FI79118C - Process for the preparation of novel therapeutically useful cyclic A-21978C peptide derivatives - Google Patents

Description

1 79118

Process for the preparation of novel therapeutically useful cyclic A-21978C peptide derivatives

The present invention relates to a process for the preparation of novel therapeutically useful cyclic A-21978C peptide derivatives of the formula 1 ΪΝΗΙΐ1 l / \ / CH3 / \ / \

Hi U

/ \ / \ / \ .0 0 = <T * CONH, „“ "R '" "• O- ^ VSVy' ^ n> - <μ 1ΐ -, - Λ ·>> u yv o = A \ = oi 20 o V H_f \ K / k / VA / vv ^ s Y i 3 HOaC ^ 2, 25 wherein R is C 1 -C 4 alkanoyl, C 1 -C 4 alkenoyl or an amino protecting group; · R 2 is hydrogen or C 1 -C 4 alkanoyl, and R 2 is hydrogen, C 1 -C 4 alkanoyl # succinyl or an amino-protecting group, provided that 1 2 1) at least one of the groups R , R and R are other than hydrogen or an amino protecting group, 1 2 2) at least one of R and R is hydrogen or an amino protecting group, 1 2 3) R, R and R contain a total of at least four carbon atom and 2 79118 1 2 4) when both R and R are, either hydrogen or the amino-protecting group R is not 8-methyldecanoyl, 10-methyl-undecanoyl, the C 1 -C 4 alkanoic acid of factor A-21978C a yl group or a C 5 and C 5 specific 5 C 12 -AHcan ° yl group, and for the preparation of their pharmaceutically acceptable salts These new derivatives of cyclic peptides have antibiotic properties. properties.

There is a great need to develop new antibiotics due to the great potential and constant threat of developing strains of pathogenic microorganisms resistant to certain antibiotics. In particular, pathogenic species of the Gram-positive genera Staphylococcus and Streptococcus are often resistant to commonly used antibiotics such as penicillin and erythromycin; see, e.g., W.O. Roye, Principles of Medicinal Chemistry 1974, pp. 684 - 686.

The novel cyclic A-21978C-peptide-20 di-derivatives of formula (1) and their pharmaceutically acceptable salts have been found to be effective antibiotics.

These novel peptide derivatives of formula (1) can be prepared by reacting an A-21978C core peptide prepared by deacylating an appropriately protected A-21978C combination or one of its suitably protected individual factors Cq, C 1, C 2, Cq , C4 or Ccj, to react with the desired acid used as a cyclizing reagent or an activated derivative thereof and, if desired, by removing any protecting groups present and / or converting the product obtained into a salt.

The cyclic A-21978C peptide derivatives of formula (1) and their pharmaceutically acceptable salts have been found to be useful semisynthetic antibacterial agents or intermediates leading to such agents.

3 79118 The following abbreviations are used in this specification, most of which are well known in the art: Ala: alanine Asp: aspartic acid 5 Gly: glycine

Kyn: kynurenine Orn: ornithine Ser: serine Thr: threonine 10 Trp: tryptophan t-BOC: t-butoxycarbonyl Cbz: benzyloxycarbonyl DMF: dimethylformamide THF: tetrahydrofuran 15 HPLC: high performance liquid chromatography NMR: 1H nuclear magnetic resonance NMR: 1H nuclear magnetic The A-21978C antibiotics are closely related to the acidic peptide antibiotics described in U.S. Patent No. 4,208,403. As described in U.S. Patent No. 4,208,403, the A-21978C antibiotic combination contains a major component, factor C, which itself is a combination of closely related factors. A-21978C factor C, called the A-21978C combination, comprises the individual factors Cq, C2, 0-2 'C3 »C4 and C8. The factors C 1, C 2 and C 8 are the main factors, and the factors Cq, and C 8 are secondary factors. The structure of A-21978C factors is shown in Formula 2.

ί 79118 L-Asp 'ϋ /

Gly 5 D ~ A, a 4 / f O-Ser L-Asp 4 ^

t 3MG

L-Orn 4 / \ L-Kyn 10 Gly q ^ \ t L-Thr t L-Asp t L-Asn 15 f L-Trp

NH

20 1

OF

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

OF

specific fatty acid moiety. The specific R groups of the factors are as follows: Factor A-21978C RN group 8 8-methyldecanoyl C2 10-methylundecanoyl C. 10-methyldodecanoyl and the like CQ-alkanoyl C2-2-alkanoyl 35 C,. C 1-6 alkanoyl * b 1 £ not further specified 5 79118

When confronted with the problem of deacylation of a peptide antibiotic, it is very difficult to know whether there is an enzyme that can be used for that purpose. Finding such an enzyme is even more difficult when the substrate antibiotic contains a cyclic peptide backbone. Because the enzymes are very specific, differences in the peptide moiety and the side chain of the substrate antibiotic affect the success of the deacylation attempt. In addition, many microorganisms produce several peptidases that affect different sites in the peptide moiety. This often leads to product mixtures that are difficult to handle.

The enzyme used to remove the acyl group from the? 2-amino group of the tryptophan fatty acid side chain can be produced by a microorganism belonging to the family Actinoplanaceae, preferably Actinoplanes utahensis NRRL 12052, or a variant thereof. To remove the acyl group, an antibiotic comprising the combination of A-21978C, the CQ, C2 / C4 of the A-21978C factors, and the C1, C2, C3, C4 and Cg of the protected A21978C factors and the protected A21978C factors. group is added to the culture of the microorganism. The terms "protected A-21978C factors" and "protected combination A-21978" refer to individual A-21978C factors or mixtures (combinations) of factors in which the amino group of organitin and / or kynurein is protected by an amino group protecting group. Preferably, the amino protecting group is located on the <? Amino group of the single factor or mixture of factors. Culture 30 is allowed to incubate with the substrate until deacylation is approximately complete. The corresponding A-21978 cyclic peptide thus obtained is separated from the fermentation broth by methods known in the art.

The 35 cyclic peptides obtained by such enzymatic deacylations and their salts are shown in formula 3, 6 79118 I NHR '\ cn, λ a 5 Ί Ye A if \ / / ΑαΛλ · ° = · \ TY / conh2> H 6 A ϊ <ff / h2 ί »h <? / A h ^ \ a '\ Α / γ * γ 7 \> A? 4 hh — I t V / H c ° 2H w

0 = / \ = 0 H

15 / —7 * iH3 0 ¥ »Λ Ϊ„ hos / A ΑΛ A / w ~ * i! μ H0a (/ 3 20 where R 'and R 0 are independently hydrogen or an amino protecting group.

By removing the acyl group from the A-21978C combination 25 or from the individual A-21978C factors CQ, C 1-4, C 1-4, C 1-4 and C 1-4. a compound of formula 3 is obtained wherein both R 'and R 0 are hydrogen. The deacylated molecule is a common cyclic peptide present in each antibiotic factor A-21978C. For simplicity, this compound is hereinafter referred to as the A-21978C backbone. This compound can also be represented by the formula 4, 79958 L-Asp /

Gl y 5 D-Ala T D-Ser L-Asp j,

t 3MG

L-Orn I

\ L-Kyn 10 r. /

Gl y o \ </ L-Thr t L-Asp t 15 L'Jsn L-Trp NHs 4 20 where 3MG is L-threo-3-methylglutamic acid.

Compounds of formula 3 in which R "or R 0 is other than hydrogen are prepared by deacylation of the appropriately protected antibiotic A-21978C factors CQ, 25 Clt C2, c3, and C1-4. For simplicity, these compounds are referred to herein as protected A-21978C. These protected compounds are useful intermediates in the preparation of certain peptides of formula 1, i.e., compounds in which at least one of R 2 and R 2 is an amino protecting group.

As will be apparent to those skilled in the art, the A-21978C backbone and the protected A-21978C backbones can be obtained as either free bases or acid addition salts. Although any suitable acid addition salt may be used, pharmaceutically acceptable salts are preferred. "Pharmaceutically acceptable salts" are salts that can be used in chemotherapy of isothermal animals.

A method for producing the A-21978C backbone 5 of formula 3 using various microorganisms is fully disclosed in our co-pending patent application (831,748). A preferred acylation enzyme, A. utahensis NRRL 12052, is available

Agricultural Research Culture Collection 10 (NRRL), Northern Regional Research Center, U.S. Pat.

Department of Agriculture, 1815 Northern University St., Peoria, Illinois 61604, USA, Accession No. NRRL 12052. Preparation Example 1 of this application illustrates the preparation of the A-21978C backbone by fermentation using A-21978C as a substrate and Actinoplanes utahensis NRRL 12052 as a microorganism. a.

Other cultures of Actinoplanaceae that can be used to prepare A-21978C strains of formula 3 are available from the Northern Regional Research Laboratory under the following accession numbers:

Actinoplanes utahensis NRRL 12052

Actinoplanes missouriensis NRRL 12053

Actinoplanes sp. NRRL 8122

Actinoplanes sp. NRRL 12065 25 Streptosporangium roseum var. hollandensis NRRL 12064

The efficiency of each specific strain of a microorganism of the family Actinoplanaceae in deacylation is determined using the following procedure. A suitable culture medium is inoculated with the microorganism. The culture is incubated at about 28 ° C for two or three days using a rotary shaker. A substrate antibiotic is then added to the culture, keeping the pH of the fermentation medium at approximately 6.5. Culture activities are monitored using the Micrococcus luteus assay. The decrease in antibiotic activity is an indication that 9 79118 microorganisms produce the enzyme required for deacylation. However, this must be confirmed by one of the following methods: 1) HPLC analysis for the detection of unchanged bodies; or 2) re-acylation with a suitable side chain (e.g. lauryl, n-decanoyl or n-dedecanoyl) to restore activity.

The process of the invention for the preparation of cyclic A-21978C peptide derivatives of formula (1) is characterized in that the cyclic A-21978C core peptide of formula (3) wherein R 'and R 0 are independently hydrogen or an amino protecting group is treated at a temperature of about 10 to 60 ° C for 1 to 24 hours with an acid of formula 5 R 6 -COOH (5) g or an activated derivative thereof, wherein R is C 8 -C 5 alkyl or C 1 -C 5 alkenyl; and optionally removing any protecting groups that may be present in the product and / or converting the resulting product to a salt, if desired.

The terms "C 1 -C 4, C 1 -C 10 alkanoyl" and "C 1 -C 4 alkanoyl" refer to 7-16, 1-10, 25 1-13, respectively. and acyl groups derived from carboxylic acids having 11 to 17 carbon atoms. When the R group is alkanoyl, the alkyl moiety is a saturated, straight-chain or branched hydrocarbon monoradical. When R is alkenoyl, the alkenyl moiety is an unsaturated, straight-chain or branched carbon-30 hydrogen monoradical containing up to three double bonds. The double bond (s) configuration of the unsaturated hydrocarbon chain may be the cis or trans configuration.

The term "amino protecting group" means known amino protecting groups that are compatible with other functional groups in the A-21978C molecule. The amino protecting groups are preferably those that can be easily removed from the acylated compound when protected. Examples of suitable protecting groups can be found in Protective Croups in Organic Synthesis-5, Theodora W. Greene, John Wiley and Sons, New York, 1981, Chapter 7. Particularly preferred amino protecting groups are t-butoxycarbonyl and benzyloxycarbonyl. .

In particular, the invention includes the following 10 preferred compounds of formula 1: a) Compounds wherein R is of the formula CH 2 Cl 2 -di-

alkanoyl and n is an integer from 5 to 14; CH0 O

1 3 il b) Compounds in which R is alkanoyl of the formula CHO (CH-) CH (CH-J -C- j z n z m and n and m independently of one another denote an integer from 0 to 12, provided that

n + m is not less than 3 and not more than 12 and that when n is 0 m is not 8 and when n is 1 m is neither 6 nor 8; O

c) Compounds in which R is cis- or trans-alkenoyl of the formula CHO (CH ~) CH = CH (CH ~) -C- 3 zn 2 m and n and m independently represent an integer from 1 to 13, provided that n + m is at least 7 3a at most 13;

O

d) Compounds wherein R is cis- or trans-alkenoyl of the formula CH2 = CH (CH2) - <ϋ- and n is an integer from 8 to 14; 25 e 'Compounds in which R is:

O

CH3 (ch2} 5 ~ C-0 ch3 (ch2) 6-c-

30 O

ch3 (ch2)? -C-O ch3 (ch2) 8-c- 0 35 ch3 (ch2) 9-c- o CH3 (CH2) 10-C- 11 79118

O

CH2 = CH- (CH2) gc-O CH3 (CH2) 11-C- 5 2 ch3 (ch2) 12-c- o CH3- (CH2) 3-CH = CH- (CH2)? - c-0 10 ch3 (ch2) 13-c- o CH3CH2CH (CH3) - (CH2) 6-C-

The compounds of formula 1 are capable of forming ground salts which are also part of this invention. Such salts are useful, for example, in the separation and purification of compounds. Particularly useful are pharmaceutically acceptable alkali metal, alkaline earth metal, amine and acid addition salts.

For example, the compounds of formula 1 have four free carboxyl groups which can form salts. Therefore, partial salts, mixed salts and complete salts formed by these carboxyl groups are part of this invention. PH values higher than 10 should be avoided when preparing these salts because the compounds are unstable in such pH ranges.

Typical and suitable alkali metal and alkaline earth metal salts of the compounds of formula 1 include sodium, potassium, lithium, cesium, rubidium, barium, calcium and magnesium salts. Suitable amine salts of the compounds of formula 1 include ammonium and primary, secondary and tertiary alkylammonium and hydroxy-C 2-6 alkylammonium salts. Examples of amine salts are those formed by the reaction of a compound of formula 1 with ammonium hydroxide, methylamine, s-butylamine, isopropylamine, diethylamine, diisopropylamine, cyclohexylamine, ethanolamine, triethylamine or 3-amino-amine. with propanol.

The alkali metal and alkaline earth metal cation salts of the compounds of formula 1 are prepared by methods commonly used in the preparation of cationic salts. For example, the compound of formula 1 is dissolved as the free acid in a suitable solvent such as warm methanol or ethanol, and then a solution containing a stoichiometric amount of the desired inorganic base in the water-methanol mixture is added. The salt thus formed can be isolated by conventional methods such as filtration and evaporation of the solvent. A suitable method for preparing salts is the use of ion exchange resins.

Salts formed with organic amines can be prepared in the same manner. For example, a gaseous or liquid amine may be added to a solution of a compound of formula 1 in a suitable solvent such as ethanol; the solvent and excess amine can be removed by brewing.

The compounds of this invention may also contain free amino groups and therefore form acid addition salts which are also part of this invention.

Typical and suitable acid addition salts of the compounds of formula 1 are those formed by conventional reaction with both organic and inorganic acids, such as, for example, sulfuric, sulfuric, phosphoric, acetic, succinic, lemon, lactic, maleic -, fumaric, palmitic, school, Pamoinin, mucus, D-glutamine, d-camphor, glutaric, glycol, phthalic, wine, lauric, stearic, salicylic, with methanesulfonic, benzenesulfonic, sorbic, picric, benzoic or cinnamic acid.

13 791 1 8

Compounds of formula 1 wherein R, R 1 or 2 R is an alkanoyl or alkenoyl group or an amino protecting group are prepared by acylation of a compound of formula 3 with the desired alkanoyl or alkenoyl side chain using conventional methods for amide bond formation. The acylation is generally accomplished by reacting the compound to be acylated with an activated derivative of a time or alkenoic acid corresponding to the desired acyl-1 2 chain (R, R or R). The term "activated derivative" means a derivative that renders the functional carboxyl group of the acylating agent sufficiently reactive to join the primary amino group to form an amide bond connecting the acyl linkage to the backbone. Suitable activated derivatives, methods for their preparation and methods for their use as acylation reagents for primary amines are known to those skilled in the art. Preferred activated derivatives are: a) an acid halide (e.g. an acid oride), 20 b) an acid anhydride (e.g. a Ioxoxic anhydride) or c) an activated ester (e.g. 2,4,5-trichlorophenyl ester). Other methods for activating a carboxyl functional group include reacting a carboxylic acid with a carbonyldiimide (e.g., N, N'-dicyclohexylcarbodiimide or N, N'-diisopropylcarbodiimide) to provide a reactive intermediate that, due to its instability, is not isolated. In such a case, the reaction with the primary amine is performed in situ.

Those skilled in the art will recognize that the compounds of formula 1 are prepared using selective acylation methods as well as with the aid of amino protecting groups.

i4 791 1 8

For example, when the starting material is a compound of formula 3 wherein R 1 and R 0 are hydrogen, both the tryptophan amino group and the ornithine β-amino group can be acylated to give β iprp, NQrn-diacyl derivatives.

To obtain monoacylated derivatives in which the o (-amino group) of tryptophan is acylated, it is preferable to acylate a compound of formula 3 in which the ε-amino group (R 0 position) of ornithine is protected by an amino-protecting group. Such starting materials are preferably obtained by protecting 10 A-21978C The aromatic amino group of kynurenine is the least reactive of the three free amino groups in the A-21978C backbone.Acylation in kynurenine requires appropriate protection of the amino groups of tryptophan and ornithine, but R or R acylation usually does not involve protection of the amino group of kynurenine.

Schemes I, II and III outline general procedures for the preparation of compounds of formula 1 wherein one of R, R and R is alkanoyl, alkenoyl or an amino protecting group. The following symbols are used in these diagrams: 1 * 7 = remainder of A-21978C Ν, ρ = c-amino group of tryptophan 25 Nq = S-amino group of ornithine N = aromatic amino group of kynurenine K 1 2 R, R, R = reported substituents Rn = natural factor acyl group B, B "* · = amino protecting groups 30 Acyl = acylation step

Deacyl = deacylation step

Block = acylation using an amino protecting group Deblock = removal of an amino protecting group

In Scheme I, General Formula 3 shows 35 N, β-monoacyl derivatives of A-21978C and General Formula 4 shows 79118 of A-21978C.

NTrp, NOrn "diacyl: derivatives · Those NTrp, NOrn" diacyls · women in which the N -acyl group is naturally occurring A-21978C

ir acyl group, shows general formula 8.

i6 791 1 8 ω d

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tn Ή% s ^ £ f * X o 0) - X χ

G h- X

<D XX

-P \ / «ΪΓ, • H Μ · | <d § / \ (0 _ 'Ö>

Xl u 0 <n 03

Γ N - * X

1 x υ o

n X O N O X X N

v-> XXX - XX

00 h- T ^ ^ h - x

I "X X X X

Ch \ I / / Q \ / ι-t ί-1 col - 5Γ

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Pj U - / H <CD / E-ι y 'Z N / XX / .. X O n

"XXX

H h- X

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O \ A <H | Ή <- *> * id 1— 'id 17 791 1 8

In Scheme II, the general formula 10 shows the ^ Trp / ^ Kyndias ^^^ i ^ derivatives of A-21978C. Those ΝΤΓρ'ΝκΥη ~ άΐ33 ^ 1: ί derivatives in which the NTrp acyl group is an acyl group of the natural factor A-21978C are shown in the general formula 12.

Compounds of general formulas 5, 6 and 7 are also present in Scheme I.

is 79118 «x o X r- cc a: x x

H- X

\ / = T oi iH | / \ υ o

_Q

Q)

Q

m

3 X ^ cTcC

Γ1 XXX

-t-1 H I M

s \ I ^

-H ^ CM

rd> a <υ>

^ O

(0

C CD

rj X

£ X O w

rS, N XXX

^ X I— 1 x: x o- x x

0 xxx \ I

71 t- ^ Γ

zv 7 = * - M

u \ /

00 IX "CVI I

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CT \ / x

rH

7 / \> f £ 1 u

H O

r— {CO

> 1 - ° X

s? - • H ^ = \ I / = X) CD -, I X X * - -K.

V- · x o x> -. o 1 I M x * „*>

pp. 1-1 U

i, ra, Η, r oi

Eh l \ o X \ \ s

M U * X2 X ° M

<X X

O h- X

ίο ϋ i9 791 1 8

In Scheme III, general formula 18 shows N-monoacyl derivatives of A-21978C, general formula 15 NKyn monoacyl derivatives of A-21978C and general formula 20 ISL ·, N-diacyl derivatives of A-21978C. The compounds represented by the general formula 6 also appear in Schemes I and II.

20 7911 8 m

•B

rt

G

G

rt «'S x - w -g N O CC cc £ ^« xo «' 2 \ / 2 XH" f "Se 0 Γ OI X \ / = CO - cm | x Γ-. x f ".. * oo! 5 OJ - * · / \ 1 υ JL o - * <C - υ

1 -O O

h rt -

M Q -Q

sL a>

O

> t N

m cc rt «- x * - n

H CD O CC CC

rrt XXX «- X

? (- yc CD O N

x Z XXX

> 1 X - '^ ^ Xw i * *. / \ / M o a 2 ~ E il M ^ ml S / N = ^ 1 / = ö sl e il 2 υ rt </ v * - X ^ S 8 £ 2 \ I / z 1 ®

S r 51 Q

0

S rt / \ CD

f · + Ι υ "" cd Χο cc U O tf Χν C ® ~ z z> 1 φ (D \ /

CO CD .M

.-3 - ^ 1

CD _ ΉI

1 - X _ / 1 1 m o «-v. / 1 • H X Z X q * / .h i- z y /> 1 \ _ / / rt T / rt - / o G / \ nl O rM |

f · U

G - Μ ω

O

S w -5 g H en Cl O ci H h 3: z x H e z T - O rt x Λ κ> · # co |

rt G rt rt i = C 4J

2i 79118

A preferred process for the preparation of compounds of formula 1 wherein one of R, R and R is alkanoyl or alkenoyl is a process using an activated ester. A compound of formula 3 wherein R '= H 5 and R 0 = t-BOC, i.e. The A-21978C-NQi_n-t-BOC backbone, or "t-BOC backbone", is a particularly preferred starting material for the preparation of compounds of formula 1. The 2,4,5-trichlorophenyl ester of the desired time or alkenoic acid is the preferred acylating agent. In this method, an excess of the active ester is reacted with the t-BOC backbone at room temperature in an inert organic solvent such as DMF, THF, diethyl ether or dichloromethane. The reaction time is not critical, although the preferred time is about 6 to 20 hours. At the end of the reaction, the solvent is removed and the residue is purified. A particularly useful purification method is the reverse phase HPLC stationary phase LP-1/18 and the solvent system H 2 O-CH 2 OH-CH 2 CH-pyridine-HOAc. The t-BOC group is removed by treatment with a mixture of trifluoroacetic acid-anisole triethylsilane or, preferably, trifluoroacetic acid-1,2-ethanedithiol for about 3-5 minutes at room temperature. After removal of the solvent, the residue is purified by reverse phase HPLC.

An alternative method of acylation is the modified Schotten-Baumann method, in which the unprotected backbone is treated with an acid chloride or alkenoic acid in a pyridine-water mixture. In this method, excess acid chloride in an inert organic solvent (such as acetone) is slowly added to a solution having a backbone in a mixture containing 90% by volume of pyridine and 10% by volume of water. The unreacted acid chloride is separated from the reaction product by extraction with a solvent (e.g. diethyl ether) immiscible with the reaction product. Final purification is performed by reverse phase HPLC as described above.

22 79118

The alkanoic and alkenic acids used as starting materials for the acylation reaction and their activated derivatives (especially acid chlorides and 2,4,5-trichlorophenyl ester) are known compounds or can be prepared from known compounds by known methods. The 2,4,5-trichlorophenyl ester is readily prepared by treating the time or alkenoic acid chloride with 2,4,5-trichlorophenol in the presence of pyridine or by treating the free time or alkenoic acid with 2,4,5-trichlorophenol N, N '~ 10 dicyclohexylcarbodiimide in the presence of a coupling reagent. 2,4,5-Trichlorophenyl ester derivatives can be purified by column chromatography.

The antibacterial activity of the compounds of formula 1 can be demonstrated in vitro. The results of studying the antibacterial activity of typical compounds of formula 1 using a conventional inhibition zone (a disk dipped in a suspended compound is placed in a Petri dish on an agar surface to which the compound then diffuses) are shown in Table I. In Table I, activity 20 is determined by its observed zone size ) in which the growth of microorganisms is inhibited by the test compound.

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The cyclic A-21978C peptides of formula 1 have been shown to have antimicrobial activity against experimental bacterial infections in vivo. When administered subcutaneously or orally to mice in exemplary infections, the effect observed with two doses of test compound was determined as the ED 2 value / effective dose in mg / kg sufficient to protect 50% of the experimental animals: see Warren Wick et al., J. Bacteriol, 31 (1961) 233 - 235 /. Typical ED 1 values of A-21978C of formula 1 are shown in Table III.

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The results of toxicity tests of some of the cyclic A-21978C peptides of formula 1 are summarized in Table IV.

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When the cyclic A-21978C peptide of this invention is used as an antibacterial agent, it can be administered either orally or parenterally. As will be appreciated by those skilled in the art, A-2] 978C is usually administered in association with a pharmaceutically acceptable carrier or diluent. The dosage of A-21978C will depend on a variety of factors, such as the nature and severity of the infection being treated. Those skilled in the art will recognize that dosage ranges or dosage units are suitable for administration. can be determined by considering the MIC and ED, .q values and available toxicity data, as well as factors such as pharmacokinetics, patient or host characteristics, and the microorganism that caused the infection.

The following examples are provided to further illustrate the invention.

Preparation Example 1 Preparation of A-21978C Body A. Fermentation of Actinoplanes utahensis 20 A stock culture of Actinoplanes utahensis NRRL 12052 is prepared and maintained on an agar slant. The medium used to make the sloping surface is one of the following:

Medium A

25 Ingredient Quantity

Pre-cooked oatmeal 60.0 g

Yeast 2.5 g K2HPO4 1.0 g

Czapek mineral source * 5.0 ml 30 tc n

Agar 25.0 g

Make up to 1 liter with deionized water

Prior to autoclaving, the pH is about 5.9; The pH is adjusted to 7.2 by adding NaOH; after autoclaving, the pH is about 6.7.

* The composition of the Czapek mineral spring is as follows: 33 791 1 8

Ingredient Quantity

FeSC> 4.7H2O (dissolved in 2 none 2 g concentrated HCl) KCl 100 g 5 MgSO4.7H2O 100 g

Make up to 1 liter with deionized water

Medium B

Ingredient Amount 10 Potato dextrin 5.0 g

Yeast extract 0.5 g

Casein enzymatic hydrolyzate * 3.0 g

Meat extract 0.5 g 15 Glucose 12.5 g

Corn starch 5.0 g

Meat peptone 5.0 g

Sugar cane molasses 2.5 g

MgSO4.7H2O 0.25 g CaCO3 1.0 g

Czapek mineral source 2.0 ml

Agar 20.0 g

Deionized water is made up to 1 liter with t-N-Z-Amine A, Humko Sheffield Chemical, Lyndhurst, NJ.

25

The slant is inoculated with Actinoplanes utahensis NRRL 12052, and the inoculated slope is incubated at 30 ° C for about 8-10 days. About half of the oblique culture is used to inoculate the culture medium (50 ml) having the following composition: 30 Ingredient Amount

Pre-cooked oatmeal 20.0 g

Sucrose 20.0 g

Yeast 2.5 g

Distillers Dried Grain * 5.0 g 35 K2HPO4 1.0 g

Czapek mineral source 5.0 ml

Make up to 1 liter with deionised water 34 7 9 1 1 8 The pH is adjusted to 7.4 with NaOH; after autoclaving, the pH is about 6.8.

* National Distillers Products Co., 99 Park Ave., New York, NY.

The inoculated culture medium is incubated in a large 250 ml Erlenmeyer flask at 30 ° C for about 72 hours using a shaker rotating at about 250 rpm with a diameter of about two inches.

This incubated culture medium can be used directly to inoculate the second stage culture medium. Alternatively and preferably, it can be stored for later use by maintaining the culture in the vapor phase formed by liquid nitrogen. For such storage, the culture is prepared in a large number of small flasks as follows. Place 2 ml of incubated culture medium and 2 ml of glycerol-lactose solution in each flask / see W.A. Dailey and C.E. Higgens, "Preservation and Storage of Microorganisms in the Gas 20 Phase of Liquid Nitrogen", Gryobiol 10 (1973) 364-367 for details. The prepared suspensions are stored in the vapor phase formed by liquid nitrogen.

The stored suspension (1 ml) thus prepared is used to inoculate the first stage culture medium (50 ml; composition described above). The inoculated first stage culture medium is incubated as described above.

To provide a larger amount of inoculum, 10 ml of incubated first stage culture medium is used to inoculate the second stage culture medium in an amount of 400 ml and the same composition as the first stage culture medium. The second stage culture medium is incubated in a 2 L wide mouth Erlenmeyer flask at 30 ° C for about 48 hours using a 35-inch diameter, two-turn arc, rotating shaker at 250 rpm.

35 79 1 1 8

The second stage culture medium (80 ml) prepared as described above, incubated, is used to inoculate a sterile production medium (10 L) which is one of the following:

5 Medium I

Ingredient Amount (g / 1)

Peanut flour 10.0

Soluble meat peptone 5.0

Sucrose 20.0 10 KH2PO4 0.5 K2HPO4 1.2

MgSO 4 '7H 2 O 0.25

Make up to 1 liter with tap water. The pH of the medium is about 6.9 at 121 ° C and after autoclaving for 45 minutes at a pressure of about 1.1 to 1.2 atm.

Medium II

Ingredient Amount (g / 1)

Sucrose 30.0 20 Peptone 5.0 k2hpo4 1.0 KCl 0.5

MgSO4.7H2O 0.5

FeSO..7H ~ 0 0.002 4 2 25 Make up to 1 liter with deionized water, adjust the pH to 7.0 with HCl; after autoclaving, the pH is about 7.0.

Medium III

30 Ingredient Amount (g / 1)

Glucose 20.0 NH4Cl 3.0

Na 2 SO 4 2.0

ZnCl2 0.019 35 MgCl2.6H2O 0.304 36 79 1 1 8 Medium III continued

Ingredient Amount (g / 1)

FeCl3.6H2O 0.062 5 MnCl2.4H2O 0.035

CuCl 2 .2H 2 O 0.005

CaCO 3 6.0 KH 2 PO 4 * 0.67

Tap water to 1 liter 10 * Sterilized separately and added aseptically.

Final pH about 6.6.

The incubated production medium is fermented in a 14 L fermentation vessel at a temperature of about 30 ° C for about 66 hours. The fermentation medium is agitated with conventional agitators at a speed of about 600 rpm and aerated with sterile air to keep the dissolved oxygen concentration greater than 30% of the air saturation value at normal atmospheric pressure.

B. Deacylation of A-21978 Fermentation of A. utahensis is performed as described in Part A using oblique medium A and production medium I and incubating the production medium for about 67 hours. The crude A-21978C combination (100 g) prepared as described in U.S. Patent 4,208,403, Figure 25, is added to the fermentation medium.

Deacylation of A-21978C is monitored by the Micrococcus luteus assay. The fermentation is allowed to continue until deacylation is complete, as evidenced by the disappearance of the effect against M. luteus, i.e. about 24 hours.

C. A-21978C Chassis Insulation

The entire fermentation broth (20 L) obtained as described in Part B was filtered using a filter aid (Hyflo Super-Cel, Johns Manville Corp.) 37 79118

The mycelial cake was discarded. The filtrate thus obtained was passed through a column containing 1.5 L of HP-20 resin (DIAION High Porous Polymer, HP-Series, Mitsubishi Chemical Industries Limited, Tokyo, Japan). The efflu-5 ent thus obtained was discarded. The column was then washed with deionized water (10 L) to remove residual filtered broth. The wash water was discarded. The column was then eluted with water-acetonitrile mixtures (10 L each was used and the ratio was 95: 5, 9: 1 and 4: 1) and 1 L fractions were collected.

Elution was monitored by paper chromatography using n-butanol-pyridine-acetic acid-water (15: 10: 3: 12) as the solvent system and UV fluorescence was used to detect the compounds. With this solvent system-15, the R-value of the A-21978C factors is about 0.56 and the R-value of the A-21978C backbone is about 0.32. The product was also examined by analytical HPLC using silica gel / CH 2 Cl 2 and a water-methanol mixture (3: 1) containing 0.1% ammonium acetate as the solvent system; the body was detected by UV light at a wavelength of 254 nm.

Fractions containing the body were combined, concentrated in vacuo to remove acetonitrile and lyophilized to give 40.6 g of a partially purified A-21978C body.

25 D. Cleaning the A-21978C Chassis

The partially purified A-21978C body (15 g) obtained as described in Part C was dissolved in 75 ml of a water-methanol-acetonitrile mixture (82: 10: 8) containing 0.2% acetic acid and 0.8% pyridine. . This solution was pum-30ed onto a 4.7 x 192 cm column containing 3.33 L of silica gel / C-g (Quantum LP-1, Quantum Industries, 341 Kaplan Drive, Fairfield, NJ 07006). At the same time, the column was developed as a solvent system. Fractions with a volume of 350 ml were collected. Separation was monitored by UV light at 35 nm at 280 nm. Fractions containing the body were combined, concentrated in vacuo to remove solvents and lyophilized to give 5.2 g of purified A-21978C body.

E. Characteristics of the A-21978C Body 5 The A-21978C body has the following characteristics: a) Shape: a white amorphous substance that fluoresces under shortwave UV light.

b) Empirical formula: cg2H83N17 ° 25 c) Molecular weight: 1465 d) Solubility: soluble in water e) Absorption peaks appear in the infrared absorption spectrum (KBr) at the following frequencies (cm: 3300 (broad), 3042 (weak), 1655 (strong) ), 15 1530 (strong), 1451 (weak), 1399 (moderate), 1222 (moderate), 1165 (weak), 1063 (weak), and 758 (between moderate and weak).

f) The UV absorption spectrum in methanol shows peaks at 223 nm (6 * 41 482) and 260 (¢ 8 687).

20 g) Potentiometric titration in 66% aqueous dimethylformamide shows the presence of four titratable groups with pK values of about 5.2, 6.7, 8.5 and cl 11.1 (initial pH 6, 12)

Preparation Example 2 Alternative Preparation of A-21978C Body The A-21978C body was prepared by the method of Preparation Example 1 except for some changes in a portion of the BA utahensis culture, initially incubated for about 48 hours, partially substrated with A-21978C combination (50 g), and incubation after substrate addition lasted about 16 hours. The broth filtrate was passed through a column containing 3.1 L of HP-20 resin. The column was washed with 10 volumes of water and then eluted with aqueous acetonitrile (95: 5). Elu-35 was monitored as in Preparation Example 1.

39 79118

After collecting 24 liters, the elution solvent was changed to water-acetonitrile (9: 1). Fractions containing the body were eluted with this solvent. These fractions were combined, concentrated in vacuo to remove acetonitrile and corner dried to give 24.3 g of a partially purified A-21978C backbone.

This partially purified A-21978C body (24.3 g) was dissolved in water (400 ml). The solution was pumped onto a 4.7 x 192 cm steel column containing 3.33 L of silica gel 10 (Quantum LP-1 / Cl 2) and prepared in a water-methanol-acetonitrile mixture (8: 1: 1) containing 0, 2% acetic acid and 0.8% pyridine. The column was developed with the same solvent at a pressure of about 140 atm and 350 ml fractions were collected. Elution was monitored at UV wavelength of 15,280 nm. The fractions containing the body were combined, concentrated under reduced pressure to remove solvents and lyophilized to give 14 g of a very pure A-21978C body.

Preparation Example 3

Nom-t-BOC-A-21978C Factors C2 and Preparation A mixture of A-21978C Factors C2 and 10 prepared as described in U.S. Patent 4,208,403 (10 g) was dissolved in water (50 mL) using sonication (200 mg). / mL). The pH of the solution was adjusted from 4.05 to 9.5 with 5N NaOH (3.6 mL). Di-t-butyl dicarbonate (3.0 ml) 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 by manual addition of 5N NaOH (6.5 mL over 2 hours).

The reaction was monitored periodically by TLC using silica gel plates and using CH 2 CN-H 2 O mixtures (7: 3 and 8: 2) as solvent systems and UV light for detection.

After about 10 minutes, the reaction solution suddenly became cloudy and base consumption increased. After 30 minutes, the rate of increase in turbidity and the rate of wear of the base decreased, indicating that the reaction was complete. Nevertheless, the reaction was allowed to proceed for another 90 minutes at 40 ° C to 79118 to ensure completion. At the end of the two-hour reaction, the reaction material was immediately freeze-dried to give 12.7 g of NO2n-t-BoC-A-21978 factors C2 and · 5 Using similar procedures, two reactions using 10 g of starting material and a reaction using 30 g of starting material were performed. . Experiments with 10 g gave 11.9 g and 12.1 g of product, respectively. The reaction with 30 g gave 35.4 g of product. j-0 Preparation Example 4 Preparation of A-21978C-NQrn-t-BOC backbone Fermentation of AA utahensis A. Fermentation of utahensis was performed as described in Preparation Example 1, Part A, using oblique surface medium A and production medium I and incubating. production medium for about 66 hours.

B. Deacylation of NQrn-t-BOC Combination A-21978C-NQrn-t-BOC combination (1185 g of crude substrate containing about 176 g of A-21978C combination) was added to the fermentation medium. The deacylation was performed as described in Preparation Example 1, Part B. Deacylation was complete by HPLC after about 24 hours.

C. Isolation of A-21978C-NO-tn-BOC Body The fermentation broth (100 L) obtained as described in Part B was filtered using a filtration aid (Hyflo Super-cel). The filtrate was passed through a 7.5 L column of HP-20 resin (DIAION), and the column was washed with water (38 L). Elution was monitored by silica gel / CH 2 HPLC 30 and using UV light at 254 nm for detection. Part of the body eluted in the washing solution. Subsequent body elution was performed using the following water-acetonitrile mixtures: (9: 5) - 40 L; (9: 1) - 40 1? and (35:15) - 100 1.

The fractions containing the body were combined, concentrated under reduced pressure to remove the solvent and lyophilized to give 298.5 g of partially purified A-21978C-N 2 O 3.

Orn t-BOC frame.

D. Purification of the A-21978C-NO-t-BOC Body The partially purified A-21978C-NO-t-BOC body (30 g) obtained as described in Part C was dissolved in a water-acetonitrile mixture (9: 1) which contained 0.2% acetic acid and 0.8% pyridine in a volume of 75 ml.

This solution was applied to a 4.7 x 192 cm steel column containing 3.33 L of silica gel (Quantum LP-1 / C1Q) equilibrated with the same solvent system. The column was developed under pressure with a water-acetone-trichloro-methanol mixture (80: 15: 5) containing 0.2% acetic acid and 0.8% pyridine, collected in 350 ml and 15 boils and UV light at 280 wavelength was used for detection. nm. The product-containing fractions were combined, concentrated under reduced pressure to remove the solvent, and lyophilized to give 18.4 g of purified A-21978C-1SL · -t-BOC backbone.

The characteristic data of the Orn 20 A-21978C-t-BOC body are as follows: a) Form: White amorphous substance which fluoresces under shortwave UV light.

b) Empirical formula N-, 0 ~ c 67 91 I7 27 25 c) Molecular weight: 1565 d) Solubility: soluble in water e) Absorption peaks appear in the infrared absorption spectrum (KBr) at the following frequencies (cm-2): 3345 (broad), 3065 (weak), 2975 (weak), 2936 30 (weak), ca. 1710 (shoulder), 1660 (strong), 1530 (strong, 1452 (weak), 1395 (moderate), 1368 (weak), 1341 (weak) ), 1250 (moderate), 1228 (moderate), 1166 (between moderate and poor) and 1063 (weak).

42 791 1 8 f) The UV absorption spectrum in 99% ethanol shows peaks at 220 nm (£ 42 000) and 260 nm (6 10 600).

g) HPLC retention time = 6 minutes using a 4.6 x 300 mm silica-5 gel / Cj, g column and using I 2 O-CH 2 CN-CH 2 OH as solvent (80: 15: 5) , containing 0.2% NH 4 OAc at a flow rate of 2 ml / min and using UV light for detection.

Preparation Example 5 10 Alternative Cleaning of A-21978C-NO-t-BOC Body

The partially purified A-21978C-NO-t-BOC body (10.8 g) obtained as described in Preparation Example 4, Part C, was dissolved in water and applied to a column containing 80 ml of Amberlite IRA-68 (Rohm and Haas, Phila- 15 Delphia, PA, acetate cycle). The column was washed with water and eluted sequentially at a flow rate of 5 ml / min with 0.05 N acetic acid (1080 ml), 0.1 N acetic acid (840 ml) and 0.2N acetic acid (3120 ml) and 120 ml fractions were collected. The column was monitored by analytical silica gel / 20 ° C HPLC using a water-acetonitrile-methanol mixture (85: 15: 5) containing 0.2% ammonium acetate as the solvent system and detecting UV light at 254 nm. The product-containing fractions were combined, the pH of the solution was adjusted to 5.8 with pyridine, and the resulting solution was concentrated under reduced pressure to a volume of about 200 ml. Water was added to the concentrated solution, and the resulting solution was reconcentrated to remove pyridine. This concentrate was lyophilized to give 3.46 g of purified A-21978C-NQrn-t-BOC backbone.

30 Examples 1-16

The preparation of various alkanoyl and alkenoyl derivatives by acylation of compounds of formula 3, which is a typical procedure for the preparation of formula 1, is shown in Table V below. The derivatives in Table V are prepared by either a modified Schotten-Bauman reaction using acid chloride as the acylation reagent or by ester method 43 791 18 using 2,4,5-trichlorophenyl ester as the acylating reagent (method B). General procedures for carrying out the acylation reactions by Method A or Method B are as follows: Method A (Modified Schotten-Bauman Reaction) This method involves the reaction of a backbone of formula 3 with time or alkenoic acid chloride corresponding to the desired acyl side chain.

The compound of formula 3 (1.95 to 2.16 g, 1.33 to 10 1.47 mmol) or the compound of formula 2 (409 mg, 0.25 mmol) is dissolved in 200 mL of an aqueous pyridine mixture (9: 1 ). The acylation reagent (18-20 mmol of excess acyl chloride dissolved in 15 ml of acetone) is added dropwise over 1-3 hours, and the reaction mixture is stirred at ambient temperature for an additional 2-3 hours. The reaction mixture is concentrated to remove acetone. The remaining aqueous layer is diluted with water to about 200 ml. The pH of this solution is adjusted to 3.0 to 3.5 with glacial acetic acid. This solution is washed 8 times with an equal volume of di-20 ethyl ether and then lyophilized.

The crude acylated derivative is purified by reverse phase HPLC as follows: A sample dissolved in water or eluent system (ca. 4-6.5 mL) is sprayed onto a 84 x 1.6 cm stainless steel column 25 filled with LP-1 / C ^ g adsorbent. The column is eluted with H 2 O-MeOH-CH 2 CN-pyridine-HOAc solvent system. Elution is performed at a pressure of about 100 to 140 atm and a flow rate of about 10 to 12 ml / min using an LDC duplex pump (Milton-Roy). The effluent is monitored under UV light using an ISCO-UA-5 detector at 280 nm. The desired fractions are combined and evaporated to dryness under reduced pressure to give the desired alkanoyl derivative. The purified product is analyzed by TLC using reversed phase plates (Whatman KCl 2) and a solvent system of H 2 -MeOH-CH 2 CN-pyridine HOAc (45: 15: 40: 2: 2).

44 791 1 8

The plates are viewed under UV light to detect the product. The products are also analyzed by UV (extinction coefficients at 220 nm and 260 nm) and subjected to amino acid analysis. Purity is determined by analytical reverse phase HPLC (C 1-4 Microbondapak, Waters Co.) using a 1β-MeOH-CH 2 CN-pyridine-HOAc solvent system and monitoring the eluent under UV light at 280 nm.

Method B (method of active 2,4,5-trichlorophenyl ester) A solution of NO 2n-t-BOC-A-21978C backbone (1.0 g, 0.64 mmol), A-21978C backbone (0 , 5 to 1.0 g, 0.34 to 0.68 mmol) or A-21978C (946 mg, 0.58 mmol), and the active trichlorophenyl acyl ester in an excess of 3.5 moles is dissolved in DMF ( 100 ml), and the solution is stirred at a temperature between room temperature and about 50 ° C for about 6-20 hours. The reaction mixture is concentrated to an oil under reduced pressure. The oil is triturated using 50 mL of Et 2 O-toluene (1: 1) and washed with Et 2 O. The monoacylated and diacylated A-21978C backbone derivative, the acylated A-20 21978C and the acylated t-BOC-A-21978c backbone are purified as described in Method A.

The acylated t-BOC-A-21978C backbone is protected using a 50 mg / ml trifluoroacetic acid-anisole-triethylsilane mixture (10: 1: 1) at a temperature of about -10 to 0 ° C and a treatment time of 3-5 minutes. This reaction mixture is concentrated under reduced pressure to an oil which is triturated using two 20 ml portions of diethyl ether. The crude acylated product is purified by reverse phase HPLC and analyzed as described in Method A.

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Example 27 NTrp - (n-decan ° yy1: i-) -A-21978C backbone (Compound 4)

The following series of reactions illustrates the large-scale preparation of compounds by the 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 in a mixture of diethyl ether (1 L) and pyridine (120 ml) is stirred for 4 hours. The reaction mixture is filtered and dried under reduced pressure. 2,4,5-Trichlorophenyl n-decanoate is purified on a silica gel column (Woelm) using toluene as eluent. The distribution is monitored by TLC using shortwave UV radiation for detection. The appropriate fractions are combined and evaporated to dryness under reduced pressure to give 10.4 g of 2,4,5-trichlorophenyl n-decanoate.

B. Acylation of NO-t-BOC-A-21978C backbone with 2,4,5-trichlorophenyl n-decanoate

A solution of N--t-BOC-A-21978C backbone (15.9 g) and 2,4,5-trichlorophenyl n-decanoate (15.0 g) in anhydrous DMF was stirred for 25 hours at ambient temperature using nitrogen as a shielding gas. The mixture is then stirred at 60 ° C for 5 hours or until TLC shows that the reaction is complete. The reaction mixture is concentrated under reduced pressure to about 200 ml and stirred in 1.2 liters of Et 2 O-toluene mixture (5: 1). The product is filtered off, washed with diethyl ether and evaporated to dryness under reduced pressure to give 15.05 g of N-prp (n-decanoyl) -Nn -t-BOC-A-21978C backbone (kaa urn 2 va 1; R = n- decanoyl, R = H, R = t-BOC).

30 C. Purification of the NTrp- (n-decanoyl) -NORn-t-BOC-A-21978C backbone The intermediate NTrp “(nc * ecanoyl) -NQrn-t-BOC-A-21978C backbone is purified as follows: Impure the product is dissolved in about 50 ml of the solvent system used in the elution and then purified by HPLC using a Waters Prep / 500 system including a cartridge filled with reverse phase C-silica gel adsorbent.

b2 79118

The system is eluted with H 2 O-MeOH-CH 3 CN (50:15:35) containing 0.2% pyridine and 0.2% HOAc. The fractions are monitored at 280 nm under UV light. The appropriate fractions are combined and evaporated to dryness under reduced pressure to give 8.56 g of pure - (n-decanoyl) -ΝφΓη-t-BOC-A-21978c body.

D. Removal of the NQrn-t-BOC Group The t-BOC group is removed by stirring the NTrp- (n-decanoyl) -NOrn-t-BOC-A-21978C backbone (1.47 g) in 15 ml of trifluoroacetic acid-1. 2-Ethanedithiol mixture (10: 1) at ambient temperature for 3 minutes. The reaction mixture is evaporated to dryness under reduced pressure and the residue is triturated using Et 2 O (50 ml). After washing (20 ml of Et 2 O) the triturate is evaporated to dryness under reduced pressure to give 2.59 g of crude N - (n-decanoyl) -A-21978C -

Trp ^ 2 backbone (Formula 1; R = n-decanoyl, R and R = H).

E. Purification of N - (n-decanoyl) -A-21978C backbone —_- * P - _ __

The crude NTrp- (n-decanoyl) -A-21978C backbone is purified by reverse phase HPlC as follows: to 4.0 mL of H 2 -MeOH-CH 2 CN-pyridine-HOAc (50:15:35 : 2: 2) a dissolved sample (2.59 g) is sprayed onto a 84 x 2.5 cm stainless steel column packed with LP-1 / C 2 g adsorbent. The column is eluted using the same solvent system. Eluation is performed at a pressure of 80 to 115 atm and a flow rate of 10 to 12 ml / min using an LDC duplex pump (Milton-Roy). The effluent is monitored with a UV detector (Isco Model UA-5, Instrument Specialist Co., 4700 Superior Avenue, Lincoln, NB 68504) using a wavelength of 30,280 nm. Fractions (20-24 ml) are collected at two minute intervals. The desired fractions, as determined by antimicrobial activity, are combined and evaporated to dryness under reduced pressure to give 1.05 g of product.

This purification procedure was repeated for 4.35 g, 35 4.25 g, 2.14 g, 2.00 g and 1.75 g of crude starting material to give a total of 5.58 g of purified starting material. NTrp- (n-decanoyl) -A-21978C backbone.

53 791 1 8

Example 28 NTrp - (n-decanoyl) -NC? - (n-decanoyl) -A-219 78C (Compound 21) N '- (n-decanoyl) -N' - (n-decanoyl) -A-21978C-iirp 2 The backbone 5 (Formula 1: R and R = n-decanoyl, R = H) is a reaction by-product in the preparation of the NTrp (trans-decanoyl) derivative of Example 27. It is isolated during the reverse phase HPLC purification described in Part E. The desired fractions were combined and evaporated to dryness under reduced pressure to give 211 mg of crude product.

The compound is purified by analytical HPLC (CH 2 Micro-Bondapak, Waters Co.) using H 2 O-MeOH-CH 3 CN-NH 4 OH-HOAc (6:23:15: 0.5: 0.5) as the elution system. times, each injected sample 500 μg) to give 4.4 g of NTrp “(n-decanoyl) -NKyn- (n-decanoyl) -A-21978C backbone, which is identified by 360 MHz proton NMR .

Example 29 NTrp "Cbz-Norn" - * - aurYyH-A-21978C backbone (Compound 20) This method comprises protecting the tryptophanamino group of the NQrn-t-BOC-A-21978C backbone with a Cbz group, followed by Removal of the -BOC group and acylation of the (β-amino group of ornithine) with trichlorophenyl lauryl ester.

25 A. Preparation of NTrp-Cbz-NO-n-t-BOC-A-21978 backbone

Nom-1: '- B® <- “' ^ - 21978C body (1.0 g) is dissolved in DMF (150 ml) and the solution is heated to 60 ° C. Add N, O-bis-trimethylsilylacetamide (0.55 mL) followed by benzyl pentachlorophenyl carbonate (257 mg). After stirring for 20 hours, the reaction mixture is concentrated to a volume of about 20-25 ml. Add water (100 mL) and adjust the pH of the solution to 6.0 with 1N NaOH. The mixture is washed with ET 2 O (six 200 mL portions) and then lyophilized.

35 The crude derivative is purified by reverse phase HPLC as follows: Into 6 ml of a mixture of H 2 O-MeOH-CH 3 CN-54 791 1 8 pyridine-HOAc (55: 15: 30: 2: 2 · the dissolved sample is injected into a stainless steel, 84 x on a 1.3 cm column packed with LP-1 / Cl 2 adsorbent The desired fractions were detected by UV absorption (280 nm) and antimicrobial activity, then combined and lyophilized to give 951 mg Nm -Cbz-N 2 -t-BOC-A-21978C backbone.

Trp Orn B. Removal of the t-BOC group The t-BOC group is removed by dissolving intermediate 10 in 50 mg / ml trifluoroacetic acid-anisole-triethylsilane (10: 1: 1) at -10 ° C and a dissolution time of 3 minutes. The reaction mixture is concentrated to an oil which is triturated using two 25 mL portions of Et 2 O to give 520 mg of crude NTrp-Cbz-A-21978C backbone.

C. Acylation of NTrp-Cbz-Ap21978C backbone The N, rrp-Cbz-A-21978C backbone is acylated using an acylation method based on active trichlorophenyl ester. NTrp? Ckz-A-21978C (520 mg) is added to a solution of 1-hydroxybenzotriazole (7 mg) and active lauryl trichlorophenyl ester (123 mg) in pyridine (150 ml). After stirring for 20 hours at 60 ° C, the reaction mixture is concentrated and the resulting residue is triturated with Et 2 O (three 25 mL portions) to give 550 mg of NTrp-Cbz-NO-lauryl-A -21978c -run-25 koa.

Claims (7)

55 791 1 8 A process for the preparation of novel therapeutically useful cyclic A-21978C peptide derivatives of formula 1 | p1 / \ / Hs / \ / \ “ϊΥ, I! 10 A Ϊ S / / \ · · ^ '"' γ? Conh9 h Y 'Yaa,"> h) “rn) V. / YV ο = γ \ = oi · —-t · CHa H H- <* 1 Ya * HOaC (1) 25 wherein R is C7-C18-alkanoyl, C1-6 (γ-alkenoyl or an amino-protecting group; R1 is hydrogen or C1-4-C1-4-alkanoyl). Q-alkanoyl, and R2 is hydrogen, C1-C13-alkanoyl, succinyl or an amino-protecting group, provided that 1 2 1. at least one of R, R and R is other than hydrogen or an amino-protecting group, 1 2 2. at least one of R and R is hydrogen or an amino-protecting group, 1 2 35 3) R, R and R contain at least four carbon atoms in total and 56 791 1 8 1 2 4. both R and R being either hydrogen or the amino-protecting group R is not 8-methyldecanoyl, 10-methyl-undecanoyl, a specific C 1-8 alkanoyl group of factor A-21978C or a specific group of C-factors A-21978C and Process according to Claim 1, characterized in that a peptide derivative of the formula (1) is prepared in which R is alkanoyl of the formula O CH, (CH 2) -d- j in which n is an integer from 5 to 14 . Process according to Claim 1, characterized in that a peptide derivative of the formula (1) is prepared in which R is alkanoyl of the formula CHO OT 3 II CH, (CH-) -CH (CH 2) -C-ό Z n Z m 15 where n and m independently represent an integer from 0 to 12, provided that n + m is at least 3 and at most 12 and that, in addition, when n is 0 m is not 8 and when n is 1 m is not 6 and not 8. Process according to Claim 1, characterized in that a peptide derivative of the formula (1) is prepared in which R is cis- or trans-alkenoyl of the formula O CH, (CH 2) CH = CH (CH ,) -1 ^ -3 Z n 2 m 25 where n and m independently represent an integer from 1 to 13, provided that n + m is at least 7 and at most 13. Process according to Claim 1, characterized in that a peptide derivative of the formula (1) is prepared in which R is cis- or trans-alkenoyl of the formula O 11 ch 2 = ch (ch 2) -C- in which n is an integer 8-14. 58 791 1 8 5. I 8 25 H · HOsC (3) wherein R 1 and R 0 are independently hydrogen or an amino protecting group are treated at a temperature of about 30 to 60 ° C for 1-24 hours with an acid of formula 5 R 6 -COOH (5) or an activated derivative thereof , wherein R 1 is C 8 -C 4 alkyl or C 10 -C 16 alkenyl; and 57 7 9118, if desired, removing any protecting groups which may be present in the product and / or, if desired, converting the product obtained into a salt. 5 C 2-8 alkyl group; and pharmaceutically acceptable salts thereof, characterized in that the cyclic Δ-21978C core peptide of formula 3 0 NHR '/ \ / *> / \ / \ YYo [N λ! . v rv- '^ ^ A CONH 15, I \ »HJK 9 NHs h / hV h Y Yy V y Y x> Y <MA VYN <> VU 20 Υ-Π * vy * 0 = / γ = 0 H yrz rd Ϊ H <. JL- ho ^ Ύ / \ / \ / \ / v \ / Process according to Claim 1 or 2, characterized in that a peptide derivative of the formula (1) is prepared in which R is n-decanoyl. Process according to Claim 6, characterized in that a peptide derivative of the formula (1) is prepared, in which R is n-decanoyl and R and R are hydrogen. 59 791 1 8