PL142112B1 - Process for preparing novel derivatives of cyclic peptides a-21978 c - Google Patents

Description

The present invention relates to a process for the preparation of novel derivatives of cyclic A-21978C peptides having antibiotic properties. Cyclic A-21978C peptides are related antibiotics described in U.S. Patent No. 4,208,403. As stated in this specification, the A-21978 antibiotic complex contains As the main component, factor C, which is itself a complex of related - factors. The factor C of the A-21978 complex, referred to as the A-21978C complex, includes the individual factors CQ, C1, C2, C-z, C6 and Cr. The main factors are C |, C2 and C, and Cq, C ^ and Cc are minor factors. The structure of factors A-21978C is shown in general formula 2, where 3MS is L-threo-3-methyl-loglutamic acid and R is a specific fatty acid group. NR in factors Cj, C2t C, CQ, C4 and Cc are respectively 8-Methylcapryoyl, 10-methyloundecanoyl, 10-methyllauroyl, CQ-alkanoyl, C12-alkanoyl and C12-alkanoyl, the latter three groups not yet identified. The abbreviations Ala, Asn, Asp, Gly, Kyn, Om, Ser, Thr and Trp are known and refer to the groups of alanine, asparagine, aspartic acid, glycine, kynurenine, ornithine, serine, threonine and tryptophan, respectively, for the sake of opportunity and of the constant danger. the emergence of strains of pathogenic microorganisms resistant to particular antibiotics, there is still a need to search for new antibiotics. In particular, the pathogenic gram-positive strains of Staphylococcus and Steeptooccus are often resistant to commonly used antibiotics, such as penicillin and erythromycin, as e.g. by W.O. Foye in "Principles of Medicinal Chemistry," pp. 684-686 (1976). It has now been found that novel derivatives of cyclic A-21978C peptides of formula 1 and their pharmacologically acceptable salts are effective antibiotics. 2 142 112. The method according to the invention produces new derivatives of the cyclic peptides A-21978C of the general formula I, in which R, R and R are the same or different and represent a hydrogen atom, an optionally substituted C2-C6-alkanoyl group, a C1-C6-alkenoyl group or an amino protecting group such as H1-tert-butoxycarbonyl or benzyloxycarbonyl, and R, R and R are hydrogen, where (1) at least one of R, R or R is not a hydrogen atom or an amino protecting group (2) at least one of R and R is hydrogen or an amino protecting group, (3) the R, R and R groups together contain at least four carbon atoms and (4) when R and R represent a hydrogen atom or an amino protecting group, then R is different from 8-methylcapryoyl, 10-methyloundecanoyl, 10-methyllauroyl, C.0-alkanoyl, CQ factor of peptide A-21978C or the C 1-4 alkanoyl groups of factors C. and Cc of peptide A- 21978C, and the pharmacologically acceptable salts of these compounds. These compounds are useful semi-synthetic antibacterials or intermediates in the preparation of such agents * The term "optionally substituted C2-C6-alkanoyl" and "C1-C8-alkenoyl" refer to acyl groups derived from of carboxylic acids having, respectively, 2-19 and 5-19 carbon atoms. • When R is an alkanoyl group, the alkyl radical which is part thereof is a monovalent, saturated, straight or branched hydrocarbon radical, optionally substituted with one hydroxyl, carboxyl group or C1-C6-alkoxy or with 1-3 halogen atoms, ie chlorine, bromine and fluorine. When R is an alkenyl group, the alkenyl radical is a monovalent, unsaturated, straight or branched hydrocarbon radical containing no more than three double bonds. A fragment or fragments with a double bond of an unsaturated hydrocarbon chain may have cis or trans configurations. Formula 1 includes: (a) compounds in which R is an alkanoyl group of the formula CHt / CH2 / - E0-, in which n is an integer of 3-17, (b) compounds in which R is an alkanoyl group of the formula CH -r / CH2 / n-C0-, where n is an integer 5-14, (c) compounds where R is an alkanoyl group of the formula CH3 / CH2 / nCT / CH, // CH2 / m-CO-, in which n and m independently denote an integer 0-14, provided that the sum of n and m cannot be less than 1 or greater than 15, and that when n is 0, then m cannot be 8 and when n is 1 , then n may not be 6 or 8, (d) a compound where R is cis- or trans-alkenoyl Words of formula CH, / CH2 / nCH »CH / CH2 / m-C0-t where n and m independently represent an integer 0-14, with the proviso that the sum of m and n may be less than 1 or more than 15, (e ) compounds in which R is a cis- or trans-alkenoyl group of the formula CH2 -CH / CH2 / n-C0-, in which n is an integer 4-15, (f) compounds in which R is groups of the formulas: CH3 / CH2 / e-C0-, CH-j / CH ^ -CO-, CH, / CH2 / 7- -C0-, CH5 / CH2 / 8-C0-, CH3 / CH2 / 9-C0-f CH ^ / CH 2/10-C0 ~ »CH2 * CH- / CH2 / Q-C0-, CH ^ / C ^ / ^ - CO-, CH3 / CH2 / 12-C0-f CH3- / CH2 / 3-CH» CH- / CH2 / 7-C0-, CH3 / CH2 / 13-C0-, CH3CH2CH / CH3 / - / CH2 / 6-CO-. The compounds of formula 1 are salt-forming and the salt-forming method is also within the scope of the invention. . Such salts are useful, for example, in the isolation and purification of compounds. Particularly useful are the pharmacologically acceptable alkali metal salts, alkaline earth metal salts, amine salts and acid addition salts. The compounds of formula I have four free carboxyl groups that can form a salt. Thus, it is within the scope of the invention to prepare partial, mixed and total salts of these carboxyl groups. When preparing these salts, a pH greater than 10 should be avoided due to the instability of the compounds in this range. Representative and suitable salts of compounds of formula I with alkali metals and alkaline earth metals include the sodium, potassium, lithium, cesium, rubidium, barium, calcium salt. and magnesium. Suitable ammonium salts of compounds of formula I are the ammonium salt and the primary, secondary and tertiary C 1 -C 6 -alkylammonium and hydroxy-C 2 -C 4 -alkyl ammonium salts. Examples of ammonium salts are those prepared by the reaction of a compound of formula 1 with ammonium hydroxide, methylamine, tertiary, -butylamine, isopropylamine, diethylamine9, diisopropylamine 9 cyclohexylamine, ethanolamine, triethylamine or 3-amino-1-propanol. they also have free amino groups and can form acid addition salts. The preparation of these salts is within the scope of the invention. Representative and suitable addition salts include the salts of organic and inorganic acids, such as, for example, hydrochloric, sulfuric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, palmitic, cholic acid. , pamoic, mucus, D-glutamine, d-camphor, glutaric, glycol, phthalic, tartaric, lauric, stearic, salicylic, methanesulfonic, benzenesulfonic, sorbic, picrin, benzoic or cinnamic. 21978C of general formula 1 and their pharmacologically acceptable salts consists in the fact that the core of a cyclic peptide of general formula 3, in which R 'and R ° are the same or different and represent a hydrogen atom or an amino protecting group having the meaning given above or its derivative protected with groups such as tert-butoxycarbonyl or benzyloxycarbonyl, optionally in the form of a farm a chemically acceptable salt, is reacted with an acylating agent of formula R -COOH, in which R is optionally substituted C1-C1Q-alkyl or C1-C1-C6-alkenyl, or with an active ester, anhydride or halide thereof acidic, then optionally removing any protecting group or groups possibly present in the product and optionally salifying the resulting compound. Compounds of formula I in which R, R or R are alkanoyl, alkenoyl or an amino protecting group are prepared by acylating factor A-21978C, protected factor A-21978C, a compound of formula III or a suitable compound of formula III containing the dC-amino group of tryptophan blocked with a predetermined alkanoyl or alkenoyl side chain. Conventional methods for amide bond formation are used. The acylation is generally carried out by reacting the selected compound with an active derivative of the alkanoic acid corresponding to the desired acyl group (R, R or R). The term "active derivative" means a derivative which confers a carboxyl group of the acylating agent with sufficient reactivity to couple with the primary amino group and form an amide linkage whereby the acyl side chain becomes attached to the core. Suitable active derivatives, methods for their preparation and use as acylating agents for primary amines are well known. The preferred active derivatives are acid halides, for example acid chlorides, acid anhydrides, for example alkoxy formic acid anhydrides, or active esters, for example the 2,4,5-trichlorophenyl ester. Another method of activating the carboxyl group is by reacting a carboxylic acid with a carbonyldiimide, for example N, N'-dicyclohexylcarbodiimide or N, N'-diisopropylcarbodiimide, to obtain an active intermediate which is not isolated due to instability. The reaction with the primary amine is then carried out in situ. The compounds of formula I are prepared by selective acylation using amino protecting groups. For example, when the starting compound is a compound of formula III, where R 'and R are hydrogen, both the α-amino tryptophan and the / 1-amino group of ornithine are acylated and the NT, NO-diacyl derivatives are formed. In order to obtain monoacylated derivatives on the αC-amino group of tryptophan, it is preferable to acylate a compound of formula III in which the ornithine group (R ° position) is blocked by a protecting group. Such starting compounds are preferably obtained by blocking factor A-21978C from acylation at this position. The aromine aminoacynurenine group is the least reactive of all three free amino groups in the A-21978C core. In acylation of kunerenin, the amino groups of tryptophan and ornithine are appropriately blocked, while acylation at the R ° and Rx positions usually does not block the amino group of kynurenine. 4 142 112 Schemes 1, 2 and 3 illustrate a general method for the preparation of compounds of formula 1, wherein 1 Either of R, R or R is an alkanoyl, alkenoyl or an amino-protecting group. In these schemes, x represents the rest of the A-21978C molecules, Nm represents the srupe = C-amino group of tryotophan, Nn represents the tT-amino group of ornithine; Nv represents T in the * -m p aromatic amino group of a kynurenine, R, R, R are the substituents defined above, Rm is the acyl group of the natural factor, and B, B are the amino protecting groups. Preferably, the NT-n-caprinoyl derivative of the compound of formula I is prepared by reacting n-capric acid or its ester, anhydride or halide with an NQ -I11-tert-butoxycarbonyl derivative of the formula III in which R = H and R o't -BOC. In Scheme 1, the NT-monoacyl derivative A-21978C shows the general formula 3 ', and the NT »NQ-diacyl derivative A-21978C shows the general formula 4'. NT derivatives, Nn-diacyl derivatives, in which the N "-acyl group is the same as in the natural factor A-21978C, is represented by the general formula 8. In Scheme 2 NT derivatives, N" -diacyl derivatives of A-21978C are represented by the general formula 10. obtained from compounds of formula 9. The NL_f NR-diacyl derivatives in which the N-acyl group is as in the natural factor A-21978C is represented by the general formula 12. These compounds are obtained after removal of the protecting group from the compound of general formula Formula 11. Compounds of general formulas 5, 6 and 7 are also shown in Scheme 1. In Scheme 3 NQ-monoacyl derivatives A-21978C shows general formula 18, NR-monoacyl derivatives A-21978C shows general formula 15 and N0rn derivatives » Nvn "diacyl A-21978C is represented by the general formula 20. Compounds of formula 20 are prepared from compounds of formula 19, prepared from the compound of formula 6, via compounds of formula 13, 16 and 17. Compounds of formula 15 are prepared from the compound of formula 6 through the compound of formula 1 Compounds of Formula 6 are also shown in Schemes 1 and 2. Compounds of Formula 1 in which one of R, R and R is an alkanoyl or alkenoyl group are preferably prepared using an active ester. A particularly preferred starting compound is the compound of formula III, wherein R "H and R" t-BOC, ie NORn-t-BOC A-219-8C core (tBOC core). The abbreviation t-BOC represents a tert-butoxycarbonyl group. A preferred acylating agent is the 2,4,5-trichlorophenyl ester of the target alkenecarboxylic acid or alkenecarboxylic acid. According to this variant of the process, excess active ester is reacted with the t-BOC core at room temperature in an inert organic solvent such as dimethyl formamide, tetrahydrofuran, ethyl ether or methylene chloride. The reaction time is not critical, but It is preferable to conduct the reaction within about 6-20 hours. After the reaction is complete, the solvent is removed and the residue is purified. A particularly preferred method of purification is reverse-phase liquid pressure chromatography (HPLC) using LP-1 / C 6 as the stationary phase and a mixture of water, methanol, acetonitrile, pyridine and acetic acid as the solvent system. The t-BOC group is removed by reaction with trifluoroacetic acid, anisole and triethylsilane or preferably with trifluoroacetic acid and 1,2-ethanediol, carried out at room temperature for about 3-5 minutes. After removal of the solvent, the residue is purified using HPLC with The acylation can also be carried out using a modified Schotten-Baumann method, in which the unprotected core is reacted with the acid chloride of the desired alkanoic or alkenecarboxylic acid in a mixture of pyridine and water. Excess acid chloride in an inert organic solvent, such as acetone, slowly added to a solution of the core in a mixture of pyridine and water (90:10). The unreacted acid chloride is isolated from the reaction product by extraction with an immiscible organic solvent, for example diethyl ether. The product is purified as described above by reverse-phase HPLC method. Salts of compounds of formula 1 are prepared in a known manner. 142 112 5 alkali metal and alkaline earth metal salts of compounds of formula 1 are prepared according to the usual methods for the preparation of cationic salts * For example, the free acid of formula I is dissolved in a suitable solvent such as warm methanol or ethanol, and then a solution of a stoichiometric amount of the inorganic base in aqueous solution of methanol is added. The resulting salt can be isolated by routine methods such as by pouring or evaporating the solvent. A good method of making salt is to use ion exchange resins. You can also make salts with organic amines in a similar way. For example, gaseous or liquid amine may be added to a solution of a compound of formula I in a suitable solvent such as ethanol, and then evaporated off the solvent and excess amine. Acid addition salts of compounds of formula I are prepared by conventional reaction with the appropriate organic acids or The cyclic peptides of the formula III used as starting compounds in the process of the invention are prepared biochemically. An antibiotic complex is prepared by the method described in US 4,208,4,039, which is then deacylated. It is extremely difficult to determine whether there is an enzyme that can be used to deacylate a peptide antibiotic. Finding such an enzyme is even more difficult when the antibiotic contains a cyclic peptide in its structure. Since enzymes exhibit a high degree of specificity, differences in the peptide moiety and in the substrate side chain affect the deacylation result. Moreover, many microorganisms produce a large number of peptidases that attack different sites of the peptide moiety. This often leads to a mixture of products. The enzyme used to deacylate the © c-amino group of tryptophan of the fatty acid side chain group may be produced by the Actinoplacaceae family, preferably by Actinoplanes utahensis NRR1 12052 or a variant thereof. any antibiotic selected from the group consisting of complex A-21978C, A-21978C, factor CQ, C 1 C 2, C 1 C 2 or C c, protected complex A-21978C and protected factors CQ, C f C2, C 1, are added to the culture of the microorganism. C or Cc of complex A-21978C, the terms "protected factors A-21978C" and "protected complex A-21978C" refer to individual factors A-21978C or mixtures of agents (complex) in which the amino group of omitin and / or kynurenine is protected. Preferably, the protecting group is located on the ornithine-amino group in an individual agent or mixture of agents. The cultures are incubated with the substrate until virtually complete deacylation. The corresponding cyclic peptide A-21978C obtained is separated from the fermentation broth using known methods. In the process of the above enzymatic deacylation, cyclic peptides of formula III are obtained, in which R 'and R independently represent a hydrogen atom or an amino protecting group, and the salts of these Cleavage of the acyl group from the A-21978C complex or the individual factors Cq, Cj, C2, C, C6 and Cc yields the compound of formula III where R 'and R ° are hydrogen. The deacylated molecule is a cyclic peptide common to each of the agents in antibiotic A-21978C. For simplicity, this compound is referred to as core C-21978C. It is a compound of Formula IV, in which 3MG is L-threo-3-methylglutamic acid. Compounds of Formula III, in which R 'or R ° are other than hydrogen, are prepared by deacylation of the corresponding protected CQ factors. , C1, C2, C1, C1 and Cc of the antibiotic A-21978C. For the sake of simplicity, the above compounds are referred to as the A-21978C secure core. Such protected compounds are useful intermediates for the preparation of various peptides of formula I, i.e. those compounds wherein at least one of R and R is an amino protecting group. 6 142 112 It is obvious to those skilled in the art that the A-21978C core and the protected core A-21978C can be obtained both as free amines and as acid addition salts. While any suitable addition salt may be used, pharmacologically acceptable salts, i.e. those useful in the chemotherapy of humans and warm-blooded animals, are preferred. The preparation of the A-21978C core of formula III using a variety of microorganisms is described in detail in the specification. U.S. Patent No. 4,482,487. Preferred deacylating enzyme-A. utahensis NRRL 12052- is available from the Agricultural Research Culture Collection (NRRL), Northern Regional Research Center, U.S. Department of Agricultur, 1815 North University St., Peoria, Illinois 61604, U.5.A., where it is registered under number NRRL 12052. Example I of this specification illustrates the preparation of the A-21978C core by a fermentation process using the substrate of the A-21978C complex and Actinoplanes utahensis NRRL 12052 as a microorganism. Other Actinoplanaceae available from the Northern Regional Research Laboratory, such as Actinoplanes utahensis (NRRL 12052), Actinoplanes missouriensis (NRRL 12053), Actinoplanes sp. (NRRL 8122), Actinoplanes sp. (NRRL 8122), can also be used to produce the A-21978C core of formula III. (NRRL 12065) and Streptosporangium roseum var. hollandensis (NRRL 12064). The suitability of a particular strain of the microorganism of the family Actinoplanaceae for desacylac.ii is determined as follows. A suitable growth medium is inoculated with the microorganism and incubated at a temperature of about 28 ° C for 2 or 3 days on a rotating jitter. A suitable antibiotic substrate is then added to the culture and the pH value of the fermentation broth is maintained at about 6.5. During fermentation, the activity is determined using the microorganism Micrococcus luteus. Loss of antibiotic activity indicates that the microorganism is producing the desired deacylation enzyme. However, this should be verified by one of the following methods: (1) HPLC analysis for the presence of an intact core, or (2) reacylation with a suitable side chain, e.g. lauroyl, n-caprinoyl or n-dodecanoyl Alkanecarboxylic and alkenecarboxylic acids and their derivatives, in particular acid chlorides and 2,4,5-trichloro-phenyl esters, used as starting compounds in the acylation reaction, are known compounds or can be prepared by known methods from known compounds . 2,4,5-trichlorophenyl esters are obtained by reacting an acid chloride of an alkane or alkenecarboxylic acid with a 2,4,5- "trichlorophenol in the presence of pyridine, or by reacting a free alkane or alkenecarboxylic acid with 2,4,5-trichlorophenol in the presence of N, N'-dicyclohexylcarbodiimide as a coupling agent. The 2,4,5-trichlorophenyl ester can then be purified by silica gel column chromatography. The cyclic A-21978C peptides prepared according to the invention can be administered orally as antibacterial agents. Compound A-21978C is administered together with a pharmacologically acceptable carrier or diluent. The dose of A-21978C depends on many factors, such as the type and severity of the infection to be treated. The appropriate dose levels or unit doses can be found in the Appendix for MIC values and EDC ~ and toxicity taking into account other factors such as pharmacokinetic data, patient characteristics and prohibition The antimicrobial activity of the compounds of formula I can be determined in vitro. The results of the tests for the antibacterial activity of representative compounds of formula I, obtained after standard diffusion tests using discs and agar plates, are summarized in Table 1. In Table 1, the activity is given as the diameter in mm of the zone in which the test compound inhibited the growth of the microorganism. the compounds were dispersed in water at a concentration of 1 mg / ml, a 7 mm diameter disk was immersed in the suspension, placed on the agar surface and incubated for 24-48 hours at 25-37 ° C. B.subtilis was grown on minimally nutrient agar. The dash, similarly to tables 2a and 3, means that the test with the given compound and the given microorganism was not performed. The abbreviation Obz stands for the group of benzyl-xycarbones Iowa.142 112 The results of the study of the antimicrobial activity of representative compounds of formula 1, obtained after standard agar dilution tests, are summarized in Tables 2 and 2a. In these tables, the antibiotic activity is presented as the minimum inhibitory concentration (MIC), that is, the lowest concentration of a compound at which inhibition of the growth of the microorganism occurs. The values for Compound Nos. 1-11 are given in Table 2 and the values for Compound Nos. 12-21 are given in Table 2a. The cyclic A-21978C peptides of formula I exhibit in vivo antimicrobial activity in experimental bacterial infections. Two doses of the test compound were administered subcutaneously or orally to experimentally infected mice, and the activity was determined as the EDcq value, or the effective dose in mg / kg, protecting 50 * test animals (see Warren Age et al., J. Bacteriol., 81). , 233-235 (1961) 7. ED5Q values for the representative compounds A-21978C of formula 1 are shown in Table 3. The results of toxicity studies of some cyclic peptides of formula I obtained after testing in mice administered intravenously with the test compounds. are shown in Table 4 The compound numbers in Tables 1-4 correspond to the compound numbers in Tables a, 8 and 10. T ab 1 ica 1 Compound R R1 R2 Inhibition zone size (mm) of Staphyl-Bacillus Microco- B. subticoceus substilis ccus lu- lis aureus ATCC teus ATCC ATCC 6633 ATCC 6633 6738P 9341 1 2 3 4 5 6 7 8 9 10 11 12 13 14 CH3 / CH2 / 5CO- CH3 / CH2 / 6CO- CH3 / CH277CO- CH3 / CH2 / 8C0- CH3 / CH2 / 9C0- CH3 / CH2 / 1QCO- CH3 / CH2 / 11C0- CH3 / CH2 / 12C0- CH3 / CH2 / 13CO- CH3 / CH2 / 14C0- CH2 «CH- / CH2 / 8CO-CH3 / CH2 / 3CH = CH / CH; ....., / tCO CH3CH2CH = CHCH2CH = CH- CH2CH = CH / CH2 / yCO- CH3CH2CH / CH3 / - / CH2 / 6C0- xi ac i a: ioi i! and: 1! HHHHHHHH - HHHHHHHHHHHHHHHH CH3C0- 17 23 20 24 19 25 21 22 20 19 21 22 15 21 15 18 "~ 16 20 19, \ 20 17 21 19 17 17 19 13 18 21 13 18 22 21 19 19 21 19 17 20 20 20 20 16 20 "~ 27 29 21 32 29 28 24 23 25 30 14 258 142 112 cd, table 1 15 j CH, aUCH / CH, / \ / CH2 / 6CO 16 {ch ^ / ch ^ cc- H HOCO / CRp / ^ I CO- ay.cl ^ / 6 J co- 17 J CH3 / CH2 / gCO 18 20 | CH3 / CH2 / 11C0 19 j ch3ch2ch / ch3 /! / ch2 / 6co! Obzx CH5 / d ^ / g | H! 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CM - CO - • every 1 • - • 1 1 bX 1 uui £ <2! 1 1 CM 1 i 1 * 1 1 CM 1 O 1 i cm ii 1 1 • »1 1 1 LAGO J 1 * 1 CM CM i 1 IA 1 t - * - lioio * A,} ——- 4 ——— ^ mii! °! ^.! 1 * 1 CM 1 1 O 1 ^ 1 ii ° • '1 1 * 1 1 4 | IA I 1 CM IO 1 f "" t " '1 1 1 LA | 1 l * | 1 CM 1 O 1 1 VO 1 (\ j 1 1 ^ ~ 1 | 1 v £ 1 OJ l! "T'" i 1 CM 1 CO 1 —I —- and VD lr 1 —f — j co i co i CM 1 CM 1 r Ir 1 «! S! ^ ioi Q i 0% i Pl ft i Pip 1 ca i ca i PIP i O 1 O | O 1 O 1 O 1 O 1 O | O 1 O 1 O 1 -PIP 1 Pi 1 P. 1 aiai U 1 U 1 fi! S! • _J142 112 <8 dPP PiS N co CCi Cti ^ Of-l C OHO d O 0) Jul OO * Cfl 00 cdi PPTJO NN * 2 - oo 9 cd cont. To cd142 112 11 Table 3 Compound i No 1 | CH ^ / CHg ^ CO- 2 I CH3 / CH2 / 6CO- j 3 j CH ^ O ^ / yCO- I- 4 and CH ^ / cWg / gCO- 5 | CH ^ CHg / gCO- 6 {CH3 / CH2 / 10CO- TT 2 i Values EE ^ (mg / kgx2)} staphylococcus Streptococcus .pyo genes • • aureus subcutaneously {oral 1.49 ~ 5.1a j - J subcutaneously H H I 2.65 I 3.75, 1.4 H H H H i i "HT" I 7 I CH3 / CH2 / 11CO- I H | I -I— I 0.5 0.28 0.37 _ I H 1 0.54 0.1.4, 0.243 j 92, 117 ----- 0.03 0.13 0.05 | 138 '- ——. ----- 0.046 45 CH3 / CH2 / 12C0- 1.17, 3.08 0.98, 0.20, <0.54 9 i CH ^ / a ^ / ^ CO- 10 j GH3 / CH ^ / 14C0- IH 8.3 -H 1.1 KO, 5, 0.18; 200 2D0 0.18 11 i CH2-CH- / CH2 / 8C0- 12 j ai3 / CH2 / 3ak CH / CHg / yCO- HH iii -r- ii - • - 0.93 0.068 0.134 138 ~ 75 163, 2D0 14 | ch3ch2ch / ch3 // c ^ / 6co- CH3C0- 1t3 15 I ch3ch2ch / cw3 // ch ^ / 6 (X) -! IIII 17 I CH3 / CH2 / 9CO- and H0C0- / Cl ^ / gCO- 6.27 1.4 JL CH-i / CHo / q "35 2, 85 aoo 200 a Two experiments b Three experiments Table 4 Compound | no i • ~ r - :: ± 1! 1 L_ »! 1 3 i 1 ^! 1 5! 1 b 1 R CH2 / CH2 / 5C0— CH3 / O ^ / gC0- CH3 / CH2 / 7C0- CH3 / CH2 / QC0- CH3 / CH2 / 9C0- CH3 / CH2 / 1QC0- I iti} iiiiiiiiiiiiii R1 "3 HHHHHH i ii ii ii i 1 1 ::: 5 :::::::::: j HHHH! 1 H! 1 H! 1 LD ^ (mg / kg) ":::::::: 5 ::: -. :::: 600 | Y 600 j 600 300; 600 144, 265a12 142 112 cont. table 4 j — 1 <1 7! 8 and | 9 1 | 10 "" 11 12 it ^ "l5 i 'i« "" 1 £ 2 1 1 CH3 / CH2 / 11C0- | gh3 / ct ^ / 12co- [CHj / CHg / ^ CO- | OT3 / C ^ 714C0- | CHg * CH- / CH ^ / qCO-! CH ^ / CHg / jCtfc CH / Ct ^ / 7CO- | I CHjCHgOl / CH ^ / CHg / gCO- I cH3ai2ai / cH3 // cH2 / 6co- I CH ^ / CHg / gCO-] I 17 [CHj / CHg / gOO- jp ::: 2 ~ ™ :::: j IH l H i HH! HH | H! H! H | H ~ "L -——.--- -———- 'HIH L _..____, | 62.5 | H \ 56 | H | ! H "| ¦ HI ri: CHjGO- T ^ 600 f | HOOO / CHg / gCO- CHj / 0H ^ / gCX- CBj / CK ^ CO-] *». J I »^« »» »M ^» «PTT3I 112.5 I <150 '| p ~ —2 ™ "" "| 50 i ≤ 500 I 450 ^ 600 J ^ 600 1 600 1 U "94 The invention is illustrated by the following examples, with Examples IV relating to the preparation of sibstrates. Example I. Preparation of A-21978C.A core * Actinoplanes utahensis fermentation * Actinoplanes utahenais stock cultures were prepared NRRL 12052 and stored on agar slants * One of the following nutrients was used to prepare the slants * Trill A «This brine rape as follows: pre-cooked oatmeal 60.0 g yeast 2.5 g KgHH ^ 1.0 g mineral nutrient Cap * * 5.0 g agar 25.0 g deionized water to 1 liter The pH value, about 5.9 before autoclaving, was adjusted to 7.2 with NsOH * B after autoclaving the pH was about 6.7 * ul. Mineral nutrient The cap had the following composition FfcSO ^ HgO 2 g (solution in 2 ml of concentrated hydrochloric acid) KO. 100g «gSO4 * 7 ^ 0 100 g deionized water up to 1 liter Phase B # This ethno label had a favorable composition: potato dextrin 5.0 g yeast extract 0.5 g enzymatic casein hydrolyzate * ' 3.0 g bovine extract 0.5 g glucose 12.5 g maize starch 5.0 g peptone monthly 5.0 g 142 112 13 molasses 2.5 g HeS04.7% 0 0.25 g CaOOj 1.0 g minerals Ciapka 2 .0-1 agar 20.0 g deionized water to 1 liter x / NZ Amine A, Humko Sheffield Chemical, Lyndhurst, NJ. Skos inoculated with Actinoplanes utahensis NRRL 12052 and incubated at 30 ° C for 8-10 days. About half of the cultivation on a slant was used to inoculate 50 µl of vegetative medium with the following composition: pre-cooked oat flour 2D90 g sucrose 20.0 g yeast 2.5 g dried grain fermentation residue x / 5.0 g KgHFO ^ 1.0 g mineral nutrient Cap 5.0 mL Demonized Water to 1 L x National Distillers Products Oo, 99 Park Ave *, New York, NT The pH was adjusted to 7.4 with HaOH. After autoclaving, the pH value was about 6.8%. The inoculated vegetative medium was incubated in a wide-necked Erlenmeyer flask with a capacity of 250 ml, at a temperature of 3 ° C, for about 72 hours, on a rotating jig with an arc diameter of about 50 mm and 250 revolutions / minute.Incubated vegetative medium can be used directly to inoculate second-stage vegetative medium * Alternatively and advantageously, these cultures can be stored for later use in liquid nitrogen vapor * From this stock, small vial cultures can be prepared as follows. Each vial is filled with 2 ml of fermented vegetable medium and 2 ml of lactose-glycerin solution [n.A. Dailey and CE * Higgens, "Prese- vation and Storage of Microorganisms in the Gas Riase of Liauid Nitrogen" Cryobiol. 10, 364-367 (LC73 / Ji). The obtained suspension is stored under liquid nitrogen vapor. * 1 ml of the stored suspension was used to inoculate 50 ml of the first stage vegetative medium with the composition given above. The inoculated first stage vegetative medium was incubated as described above. To prepare a large volume of inoculum, 10 ml of the first stage incubated vegetative medium was used to inoculate 400 ml of the second stage vegetative medium of the same composition as the first. The second stage tuber was incubated in a 2-liter wide-necked Erlenmeyer flask for about 48 hours at 30 ° C, on a rotating glass with an arc diameter of about 50 mm and 250 rpm. Incubated second-stage vegetative medium (80 ml), prepared as above, 10 liters of one of the sterile media below were inoculated. I, E This badge had the following composition, with the amount of ingredients given in g / liter. peanut flour 10.0 soluble meat peptone 5 * 0 sucrose 2o.0 KI ^ PO ^ 0.5 I ^ HPO ^ 1.2 MgSO ^ .7H20 0.25 tap water up to 1 liter The pH of the nutrient solution was up to 6.9 after uncooked in an autoclave at 121 ° C, within 45 minutes, under a pressure of approximately 110-123 kFa. 14 142 112 B patch II. The nutritional formula was as follows, with the amounts of ingredients given in g / liter * sucrose 30.0 peptone 5 f0 K2HP04 1 f0 KO. 0.5 MgSO 4, 7th) 0.5 FeSO 4 H 2 0.002 deionized water to 1 liter The pH was adjusted to 7.0 with hydrochloric acid * R after autoclaving the pH was about 7.0. R) Medium III. The formula was as follows, with the amounts of ingredients given in g / liter. glucose 20.0 mkd 3.0 Na ^ O ^ 2.0 ZnCl2 0.019 Mga2.6jy) 0.304 Fed5 »6H ^ 0 0.062 MnCl2.4fy) 0.035 CuCl2.2H ^ 0 0.005 CaCO ^ 6.0 Kl ^ P04 ** 0 , 67 tap water up to 1 liter. The final pH value was around 6.6. x) Separately quilted and added under sterile conditions. The inoculated production medium was fermented in a 14 liter fermentor at approximately 30 ° C for approximately 66 hours. The fermentation broth was mixed with standard agitators (about 600 rpm) and aerated with sterile air, keeping the dissolved oxygen level above the air saturation 30% at atmospheric pressure. B. Deacylation of A-21978C. A. Utahensis fermentations were carried out as in point A, using slanting medium A and production medium I, and incubating for about 67 hours. 100 g of crude A-21978C complex, prepared by the method described in US Patent No. 4,208,4} 3, was added to the fermentation environment. The deacylation process of the A-21978C complex was monitored by testing against Micrococcus luteus. The fermentation was continued until the deacylation was complete, as indicated by the disappearance of activity against M. luteus after approximately 24 hours. Extraction of the A-21978C core. The entire fermentation broth (20 liters) obtained at point B was filtered using a filter aid (Hyflo Super-Cel, John Manville Corp.). The mycelia were discarded and the filtrate passed through a column containing 1.5 liters of HP-20 resin (Diaion High Fbrous Palymer, HF-Series, Mitsubishi Chemical Jhdustries Limited, Tokyo, Japan). The column effluent was discarded and the column was washed with 10 liters of deionized water to remove residual filtered wort, and the water was discarded. The column was eluted sequentially with 10 liters of mixtures of water and acetonitrile (95: 5, 9: 1 and 4: 1), collecting fractions of 1 liter. The elution was monitored by paper chromatography, developing a mixture of n-butanol, pyridine, acetic acid and water (15: 10: 3: 12) and compounds were detected by UV fluorescence. In this system, factors A-21978C have an Rp of about 0.56 and core A-21978C of about 0.32. The product can also be detected using analytical HPLC on silica gel / 0.q and eluting with a mixture of water and methanol (3: 1) containing 0.196 ammonium acetate and the core was detected under UV radiation at 254 nm. removal of acetonitrile and lyophilized to give 40.6 g of semi-purified A-21978C.D core. Purification of the core of A-21978C. 15 g of the semi-purified core of A-21978C obtained at point C was dissolved in 75 ml of a mixture of water, methanol and acetonitrile (82: 10: 8) containing 0.2% acetic acid and 0.896 pyridine. The solution was pumped onto a 4.7 x 192 cm column containing 3.33 liters of silica gel (CU8) Quantum LP-1, Quantum Industries, 341 Kaplan rrive, Fairfield, NJ 07006) • For elution, the above mix is used Nine solvents and 350 ml fractions were collected. The course of the elution was monitored with a UV monitor at 280 nm. The faded fractions containing the core were concentrated in vacuo to remove solvents and then lyophilized to give 5.2 g of purified A-21978C.E core. A-21978C core characteristics. a) Fbstaci white amorphous solid, fluorescent under short wave UV radiation # b) Empirical formula: C62H83N17 ° 25 c) Molecular mass: 1465 d) Solubility: water-soluble substance e) Maximum absorption in infrared (KBr) occurs 3300 (wide band), 3042 (weak), 2909 (weak), 1655 (strong), 1530 (strong), 1451 (weak), 1399 (medium), 1222 (medium), 1165 (weak), 1063 (weak) i 758 (moderate to weak) cm "1 f) There are maxima in the ultraviolet absorption spectrum (methanol) at 223 nm (£ s41482) and 260 wounds (£ = 8687). g) Electrometric titration in 6896 in aqueous dimethylformamide shows the presence of four titratable groups with pK values of around 5.2, 6.7, 8.5 and 11t1 (initial pH 6.12). Pre clade II. An alternative method of making the A-21978C core. The A-21967C core was prepared in this way as in example I, introducing only some changes in the point Bi. A. utahensis cultures were initially incubated in agu for approximately 48 hours, the semi-purified complex A-21978C (50 g) was used as the substrate and incubated for approximately 16 hours after the addition of substrate. The wort drainer was passed through a column containing 3.1 liters of HP-20 resin. The column was washed with 10 volumes of water and then a mixture of water and acetonitrile (95: 5) was eluted, controlling its course as in Example I. R), the collection of 24 liters was changed to a mixture of water and acetonitrile (9: 1). eluting the core, the faded fractions containing the core were sedimented under vacuum to remove acetonitrile and freeze-dried to give 24.3 g of a semi-purified A-21978C core and 24.3 g of a semi-purified A-21978C core dissolved in water (400 ml) were fed by a pump. on a steel column 4.7 x 192 cm, containing 3.33 liters of silica gel (Quatum LF-1 / co) in a mixture of water, methanol and acetonitrile (8: 1: 1) containing 0.296 acetic acid and 0 8% pyridine. The column was eluted with the same solvent under a pressure of about 13,720 kPa, collecting 350 ml fractions. The course of the elution was monitored at 280 μl The pooled core fractions were concentrated under reduced pressure to remove solvents and freeze-dried to give 14 g of high purity A-21978C core. Example III. Preparation of factors C2 and C ^ NQrn-t-BOC-A-21978C. 10 g of a mixture of factors O, and C ~ A-21978C, prepared as described in U.S. Patent No. 4,208,4Q3, was dissolved in 50 ml of water. The pH of the solution was adjusted to pH 4.05 with 3.6 ml of 5N sodium hydroxide to 9.5. Then 3.0 ml of di-tert-butyl bicarbonate was added and the mixture was stirred for 2 hours at room temperature. The pH was kept at 9.5 while adding 6.5 ml of 5N sodium hydroxide solution during this time. The course of the reaction was checked periodically by thin layer chromatography on silica gel, using a mixture of acetonitrile and water (7: 3 and 8: 2) for development and detecting the product under UV.16 142 112 R after about 10 minutes, the solution rapidly turned loose and the consumption of base increased. Fb 30 minutes elapsed, the degree of increase in the turbidity and consumption of the malal base, which means the reaction was complete * Stirring but continued for an additional 90 minutes * R the completion of the reaction, a total time of 2 hours, the mixture was immediately lyophilized to obtain 12.7 g of a factor mixture of C ^ and C, N0rn-t-B0C-A-21978C * Using a similar procedure, two 10 g and one 30 g portions were reacted. «In each case the reaction time was only 40 minutes * 10 g portions yielded 11.9 and 12.1 g and a portion of 30 g gave 35f4 g of the product. Example IV. Core preparation N0rn-t-B0G-A-2197BC A. Fermentation of A. utahensis. A. utahensls fermentations were carried out as described in point A of example I, using slant growth medium A and production medium I, and incubating production medium for approximately 66 hours. B. Deacylation of the NQ ^ n-t-BOC complex. 1185 g of crude A-21978C-N-n-t-BOC complex containing about 176 g of A-21978C complex was added to the fermentation environment. The deacylation steps were performed as in item B of example I. The deacylation was complete after approximately 24 hours, as determined by HFLC.C. Isolation of the core N0rn-t-BOC-A-21978C * 100 liters of fermentation broth, obtained as described under B, was cleared through a filter aid (ityflo Super-cel). The ft * trap was passed through a column containing 7.5 liters of HP-20 resin (Dlaion), and the column was washed with 38 liters of water. KLuts were monitored by HPLC on silica gel / OQf using UV detection at 254 nm. Some core salt was found in the water. Then the core was eluted sequentially with 40 liters of a mixture of water and acetonitrile (95: 5), 40 liters of a mixture of water and acetonitrile (9: 1) and 100 liters of a mixture of water and acetonitrile (85:15) * the fused fractions containing the core were concentrated under reduced pressure to remove the solvent and freeze-dried. 298.5 g of the semi-purified core of NOrn-t "BOC-A" 21978C # 'D were obtained. Purification of the core of NOrn-t-B0OA-21978C. 30 g of the semi-purified N Cl -t -B0OA-21978C core obtained under point C was dissolved in 75 ml of a mixture of water and acetonitrile (9: 1) containing 0.2% acetic acid and 0.8% pyridine. The solution was applied to a 4.7 × 192 cm steel column, containing 3.33 liters of silica gel (Quantum UM / C-o), equilibrated with the same solvent system. The column was eluted under pressure with a mixture of water, acetonitrile and methanol (80: 15: 5) containing 0.2% acetic acid and 0.8% pyridine. Fractions of 350 ml were collected and the product was detected by UV at 280 nm. The pale product fractions were concentrated in vacuo to remove the solvent and lyophilized. There was obtained 18.4 g of purified Nq ^ -t-B0OA-21978C core with the following characteristics. a) Rstac: white, amorphous solid, fluorescent under short wave UV radiation. b) Empirical formula: ^ 7 ^^ 7027 c) Molecular mass: 1565 d) Solubility: water-soluble substance e) In the infrared absorption spectrum (KBr) there are absorption maxima at 3345 (wide band), 3065 (weak), 2975 (weak), 2936 (weak), <^ 1710 (inflection), 1660 (strong), 1530 (strong), 1452 (weak), 1395 (average), 1368 (weak). 1341 (weak), 1250 (average), 1228 (average), 1166 (average to weak), and 1063 (weak) cm ". F) The ultraviolet absorption spectrum (90% ethanol) has peaks at 220 nm ( ≤4.22,000) and 260 nm (≤10,600), g) The retention time in HPC is 6 minutes Column dimensions 4.6 x 300 nm, silica gel / Oiq, elution with a mixture of water, acetonitrile and methanol (80: 15: 5) containing 0.2% ammonium acetate, fast flow rate 2 ml / minute, detection in W.142 112 17 Example V, Alternative purification method for Nq ^ -t-BOC-A-21978C core. 10.8 g of a semi-purified Nc1-t-BOC-A-21978C core, prepared as described in C in example IV, was dissolved in water and applied to a column containing 80 ml of Amberlite IRA-68 (Rohm and Hass, Fhiladelphia, PA, Acetate form) The column was washed with water 1 at a rate of 5 ml / minute, eluted sequentially with 1060 ml 0.05 acetic acid. 840 ml 0.1N acetic acid and 3120 ml 0.2N acetic acid. 120 ml fractions were collected and the course of elution was monitored by HPLC on silica gel / Gjg with a mixture of water, acetonitrile and methanol (80.115: 5) containing 0.2% ammonium acetate and the product was detected by UV at 254 nm. the faded fractions containing the product, after adjusting the pH to 5.8 with pyridine, were concentrated under reduced pressure to a volume of about 200 ml. Water was added to the residue, and the resulting solution was concentrated to remove pyridine. Lyophilized, 3.46 g of the purified core NQ ^ nt-B0C-A-21978C was obtained. Example VI-XXXI Compounds of formula 1 are prepared by acylating compounds of formula 3 via the modified Schotten-Baumann fraction using an acid chloride as the acylating agent ( method A), alternatively using an active ester, i.e. 2,4,5-trichlorophenyl ester, as acylating agent (method B). The general procedure for carrying out the acylation reaction method A and method B are described below. Method A - Modified Schotten-Baumann reaction. The method consists in reacting the core of formula III with an alkane or alkenecarboxylic acid chloride corresponding to the given acyl side chain. Compound of formula 3 (1.92-2.16 g, 1.33-1.47 mmol) or a compound of formula 2 (409 mg, 0.25 mmol) is dissolved in 200 ml of a mixture of pyridine and water (9: 1), then the acylating agent (excess 18-20 mmol of acyl chloride in 15 ml) is added dropwise to the solution over 1-3 hours. The reaction mixture was stirred for an additional 2-3 hours at room temperature and then concentrated to remove acetone. The residual aqueous phase is diluted with water to about 200 ml, the pH value is adjusted to pH 3.0-3.5 with glacial acetic acid, washed 8 times with an equal volume of ethyl ether and lyophilized. The crude acyl derivative is purified by liquid pressure liquid chromatography. (HPLC) with reversed phase. For this purpose, the sample is dissolved in 4-6.5 ml of water or elution mixture and injected onto a column of approximately 84-1.6 cmf in size made of stainless steel and filled with LP * / c ^ g gel. The column is eluted with a mixture of water, methanol, acetonitrile, pyridine and acetic acid at a rate of about 10-12 ml / minute and a pressure of about 10 290 - 13 720 kPa using an LDC duplex pump (Milton-Roy). The leakage is monitored under UV at 280 nm using an ISC0-UA-5 detector. The desired fractions are combined and evaporated to dryness under reduced pressure to give the desired alkanoyl derivative. The purified product is analyzed by thin layer chromatography using reversed-phase plates (Whatman KC ^ Q) and for development with a mixture of water, methanol, acetonitrile, pyridine and acetic acid (45: 15: 0: 2: 2). is detected by observing the plates under UV light. The product can also be analyzed by measuring the extinction coefficients at 22 ° and 2G0 nm and with an amino acid analyzer. Purity is determined by reverse-phase HPLC (C11 Microbondapak, Waters Co.) using a mixture of water, methanol, acetonitrile, pyridine and acetic acid and monitoring the UV eluate at 280 nm. Method B - Use of active ester. 2f4,5-trichlorophenyl. NQrn-t- -B0C-A-21978C core (1.0 g, 0.64 mmol), A-21978C core (0.5-1.0 g, 0.34-0.68 mmol) or A-21978C (946 mgf 0.58 mmol) and a 3.5 molar excess of active trichlorophenyl ester are dissolved in 100 ml of dimethylformamide and stirred at about room temperature to about 50 ° C for about 6-20 hours. The reaction mixture is then concentrated under reduced pressure. The oily residue is triturated with 50 ml of a mixture of diethyl ether and toluene (1: 1) and washed with ether. The monoacyl and diacyl derivatives of the A-21978C core, the acyl A-21978C1 and the acylated T-B0C-21978C core are purified as in method A.18 142 112 The acylated t-B0C-A-21978C core is deblocked using a 50 mg / ml kwa mixture ¬ su trifluoroacetic acid, anisole and triethylsilane (10: 1: 1) and carry out the reactions at a temperature of about -10 to about 0 ° C for 3-5 minutes. The reaction mixture is concentrated under reduced pressure and the oily residue is rubbed twice with 20 ml of diethyl ether. The crude acylated product is purified using reverse-phase HPLC, analyzing as in method A. Conditions and parameters of the process for the preparation of the N, N-acyl-one derivatives and the N-f Ng-diacyl cyclic peptides A-21978C and the N-, N derivative. - diacyl compounds are given in Tables 5, 7 and 9f, respectively, while the physicochemical properties of Cyeh derivatives are given in Tables 6, 8 and 10, respectively. In Examples VI-XXI (Table 5), compounds of formula 1 are prepared, where R and R are hydrogen atoms. The compound N · N-acyl-H · t -BOC is used as the substrate. In Example XXIV (Table 7) the product is prepared by treating the core with succinic anhydride and pyridine 9096 (method C). In Example XXIX the product is prepared by using tert-butoxycarbonyl anhydride in water (method D). The R values in Tables 6, 8 and 10 were obtained by reverse phase thin layer chromatography analysis on silica gel (Whatman KC-g with indicator), using a mixture of water, methanol in acetonitrile (45:15:40) with the addition of 0.2% pyridine and 0.2% acetic acid as a developing system. Table 5 µm. and No. j 1 VI VII l ~ | Method I for the synthesis of CH ^ CH ^ CO- CH ^ CHg / gCO- Hosc of I core j (mg) | a i Amount i Time I HPLC reaction eluent for more (hours} intermediate substrate J 1028 1950 ka (mg) 800 and 20 3421 | i 3 "zk ¦ + '55:15:20 50:15:35 Product eluent J in HPLC i 50:15:35 55:15:30 amount (mg) 205 355" 264 "" 212 240 334 3921 VIII CH ^ / CHg / gCO- IX CH ^ / CHg / yCO- 1000 2000 350! 882 John 2 115: 15: 64! 115: 15: 64 \ untreated 50:15:35 CH3 / CH2 / 7CO- 1000 400 J 20 untreated 50:15:35 XI CH ^ / C ^ / gCO- 2160 4595 5 22 50:15 : 35 45:15:40 XII CH2 / CHg / cCO- 1000 288 XIII untreated! 45:15:40 i— 1 CH5 / CH2 / 10CO- 1000 3406 | 1 800 1 1 2 24 "45:15:40 j 45:15: to 1 1 40:15:45 370" 317 267 215 A 337.8 XIV a ^ / a ^ / ^ co- I b 1000 10:15 : 75 ¦ + - 1 1: 0: 1 ° + j 50:15:35 J 35: 10: 55 ° XV CH3 / CG2 / 12C0- | B 1000 812 22c 50:15:35 t- XVI XVII ch ^ o ^ / ^ co- CH ^ / CHg / ^ CO- B 1000 1000 900 1031 20 22L 60:15:35 not cleaned * i- XVIII | CHj / CHg / ^ CO- B j 1000 744 18L 1 ——1 XIX x not clean- J 2: 0: 3 on 1} cH ^ cH-Za ^ / gCO- r- •• B! 1000 237 20 20:15:65 T45: 15: * 0 257 290 XX CT ^ / CH ^ CH. CH / O ^ / yCO- XXI B J 1000 -i-- 1- CH, CHpCH = CHCHpCH = CH2CHOLCH / CHg / yCO- B j 1000 400 400 48 28 not purified! 40:15:45 35:15:50 j 35:15:50 1 1 1 —1- 1 1 1 215 283 142 112 19 data from table 5 a mixture of the given volumes of water with methanol and acetonitrile, containing 0.2% pyridine and 0, 2% acetic acid b reaction temperature 5 ° C c mixture containing 1% pyridine and 1% acetic acid Table 6 and Compound L. — __—- 1 1 p ———— 1 [5 r * i _ i! 8! 9, 1 1! 10! and i! R GH ^ CHg / jCO- CH ^ / CHg / gCO- CH ^ CHg / yCO- CH ^ / CHg / gCO- CHy ^ CH ^ CO- CH ^ / CHg / ^ CO- O ^ / O ^ / ^ CO- O ^ / O ^ / ^ CO- O ^ / O ^ / ^ CO- O ^ / O ^ / ^ CO-! 11 and CH 2 CH-Z 2 Cl 2 CO-1 and 12 and CH 2 CH 2 CH. CH /! | | / cV7co- i 13! CH 2 CH 6 CLCH CH 8 CH 2 CH-CH 8 OH | 1 / CT ^ / yCO- 1! H 1 H 1 H! h! H H H H H H H H H 1 j 1 1! 1 1 1 —t — ¦ 1 1 1 —U— 1 1 1 1 I j 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 R2 HHHHH "ii HHHHHHH i 1 j Product characteristics | R" J eluent w 1 1 analytical HPLC 1 0.85 j 60:15:25 | 0.80 J 60:15:25 | 0.75 "45:15: to t l_t j 0.70 S 45:15:40 0 , 65 and 45:15:40 0.59 1 45:15: ^ 0 0.53 1 45:15:40 0.42] 50: 0: 49. j 0.34 and 1 1: 0: 1 °; 1 1 0.25] 39: 0: 65 "| 1 1 0.70 | 45:15:40 |, i J 1 1 1 1 0.54 | 45: 15: ^ 0 j 1 1 ii 1 1 0, 50 j 45: 15: 40b | 1 1! UY 1 tjt max 220 nm | 48 100 | 46 900 46 000 46 500 46 200 44 000 48 500 48 447 .j 50 172 | i 54 000 ii 44 000 | i; 45 000 | i 1 1 1 50 000 1 1 1 1 1 <fj max i! C 260 nm J! 11 400 1, -1 10 500 j _J 11 000 j 11 000 j 10 400 j 9 500 j 9 800 I 9 170 j 1 9 751 | j 12 000 | 9 200} 1 1 9 600 j 11 600 | a mixture of the stated volumes of water, methanol and acetonitrile containing 0.296 pyridine and 0.2% acetic acid b mixture containing 1% pyridine 1 '\% acetic acid1tó 112 J- I ° P - SP H * «3 S • H | ON oj co p ^ HOxH O ^ O p SMM HO 01 WP ^ id i • oa CM 5 00 8 s% io ffi iiiiiiii K—— IT * I (0 I iH I ^ Lh ¦1 • 5 I ir \ 5 mo JO iiiiivia! S 5 i- i cm IfN ilil 12 II 15 I - imiv I •• - vO -I — ¦ + - ar * iiiii - ^ I CM I 81 io I KY I LTv IIIII 18 ITIOO mii 13 IOIIII 18 I r- ON 15 II CM I T - * - O ~ h- + 00 every IIII -I -I— C * 1 -I—. O ! 8 I r- m In O o a? ABOUT . o 18 I X I T ~ r * ii O I U vo i. er * JO | K »H- + lithium PQ PQ -u. i W I W U v0 | U vO Cl! Cl 8 4 -.- MHM LB- «A ii UIO III IIIII! I I 0 1 ¦ OOIU o I * - l KIK \ 15 15 II Ir- ¦ ii -r-— iiiii -tH II -4- ii I CM '• 8 18 I * - IIIU ioi t- ff \ «\ #! aT 15 \ i se ii I Ih IB IIIII IM a Ib is is is eiii £ 4-1 i II -o • oo I • oo as oomoc 1 N 3 0) (0 (U cd = 3 1 U142 112 21 Table 8 Compound i no. Iiiii 14 | CH3O ^ CH / CH3 / j / c ^ / 6co- 15! Ch3o ^ ch / ch3 / 16 j ch3 / o ^ / 6co- j 17 j CH3 / CH2 / 9CO- I -? - I 18 | cH3 / a ^ / 11co- CH3CO- -t HOCO / CHg / gCO CH3 / CH2 / 6CO- CH3 / CH2 / 9C0- CH3 / CH2 / 11CO- -4 Product characteristics S eluent in anallytic HPLC * 0.69 I 45:15:40 0f75 0.67 | 0.31 l- 45:15:40 45: 15: 40 * 4 30: 15: 55 ^ J 45: 15: 40D 0.03 | 45: 15: ^ 0B "" t IXmax la 220 nmj 260 46,000! 10,400 max 47,000 | 11,000 45,000 j 9,000 1 48 200 8,000 -i 49,000! 9,000 I 1 19 Sa ^ CHgCH / CHj /! I / CH ^ CO-; -! 20 I 4 .—— | Cbz I - * -—. HH t-BOC 1 l 1 1 - • - CH3 / CH2 / 10CO- j 0.58 | I Example No. and XXXI 1 1 1. a mixture of the given volumes of water, methanol and acetonitrile, containing 0.2% pyridine and Of 2, acetic acid, a mixture containing 1% pyridine and 1% acetic acid Table 9 1 1 1 1 1 1 1 -JR, R1 and C ^ / CHg / g - + - co-! Synthesis method "Quantity of substrate (g) -l- 15! hosc of acylating agent (g) 15 r j Reaction time (hours) Eluent in j Product HPLC for. , t-BOC- J eluent, j amount of "diacyl in HPLC j (mg) of the derivative 30 150: 15: 35 1 50:15:35! 211 a see example XXXIII b mixture of the stated volumes of water, methanol and acetonitrile" , containing 0.296 pyridine and 0.2% acetic acid Table 10 I Compound I | nr | i 1 1 1 i 21! a | CH3 / CH2 / 8C0- i 1 1! R! CHj / CHg / gCO-! R2 H i ! RF i 0.66 Product characteristics l 1 im 1 analytical £ Amax l L / lmax j n HPLCa J 220 nm j 260 nm 1 1 1 and 61: 15: 23: 1! 41 800, '11 500 1 1 1 I a mixture of the stated amounts of water, methanol, aceonitrile and ammonium acetate, containing 0.2% pyridine and 0.2% acetic acid 22 142 112 Example XXXII. Preparation of the core N ^ - / n-caprinoyl / -A-21978C (compound No. 4). The procedure described below illustrates the large-scale preparation of the compounds using an active ester. A. Preparation of 2,4,5-trichlorophenyl n-caprate. A solution of 5.6 ml of caprinoyl chloride (Pfaltz and Bauer) and 5 6 g of 2,4,5-trichlorophenol in 1 liter of diethyl ether and 120 ml of pyridine are stirred in iag 4 hours. The mixture is filtered and evaporated under reduced pressure. The obtained 2,4,5-trichlorophenyl n-caprate is purified on a silica gel column (Woelm), eluting with toluene, monitoring the fractions by thin-layer chromatography and using for detection. short-wave UV radiation. The faded product fractions were evaporated under reduced pressure to give 10.4 g of 2f4,5-trichlorophenyl n-caprate. B. Acylation of the core with N-, -t-BOC-A-21978C-n-caprate with 2,4,5-trichlorophenyl. A solution of 15.0 g of the N, t-BOC-A1, fsc core and 15.0 g of 2f495-trichlorophenyl n-caprate in 500 ml of anhydrous dimethylformamide was stirred under nitrogen at room temperature for 25 hours. The mixture is stirred for 5 hours at 6 ° C until the reaction is complete as determined by thin layer chromatography. The reaction mixture is concentrated under reduced pressure to a volume of about 200 ml and mixed with 1.2 liters of a mixture of ethyl ether and toluene (5: 1). The product is filtered off, washed with ether and dried in vacuo to give 15.05 g of crude N- (n-caprinoyl) -N-, nt-BOC-A-21978C core (Formula 1, ftsn-caprinoyl, R1). H, R2, t-BOC). C. N ™ Core Cleanup. - / n-caprinoyl / -N0rn-t-B0C-A-21978C. The crude core of N- (n-capricoyl) -NQrn-t-BOC-A-21978C is purified as follows: • The crude preparation is dissolved in approximately 50 ml of elution mixture and purified by HPLC using the Waters Prep system / 500 containing charges of a reverse phase adsorbent with Gogel silica gel. A mixture of water, methanol and acetonitrile (50:15:35) containing 0.2% pyridine and 0.2% acetic acid is used for elution. The fractions are monitored under UV at 280 nm. The appropriate fractions are combined and evaporated under reduced pressure to give 8.56 g of purified H_- / n-caprinoyl / -NQrn-t-BOC-A-21978C.D * core. Removal of the NOrn-t-BOC group. The clump of t-BOC is removed by stirring 1.47 g of the NQ2- (n-caprinoyl) -NQ4-t-BOC-A-21978C core with 15 ml of a mixture of trifluoroacetic acid and 1,2-ethanediole (10: 1) at room temperature for 3 minutes. The mixture is evaporated under reduced pressure and the residue is triturated with 50 ml of diethyl ether. By washing with 20 ml of ether and drying in vacuo, 2.59 g of a crude N- core are obtained. n-caprinoyl / -A-21978C (compound of formula 1, Rsn-caprinoyl, R1SH, RcH). E. Purification of the core No. 1- / n-caprinoyl-A-21978C »Crude N2- / n-caprinoyl core - -A-21978C is purified by reverse phase HPLC as follows: 2.59 g of sample is dissolved in 4.0 ml of a mixture of water, methanol, acetonitrile, pyridine and acetic acid (50: 15: 2: 2) and injected on a stainless steel column with dimensions of approximately 84 x 2.5 cm filled with LF-1 / oq adsorbent »The column is eluted with the same mixture of solvents. Elution is carried out at a rate of 10-12 ml / minute under pressure m about 8230-11660 klfc, using an LDC duplex pump (Milton-Roy). Leakage is monitored with a UV detector (ISO Model UA-5, Instrument Specialist CO., 4700 Superior Avenue. Lincoln, NB 6850 4) at 280 nm. Fractions of 20-24 ml are collected for two minutes. The fractions determined for antimicrobial activity are pooled and evaporated under reduced pressure to give 1.05 g of product. The purification is repeated using 4.35 g, 4.25 g, 2.14 g, 2.00 g and 1.75 g, respectively. g of crude starting derivative, yielding a total of 5.58 g of purified N-core, - - / n-capricin W-A-21 978C.142 112 23 Example XXXIII, Yfproduction of HCrp- / n-caprinoyl / -NK- / n-capricoyl / -A-21978C (compound No. 21) # N- - / n-caprinoylZ-N ^ - / n-caprinoyl / -A-21978C core (compound of formula 1f "1 2 \ Ky ** R and R s = n -caprinoyl (RsH) is a minor reaction product of the reaction for the preparation of the N-caprinoyl derivative in Example XXXII. It was isolated during the purification by means of reverse-phase HPLC described in section E. The desired fractions are combined and evaporated under reduced pressure. pressure to give 211 mg of crude product. The compound is purified by an analytical HPLC (C18 Microbondapak, Waters Go.) using a mixture of water, methanol, acetonite for elution. ryl, ammonium hydroxide and acetic acid (6x23: 15: 0 * 5: 0 * 5). After purification (32 times using a 500 µg sample each time), 4.4 mg of N2- (n-caprinoyl) - -Njin- / n-caprinoyl) -A-21978C core, identified by the 1H NMR spectrum ( 360 MHz). Example XXXIV. Preparation of the N2 -Cbz-NOrn-lauroyl-A-21978C core (compound No. 2o). The method consists in protecting the o-amino group of tryptophan in the core Ng1-t-BOO-A-21978C the Cbz group, and then removing the t-BOC group and acylating the ornithine -1-amino group with trichlorophenyl laurate. Preparation of the N2 -Cbz-NOrn * T-B0C-A-21978C core. 1.0 g of the NOH-t-BOC-A1β / fSC core is dissolved in 150 ml of dimethylformamide and the solution is heated to 6 ° C. 0.55 ml of NfO-bis-trimethylsilylacetamide and then 257 mg of benzyl-pentachlorophenyl carbonate are added to the solution. The reaction mixture is stirred for 20 hours, then concentrated to a volume of about 20-25 ml, mixed with 100 ml of water and the pH is adjusted to 6.0 with 1N sodium hydroxide solution. The mixture is washed with 6 x 2 ml of diethyl ether and lyophilized. The crude derivative is purified by reverse phase HPLC as follows. The sample is dissolved in approximately 6 ml of a mixture of water, methanol, acetonitrile, pyridine and acetic acid (55:15:30: 2: 2) is placed on a stainless steel column about 84 x 1.27 cm, filled with LF-1 / CLi carrier. The desired fractions, assessed by UV absorption at 280 nm and antimicrobial activity, pooled and lyophilized to give 951 mg of the core derivative ^ rp ^ bz-N0rn-t-BOC-A-21978C.B. Removal of the t-B0C group. The t-BOC group is removed by dissolving the intermediate in a mixture of trifluoroacetic acid, anisole and triethylsoulas (L0: 1: 1) at 50 mg / ml. After 3 minutes at -100 ° C, the mixture is concentrated and the remaining oil is triturated with diethyl ether (2 x 25 ml), yielding 520 mg of crude N, ^ -Cbz-A-21978C # C core. Acylation of the N ^ core - Cbz-A-21978C. The NTr1 -Cbz-A-21978C acyl core is reacted with the active trichlorophenyl ester. For this purpose, 520 mg of R-CBZ-A-21978C are added to a solution of 7 mg of 1-hydroxybenzotriazole and 123 mg of trichlorophenyl laurate in 150 ml of pyridine. The reaction mixture is stirred for 20 hours at 6 ° C and then it is concentrated, and the residue is triturated with ethyl ether (3 x 25 ml) to obtain 350 mg of the NQ-lauroyl-A- -21978C core. Patent claims 1. Method for the preparation of new derivatives of cyclic A-21978C peptides of general formula 1, in which R, R and R are the same or different and represent a hydrogen atom, an optionally substituted C1-O11-alkanoyl group, a C1-C1-n-alkenoyl group, or an amino protecting group such as a tert-butoxycarbonyl group, or benzyloxycarbonyl, and R, R and R 1 2 are hydrogen, with at least one of R, R or R not being hydrogen or an amino protecting group, at least one of R 12 and R 12 being hydrogen or an amino protecting group, R, R and R together contain - and R are at least four carbon atoms, and when R and R are hydrogen or an amino protecting group, then R is different from 8-methylcapryoyl, 10-methyloundecanoyl, 10-methyllauroyl, The C10-alkanoyl of factor C. of the peptide 24 142 112 A-21978C or the C12 "groups of the alkanoyl groups of the factors C1 and Cc of the peptide A-21978C, and pharmacologically acceptable salts of these compounds, characterized in that the core of the cyclic peptide of general formula 3, wherein R ' and R ° are identical or different and represent a hydrogen atom or an amino protecting group as defined above, or a derivative thereof protected by groups such as a tertiary -butoxycarbonyl or benzyloxycarbonyl group, possibly in the form of a pharmacologically acceptable salt, is reacted with an acylating agent of the formula R -COOH, in which R is an optionally substituted O-O 8 -alkyl or C4-G13-alkenyl group, or with an active ester, anhydride or an acid halide, and then optionally removing any protecting group or groups possibly present in the product, and the resulting compound is optionally salified. 2. A process according to claim 1, characterized in that in the preparation of a U-n-caprinoyl derivative of a compound of formula 1, n-capric acid or its active ester, anhydride or acid halide is reacted with the NQ-n-1-t-butoxycarbonyl-derivative of the A-21978C core of formula 3f in which R ° is the tertiary group -butoxycarbonyl and R 'is hydrogen. 3r1 ho2cihYoH3 IH H02C hN CH 0 H02C Formula 1142 11L L-Asp D-Ala l L-Asp t L-Orn \ Gly \ Gly \ D-Ser l 3MG l L-Kyn O / L-Thr l L-Asp L- Asn t L-Trp l NH RN Formula 2 z ^ 4 D-Ala t L-Asp l L-Orn \ Gly -Ser L MG Lyn Gly O \ / L-Thr t L-Asp t L-Asn l L-Trp I NH; Formula U HO HN O NHR 'NH 0 = ,, CONH2 0 <CH20 h / NH C02H H02C H-N7 ^ 9 ^ H ~ N = 0 vVS * H ^ -NR ° OA ^ o- H02C Formula 3142 112 AHRN aa MRN NKH NKH2 Formula B Formula T NTH2, NTHR * j NtHR DezocytoW} n, e - CI ^ Hi (g £ M ^ StW ^ NKH2 NKHj NKHj Acylation using the formula 2 'V secondary3' formula V protecting group \ Removal of the NKH2 protecting group NKH2 NKH2 Wabr5 Formula 6 Formula 6 1 NtHR ^ NjHRm NKHR removal of NKHR1 Acylation Formula 11 Formula 12 NTH2 NTHR NTHR, / NjHRn Desacyl- / "* Acylo-",. Agto ^ NKH2 N ^ j NKH2 NKHR! LKn2 Formula 5 Formula 7 NtHR Group removal _ ^ 'protecting I \ O.2 NKHR was taken Scheme 2 Formula 9142 112 NyHB1 Removal of group (*)' - N0HR2.... '\ u, protecting nkhr' ^ - Acylation (*) / NTH2 NoHR2 Formula 19 N NKHRt Formula 20 y NTH2 Removal of NjHB1 Selective nthb 'groups (*) - NQHB *' ,! "¦. 1 l \ u protecting- N ^ H2 encapsulating Formula 6 Removal of t * \ / n group removal (*) - N0HB of Acylo group- NtH1 N Protecting NNKH2 Formula 16 NKH2 Formula 17m - (*) - NOHR 2 • W Formula 18 y NTH2 Scheme 3 NyHB1 Removal of NKHR Formula 14 Formula 15 NKHR PL

Claims (2)

Claims 1. Process for the preparation of new derivatives of cyclic peptides A-21978C of the general formula 1, in which R, R and R are the same or different and represent a hydrogen atom, optionally substituted C1 -O11-alkanoyl group, Cc group -C 1-6 n -alkenoyl or an amino-protecting group such as tert-butoxycarbonyl or benzyloxycarbonyl, and R, R and R 12 are hydrogen, with at least one of R, R or R being as other than hydrogen or an amino protecting group, at least one of R 12 and R 12 is hydrogen or an amino protecting group, R, R and R together contain at least four at least four atoms, and when R and R represent a hydrogen atom or an amino protecting group, then R is different from the 8-methylcaprynooyl group, 10-methyloundecanoyl group, 10-methyllauroyl group, C10-alkanoyl group of the C factor peptide 24 142 112 A-21978C or C12 "alkanoyl agents C1 and Cc of the A-21978C peptide, and the pharmacologically acceptable salts of these compounds, characterized in that the core of the cyclic peptide of general formula 3, in which R 'and R ° are the same or different and represent a hydrogen atom or an amino protecting group having the above-mentioned meaning, or its derivative protected with groups such as tert -butoxycarbonyl or benzyloxycarbonyl, optionally in the form of a pharmacologically acceptable salt, is reacted with an acylating agent of formula R -COOH, in which R is an optionally substituted O group. -Ojg-alkyl or C4-G13-alkenyl, or with its active ester, anhydride or acid halide, then optionally removing any protecting group or groups possibly present in the product and the resulting compound optionally salifying, 2. A process according to claim 1, characterized in that in the preparation of the U-n-caprinoyl derivative of the compound of formula I, n-capric acid or its active ester, anhydride or acid halide is reacted with NQ ^ n-1II- a tertiary butoxycarbonyl derivative of core A-21978C of formula IIIf where R ° is tert-butoxycarbonyl and R 'is hydrogen. 3r1 ho2cihYoH3 IH H02C hN CH 0 H02C Formula 1142 11L L-Asp D-Ala l L-Asp t L-Orn \ Gly \ Gly \ D-Ser l 3MG l L-Kyn O / L-Thr l L-Asp L- Asn t L-Trp l NH RN Formula 2 z ^ 4 D-Ala t L-Asp l L-Orn \ Gly -Ser L MG Lyn Gly O \ / L-Thr t L-Asp t L-Asn l L-Trp I NH; Formula U HO HN O NHR 'NH 0 = ,, CONH2 0 <CH20 h / NH C02H H02C H-N7 ^ 9 ^ H ~ N = 0 vVS * H ^ -NR ° OA ^ o- H02C Formula 3142 112 AHRN aa MRN NKH NKH2 Formula B Formula T NTH2, NTHR * j NtHR DezocytoW} n, e - CI ^ Hi (g £ M ^ StW ^ NKH2 NKHj NKHj Acylation using the formula 2 'V secondary3' formula V protecting group \ Removal of the NKH2 protecting group NKH2 NKH2 Wabr5 Formula 6 Formula 6 1 NtHR ^ NjHRm NKHR removal of NKHR1 Acylation Formula 11 Formula 12 NTH2 NTHR NTHR, / NjHRn Desacyl- / "* Acylo-",. Agto ^ NKH2 N ^ j NKH2 NKHR! LKn2 Formula 5 Formula 7 NtHR Group removal _ ^ 'protecting I \ O.2 NKHR was taken Scheme 2 Formula 9142 112 NyHB1 Removal of group (*)' - N0HR2.... '\ u, protecting nkhr' ^ - Acylation (*) / NTH2 NoHR2 Formula 19 N NKHRt Formula 20 y NTH2 Removal of NjHB1 Selective nthb 'groups (*) - NQHB *' ,! "¦. 1 l \ u protecting- N ^ H2 encapsulating Formula 6 Removal of t * \ / n group removal (*) - N0HB of Acylo group- NtH1 N Protecting NNKH2 Formula 16 NKH2 Formula 17m - (*) - NOHR 2 • W Formula 18 y NTH2 Scheme 3 NyHB1 Removal of NKHR Formula 14 Formula 15 NKHR PL