FI79545B - NYA CYCLISKA A-21978C-KAERNPEPTIDER OCH FOERFARANDE FOER DERAS FRAMSTAELLNING. - Google Patents

Description

The present invention relates to novel cyclic A-21978C core peptides of formula (1 ') 0 NH. ! i 10 / CH3 / \ / \; i Y ”! I l

λ). V

0 = / Vvv. conh2 15 / - / · h ° l, r k? HO Η-Νχ η3 </ y y Y Y.

/ “0 ° * \ V 8 H- {S \ \ NH C0oH · · i

Vh / 2 Χγ \ /

20 O * · '' Sa.O H

/ —Γ fH3 P f / 1 2 YA / Yyv · '• -ill u'

0 H * O

25 HOjC

wherein R 2 is hydrogen or an amino protecting group, and salts thereof, and their preparation.

The novel compounds of formula (1 ') and their salts are useful as intermediates in the preparation of semi-synthetic antibiotic derivatives.

There is a great need to develop new antibiotics because there is a very possible and constant threat of developing strains of 35 microorganisms that are specifically resistant to antibiotics. In particular, pathogens belonging to the gram-positive 2,79545 genera Staphylococcus and Streptococcus are often resistant to commonly used antibiotics such as penicillin and erythromycin [see e.g., W.O. Foye, Principles of Medicinal Chemistry (1974), 5 ss. 684-686].

The novel derivatives of the cyclic A-21978C peptides disclosed in co-pending patent applications 831746, 831747 and 831756 and their pharmaceutically acceptable salts have been found to be effective drugs.

These novel peptide derivatives disclosed in the above-mentioned co-pending patent applications can be prepared by reacting a novel A-21978C core peptide of formula (1 ') or a salt thereof with a desired acylating agent or an activated derivative thereof.

According to the invention, the novel cyclic A-21978C core peptides of formula (1 ') and their salts can be prepared such that the A-21978C complex, the A-21978C factor C0, C3, C2, C3, C4 or C5, a blocked A-21978C complex or a blocked A-21978C factor C0, C3, C2, C3, C4 or C5, having the formula (1)! p. »Αγ“ ΜΛ .. Am k / -AH 'yv. · «M".

- <* -cyw'i · (Y \ / 35, υ HOac / 3 79545 where R is 8-methyldecanoyl, 10-methylundecanoyl, 10-methyldodecanoyl, a Cx0 alkanoyl group characteristic of factor C0 of A-21978C or A-21978C: The C: 2 alkanoyl group characteristic of C4 and C5, and R1 and R2 are independently hydrogen or an amino protecting group, is enzymatically deacylated in an aqueous medium by a deacylating enzyme produced by the microorganism Actinoplanaceae. utahensis NRRL 12052, Streptosporangium roseum var. hollandensis NRRL 10 12064, Actinoplanes missouriensis NRRL 12053, Actinoplanes sp. NRRL 12065 or Actinoplanes sp. NRRL 8122, if desired the amino protecting group R2 is removed from the product thus obtained and, if desired, the product obtained is salted.

Compounds of formula (1) in which R and R2 are amino protecting groups are inhibited factors C0, Clf C2, C3, C4 and C5 of the antibiotic A-21978C.

The compound of formula (1 ') wherein R 2 is hydrogen is a common cyclic peptide in the antibiotic A-21978C factors C0, C3, C2, C3, C4 and C5. For convenience, this compound is referred to herein as the A-21978C core. This compound can also be represented by formula (2): L-Asp? - 25 K ^ D-Ala Gly

i * V

L-Asp D-Ser -p i /

L-Orn 3MG

L-KYN

Gly / (27 30 ^ L ~ Thr ^

T

L-Asp t. L-Asn

A

L-Trp * nh2 35 4 79545 where 3MG means L-threo-3-methylglutamic acid.

Naturally occurring A-21978C factors share a common peptide backbone, but each has a different fatty acid moiety. The method of the present invention for removing fatty acid groups 5 from A-21978C and inhibited factors comprises exposing the antibiotic or inhibited antibiotic in an aqueous medium to an enzyme produced by a microorganism of the Actinoplanaceae group, such as deacylation is substantially accomplished.

The preferred method of this invention uses an enzyme produced by the microorganism Actinoplanes utahensis NRRL 12052 to cleave the fatty acid side chain. Deacylation is usually accomplished by adding a suitable antibiotic or blocked antibiotic to A. utahensis culture and incubating the culture until deacylation occurs. The cyclic A-21978C peptide or blocked peptide thus formed is separated from the fermentation broth by known methods.

Cyclic A-21978C peptides of formula (1 ') are useful in that they can be acylated to provide new antibiotics.

The attached infrared absorption spectra determined in KBr are as follows:

Figure 1 - A-21978C core (Formula 1 ', R 2 = H) Figure 2 - A-21978C-NO-t-BOC core (Formula 1, R * H, R 2 = tert-butyloxycarbonyl) The following are used in this definition. abbreviations: Ala: alanine Asp: aspartic acid 30 Gly: glycine

Kyn: kynurenine Orn: ornithine Ser: serine Thr: threonine 35 Trp: tryptophan s 79545 t-BOC: tert-butyloxycarbonyl Cbz: benzyloxycarbonyl DMF: dimethylformamide THF: tetrahydrofuran 5 HPLC: high performance liquid chromatography A9 chromatography: TLC: thin layer chromatography antibiotics are closely related acidic peptide antibiotics and are described in U.S. Patent No. 4,208,403. As described in U.S. Patent No. 4,208,403, the antibiotic complex A-21978C contains a major component, factor C, which in turn is a structurally related factor. complex. Factor C of A-21978C, called the A-21978C complex, contains the individual factors C0, C2, C2, C3, C4, and C5. Factors Cx, C2, and C3 are major factors and factors C0, C4, and C5 are minor factors. The structure of A-21978C factors is shown in formula (3): 20, L ~ * sp \

Mia G'y A * f D-Ser L-Asp

25 t 3MG

L-0rn ^ \ L-Kyn G, y 0 / \ / (3) L — Thr 30 f L-Asp t L-Asn f L — Trp

35 r, H

RN

6,79545 wherein 3MG represents L-threo-methylglutamic acid and R "represents a fatty acid group. The characteristic β-groups are as follows: A-21978C factor R" group 5 C3 8-methyldecanoyl C2 10-methylundecanoyl C3 10-methyldodecanoyl alkanoyl * C4 C: 2-alkanoyl * 10 C5 2-alkanoyl * * Not yet identified

When the problem is deacylation of a peptide antibiotic, it is very difficult to know if there is an enzyme that can be used for this purpose. Finding such an enzyme is even more difficult when the substrate antibiotic contains a cyclic peptide structure. Because the enzymes are highly specific, differences in the peptide backbone and side chain of the substrate affect the success of the deacylation attempt. In addition, many micro-20 organisms produce a large number of peptidases that affect different parts of the peptide structure. This often leads to product mixtures that are difficult to handle.

In each A-21978C antibiotic (Formula 3), the fatty acid side chain (R ") is attached to the α-25 amino group of the tryptophan group. We have found that the fatty acid side chain can be cleaved by an enzyme without affecting the chemical structure of the other A-21978C peptide.

The term "amino protecting group" means a known 30 amino protecting group that is compatible with other functional groups on the A-21978C molecule. Preferably, the amino protecting groups are those that can be readily removed later. Examples of suitable protecting groups can be found in Theodora W. Greene, 35 Protective Groups in Organic Synthesis, John Wiley and 7,79545

Sons, New York, 1981, Chapter 7. Particularly preferred amino protecting groups are tert-butyloxycarbonyl and benzyloxycarbonyl groups.

With the A-21978C core, i.e. the compound of formula 1, wherein R 2 is hydrogen, which is alternatively also described by formula 2, has the following properties:

Form: white amorphous solid that fluoresces in shortwave UV

Empirical formula: C62H83N17 02 5 10 Molecular weight: 1465

Solubility: water soluble

Infrared Absorption Spectrum (KBr): shown in Figure 1 of the accompanying drawings; absorption maxima are observed at the following frequencies (cm-1): 15 3300 (wide), 3042 (weak), 2909 (weak), 1655 (strong), 1530 (strong), 1451 (weak), 1399 (medium), 1222 (medium) , 1165 (weak), 1063 (weak) and 758 (moderate to weak).

Ultraviolet absorption spectrum (methanol): UV maxima 223 nm (6 41 482) and 260 nm (6-8 687).

Potentiometric titration (66% aqueous dimethylformamide) shows four titratable groups with pKa values of about 5.2, 6.7, 8.5, and 11.1 (initial pH 6.12).

A particularly useful cyclic peptide of formula 1 'is a compound wherein R 2 is tert-butyloxycarbonyl. This compound is hereinafter referred to as the "t-BOC core" for convenience. The A-21978C-tBOC core has the following properties: 30 Form: white amorphous solid that fluoresces in shortwave UV

Empirical formula: C6 7 H, 3 Nx 7 02 7 Molecular weight: 1565

Solubility: water soluble 35 Infrared Absorption Spectrum (KBr): shown in Figure 2 of the accompanying drawings; absorption maxima are observed at the following frequencies (cm * 1): 3345 (wide), 3065 (weak), 2975 (weak), 2936 (weak), 1710 (shoulder), 1660 (strong), 1530 (force-5 kas), 1452 ( weak), 1395 (medium), 1368 (weak), 1341 (weak), 1250 (medium), 1228 (medium), 1166 (medium to weak), and 1063 (weak).

Ultraviolet absorption spectrum (90% ethanol): UV maxima 220 nm (42,000) and 260 nm (10,600) 10 High performance liquid chromatography: residence time = 6 min 4.6 x 300 mm silica gel-Cj8 column using H 2 O / CH 3 CN / CH 3 OH (80: 15: 5) containing 0.2% NH 4 OAc at a flow rate of 2 ml / min and a UV detector as solvent.

Blocked A-21978C factors of this invention include compounds of formula 1 wherein R 2 is an amino protecting group and R is 8-methyldecanoyl, 10-methylundecanoyl, 10-methyldodecanoyl, C30 specific for C-factor C-30978C. alkanoyl group or one of the C32 alkanoyl groups characteristic of factors C4 and C5 of A-21978C.

The blocked factors and cyclic peptides of A-21978C of this invention may form salts which are also part of this invention. Such salts are useful, for example, in the separation and purification of compounds. Pharmaceutically acceptable alkali metal salts, alkaline earth metal salts, amine salts and acid addition salts are particularly useful. "Pharmaceutically acceptable" salts are salts that can be used for chemotherapy in warm-blooded animals.

For example, cyclic A-21978C peptides of formula 1 'have four free carboxyl groups that can form salts. Partial and mixed salts of these carboxyl groups, as well as complete salts, are therefore contemplated as part of this invention. When preparing 35 9 79545 these salts should be avoided at pH values higher than 10 because the compounds are unstable at such pH values.

Representative examples and suitable alkali metal and alkaline earth metal salts of cyclic A-21978C peptides of formula 1 'include sodium, potassium, lithium, cesium, rubidium, barium, calcium and magnesium salts. Suitable amine salts of cyclic A-21978C peptides include ammonium salts and primary, secondary and tertiary C 1-4 alkylammonium and hydroxy-C 1-4 alkylammonium salts. Examples of amine salts include those obtained by reacting cyclic A-21978C peptide with ammonium hydroxide, methylamine, sec-butylamine, isopropylamine, diethylamine, diisopropylamine, ethanolamine, triethylamine and 3-amino-1-propanol. with.

The alkali metal and alkaline earth metal salts of the cyclic A-21978C peptides of formula 1 'are prepared by methods commonly used in the preparation of cationic salts. For example, the free acid form of cyclic A-21978C peptide is dissolved in a suitable solvent such as warm methanol or ethanol, then a solution containing a stoichiometric amount of the desired inorganic base in aqueous methanol is added. The salt thus formed can be isolated by conventional methods such as filtration or evaporation of the solvent.

Salts formed with organic amines can be prepared in the same manner. For example, a gaseous or liquid amine may be added to a cyclic A-21978C-30 peptide dissolved in a suitable solvent such as acetone; the solvent and excess amine can be removed by evaporation.

The cyclic A-21978C peptides of this invention also have free amino groups and can thus form acid addition salts, which are also part of this invention. Representative examples and suitable acid addition salts of cyclic A-21978C peptides include, but are not limited to, salts formed by standard reactions with organic and inorganic acids such as hydrochloric, sulfuric, phosphoric, acetic, succinic, lemon, dairy. , maleic, fumaric, paltaminic, school, pamoin, mucus, D-glutamine, d-camphor, glutar, glycol, phthalic, wine, lauric, stearic, salicylic with methanesulfonic, benzenesulfonic, sorbic-10-nitric, picric, benzoic and cinnamic acids.

Preparation of cyclic A-21978C peptides

Cyclic A-21978C peptides of formula 1 'are obtained by deacylation of a peptide antibiotic selected from the group consisting of A-21978C factors C0, C: C2, C2, C3, C4 and C5 and blocked A-21978C factors C0 , Cx, C2, C3, C4 and C5.

I. Preparation of Substrates C-factors C0, Ct, C2, C3, C4, and C5 of A-21978C are prepared as described in U.S. Patent 4,208,403. These factors are components of the A-21978C complex, which is part of the A-21978C complex. 21978 complex.

The A-21978 complex can be produced by culturing Streptomyces roseosporus NRRL 11379 (deposited August 29, 1978) under aerobic submerged culture conditions when a substantial level of antibiotic activity has developed. The A-21978 complex is separated by filtration of the culture broth, lowering the pH of the filtrate to about 3, allowing the complex to precipitate, and separating the complex by filtration. The separated complex can be further purified by extraction techniques 30. Chromatographic separation methods are required to isolate the separate A-21978 complex and factors.

The blocked A-21978C complex and blocked A-21978C factors C0, C3, C2, C3, C4 and C5 of the present invention (compounds of formula 1 wherein R is selected from the group consisting of 8-methyldecanoyl, -methyl 79545 lundecanoyl, 10-methyl ± dodecanoyl and C10, C4 and C5 C10 and Cx2 alkanoyl groups) are prepared using methods used to protect the amino group of peptides. The protecting groups are selected from various known amyl-5 protecting groups, for example, benzyloxycarbonyl, tert-butyloxycarbonyl, tert-amyloxycarbonyl, isobomyloxycarbonyl, adamantyloxycarbonyl, o-nitrophenylthio, diphenylphosphinothioyl, diphenylphosphinothioyl.

The blocked A-21978C complex is particularly preferred as a substrate. It is prepared from the A-21978C complex, thus avoiding the steps required to separate the individual A-21978C factors, but when deacylated, it gives a single product, i. suitably blocked core. 15 II. Method of Deacylation A. Preparation of Enzyme 1. Microorganisms A-21978C enzymes useful in the deacylin-20 of C0, Ct, C2, C3, C4 and C5 and blocked factors C0, Cx, C2, C3, C4 and C5 can be prepared in certain by microorganisms of the genus Actinoplanaceae, preferably with the microorganism Actinoplanes utahensis NRRL 12502 (recorded October 9, 1979). Although Example 1 describes a preferred method for culturing A. utahensis NRRL 12052 to produce this enzyme, it will be apparent to one skilled in the art that other methods may be used.

Actinoplanaceae is a family of the microorganism group Actinomy-cetales. This tribe was found in 1955, it was described as en-30 slum by Dr. John N. Couch [Elisha Mitchell Sei. Soc. 71, 148-155 (1955)]. The characteristics of the tribe and several individual genera are given in "Bergey's Manual of Determinative Bacteriology", 8th edition, ed. R. E. Buchanan and N. E. Gibbons, The Williams and Wilkins Co., 35 Baltimore, Md., 1974, pp. 706-723. Thus, ten genera of i2 79545 have been distinguished: I. Actinoplanes (type genus and thus by far the most common genus); II. Spirillospora; III. Strep-tosporangium; IV. Amorphosporangium; V. Ampullariella; VI. Pilimelia; VII. Planomonospora; Vili. Planobispora; IX.

5 Dactylosporangium; and X. Kitasatoa.

Some species and variants isolated and characterized to date include: Actinoplanes philippinensis, Actinoplanes armeniacus, Actinoplanes utahensis, and Actinoplanes missouriensis; Spirillospora albida; Strep-10 tosporiangium roseum, Streptosporangium vulgare, Strepto-sporangium roseum var. hollandensis, Streptosporangium album, Streptosporangium viridialbum, Amorphosporangium auranticolor, Ampullariella regularis, Ampullariella cam-panulata, Ampullariella lobata, Ampullariella digitata, Planimelia terevara, Pilimelia anulata, Planomonospora pa-rontospora and Dactylosporangium thailandense.

The genus Actinoplanes is a preferred source of the enzyme useful in this invention. In the genus Actinoplanes, the species Actinoplanes utahensis is a particularly preferred source of this enzyme.

Other cultures of typically useful species are available to the public from the Northern Regional Research Center, Agricultural Research Culture Collection (NRRL), U.S. Pat. Department of Agriculture, 1815 North University St., Peoria, Illinois 61604, U.S.A., under the following accession numbers:

Actinoplanes utahensis NRRL 12052 30 (deposited October 9, 1979)

Actinoplanes missouriensis NRRL 12053 (deposited October 9, 1979) Actinoplanes sp. NRRL 8122 (deposited October 17, 1975) 35 i3 79545

Actinoplanes sp. NRRL 12065 (deposited November 5, 1979) Streptosporangium roseum var. hollandesls NRRL 12064 5 (deposited November 5, 1979) A. utahensls NRRL 12052 was developed from a mother culture also deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852 (A. utahensls ATCC 14539). A culture of A. utahensls ATCC 10 14539 can also be used as a source of enzyme.

A. missouriensis NRRL 12053 was developed from a culture that was also deposited with the ATCC (A. missouriensis ATCC 14538) and is also a source of enzyme.

The potency of any microorganism strain belonging to the group Actinoplanaceae in the deacylation of this invention is determined by the following method. A suitable medium is inoculated with the microorganism. The culture is incubated at about 30 ° C for two or three days on a rotary shaker. One substrate antibiotic is then added to the culture while maintaining the pH of the fermentation medium at about 7.0. Culture activity is monitored using the Micrococcus luteus assay. This procedure is described in Section D. The decrease in the potency of the antibiotic is an indication that the microorganism produces the enzyme required for deacylation. However, this can be confirmed by one of the following methods: 1) HPLC analysis to detect the presence of an unchanged nucleus, or 2) re-acylation with a suitable side chain (e.g., lauroyl, n-decanoyl, or n-dode-30 canoyl) to restore activity. When deacylating blocked A-21978C, it is difficult to detect a decrease in antibiotic activity because the addition of a blocking group causes a decrease in antibiotic activity by 80-90%.

35 i4 79545 2. Conditions for production of the enzyme

Enzyme formation occurs under conditions suitable for the growth of Actinoplanaceae, i. at a temperature in the range of about 25 ° C to about 30 ° C and in the pH range of about 5.0 to about 8.0 with stirring and aeration. The culture medium should contain a) an assimilable carbon source such as cane sugar, glucose, glycerol or the like, b) a nitrogen source such as peptone, urea, ammonium sulphate or the like, c) a phosphate source such as soluble phosphate salt 10 and d) inorganic salts which are generally found to promote the growth of microorganisms. An effective amount of enzyme is generally obtained about 40 to about 60 hours after the start of the growth phase and is generally maintained for some time after effective growth is achieved. The amount of enzyme formed varies depending on the species of microorganism and the growth conditions.

As will be apparent to those skilled in the art, enzyme-producing microorganisms such as Actinoplanes utahensis NRRL 12052 are susceptible to transformation. 20 Artificial modifications and mutants of these strains are obtained, for example, by various known mutagens such as ultraviolet rays, X-rays, high frequency waves, radioactive radiation and chemicals. All naturally occurring and artificial variants and mutants obtained from the group Actinoplanaceae that produce the enzyme can be used in accordance with this invention.

B. Deacylation Conditions The starting substrate is preferably added to the Actinoplanaceae culture after incubation for about 48 hours. The concentration of the substrate in the reaction medium can vary widely. However, in order to use the enzyme as efficiently and practically as possible to achieve complete deacylation within three hours, a range of about one to about three mg / ml is usually used. Concentrations lower than 79545 may be used, but do not give maximum benefit from the enzyme; higher concentrations can also be used, but the substrate will not be completely deacylated if the fermentation time is not extended.

The conversion of a substrate antibiotic to the A-21978C core in accordance with the present invention is best accomplished when the pH of the fermentation medium is maintained in the range of about 7.0 to about 7.2. Below pH 7, deacylation occurs slowly; when the pH rises above 7.2, the formed nucleus is increasingly exposed to alkaline hydrolysis. In stirred fermenters, the pH can be adjusted with tactile controls. Where this is not possible, as in bottle fermentors, the pH can be controlled by adding a 0.1 molar phosphate buffer to the medium before adding the substrate.

After the substrate is added, the incubation of the culture should be continued for another 3-6 hours. The purity of the substrate affects the rate of deacylation. For example, a substrate with a purity greater than 50% is deacylated at a rate of about 2.5 to 20 mg antibiotic / ml in 3 hours. When more impure substrates are used, deacylation occurs somewhat more slowly.

The substrate can be added in several batches. For example, 0.75 mg antibiotic / ml may be added every 24 hours in at least five or more additions.

The deacylation can be performed over a wide temperature range, e.g. from about 20 to about 45 ° C. However, it is recommended to perform deacylation at a temperature of about 30 ° C due to optimum deacylation and optimum substrate and core stability.

C. Substrate

It is preferred but not necessary to use a purified antibiotic as a substrate. Because the purified substrate is soluble in water or buffer, it can be handled more easily. In addition, when the purified substrate ie 79545 is used, deacylation occurs more rapidly. Partially purified substrates containing up to only 15% of the starting antibiotic have been successfully deacylated.

5 Substrate antibiotics have antibacterial activity.

As a result, although substrate materials (especially those of low purity) can act as carriers for bacterial cells or spores that are thought to grow in the deacylation medium and affect the de-10 acylol reaction or the structure of the starting antibiotic or product. Therefore, it is not necessary for the substrates to be sterile, especially in the case of short deacylation steps.

15 D. Monitoring of deacylation Factors C0, Cx, C2, C3, C4 and C5 of A-21978C are antibacterial agents that are particularly effective against Micrococcus luteus. For this reason, any experiment using M. luteus should be recommended for the determination of substrate amounts. The A-21978C core formed is water-soluble but biologically ineffective. The decrease in biological activity is therefore a rapid, approximate experiment on deacylation.

The amount of nucleus formed can be determined quantitatively according to the system described below by HPLC analysis.

E. Use of resting forms of cells

An alternative method of deacylation involves removing Actinoplanaceae cells from the culture medium, suspending the cells in buffer, and performing deacylation as described in B. When using this method, the enzymatically active mycelium can be reused. For example, A. utahensis NRRL 12052 mycelium retains its deacylation activity when stored for one month or longer under refrigeration i7 79545 (4-8 ° C) or frozen (-20 ° C). The preferred buffer solution is a 0.1 molar phosphate buffer.

F. Immobilized enzymes

Another way to perform deacylation is to immobilize the 5 enzymes by methods known in the art (see, e.g., "Biomedical Applications of Immobilized Enzymes and Proteins", ed. Thomas Ming Swi Chang, Plenum Press, New York, 1977; Vol. 1). The immobilized enzyme can then be used in a column (or other suitable type of reactor) to effect deacylation.

In addition, the microorganism itself can be immobilized and used to catalyze the deacylation reaction.

Usefulness of Cyclic A-21978C Peptides Cyclic A-21978C peptides and their salts are useful intermediates in the preparation of semisynthetic antibacterial compounds.

The chemotherapeutically useful compounds described in our co-pending patent application 831 756 have the general formula (1) wherein R is C7-C36 alkanoyl, Cx3-Cx7 alkenoyl or an amino protecting group;

R 1 is hydrogen or C 1 -C 10 O-alkanoyl; and R 2 is hydrogen, C 1 -C 3 alkanoyl, succinyl or an amino protecting group; Provided that 1) at least one of R, R1, R2 is other than hydrogen or an amino protecting group, 2) at least one of R1 and R2 is hydrogen or an amino protecting group, 3) R, R1 and R2 together contain at least four carbon atoms and 4) when both R1 and R2 are either hydrogen or an amino protecting group, R is not 8-methyldecanoyl, 10-methyl-undecanoyl, O-alkanoyl-35 group specific for factor A-21978C or a C3 2 to 79545 18 alkanoyl group specific for C4 and C5 of A-21978C.

The terms "C7-Cx6-alkanoyl" and "C3-Cx7-alkenoyl" refer to acyl groups derived from carboxylic acid-5-carbons having 7 to 16 and 11 to 17 carbon atoms, respectively.

When the group R is alkanoyl, the alkyl moiety is a monovalent saturated unbranched or branched hydrocarbon group. When R is alkenoyl, the alkenyl moiety is a monovalent unsaturated straight or branched hydrocarbon group having no more than three double bonds. The double bond moiety (ies) of the unsaturated hydrocarbon chain may be in either the cis or trans configuration.

The term "amino protecting group" means a known amino protecting group as defined above.

The following are preferred embodiments of the compounds of formula (1) disclosed in patent application 831756: a) Compounds wherein R is alkanoyl of formula CH 3 (CH 2) n -? - and n is an integer from 5 to 14; CH, 0

I 3 II

B) compounds wherein R is of the formula CH3 (CH2) n CH (CH2) mC- alkanoyl and 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 n when n is 0, m is not 8 and when n is 1, m is neither 6 nor 8; 0

II

C) Compounds wherein R is cis- or trans-alkenoyl of the formula CH3 (CH2) nCH «CH (CH2) n -C and n and m independently represent an integer from 1 to 13, provided that n + m is at least 7 and up to 13; D) Compounds wherein R is cis- or trans-alkenoyl of the formula CH 2 • CH (CH 2) n - & - and n is an integer from 8 to 14; e) Compounds in which R is: 0 ch3 (ch2) 5 -Ϊ-35 i9 79545 o ch3 (CH2) 6-fc-o

ch3 (CH2) 7 -c-5 O

CH3 (CH2) eC- o ch3 (CH2) 9 -C-O 10 CH3 (CH2) 10-c- o CH2 = CH- (CH2) 8-C-O CH3 (CH2) n -C-15 o CH3 ( CH2) i 2 -Ä- o ch3 - (CH2) 3 -CH = CH- (CH2) 7 -i- .V 0 20 CH3 (CH2) 13- <ϋ- 0 ch3 ch2 CH (ch3) - (ch2) 6 -Ϊ-

The chemotherapeutic compounds described in our co-pending patent applications 831,746 and 831,747 have the general formula (1) wherein R 1 is hydrogen.

The derivatives of formula (1) described in patent application 831 747 are those in which R is hydrogen, 8-methyldecanoyl, 10-methyldecanoyl, 10-methylundecanoyl, a C20 alkanoyl group specific for A-21978CO or an A-21978C factor. C4 and C5 specific C12 alkanoyl groups, amino protecting group, aminoacyl group,

O

35 of the formula -C-Q-NH2, wherein Q is C7-6 alkylene, or an N-alkanoylaminoaminoacyl group of the formula -W- li-R2 wherein W is a bivalent aminoacyl group of the formula

5 Q

a) -ϋ-Α-ΝΗ- wherein A is C 3-10 alkylene or C 5-6 cycloalkylene; 10 0 R3

il I

b) -C-CH-NH- wherein R 3 is hydroxymethyl, hydroxyethyl, mercaptomethyl, mercaptoethyl, methylthioethyl, 2-thienyl, 3-15 indolymethyl, phenyl, benzyl or substituted phenyl or substituted benzyl whose benzene ring is substituted by chlorine, bromine , iodine, nitro, C 1-3 alkyl, hydroxy, C 3-3 alkoxy, C 1-3 alkylthio, carbamyl or C 1-3 alkylcarbamyl; 20

X X

; Wherein X is hydrogen, chlorine, bromine, iodine, amino, nitro, C3-3 alkyl, hydroxy, C3-3 alkoxy, mercapto, C3-3 alkylthio, carbamyl or C3-3 alkylcarbamyl; 30 1 X1 -CT of NH_. ~ c ~ r | or kx> <L-NH- ^ X1 X X1

35 X

2i 79545 wherein X 1 is chlorine, bromine, iodine, amino, hydroxy, C 1-3 alkyl or C 2-3 alkoxy;

-tjJH

5 (e) L / \ Jy \.

i I! or 7 il or - ^ V v # 10 (f) s / * \. . wherein B is a divalent radical of formula - (CH 2) n - and n is an integer from one to three; -CH = CH-; -CH = CH-CH2- or? -CH2 NHC-; R2 'is C2. x 7 -alkyl or C 2-27 -alkenyl and R 2 is hydrogen, a 20 amino protecting group, an aminoacyl group of the formula -C - & - NH 2 as defined above, or N-alkanoylaminoacyl, 0 of the formula -WC-R 2 'as defined above provided that when R is other than aminoacyl or N-alkanoylaminoacyl, R 2 must be aminoacyl or N-alkanoylaminoacyl and when R 2 is an amino protecting group, R must be aminoacyl or N-alkanoylaminoacyl, or a pharmaceutically acceptable salt thereof.

The terms "alkylene", "alkyl", "alkoxy", "alkylthio" and "alkenyl" include both straight and branched chain hydrocarbon chains. "Alkyl" means a monovalent saturated hydrocarbon group. "Alkenyl" means a monovalent unsaturated hydrocarbon radical containing one, two or three double bonds, which may be in the cls or trans configuration. "Alkylene" means a divalent saturated hydrocarbon radical. "Cycloalkylene" means a divalent saturated hydrocarbon radical.

Examples are Cx.20 or C3. Preferred 26-alkylene groups include R5 -CH2-; Wherein R 5 is C 2-4 alkyl (i.e. methyl, ethyl, n-propyl, i-propyl, n-butyl, tert-butyl, i-butyl or 1-methylpropyl); - (CH2) b-, where m is an integer from two to ten and CH3- (CH2) q-CH- (CH2) -, where p is an integer from one to eight and q is an integer from zero to seven, provided that p + q must not be greater than 8.

Examples of preferred C 2-27 alkyl groups include: (a) CH 3 -; (b) - (CH 2) n CH 2, where n is an integer from one to sixteen; and CH3 (c) - (1ξη2) rCH (CH2), CH3, where r and s are each independently an integer from zero to fourteen, provided that r + s cannot be greater than 14.

Examples of C2-17 alkenyl groups which are preferred are: (a) - (CH2) t-CH * CH- (CH2) u-CH, where t and u are each independently an integer from zero to fourteen, provided that t + u is not may be greater than 30 14.

(b) - (CH2) v -CH-CH- (CH2) r -CH-CH- (CH2) t -CH3, where v and z are each independently an integer from zero to eleven and y is an integer from one to twelve, provided that v + y + z cannot be mouth-35 higher than 12.

23 79545

In particular, the following C 3-7 alkyl groups are preferred: ch 3 -ch 3 (ch 2) 5 -CH 3 (CH 2) 6 -ch 3 (ch 2) 8 -CH 2 CCH; > „- CH3 (CHj) t 2 -ΟΗ, (ΟΗ,) 1 (- 10 CHS (CH;) 1S-

In particular, the following C 2-17 alkenyl groups are preferred: cis-CH 3 (CH 2) 5 CH = CH (CH 2) 7 -trans-CH 3 (CH 2) s CH = CH (CH 2) 7 -15 cis-CH 3 (CH 2) 10 CH = CH (CH 2) 4 - trans-CH3 (CH2) 10CH = CH (CH2) 4 -cis-CH3 (CH2) 7CH = CH (CH2) 7-trans-CH3 (CH2) 7CH = CH (CH2) 7 -cis-CH3 (CH2) 5CH = CH (CH2) 9-20 trans-CH3 (CH2) 5CH = CH (CH2) 9-. cis, cis-CH3 (CH2) 4CH = CHCH2CH = CH (CH2) 7 - trans, trans-CH3 (CH2) 4 CH «CHCH2 CH-CH (CH2) 7 -: '. cis, cis, cis-CH3 CH2 CH-CHCH2 CH-CH-CHCH2CH-CH- (CH2) 7.

25 When W is the formula V \

Tf \ / 6

30 V

is a person skilled in the art

II

it will be appreciated that the -C and -NH groups may be attached at the benzene ring at the ortho, meta or para positions 24 79545 relative to each other. The substituent represented by X may be attached to any free position on the benzene ring. Preferred embodiments are compounds wherein

O

II

X is hydrogen and the groups -C- and -NH- are in the para position relative to each other.

The attributes "substituted phenyl" and "substituted benzyl" included in the definition of the symbol R 3 of formula (1) comprise a substituent attached to any available position on the benzene ring, i. the substituent may be in the ortho, meta or para position. The term "C 1-3 alkyl" included in the definitions of the symbols R 3 and X in the formula (1) includes methyl, ethyl, n-propyl and i-propyl groups.

The derivatives of formula (1) described in patent application 831746 are those in which R is a substituted benzoyl group of formula g A,> 20 i-4- - | - ^ 5 nx wherein X is hydrogen, chlorine, bromine, iodine, nitro C 1-3 alkyl, hydroxy, C 1-3 alkoxy or C 13 alkylthio; R 2 is hydrogen or an amino protecting group and R 2 'is C 815 alkyl; or a pharmaceutically acceptable salt thereof.

In a substituted benzoyl group, the groups 0-16 -C and -OR2 may be in the ortho, meta or para position relative to each other. The association of these groups at the para positions is preferred. The substituent represented by X may be attached to any available position on the benzene ring which does not have either of these two groups.

35 The term "alkyl" embraces both straight and branched chain hydrocarbon chains.

Examples of C 1-2 5 alkyl groups which are preferred as R 2 'are (a) - (CH 2) n CH 3, where n is an integer from seventh to fourteen, and ch 3 (b) - (CH 2) γ ^ Η (ΟΗ2) , CH3, where r and s are each independently an integer from zero to twelve, provided that r + s cannot be greater than 12 and 10 cannot be less than 5.

: Em. the compounds of formula 1 disclosed in patent applications 831,746, 831,747 and 831,756 can be prepared by acylation of a compound of formula 1 with a suitable acyl side chain using methods conventional in the art for forming an amide bond. The acylation is usually carried out by reacting the selected compound against the desired side chain with an activated derivative of the acid.

The term "activated derivative" means a derivative that reactivates the carboxyl group of an acylating agent to join a primary amino group to form an amide bond that connects the acyl side chain to the core. Suitable activated derivatives, their preparation methods and their use for the acylation of primary amines are known to those skilled in the art. Preferred activated derivatives are: (a) an acid halide (e.g., acid chloride), (b) an acid anhydride (e.g., alkoxy formic anhydride or aryloxy formic anhydride), or (c) an activated ester (e.g., 2,4,5-trichlorophenyl ester). Other methods of activating a carboxyl group include the reaction of a carboxylic acid with a carbonyl diimide (e.g., N, N'-dicyclohexylcarbodiimide or N, N'-diisopropylcarbodiimide) to give a reactive intermediate that is not isolated due to its instability. . Such a reaction with the primary amine is carried out in situ.

26 79545

A preferred process for the preparation of the above compounds of formula 1 is the use of an activated ester. The 2,4,5-trichlorophenyl ester of the desired acid is the preferred acylating agent. In this process, an excess of the active ester 5 is reacted with a compound of formula 1 'at room temperature in an inert organic solvent such as DMF. The reaction time is not critical, although a reaction time of about 6 hours to about 20 hours is recommended. At the end of the reaction, the solvent is removed and the residue is purified by a known method such as column chromatography. The t-BOC groups can be removed by treatment with trifluoroacetic acid / anisole / triethylsilane or preferably trifluoroacetic acid / 1,2-ethanediol for about three to about five minutes at room temperature. After removal of the solvent, the residue can be purified by reverse phase HPLC.

2,4,5-Trichlorophenyl esters of the desired acids can be readily prepared by treating the corresponding acid with 2,4,5-trichlorophenol in the presence of a coupling agent such as N, N'-20 dicyclohexylcarbodiimide. Other methods for preparing acid esters will be apparent to those skilled in the art.

The alkanecarboxylic acids and alkenecarboxylic acids and their activated derivatives (especially acid chlorides and 2,4,5-trichlorophenyl esters) used as starting materials in the preparation of the derivatives of formula (1) disclosed in Patent Application 831,756 are known compounds or can be prepared from known compounds by known methods. 2,4,5-Trichlorophenyl esters 30 are prepared simply by treating an acid chloride of an alkoxy or alkenecarboxylic acid with 2,4,5-trichlorophenol in the presence of pyridine or by treating the free alkoxy or alkenecarboxylic acid with 2,4,5-trichlorophen-1111a N, N'-dicyclohexylcarbodiimide acid. The 2,4,5-trichlorophenyl ester derivative can be purified by flash chromatography on silica gel.

The N-alkanoylamino acids or N-alkenoylamino acids 5 used as starting materials in the preparation of the derivatives of formula (1) disclosed in Patent Applications 831,746 and 831,747 are known compounds or can be prepared by acylation of a suitable amino acid with a desired alkanoyl or alkenoyl group according to conventional methods. A preferred way to prepare N-alkanoylamino acids is by treatment of the appropriate amino acid with alkanecarboxylic acid chloride in pyridine. The time or alkenecarboxylic acids used, their activated derivatives and amino acids are known compounds or can be prepared by known methods or modifications of known methods which will be apparent to a person skilled in the art.

If the amino acid in question contains an acylatable functional group in addition to the amino group, it will be apparent to one skilled in the art that such a group must be protected before the amino acid is reacted with the N-alkanoyl group or the reagent used to attach the N-alkenoyl group. Suitable protecting groups are all groups known in the art to be used in peptide synthesis to protect a side chain functional group. Such groups are well known and the selection of the group in question and its mode of use will be readily made by one skilled in the art, [see, e.g., "Protective Groups in Organic Chemistry", M. McOmie, ed., Plenum Press, N.Y., 1973].

Certain amino acids used in the synthesis of these products may exist in optically active forms. Both the naturally occurring configuration (L-configuration) and the non-naturally occurring configuration (D-configuration) are useful as starting materials.

The substituted benzoic acids and their activated derivatives used as starting materials in the preparation of a few A-21978C derivatives are known compounds or can be prepared from known compounds by methods known in the art. Alkoxybenzoic acids can be prepared simply from the appropriate hydroxybenzoic acid by reacting the appropriate alkyl halide with the disodium salt of the appropriate hydroxybenzoic acid.

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

Derivatives of the intermediates of this invention, i.e. the novel compounds of formula 1 ', i. the compounds disclosed in patent applications 831,746, 831,747 and 831,756 inhibit the growth of pathogenic bacteria as demonstrated by standard biological experiments. Compounds 15 are therefore useful for inhibiting bacterial growth on environmental surfaces (as an antiseptic) or for treating bacterial infections. The antibacterial activity of the compounds has been demonstrated in in vitro agar plates in strip diffusion experiments and agar dilution experiments, as well as in vivo experiments in mice infected with Staphylococcus aureus and Streptococcus pyogenes. The compounds are particularly useful in the treatment of infections caused by gram-positive organisms.

When the above cyclic A-21978C peptide derivatives are used as an antibacterial agent, they can be administered either orally or parenterally. As will be appreciated by those skilled in the art, A-21978C is usually administered in association with a pharmaceutically acceptable carrier or diluent. The dosage of A-21978C depends on various factors, such as the nature and severity of the infection being treated. As will be appreciated by those skilled in the art, appropriate dosage ranges and / or dosage units can be determined from the MIC and ED50 values and toxicity data provided, in conjunction with factors such as the pharmacokinetics and characteristics of the patient or microbial carrier or infectious microorganism.

The following examples are provided to more fully illustrate the invention.

5 Example 1 Preparation of A-21978C core A. Fermentation of Actinoplanes utahensis

A stock culture of Actinoplanes utahensis NRRL 12052 is prepared and stored on an agar slant.

10 The medium used for the preparation of the slant medium is selected from the following:

Medium A

Ingredient Quantity

Pre-cooked oatmeal 60.0 g 15 Yeast 2.5 g K2HPO4 1.0 g

Czapek Mineral Nutrition Solution * 5.0 ml

Agar 25.0 g of deionized water is made up to 1 liter with a pH of about 5.9 before being placed in an autoclave; adjusted to pH 7.2 by adding NaOH; after autoclaving, the pH is about 6.7.

* Czapek mineral nutrient solution has the following composition:

Ingredient Quantity

FeSO 4 .7H 2 O (dissolved in 2 ml of concentrated HCl) 2 g KCl 100 g 30 MgSO 4. 7H2 0 100 g

Deionized water is made up to 1 liter of Medium B

Ingredient Quantity

Potato dextrin 5.0 g 35 Yeast extract 0.5 g i.e. 79545

Casein enzymatic hydrolyzate * 3.0 g

Meat extract 0.5 g

Glucose 12.5 g 5 Corn starch 5.0 g

Meat peptone 5.0 g

Sugar cane molasses 2.5 g

MgSO 4. 7H 2 O 0.25 g

CaCO3 1.0 g 10 Czapek mineral nutrient solution 2.0 ml

Agar 20.0 g

Deionized water is made up to 1 liter * N-Z-Amine A, Humko Sheffield Chemical, Lyndhurst, N.J.

The oblique culture is inoculated with Actinoplanes utahensis 15 NRRL 12052 and the inoculated oblique culture is incubated at 30 ° C for 8-10 days. Approximately half of the oblique crop is inoculated with 50 ml of medium having the following composition:

Ingredient Amount 20 Pre-cooked oatmeal 20.0 g

Cane sugar 20.0 g

Yeast 2.5 g

Distiller's Dried Grain * 5.0 g K2HPO4 1.0 g 25 Czapek Mineral Nutrition Solution 5.0 ml

Make up to 1 liter with deionised water Adjust the pH to 7,4 with NaOH; after autoclaving, the pH is about 6.8.

♦ National Distillers Products Co., 99 Park Ave., New York, 30 N.Y.

The inoculated plant medium is incubated in a 250 ml wide conical flask at 30 ° C for about 72 hours on a shaker rotating in a 5.1 cm diameter arc at 250 rpm.

35 This incubated plant medium can be used 3i 79545 directly for inoculation of a second lie plant medium. Alternatively and preferably, it can be stored for later use by storing the culture in a liquid nitrogen atmosphere. For such storage, the culture is prepared in several small flasks as follows: Transfer to each flask 2 ml of Incubated Growth Medium and 2 ml of Glycerol-Lactose Solution [see WA Dalley and CE Higgens, Preservation and Storage of Microorganisms In the Gas Phase of Llguld Nitro-10 for details. gen, Cryobiol 10, 364-367 (1973)]. The prepared suspensions are stored in a liquid nitrogen atmosphere.

The stored suspension thus prepared (1 ml) is used for inoculating 50 ml of the first stage plant growth medium (having the composition previously described). The inoculated growth medium of the first 15 steps is incubated as described above.

To obtain larger amounts, inoculate 10. . ml of the first stage incubated medium 400 ml of the second stage medium having the same composition as the first stage plant medium. The second stage medium is incubated in a wide-mouthed two-liter Erlenmeyer flask at 30 ° C for about 48 hours on a shaker rotating in a 5.1 cm diameter arc at 250 rpm.

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

Culture medium I

30 Ingredient Amount (g / 1)

Peanut flour 10.0

Soluble leptapeptone 5.0

Cane sugar 20.0 KH2PO4 0.5 35 K2HPO4 1.2 32 7 9 5 4 5

MgSO0.7H, 0.25

Make up to 1 liter with tap water

The pH of the medium is about 6.9 after sterilization in an autoclave at 121 ° C for 45 minutes at a pressure of about 1.10 to 1.24 bar.

Growth medium II

Ingredient Amount (g / 1)

Cane sugar 30.0

Peptones 5.0 10 K2HPO4 1.0 KCl 0.5

MgSO 4. 7H2 0 0.5

FeSO4.7H2 0 0.002

Deionized water made up to 1 liter Adjust the pH to 7.0 with HCl; after autoclaving, the pH is about 7.0.

Growth medium III

Ingredient Amount (g / 1)

Glucose 20.0 20 NH4Cl 3.0

Naa S04 2.0

ZnCl 2 0.019

MgCl2.6H2O 0.304

FeCl3. 6H2 0 0.062 25 MnCl2.4H2O 0.035

CuCl2 .2H2 0 0.005

CaCO 3 6.0 JH 2 PO 4 * 0.67

Make up to 1 liter with tap water * Sterilize separately and add aseptically. Final pH about 6.6.

The inoculated production medium is fermented in a 14-liter fermentation tank at a temperature of about 30 ° C for about 66 hours. The fermentation mixture is agitated with conventional agitators at about 600 rpm and is aerated with sterile air to keep the dissolved oxygen concentration above 30% of the air-saturated normal pressure.

B. Deacylation of A-21978C 5 A. utahensis is fermented as described in A using sloping medium A and production medium I and incubating the production medium for about 67 hours. Crude A-21978C complex (100 g) is added to the fermentation broth.

Deacylation of the A-21978C complex is monitored experimentally against Micrococcus luteus. The fermentation is allowed to continue until deacylation is complete, which is manifested by the loss of potency against M. luteus, a period of about 24 hours.

This same method was used to examine whether other microorganisms would produce the desired A-21978C core. In particular, strains of Actinoplanes missouriensis NRRL 12053, Actinoplanes sp. NRRL 8122, Actinoplanes sp. NRRL 12065 and Streptosporangium roseum var. hollandesis 20 NRRL 12064 was found to produce the desired core when used in place of the Actinoplanes utahensis NRRL 12052 strain in the method described above. HPLC comparative experiments using as authentic samples the product obtained by using A. utahensis strain as deacylation enzyme in this method confirmed that these other microorganisms produced the A-21978C core.

C. Isolation of the A-21978C core

The entire fermentation broth (20 liters) obtained as described in Section B was filtered through a filter (Hyflo Super-Cel, Johns Manville Corp.). The mycelium cake was removed. 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 liquid that flowed through was discarded. The column was then washed with deionized water (10 L) to remove residual filtrate broth. This wash water was discarded. The column was then eluted with water / acetonitrile mixtures (10 L each, 95: 5, 9: 1 and 4: 1) and collected in 1 L fractions.

Elution was monitored by analytical HPLC using silica gel / CH 2 Cl 2 and a solvent system of water / methanol (3: 1) containing 0.1% ammonium acetate, the desired core was detected by UV detection at 254 nm. The core-containing fractions were combined, 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 C was dissolved in 75 ml of a 15 water / methanol / acetonitrile mixture (82: 10: 8) containing 0.2% acetic acid and 0, 8% pyridine. This solution was pumped onto a 4.7 x 192 cm column containing 3.33 L of silica gel (Quantum LP-1) / C18. The column was developed with the same solvent system. Fractions with a volume of 350 ml were collected. Separation was monitored at 280 nm with a UV detector. The fractions containing the core were combined, concentrated in vacuo to remove solvents and lyophilized to give 5.2 g of purified A-21978C core.

25 Example 2

Alternative Method for Preparing the A-21978C Core The A-21978C core was prepared according to the method of Example 1, except for some modifications in Step B.

The A. utahensis culture was initially incubated for about 48 hours; Substrate 30 was a semi-pure A-21978C complex (50 g); and incubated for about 16 hours after substrate addition. The nutrient solution filtrate was passed through a column containing 3.1 liters of HP-20 resin. The column was washed with 10 volumes of water and then eluted with water / acetone (95: 5). The elution was followed as in Example 1. After collecting 35 79545 liters, the elution solvent was converted to a water / acetonitrile mixture (9: 1). The core-containing fractions eluted with this solvent. These fractions were combined, concentrated to remove acetonitrile and freeze-dried to give 24.3 g of semi-pure A-21978C core.

This semi-pure A-21978C core (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 liters of bridge gel (Quantum 10 LP-1) / C1B prepared in a water / methanol / acetonitrile mixture (8: 1: 1). containing 0.2% acetic acid and 0.8% pyridine. At the same time, the column was developed as a solvent at a pressure of about 138 bar and collected in 350 ml fractions. Elution was monitored by UV at 280 nm. Fractions containing the desired core were combined, concentrated in vacuo to remove solvents and lyophilized to give 14 g of high purity A-21978C core.

Example 3 Preparation of N, __-t-BOC-A-21978C Factors C, and O A mixture of A-21978C Factors C2 and C3 (10 g) prepared as described in U.S. Patent 4,208,403. was dissolved in water (50 ml) in an ultrasonic stirrer (200 mg / ml). The pH of the solution was adjusted from 4.05 to 9.5% with 5N NaOH (3.6 mL). Di-tert-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 manually adding 5N NaOH (6.5 mL added over 2 hours).

The reaction was monitored periodically by TLC on silica gel using CH 3 CH / H 2 O solvent systems (7: 3 and 8: 2) and a UV detector.

After about 10 minutes, the reaction mixture suddenly became cloudy and base consumption increased. After 30 minutes, the rate of increase in turbidity 35 and the rate of base consumption decreased by 36,79545, indicating that the reaction was complete. However, the reaction was continued for another 90 minutes to ensure completion of the reaction. After the reaction for 2 hours, the reaction product was immediately lyophilized to give 12.7 g of 5 NO-t-BOC-A-21978 factors C2 and C3.

Using similar methods, two 10 gram reactions and a 40 gram reaction were performed. In all of these, the reaction time was only 40 minutes. Two 10 g experiments gave 11.9 and 12.1 g of product, respectively. The 30 g reaction gave 35.4 g of product.

Example 4 Preparation of A-21978C-N ', n-t-BOC core Fermentation of A. utahensis A. utahensis was fermented as described in Example 15 (A) using oblique culture medium A and production medium I and incubating the production medium for about 66 hours.

B. Deacylation of the N-tert-BOC Complex A-21978C-N-rn-t-BOC complex (1185 g of impure 20 substrate containing about 176 g of A-21978C complex) was added to the fermentation broth. Deacylated as described in Example 1, Section B. Deacylation 011 complete after about 24 hours, which was detected by HPLC.

25 C. Isolation of A-21978C-N „rn-t-BOC core

The fermentation broth (100 L) obtained as described in B was filtered through a filter (Hyflo Super-cel). The filtrate was passed through a column containing 7.5 L of HP-20 resin (DIAION), the column was washed with water (38 L).

Elution was monitored by silica gel / C 8 O 8 HPLC as a detector at UV 254 nm. Some core eluted in the wash. Elution of the nucleus after washing was performed with water / acetonitrile mixtures as follows: (95: 5) 40 L; (9: 1) to 40 L and (85:15) to 100 L. The core-containing fractions were combined, concentrated in vacuo to remove the solvent, and freeze-dried to give 79845 g of 208.5 g of semi-pure A-21978C-NO 2. BOC core.

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

The semi-pure A-21978C-NO-t-BOC core (30 g) obtained as described in C was dissolved in a water / acetonitrile mixture (9: 1) containing 0.2% acetic acid and 0, 8% pyridine (75 ml). This solution was run on a 4.7 x 192-cm steel column containing 3.33 L of silica gel (Quantum LP-1) / Cie equilibrated with the same -10a solvent system. The column was developed under pressure with water / acetonitrile / methanol (80: 15: 5) containing 0.2% acetic acid and 0.8% pyridine to collect 350 ml fractions and the product was detected by UV at 280 nm. :in. Fractions containing product were combined, concentrated in vacuo to remove the solvent, and lyophilized to give 18.4 g of pure A-21978C-NO-t-BOC core.

Example 5

Alternative Purification of the A-21978C-N'--t-BOC Core The semi-pure A-21978C-N-rn-t-BOC core (10.8 g) obtained as described in Example 4 (C) was dissolved in water and run to a column containing 80 ml of Amberlite IRA-68 (acetate step). The column was washed with water and eluted successively with 0.05 N acetic acid (1080 ml), 0.1 N acetic acid (840 ml) and 0.2 N acetic acid (3120 ml) at a flow rate of 5 ml / min, and collected in 120 ml fractions. . The column was monitored by analytical HPLC on silica gel / C18 using a water / acetonitrile / methanol system (80: 15: 5) containing 0.2% ammonium acetate and detecting the product at UV 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 in vacuo to a volume of about 200 ml. Water was added to the concentrate and the resulting solution was reconcentrated to remove pyridine. This concentrate was lyophilized to give 3.46 g of purified A-21978C-NO-t-BOC core.

Example 6

The following process illustrates the preparation of compounds of formula 1 disclosed in Patent Application 831,756 by a process using an "active ester". The individual compounds prepared by this method are compounds of formula 1 wherein R is CH 3 (CH 2) 8 CO- (n-decanoyl), R 1 is hydrogen and R 2 is t-BOC or hydrogen.

Preparation of NT r p- (n-decanoyl) -A-21978C nucleus A. 2,4,5-trichlorophenyl-n-decanoate

A solution of decanoyl chloride (Pfalz and Bauer, 5.6 ml), 2,4,5-trichlorophenol (5.6 g), diethyl ether (1 L) and pyridine (120 ml) is stirred for 4 hours. The reaction mixture is filtered and dried in vacuo.

2,4,5-Trichlorophenyl n-decanoate is purified on a silica gel column (Woelm) using toluene as eluent. Fractions are monitored by TLC using short UV-20 UV. The correct fractions are combined and dried in vacuo to give 10.4 g of 2,4,5-trichlorophenyl decanoate.

: Acylation of B, N'r_-t-BOC-A-21978C Core with 2,4,5-Trichlorophenyl-n-decanoate 25 A solution of N-rn-t-BOA-A-21978C core (15.0 g) and 2,4,5-trichlorophenyl n-decanoate (15.0 g) in dry DMF (500 ml) are stirred under nitrogen at room temperature for 25 hours. The mixture is then stirred at 60 ° C for 5 hours, i.e. until TLC shows that the reaction is complete. The reaction mixture is concentrated in vacuo to about 200 mL and stirred in 1.2 liters of Et 2 O / toluene (5: 1). The product is filtered off, washed with Et 2 O and dried in vacuo to give 15.05 g of NTrp- (n-decanoyl-1) -NOrn-t-BOC-A-21978C nuclear intermediate (Formula 1: R * 35 n-decanoyl, R1, H, R2 = t-BOC).

39 79545 C. Purification of N, r p- (n-decanoyl ±) -N „r nt-BOC-A-21978C nucleus NT r p- (n-decanoyl) -NO rn-t-BOC-A-21978C - the core intermediate is purified as follows: The crude product is dissolved in about 50 ml of an elution solvent system and this is purified by HPLC using a Waters Prep 500 system containing a cartridge packed with reversed phase C 6 e 6 silica gel. 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 by UV at 280 nm. The correct fractions are combined and dried in vacuo to give 8.56 g of purified NTrp- (n-decanoyl) -NOrn-t-BOC-A-21978C core.

D. N '. Removal of the t-BOC group The t-BOC group is removed by stirring the NT rp - (undecanoyl) -NOrn- (BOC-A-21978C core (1.47 g) in 15 ml of trifluoroacetic acid / - 1,2-ethanediol (10: 1) at room temperature for 3 minutes, the reaction mixture is dried in vacuo and the residue is triturated with Et 2 O (50 mL) After washing the tri-20 turate with 20 mL of Et 2 O, it is dried in vacuo. to give 2.59 g of NTrp- (n-decanoyl) -A-21978C core as a crude product (formula 1: R = n-decanoyl; R1 and R2 = H).

E. Purification of NT r p- (n-decanoyl) -A-21978C Core The crude NIrp- (n-decanoyl) -A-21978C core is purified by reverse phase HPLC as follows: Sample (2.59 g) dissolved in 4.0 ml of a mixture of H 2 O / MeOH / CH 2 -CN / pyridine / HOAc (50: 15: 35: 2: 2), sprayed onto an 84- x 2.54-cm stainless steel column with LP-1 / C18 absorbent. The column is eluted with the same solvent system. Elution is performed at a pressure of 82.7 to 117.2 bar at a flow rate of 10-12 ml / min using a LOC duplex pump (Milton-Roy). The effluent is monitored by a UV detector (Inco Model UA-5, Instrument Spe-35 cialist Co., 4700 Superior Avenue, Lincoln, Nebraska 40 7 9 5 4 5 68504) at 280 nm. Fractions (20.24 ml) are taken every other minute. Fractions found to be desirable based on antimicrobial activity are combined and dried in vacuo to give 1.05 g of product.

This purification procedure was repeated using 4.35 g, 4.25 g, 2.14 g, 2.00 g and 1.75 g of crude starting material derivative to give a total of 5.58 g of purified NT r p- (n-decanoyl) -A-21978C core.

Example 7 This example illustrates the preparation of compounds of formula 1 disclosed in patent applications 831746 and 831747. The individual compounds prepared by this method are compounds of formula 1 wherein R is N- (n-decanoyl) -L-phenylalanyl and R 2 is t-BOC or hydrogen.

Preparation of NT r - - [N- (n-decanoyl) -L-phenylalanyl] -A-21978C core A. Preparation of N- (n-decanoyl) -L-phenylanyl-2,4,5-trichlorophenolate A solution of N- (n-decanoyl) -L-phenylalanine (31.9 g, 0.1 mol) and 2,4,5-trichlorophenol (19.7 g, 0.1 mol) in 1 liter of anhydrous ether, treated with N, N'-dicyclohexylcarbodiimide (20.6 g 0.1 mol). The reaction mixture was stirred overnight at room temperature. The precipitated N, N * -dicyclohexylurea was removed by filtration. The filtrate was evaporated to dryness in vacuo. The resulting residue was triturated with ether and the solid (residual cyclohexylurea) was removed by filtration. The filtrate was evaporated to dryness under reduced pressure.

The residue was crystallized from acetonitrile to give 36.9 g of crystalline N- (n-decanoyl) -L-phenylalanyl-2,4,5-trichlorophenolate, m.p. 122-124eC.

B. Preparation of N, r p- [N- (n-decanoyl) -L-phenylalanyl] -N'R nt-BOC-A-21978C core 35 A solution of N- (n-decanoyl) -L-de 4i 79545 Nylalanyl 2,4,5-trichlorophenolate (10 g, 0.02 mol) and NOrn-t-BOC-A-21978C core (10 g, 01006 mol) in anhydrous DMF (1 L) were stirred at room temperature for 96 hours under nitrogen. The solvent was removed by evaporation under reduced pressure. The residual material was stirred in a mixture of diethyl ether (800 ml) and chloroform (200 ml) for 2 hours. The product was isolated by filtration to give a light brown powder (10.3 g). This material (9.9 g) was dissolved in methanol (200 mL) and purified by preparative HPLC using a "Prep LC / System 500" PrepPak-500 / C18 column as the stationary phase. The column was eluted isocratically using water / methanol / acetonitrile (2: 1: 2) as the solvent system and 250 ml fractions were collected at a rate of one fraction per minute. The desired compound eluted in fractions 9-22.

Fractions were combined by TLC [reverse phase silica gel / C18; development with water / methanol / acetonitrile (3: 3: 4); expression by Van Urk spray]. The combined fractions were examined by bioautography [silica gel-20 TLC, solvent system acetonitrile / acetone / water (2: 2: 1) and Micrococcus luteus as detection organism] and proved to consist of a single bioactive component. This method gave 6.02 g of NTrp- [N- (n-decanoyl) -L-phenylalanyl] -NOrn-t-BOC-A-21978C core compound of formula 1: R = N- (n-decanoyl-L phenylalanyl); R2 - t-BOC].

Preparation of C._NT pp- [N- (n-decanoyl) -L-phenylalananyl] -A-21978C core

The flask (100 mL) was cooled to 5 ° C in an ice bath. NTrp- [N- (n-decanoyl) -L-phenylalanyl] -N-rn-t-BOC-A-21978C nucleus (6.02 g, 0.008 mol) prepared as described in B and then anhydrous trifluoroacetic acid containing 2% anlsol (50 ml) was added to the flask. The mixture, which became a solution in about two minutes, was stirred under nitrogen for 10 minutes. The solution was evaporated to dryness <2 79545 under reduced pressure below 40 ° C to give a gummy solid which was triturated twice with diethyl ether / dichloromethane (4: 1) (two 100 mL portions). The solid was collected by filtration and washed with diethyl ether to give the TFA salt. This was dissolved in water (50 ml) and the pH of the solution was adjusted to 5.4 with pyridine. The solution was then lyophilized to give 6.1 g of an off-white lyophilisate.

The lyophilisate dissolved in methanol (35 mL) was purified using a reverse phase C18 silica gel column (Waters Associates, Prep 500) eluting with a step gradient using solvent systems H 2 O / CH 3 OH / CH 3 CN containing 0.1% pyridinium acetate and solvent ratios of 3 v / v. : 1: 2, 2: 1: 2 and 1: 2: 2 and 250 ml fractions were collected. The desired product was eluted with a solvent ratio of 2: 1: 2. Fractions containing product were lyophilized to give 2.23 g of a cream-colored NTrp- [N- (n-decanoyl) -L-phenylalanyl] -A-21978C core (compound of formula 1: R = N- (n-decanoyl). ) -L-phenylalanyl; R2 (H).

The product was evaluated by analytical HPLC [reverse phase C18-silica gel column, MeOH / CH3 CN / H2O / PyOAc (15: 35: 49: 1) as solvent and UV detection at 230 nm, TLC [reverse phase -Cj 8 silica gel plates (Whatman), H 2 O / CH 3 OH / -25 CH 3 CN (3: 3: 4) as solvent and Van Urk spray and shortwave UV detection] and bioautography [silica gel TLC (Merck), solvent H 2 O / CH 3 CN / acetone (1: 2: 2) and Micrococcus luteus as a detection organism]. Each method showed the product to be homogeneous. The substitution at the N-terminus 30 of tryptophan was confirmed by 360 MHz PMR. Amino acid analysis confirmed that the product contained one equivalent of L-phenylalanine.

Example 8

The following method illustrates the preparation of other compounds of formula 1 disclosed in U.S. Patent Nos. 4,317,446 and 8,371,747. The individual compounds prepared by this method are compounds of formula 1 wherein R is p- (n-dodecyloxy) -benzoyl and R 2 is t-BOC or hydrogen.

Preparation of 5-N-dodecyloxy) benzoyl-N, N-t-BOC-A-21978C nucleus

A solution of p- (n-dodecyloxy) -benzoyl-1,4,5-trichlorophenolate (0.9 g / mol) and A-21978C-t-BOC nucleus (0.9 g, 1 mol) in 400 ml in anhydrous dimethylformamide, was stirred at room temperature for 120 hours under nitrogen. The solvent was removed by evaporation under reduced pressure. The residue was stirred with a mixture of diethyl ether (400 ml) and chloroform (400 ml) for 2 hours. The product was filtered off and dried to give a light brown powder (0.062 g). A portion of this material (0.78 g) was dissolved in methanol (200 mL) and purified by preparative HPLC using a "Prep LC / System 500" unit (Waters Associates, Inc. Milford Mass.) And Prep Pak-500 / C18. column (Waters Associates) as the statio-20 female phase. The column was used isocratically with water / methanol / acetonitrile (2: 1: 2) as the solvent system and 250 ml fractions (1 fraction / min) were taken. The desired product was eluted into fractions 2-6.

Fractions were pooled by TLC [reverse phase 25 Si / C18 silica gel, development with solvent system water / methanol / acetonitrile (3: 3: 4), detection by Van Urk spray]. Bioautography of the pooled fractions using yesterday's gel TLC, acetonitrile / acetone / water (2: 2: 1) as solvent and Staphylococcus aureus as the detection organism, showed that the product was a single bioactive component. This method gave 0.421 g of NTrp-p- (n-dodecyloxy) benzoyl-NO-n-t-BOC-A-21978C core.

Preparation of Bj._NT rr -p- (n-dodecyloxy) benzoyl-A-21978C core 35 NT r pp- (n-dodecyloxy) benzoyl-N0 r nt-BOC-44 79545 A21978C core (230 mg) was dissolved in 5 ml in trifluoroacetic acid containing 2% anisole and stirred for 5 minutes at 0 ° C. The solution was concentrated to an oil in vacuo and the oil was triturated with Et 2 O (100 mL). The solids were separated, air dried and taken up in water (10 mL). The pH of this solution was adjusted from 3.25 to 7 by the addition of pyridine. The resulting solution was lyophilized to give 179 mg of a white amorphous NTrp-β- (n-dodecyloxy) -benzoyl-A-21978C core. This compound has an Rf value of about 0.78 on silica gel TLC using the acetonitrile / acetone / water (2: 2: 1) solvent system and Van Urk spray for detection.

Example 9

The antibacterial activity of the compounds of formula 1 disclosed in patent applications 831,746, 831,747 and 831,576 can be demonstrated in vitro on agar plates in standard strip diffusion and agar dilution assays and in in vivo standard experiments in mice to show efficacy against systemic bacterial infection. . The results obtained when examining the antibacterial activity of the exemplified compounds of formula 1 are shown in Tables I, II and III.

Table I shows the results obtained by studying the compounds of Examples 6-8 in vitro on agar-25 by strip diffusion methods.

Table II shows the results obtained when testing the compounds of Examples 6-8 in standard agar dilution experiments. In Table II, activity is reported as the Millimeter Concentration (MIC), i.e. at the lowest concentration at which the compound inhibits the growth of the microorganism.

The results obtained in in vivo experiments evaluating the efficacy of the compounds against experimentally induced bacterial infections in mice are given in Table III. In these experiments, two doses of test compound-35 were administered subcutaneously or orally to mice with an infection of the example. The observed activity was measured as the ED 50 value [effective dose in mg / kg to protect 50% of the experimental animals, see Warren Wick, et al. J. Bacteriol. 81, 233-235 (1961)].

The toxicity of the exemplified compounds of formula 1 is shown in Table IV.

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Claims (5)

50 79545 A process for preparing a cyclic A-21978C core peptide of formula (1 ') 5 0 NH? 1 i / CH / \ / \ HO-C \ / J f Y N · Il! V '/ Χ / Ν. / Χ O 0 = /, V C ° NH2 h \ 1 β o H X n2 I «, <7. > - X iv '>, /' wX / 1 ° * ν \ = * oh, • —7 · pH3 OH HN S ho2c γΛΛ / νν- »2 o 1 I * ho2c 25 wherein R2 is hydrogen or an amino protecting group, and for the preparation of its salts, characterized in that the A-21978C complex, the A-21978C factor C0, Cx, C2, C3, C4 or C5, the blocked A-21978C complex or the blocked A-21978C factor C0, C ,, C2, C3, C4 or C5 of formula (1) si 79545 O NHR2 5 / \ / H3 / V \ il! ! 'ν' ίο h / C0NH2 h> ”-> y * w xi y YYYYvV HOsci wherein R is 8-methyldecanoyl, 10-methylundecanoyl, 10-methyldodecanoyl, a Cx0 alkanoyl group having the characteristic C0 of A-21978C or a C12 alkanoyl group having the characteristic of C4 and C5 of A-21978C, and R2 is or an amino protecting group, is deacylated enzymatically in an aqueous medium by a deacylating enzyme produced by a microorganism of the family Actinoplanaceae, which is Actinoplanes utahensis NRRL 12052, Streptosporangium ro-seum var. hollandensis NRRL 12064, Actinoplanes missou-riensis NRRL 12053, Actinoplanes sp. NRRL 12065 or Actinoplanes sp. NRRL 8122, if desired, the product thus obtained is deprotected with amino 35 R2, 52 79545 and, if desired, the product obtained is converted into a salt. The method according to claim 1, characterized in that the enzyme is present in a culture of the microorganism Actinoplanaceae producing it. Process according to Claims 1 or 2, characterized in that a compound of the formula (1 ') in which R 2 is hydrogen or a pharmaceutically acceptable salt thereof is prepared. Process according to Claim 1 or 2, characterized in that a compound of formula (1 ') is prepared in which R 2 is an amino-protecting group, or a pharmaceutically acceptable salt thereof. A compound of formula (1 ') as defined in claim 1. 53 79545