factors are generated. These compounds are active on S. aureus and have similar mobility in the system used in thin-layer chromatography. It has been found that the following compounds of formula 1 are formed (in each case R ¹ is hydrogen).

Table Preparation of Schiff bases and reduced ones Schiff bases Reaction A-21978C p Product R R ² Activity> on S. aureus ⁹

heptaldehyde СНз [СН2) 5СН = Н пе СНз (СН2) б— Н апо СЫз (СН2) 5СН = СНз (СН2) 5СН = пе СНз [С ‰] б— СНз (СН2] б— апо octylaldehyde СНз (СН2 JgCH) н пе СНЗ (СН2) 7— н апо СНз [СН2) вСН = СНз (СН2) бСП = пе СНз (СН2) 7— СНз (СН2] 7— апо decylaldehyde СНз (СН2) 8СН = н пе СНз [СН2) 9— н апо СНз (СН2) 8СН = СНЗ (СН2) 8СН = пе СНз (СН2) 9— СНз (СН2) 9— апо undecylaldehyde СНз [СН2) 9СН = н пе СНз (СН2) ю— н апо СНз [СН2] 9СН = СНз (СН2) 9СН = пе СНз [СН2) ю— СНз (СН2] 10— апо

^and Schiff base inactivity in this assay may be due to instability under assay conditions.

Example 31

Preparation No. _lT -laurylaldehydových Schiff bases and N _Trp, N _O nrdllaurylaldehydových Schiff bases and their reduced Schiff's bases (compounds 22 and 23]

The A-21978C core (1 g) was dissolved in water (50 mL) and dodecylaldehyde [588 µl] in methanol (200 mL) was added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was then reduced for 50 minutes by addition of NaBH 3 CN (291 ing). The reaction mixture was filtered under vacuum using Whatman # 1 paper. The supernatant was concentrated, then the aqueous solution was lyophilized to give 1.256 g of product. This product is evaluated by analytical high pressure liquid chromatography and purified in portions by preparative reverse phase liquid chromatography.

Each portion (350 mg) was dissolved in 50% aqueous methanol (6 mL) and heated to dissolve the material. The solution was then poured onto an 80 cm X 1.5 cm column with LPi-C 18 support and eluted with a mixture of methanol, acetonitrile, water (25: 40: 35) containing 0.2% pyridine and 0.2% acetic acid at a rate 7.5 ml / min. Elution is monitored by a UV detector at 280 nm. The fractions containing the desired products were combined to give a total of 104 mg of N _T r _P - (n-dodecyl) -A-21978C core [compound 22] and 19.2 mg of N _Trp - (n-dodecyl) _-Noin - ( ⁿ (dececyl) -A-21978C core (compound 23).

Example 32

The antibacterial activity of the compounds of formula I can be demonstrated by in vitro assays. The results of antibacterial tests of representative compounds of formula I using standard diffusion assays on agar plates are shown in Table VII. In Table VII, activity is measured by the size of the zone (diameter in mm) in which the growth of the microorganism is inhibited by the test compound.

Antibacterial activity of compounds of formula I determined by an agar plate diffusion assay

hill; incubation: 24 to 48 hours at 25 to 37 ° C. Growth on minimal nutrient agar tr = traces

AND

The results of antibacterial tests of representative compounds of formula I by a standard agar screening test are summarized in Table VIII. The activity shown in Table VIII is measured as the minimum inhibitory concentration of the compound at which the microorganism is inhibited by the test compound.

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Antibiotic activity of A-21978C cyclic peptides см

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continuation of Table VIII

Test organism MIC values of test compounds ¹

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44

The A-21978С cyclic peptides of formula I exhibit in vivo antibacterial activity on two experimentally induced bacterial infections. When two doses of test compound are administered subcutaneously or orally to mice with induced infection, the activity obtained is measured as ED 50 values (effective dose in mg / kg that protects 50% of test animals, see Warren Wick et al. J. Bacteriol. 81 , 233-235 (1961)] The observed ED50 values of representative A-21978C compounds of Formula I are shown in Table IX.

- 5 7 7 6 6> ω rS * 3 o

Table IX

In vivo activity of A-21978C cyclic peptides

Compound - Compound of Formula IR ¹ R ² ED 50 of ³ nin # R Staphylococcus Streptococcus pyogenes> CD

O

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CO CM 00 C

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The results of the toxicity tests of some A-21978C cyclic peptides of formula I are summarized in Table X.

Table X

Toxicity of A-21978C cyclic peptides

Compound C. Compound of Formula I R R ¹ R ² LD 50 (mg / kg) in mice ³ 1 СНз (СН2) 5СО— н н > 600 2 СНз (СН2) бСО— н н > 600 3 СНЗ (СН2) 7СО— н н > 600 4 СНз (СН2) 8СО— н н > 300 5 СНз (СН2] 9СО— н н > 600 6 СНз (СН2) 10 СО— н н 144; 265 ^p 7 СНз (СН2) 11СО— н н 112.5 8 СНз (СН2) 12СО— н н 62.5; <150 9 СНЗ (СН2) 13СО— н н 56, 25 10 СН5 (СН2) 14СО— н н 50 11 СН2 = СН— (СН2) 8 СО— н н L- 500 12 С Нз (СН2) зСН = CH (СН2) 7СО- н н 450 14 СНзСНгСН (СНз и СН2) 6 С О— н СНзСО— > 600; 600 15 Dec СНзСНгСН (СНз] (СНг) бСО— н НОСО (СН2) 2СО— 450; 600 16 СНз (СН2) бСО— н СНз (СН2) бСО— > 600 17 СНз (СН2) 9СО— н СНз (СН2) 9СО— 94 22nd СНз (СН2) 11СО— н н '> 300

³ intravenous applications, ^b two trials

Claims (7)

I will invent the subject 1. A process for the preparation of a derivative A-21978C cyclic peptides of formula I wherein R ¹ , R ¹ and R ² are each independently hydrogen, C 2 -C 19 alkanoyl optionally substituted with carboxyl, C 5 -C 19 alconoyl or an amino protecting group selected from carbobeinzyloxy or tert-butoxycarbonyl, R ³ , R ⁴ and R ⁵ are hydrogen atoms, wherein (1) at least one of R, R ¹ or R ² must be other than hydrogen or an amino protecting group as defined above; 2. at least one of R ¹ or R ² must be a hydrogen atom or an amino protecting group as defined above, The third of R, R ¹ and R ² groups together must contain at least four carbon atoms and 4. when both R ¹ and R ² are hydrogen or an amino protecting group as defined above, R cannot be 8-methyldecanoyl, 10-methylundecanoyl, 10-methyldodecanoyl, a specific C 10 alkanoyl group A-21978C or a specific alkanoyl group having 12 carbon atoms of A-21978C factors C4 and C5 and pharmaceutically acceptable salts of these peptides, characterized in that the A-21978C cyclic peptide core of formula III Or a correspondingly protected derivative thereof, wherein R 1 and R 0 are independently hydrogen or an amino protecting group as defined above, or a corresponding pharmaceutically acceptable salt thereof, is reacted with an acylating agent of formula V ^Three R-COOH (V) or an activated derivative thereof, wherein R ⁶ is alkyl having 1 to 18 carbon atoms optionally substituted with carboxyl, or alkenyl having 4-18 carbon atoms, and optionally removing any protecting group that can be present in of the above reaction. 2. A process according to claim 1, wherein said starting compound is reacted to form a compound of formula (I) wherein R is alkanoyl of formula (I). C II CH 5 (CH 2) n - He is an integer from 3 to 17. 3. A process according to claim 1, wherein said starting compound is reacted to form a compound of formula (I) wherein R is an alkanoyl of formula (I). СНз O I ΊΙ СНз (CH 2) CH - (CH 2 j c - and n and m are independently integers from 0 to 14, wherein -n + m shall not be less than 1 and not more than 15, and furthermore if n is 0, m shall not be 8 and if n is 1, m shall not be 6 or 8. 4. A process according to claim 1, wherein said starting compound is reacted to form a compound of formula I wherein R is cis or trans-alkenoyl of formula O AND! СНз (СН2) “- CH - CH— (СНг) ·. — C— where n and m are independently integers from 0 to 14, where n 4- m must not be less than 1 and not more than 15. 5. The process of claim 1, wherein the above starting compound is reacted to form a compound of formula I wherein R is cis or trans-alkenoyl of formula O II CH 2 = CH (CH 2) n - c - wherein n is an integer from 12 to 16. 6. The process of claim 1, wherein the above starting compounds are reacted to form a compound of formula I wherein R is n-decanoyl. 7. A method according to claim 6, characterized by reacting the above starting compounds to give compounds of formula I wherein SK is n-decanoyl, and R ^1, R ^2, R ^3, R ⁴ and R ⁵ are hydrogen.