KR860002194B1 - Process for the preparation of a-21978c cyclcic peptides derivatives - Google Patents

Abstract

Title compds. I [R, R1, R2=H, C4-14 alkyl, C2-19 alkanoyl, C5-19 alkenoyl, protected amino; R3, R4. R5=H, C4-14 alkyliden!, useful as antibiotics, were prepd. Thus, 10l culture broth consisting of 100g peanut meal, 50g peptone, 200g sucrose, 5g KH 2PO4, 12g K2HPO4, and 2.5 g MgSO4.7H2O in 14 liter-fermentor was inoculated with A. utaensis and cultivated at 30≰C for 66 hrs. The resulting broth was mixed with 100 g crude A-21978 complex. The culture broth was filtered, passed through HP-20 column, and eluted with a mixt. contg. H2O and acetonitrile to give A-21978C nucleus.

Description

A-21978C Preparation of Cyclic Peptide Derivatives

The present invention relates to novel derivatives of cyclic peptides having antibiotic properties, in particular the A-21978C cyclic peptide derivatives of the following general formula (1) and semisynthetic methods for the preparation of pharmaceutically acceptable salts of these peptides.

Wherein R, R ¹ and R ² are independently hydrogen, C ₄ to C ₁₄ -alkyl, optionally substituted C ₂ to C ₁₉ -alkanoyl, C ₅ to C ₁₉ -alkenoyl or amino protecting group; R ³ , R ⁴ and R ⁵ are all hydrogen or R ³ and R ¹ , R ⁴ and R, or R ⁵ and R ² together represent a C ₄ to C ₁₄ -alkylidene group; Provided that 1) at least one of R, R ¹ or R ² is not a hydrogen or amino protecting group, and 2) at least one of R ¹ or R ² is a hydrogen or amino protecting group, and 3) R, R ¹ And the R ² groups together must contain at least 4 carbon atoms, and 4) if both R ¹ and R ² are selected from hydrogen or an amino protecting group, then R is 8-methyldecanoyl, 10-methylundecanoyl, 10- Methyldodecanoyl, a specific C ₁₀ -alkanoyl group of A-21978C factor C ₀ or a specific C ₁₂ -alkanoyl group of A-21978C factors C ₄ and C ₅ .

Due to the high likelihood and the constant threat of the development of antibiotic-specific resistant strains of pathogenic microorganisms, the development of new antibiotics is urgently needed. In particular, pathogens belonging to the genus Gram-positive bacteria Staphylococcus and Streptococcus are sometimes resistant to commonly used antibiotics such as penicillin and erythromycin. See WO Foye, Principles of Medicinal Chemistry, pp. 684-686 (1974).

In accordance with the present invention, novel derivatives of A-21978C cyclic peptides and pharmaceutically acceptable salts thereof have been found to be effective antibiotics.

These novel derivatives are suitable acylating agents for A-21978C nuclei prepared by deacylating a properly blocked 4A-21978C complex or an appropriately blocked individual C ₀ , C ₁ , C ₂ , C ₃ , C ₄ or C ₅ . Or it may be prepared by reacting with an active derivative thereof, or in the case of alkylated derivatives may be prepared by forming a suitable Schiff base, and then reducing the base imine bond.

In addition, the present invention provides the A-21978C cyclic peptide nucleus of the general formula (3) or a derivative thereof, wherein the derivative includes a protected derivative thereof and a pharmaceutically acceptable salt thereof. And an activating derivative thereof) to prepare an A-21978C cyclic peptide derivative of the following general formula (1).

Wherein R, R ¹ and R ² are independently hydrogen, C ₄ to C ₁₄ -alkyl, optionally substituted C ₂ to C ₁₉ -alkanoyl, C ₅ to C ₁₉ -alkenoyl or aminoprotecting group, R ³ , R ⁴ and R ⁵ are all hydrogen, or R ³ and R ¹ , R ⁴ and R, or R ⁵ and R ² together may represent a C ₄ to C ₁₄ -alkylidene group, and R ¹ and R ⁰ Is independently hydrogen or an amino-protecting group, and R ⁶ is optionally substituted C ₁ to C _18- , alkyl or C ₄ to C ₁₈ -alkenyl, provided that 1) at least one of R, R ¹ or R ² Must not be a hydrogen or amino protecting group, 2) at least one of R ¹ or R ² must be a hydrogen or amino protecting group, and 3) the R, R ¹ and R ² groups together contain at least 4 carbon atoms; 4) when R ¹ and R are both selected from hydrogen or an amino protecting group, R is 8-methyldecanoyl, 10-methylundecanoyl, 10-methyldodecanoyl, It cannot be a specific C ₁₀ -alkanoyl group of A-21978C factors C ₀ or a specific C ₁₂ -alkanoyl group of A-21978C factors C ₄ and C ₅ .

According to the present invention, the A-21978C cyclic peptides of formula (1) and pharmaceutically acceptable salts of these peptides have been found to be useful as semisynthetic antibacterial agents or intermediates of such agents.

In the present specification, the following abbreviations are used, most of which are commonly known in the art.

Ala: Alanine

Asn: Asparagine

Asp: Aspartic Acid

Gly: glycine

Kyn: Kynurenine

Orn: Ornithine

Ser: Serine

Thr: Threonine

Trp: Tryptophan

t-BOC: tert-butoxycarbonyl

CbZ: benzyloxycarbonyl

DMF: Dimethylformamide

THF: Tetrahydrofuran

HPLC: High Performance Liquid Chromatography

NMR: ¹ H nuclear magnetic resonance

TLC: thin layer chromatography

UV: UV

A-21978C antibiotics are closely related to the acidic peptide antibiotics described in US Pat. No. 4,208,403, which is incorporated herein by reference. As described in US Pat. No. 4,208,403, supra, the A-21978C antibiotic complex contains Factor C, which is itself a complex of closely related factors as its main component.

A-21978 Factor C, called A-21978C Complex, contains the individual C ₀ , C ₁ , C ₂ , C ₃ , C ₄ and C ₅ . Of these, factors C ₁ , C ₂ and C ₃ are major factors, and factors C ₀ , C ₄ and C ₅ are minor facotr. The structure of A-21978C factor is shown in the following general formula (2).

Wherein 3MG represents L-threo-3-methylglutamic acid and R ^N represents a specific fatty acid residue.

The specific R ^N group of each factor is as follows:

* Not yet confirmed

When faced with the problem of deacylation of peptide antibiotics, it is extremely difficult to find an enzyme that can be used for this. Especially when antibiotics as substrates contain cyclic peptide nuclei, such enzymes are more difficult to find. Since enzymes have a high degree of specificity, differences in peptide sites and side chains of substrate antibiotics affect the outcome of deacylation. In addition, many types of microorganisms produce a number of peptidases that act on other sites in the peptide moiety. As a result, the product may be obtained in the form of a mixture which is difficult to handle.

The enzyme used to cause deacylation from the α-amino group of the tryptophan of the fatty acid side chain is a microorganism with the Actinoplanaceae, preferably Actinoplanes utahensis NRRL12052 [Korea Accession No .: KFCC-10054, Korean Depositary Organization: Korean spawn association, Deposited: August 17, 1983] or a variant thereof. To perform deacylation, A-21978C complex, A-21978C factor C ₀ , C ₁ , C ₂ , C ₃ , C ₄ and C ₅ , blocked A-21978C complex and blocked A-21978C factor C ₀ , C Antibiotics selected from ₁ , C ₂ , C3, C ₄ and C ₅ are added to the culture of the microorganism. The terms "blocked A-21978C" and "blocked A-21978C complex" mean a complex of individual A-21978C factors or mixtures of factors in which the amino group of ornithine and / or kynurenine is blocked with an amino protecting group. . Preferably, the amino protecting group is located at the δ-amino group of ornithine in the individual factor or mixture. The culture is incubated with the substrate until deacylation is almost complete. The corresponding A-21978C cyclic peptide thus obtained is isolated from fermentation broth by methods known in the art.

The cyclic peptide and its salt obtained by such enzymatic deacylation are represented by the following general formula (3).

Wherein R ¹ and R ⁰ are independently hydrogen or an amino protecting group.

A compound of formula (3) wherein R ¹ and R ⁰ each represent hydrogen when the acyl residue is removed from A-21978C complex or A-21978C individual factors C ₀ , C ₁ , C ₂ , C ₃ , C ₄ and C ₅ Is generated. Deacylation molecules are common cyclic peptides present in each antibiotic A-21978C factor. For convenience, this compound is called the A-21978C nucleus. This compound can also be represented by the general formula (4).

Wherein 3MG represents L-threo-3-methylglutamic acid.

Compounds of formula (3) wherein R ¹ or R ⁰ are not hydrogen are prepared by deacylating the appropriately blocked antibiotics A-21978C factors C ₀ , C ₁ , C ₂ , C ₃ , C ₄ and C ₅ .

For convenience, these compounds are referred to as blocked A-21978C nuclei. These blocked compounds are useful as intermediates for peptides of formula (1), ie compounds in which at least one of R ¹ and R ² is an amino protecting group.

As will be apparent to those skilled in the art, the A-21978C nucleus and blocked A-21978C nuclei can be obtained in free amine or acid addition salt form. Any acid addition salt may be used, but pharmaceutically acceptable salts are preferred. "Pharmaceutically acceptable" salts are salts that are useful as chemotherapeutic agents in warm-blooded animals.

The method for preparing A-21978C nuclei of general formula (3) using various microorganisms is fully described in the applicant's pending patent application (Docket No. X-5279). A. which is a preferred deacylating enzyme. Utahensis NRRL 12052 is deposited from the Agricultural Research Culture Collection (NRRL) (Nothern Regional Research Center, USDepartment of Agriculture, 1815 North University St., Peoria, Illinois 61604, USA), accession number NRRL 12052 (Korea Deposit No .: KFCC-10054). , Korean Depositary Organization: Korean spawn association, Deposited Date: 1983.8.17).

Preparation Example 1 uses the A-21978C complex as a substrate, and actinoides utahensis NRRL 12052 (Korea Deposit No .: KFCC-10054, Korea Deposit Organization: Korea spawn association, Deposit Date: 1983.8.17) as a microorganism. It describes the method of making A-21978C nuclei by fermentation using.

Other actinoplanacea cultures that can be used to prepare A-21978C nuclei of Formula (3) are available from the Northern Regional Research Laboratory as the following accession number:

Actinoplanes Utahensis NRRL 12052 (Korea Deposit No .: KFCC-10054, Korea Deposit Organization: Korean spawn association, Deposit Date: 1983.8.17), Actino Plains Missouri NRRL 12053 (Korea Accession No .: KFCC-10056, Korean Deposit Institution: Korean spawn association, deposited date: 1983.8.17), Actino Plains NRRL 8122 (Korea Deposit Number: KFCC-10052, Korean Deposit Organization: Korean spawn association, deposited date: 1983.8.17), Actinoplane Species NRRL 12065 (Korea Deposit No .: KFCC-10053, Korea Deposit Organization: Korean spawn association, Deposit Date: 1983.8.17), Streptosporanium roseum variety Hollandensis NRRL 12064 (Korea Deposit No .: KFCC-10055, Korea Depositary Organization: Korean spawn association, Depositary Date: 1983.8.17).

The deacylation performance of certain microbial strains belonging to the Actinoplanacea family is measured by the following procedure. Inoculate the microorganisms into the appropriate growth medium. Cultures are incubated in a rotary shaker at about 28 ° C. for 2 or 3 days. One of the substrate antibiotics is added to the culture and the pH of the fermentation broth is maintained at 6.5. The activity of the cultures is monitored using the Micrococcus luteus assay. Loss of antibiotic activity indicates that the microorganism produced the enzymes needed for deacylation, which can be demonstrated by one of the following methods.

1) HPLC analysis for the presence of complete nuclei; Or 2) activity recovery by acylation with the appropriate side chain (eg lauroyl, n-decanoyl or n-dodecanoyl).

The present invention is prepared by acylating A-21978C nucleus [compound of formula (3)], A-21978C factor or blocked A-21978C factor, and then, in the process for preparing a novel compound of formula (1) or a salt thereof. Is about:

Wherein R, R ¹ and R ² are independently hydrogen, C ₄ to C ₁₄ -alkyl, optionally substituted C ₂ to C ₁₉ -alkanoyl, C ₅ to C ₁₉ -alkenoyl or amino protecting group; R ³ , R ⁴ and R ⁵ are all hydrogen, or R ³ and R ¹ , R ⁴ and R, or R ⁵ and R ² together may represent a C ₄ to C ₁₄ -alkylidene group; Provided that 1) at least one of R, R ¹ or R ² is not a hydrogen or amino protecting group, 2) at least one of R ¹ or R 2 is a hydrogen or amino protecting group, and 3) R, R ¹ and The R ² groups together must contain at least 4 carbon atoms, and 4) if both R ¹ and R ² are selected from hydrogen or an aminoprotecting group, then R is 8-methyldecanoyl, 10-methyl undecanoyl, 10-methyl Dodecanoyl, a specific C ₁₀ -alkanoyl group of A-21978C factors C ₀ or a specific C ₁₂ -alkanoyl group of A-21978C factors C ₄ and C ₅ .

의 그룹을 의미하는 것이며, 여기에서 R³및 R⁴는 수소 또는 탄소원자 3내지 13의 알킬그룹이고, 단 R³및 R⁴중의 하나는 수소가 아니어야 하며, R³및 R⁴는 탄소원자의 합은 13이하여야 한다. R, R¹또는 R²중의 하나가 C₄내지 C₁₄-알킬리데닐인 화합물은 쉬프염기로 알려져 있다.The term "C ₄ to C ₁₄ -alkylidedenyl" is of the general formula

Wherein R ³ and R ⁴ are hydrogen or an alkyl group of 3 to 13 carbon atoms, provided that one of R ³ and R ⁴ is not hydrogen, and R ³ and R ⁴ are The sum must be 13 or less. Compounds wherein one of R, R ¹ or R ² are C ₄ to C ₁₄ -alkylidedenyl are known as Schiffbase groups.

The term "C ₄ to C ₁₄ -alkyl" refers to a monovalent saturated straight or branched chain alkyl group of 4 to 14 carbon atoms. R, R ¹ or R ² is C ₄ to C ₁₄ - alkyl is a R, R ¹ or R ² group, C ₄ to C ₁₄ -, and prepared by reducing the corresponding compound of alkylidenyl, in the present specification " Reduced Schiffbase ".

The terms "optionally substituted C ₂ to C ₁₉ -alkanoyl" and "C ₅ to C ₁₉ -alkenoyl" refer to acyl groups derived from carboxylic acids containing 2 to 19 and 5 to 19 carbon atoms, respectively. When the R group is alkanoyl, the alkyl moiety is a monovalent saturated straight or branched chain hydrocarbon group, which contains one hydroxyl, carboxyl or C ₁ to C ₄ -alkoxy group, or selected from chlorine, bromine and fluorine. To 3 halo substituents.

If R is alkenoyl, the alkenyl moiety is a monovalent unsaturated straight or branched chain hydrocarbon group containing up to three double bonds. The double bond site of an unsaturated hydrocarbon chain may be present in cis or trans configuration.

The term "amino protecting group" refers to an amino protecting group recognized to be independent of other functional groups in the A-21978C molecule. It is preferred that the amino protecting group can be easily removed from the acylated compound. Examples of WEJR combined protection groups are described in the literature ("Protective Groups in Organic Synthesis" by Theodora W. Greene, John Wiley and Sons, New York, 1981, Chapter 7). Particularly preferred amino protecting groups are tert-butoxycarbonyl and benzyloxycarbonyl groups.

In an ancillary aspect, the present invention includes preferred embodiments of the following general formula (1):

의 알카노일이며, n이 3내지 17의 정수인 화합물;(a) R is a general formula

An alkanoyl compound wherein n is an integer of 3 to 17;

의 알카노일이며, n이 5내지 14인 화합물 ;(b) R is a general formula

An alkanoyl compound wherein n is 5 to 14;

의 알카노일이며, n 및 m은 각각 독립적으로 0내지 14의 정수이고, 단 n과 m의 합은 1이상이 15이하이어야 하며, 또한 n이 0이면 m은 8일 수 없고, n이 1이면 m은 6 또는 8일 수 없는 화합물;(c) R is a general formula

Is an alkanoyl of n and m are each independently an integer of 0 to 14, provided that the sum of n and m is 1 or more and 15 or less, and if n is 0, m cannot be 8 and n is 1 m is a compound that cannot be 6 or 8;

의 시스 또는 트란스 알케닐이고, n 및 m이 각각 독립적으로 0내지 14의 정수이며, 단 n과 m의 합은 1이상, 15이하이어야 하는 화합물;(d) R is a general formula

Is a cis or trans alkenyl, n and m are each independently an integer of 0 to 14, provided that the sum of n and m is 1 or more and 15 or less;

의 시스 또는 트란스 알케닐이며, n은 4내지 15의 정수인 화합물 ;(e) R is a general formula

Is cis or trans alkenyl, n is an integer of 4 to 15;

(f) a compound wherein R is alkyl of the general formula CH ₃ (CH ₂ ) _n −, and N is an integer from 5 to 12; And

(g) Compounds in which R is of

Compounds of formula (1) may form salts, which are also part of the present invention. Such salts are useful, for example, for the separation and purification of compounds. Pharmaceutically acceptable alkali metal salts, alkaline earth metal salts, amine salts and acid addition salts are particularly useful.

For example, the compound of formula (1) has four free carboxyl groups capable of forming salts. Therefore, partial, mixed and complete salts of these carboxyl groups are also included in the present invention. When preparing such salts, the pH must be below 10, because at pH above 10 the compound is unstable.

Representative and suitable alkali and alkaline earth metal salts of the compounds of formula (1) include sodium, potassium, lithium, cesium, rubidium, barium, calcium and magnesium salts. Suitable amine salts of compounds of formula (1) include ammonium and primary, secondary and tertiary C ₁ to C ₄ -alkylammonium and hydroxy-C ₂ to C ₄ -alkylammonium salts. Specifically, the amine salt includes a compound of formula (1) and ammonium hydroxide, methylamine, secondary-butylamine, isopropylamine, diethylamine, di-isopropylamine, cyclohexylamine, ethanolamine, triethylamine Or salts formed by reaction with 3-amino-1-propanol.

Alkali metal and alkaline earth metal cationic salts of the compound of formula (1) are prepared according to the methods commonly used for the preparation of cationic salts. For example, an advantageous form of the compound of formula (1) is dissolved in a suitable solvent such as warm methanol or ethanol and an aqueous methanol solution containing a stoichiometric amount of the desired inorganic base is added. The salts thus formed can be separated by conventional methods such as filtration or evaporation of the solvent. It is a preferred method to prepare salts using cation exchange resins.

Salts formed with organic bases can also be prepared by similar methods. For example, a gaseous or liquid amine can be added to a solution of the compound of formula (1) in a suitable solvent such as ethanol and the solvent and excess amine can be removed by evaporation.

The compounds of the present invention also have free amino groups that can produce acid addition salts, which are also part of the present invention. Representative suitable acid addition salts of compounds of formula (1) include hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, succinic acid, citric acid, lactic acid, maleic acid, fumaric acid, palmitic acid, cholic acid, pamoic acid, muxic acid, D-glutamic acid, d- Standard reactions with organic and inorganic acids such as camphoric acid, glutaric acid, glycolic acid, phthalic acid, tartaric acid, lauric acid, stearic acid, salicylic acid, methanesulfonic acid, benzenesulfonic acid, sorbic acid, picric acid, benzoic acid and cinnamic acid Included salts are

A compound of formula (1) wherein R, R ¹ or R ² is an alkanoyl, alkenoyl or amino protecting group can be used to form A-21978C factor, blocked factor, general A compound of formula (3) or a suitable compound of formula (3) in which the α-amino group of tryptophan is blocked is prepared by acylation with the desired alkanoyl or alkenoyl side chains. Generally, the acylation reaction is carried out by reacting the selected compound with an active derivative of alkanoic acid or alkanoic acid corresponding to the desired acyl side chain group (R, R ¹ or R ² ). The term "activating derivative" refers to a derivative containing a carboxyl functional group of an acylating agent which is reactive to form an amide bond linking an acyl side chain to the nucleus by coupling with a primary amino group. Suitable activated derivatives, methods for their preparation and their use as acylating agents for primary amines will be well known to those skilled in the art. Preferred activated derivatives are: (a) acid halides (eg acid chlorides) (b) acid anhydrides (eg alkoxyformic anhydrides) or (c) activated esters (eg 2, 4, 5-trichlorophenyl esters) ). Other methods of activating carboxyl functionalities include the reaction of carboxylic acids with carbonyldiimides (eg, N, N'-dicyclohexylcarbodiimide or N, N'-diisopropylcarbodiimide). The result is a reactive intermediate that does not separate because it is unstable. In this case, the reaction with the primary amine is carried out in the reaction system.

One skilled in the art will understand that the amino a protecting group is used to prepare the compound of formula (1) in a selective acylation process. For example, when starting from a compound of the general formula (3) wherein R 'and R ⁰ are hydrogen, acylation occurs in both the α-amino group of tryptophan and the δ-amino group of ornithine, and thus N _Trp , N _Orn Diacyl derivatives are produced. Therefore, in order to obtain an acylated derivative of only the α-amino group of tryptophan, acylating the compound of formula (3) in which the δ-amino group of the ornithine (R ⁰ position) is blocked by an amino protecting group is required. desirable. This starting material is preferably obtained by blocking its position before deacylating the A-21978C factor. The aromatic amino group of kynurenine is the least reactive of the three free amino groups in the A-21978C nucleus. Acylating kynurenine should block the amino groups of tryptophan and ornithine properly, but for acylation at the R or R ¹ position, it is not necessary to block the amino group of kynurenine.

Schemes I, II and III summarize the general process for the preparation of compounds of formula (1) wherein one of R, R ¹ or R ² is an alkanoyl, alkenoyl or amino protecting group. The following markers are used in these schemes.

(*) = Rest of A-21978C

N _T = α-amino group of tryptophan

N _O = α-amino group of ornithine

N _K = α-amino group of kynurenine

R, R ¹ , R ² = substituents as described above

R _N = acyl group of natural factor

B, B ¹ = amino protecting group

Acyl = Acylation Step

Deacyl = Deacylation Step

Block = blocking by amino protecting group

Deblock = Remove Amino Protection Group

In Scheme I, the N _Trp -monoacyl derivative of A-21978C is represented by formula (3), and the N _Trp , N _Orn -diacyl derivative of A-21978C is represented by formula (4). N _Trp , N _Orp -diacyl derivatives in which the N _Trp -acyl group is identical to the acyl group of the native A-21978C factor are represented by the general formula (8).

Scheme I: _{Preparation of} N _Trp -monoacyl and N _Trp , N _Orp -Diacyl-A-21978C derivatives

In Scheme II, N _Trp , N _Kyn -diacyl derivatives of A-21978C are represented by general formula (10). N _Trp , N _Kyn -diacyl derivatives in which the N _Trp -acyl group is identical to the acyl group of the native A-21978C factor are represented by general formula (12).

In addition, the compounds of the general formulas (5), (6) and (7) are as described in Scheme I.

Scheme II: _{Preparation of} N _Trp , N _Kyn -Diacyl-A-21978C Derivatives

In Scheme III, the N _Orn -monoacyl derivative of A-21978C is represented by formula (18), the N _Kyn -monoacyl derivative of A-21978C is represented by formula (15), and N _Orn , N _Kyn -diacyl derivative is represented by General formula (20). Compounds of formula (6) are as described in Schemes I and II.

Scheme III: _{Preparation of} N _Orn - _Monoacyl , N _Kyn - _Monoacyl and N _Orn , N _Kyn -Diacyl-A-21978C Derivatives

Compounds of formula (1) in which at least one of the amino groups is substituted with alkylidedenyl (schief base) or alkyl group (reduced Schiff base) are prepared by known methods for preparing the Schiff base and the reduced Schiff base, respectively. can do. Thus, the Schiff base is prepared by reacting the primary amino group of tryptophan, ornithine or kynurenine of A-21978C with the appropriate aldehyde or ketone in an appropriate solvent. Reduction of the imine bond of the Schiff base to yield the corresponding compound of formula (1) wherein R, R ¹ or R ² is alkyl can be carried out by known selective reduction processes. Preferred reducing agents for this reaction are sodium cyano borohydride.

Under the in vitro test conditions used, the Schiff base does not exhibit antimicrobial activity because it is unstable in the test medium. However, they are useful as intermediates for producing reduced Schiff bases. If a Schiff base is used as an intermediate, there is no need to separate the intermediate before reducing to form a reduced Schiff base.

A preferred method for producing the compound of formula (1) wherein one of R, R ¹ or R ² is alkanoyl or alkenoyl is an active ester method. Formula (3) wherein R 'is hydrogen and R ⁰ is t-BOC is a compound, ie A-21978C N _Orn- t-BOC nucleus or "tBOC nucleus" is a particularly preferred starting point for preparing compounds of formula (1) It is a substance. Preferred acylating agents are 2, 4, 5-trichlorophenyl esters of the desired alkanoic acid or alkenoic acid. In this method, the excess active ester is reacted with the t-BOC nucleus at room temperature in an unreactive organic solvent such as DMF, THF, diethyl ether or dichloromethane. The reaction time is not critical but is preferably about 6 to about 20 hours. Upon completion of the reaction, the solvent is removed and the residue is purified. A particularly useful purification method is reverse phase HPLC using a mixture of LP-1 / C ₁₈ as stationary phase and H ₂ O / CH ₃ OH / CH ₃ CH / pyridine / HOAC as solvent system. The t-BOC group is removed by treatment with trifluoroacetic acid / anisole / triethylsilane or preferably tri-fluoroacetic acid / 1,2-ethanedithiol at room temperature for about 3 to about 5 minutes. After removing the solvent, the residue is purified by reverse phase HPLC.

The modified acylation method is a modified Schotten-Baumann process, in which the nucleus, which is not blocked, is treated with the acid chloride of the desired alkanoic acid or alkenoic acid in a pyridine / water mixture. In this method, excess acid chloride in an unreactive organic solvent (eg acetone) is slowly added to a solution of the nucleus in 90% pyridine / 10% water (volume). Unreacted acid chloride is extracted from the reaction product by extraction with an immiscible organic solvent (eg diethyl ether). The final purification step is carried out by reversed phase HPLC as described above.

Alkanoic and alkenoic acids and their active derivatives (particularly acid chlorides and 2, 4, 5-trichlorophenyl esters) used as starting materials in acylation reactions are known compounds or are known from known compounds by known methods. It can manufacture. 2,4,5-trichlorophenyl esters can be treated by treating acid chlorides of alkanoic acid or alkenoic acid with 2,4,5-trichlorophenol in the presence of pyridine, or by using free alkanoic acid or alkenoic acid as coupling agents. Prepared conveniently by treatment with 2,4,5-trichlorophenol in the presence of '-dicyclohexylcarbo-diimide. The 2,4,5-trichlorophenyl ester derivative can be purified by column chromatography on silica gel.

When the A-21978C cyclic peptide of the present invention is used as an antimicrobial agent, it can be administered orally or parenterally. As will be appreciated by those skilled in the art, the A-21978C compound is administered in combination with conventional pharmaceutically acceptable carriers or diluents. A-21978C The dosage of a compound depends on various conditions, such as the type and severity of the particular infection that requires treatment. Appropriate dosage ranges and / or dosage units at the time of administration are determined in conjunction with factors such as the pharmacokinetics of the MIC and ED ₅₀ values and toxicity data herein, the characteristics of the patient or host and the infecting microorganism.

The following non-limiting examples illustrate the invention in more detail.

Preparation Example 1

Manufacture of A-21978C nucleus

A. Fermentation of Actinoplanes Utahensis Actinoplanes Utahensis NRRL 12052

The storage culture of (Korean Deposit No .: KFCC-10054, Korea Depositary Organization: Korean spawn association, Deposit Date: 1983.8.17) is prepared and maintained on sloped agar. The medium used to prepare the slope culture is selected from the following:

Badge A

The pH before autoclaving is about 5.9, adjusted to pH 7.2 by addition of sodium hydroxide; The pH after autoclaving is about 6.7.

* The composition of Zapek Mineral Stock is as follows.

Badge B

[* N-Z-amine, A, Humko Sheffield Chemical, Lyndhurst, NJ.]

Slope cultures were inoculated with Actino Plains Utahensis NRRL 12052 (Korea Deposit No .: KFCC-10054, Korea Deposit Organization: Korean spawn association, Deposit Date: 1983.8.17), and the inoculated culture was inoculated at about 8 ° C at about 8 ° C. Incubate for 10 days.

About half of the sloped proliferation is used to inoculate 50 ml of proliferative medium with the following composition:

The pH is adjusted to 7.4 using sodium hydroxide, and the pH after autoclaving is about 6.8.

[* National Distillers Products Co., 99 park Ave., New York, NY.]

The inoculated caustic medium is transferred to a 250 ml wide-mouth triangle flask and incubated for about 72 hours at 30 ° C. on a rotary shaker drawing an arc 2 inches in diameter at a speed of 250 RPM.

The proliferative medium cultured as described above can be used directly to inoculate the proliferative medium of the second step. It is also possible to keep the culture in the vapor phase of liquid nitrogen for later use. For this storage, the cultures are prepared in several small vials as follows: Each vial contains 2 ml of cultured proliferative medium and 2 ml of glycerol-lactose solution. W.A. Dailey and C.E. Higgens, "Preservation and Storage of Microorganisms in the Gas Phase of Liquid Nitrogen, Cryobiol 10, 364-367 (1973)]. The prepared suspension is stored in the vapor phase of liquid nitrogen.

50 ml of the first stage proliferative medium of the composition as described above was inoculated using the thus prepared storage suspension (1 ml). The inoculated first stage proliferative medium is cultured as described above.

To obtain a large amount of inoculum, 400 ml of the first stage proliferative medium having the same composition as the first stage proliferative medium was inoculated using 10 ml of the cultured first stage proliferative medium. The second stage medium is transferred to a 2-liter Erlenmeyer flask and incubated at 30 ° C. for about 48 hours on a rotary shaker drawing a 2 inch diameter arc at 250 RPM.

Inoculate 10 L of the sterile culture solution selected from the following using a cultured second stage proliferative medium (80 ml) prepared as described above:

Badge Ⅰ

After autoclaving at 121 ° C. for about 16-18 psi for 45 minutes, the pH of the medium is about 6.9.

Badge Ⅱ

The pH is adjusted to 7.0 using hydrochloric acid, and the pH after autoclaving is 7.0.

badge

* Sterilize separately and add aseptically.

The final pH is about 6.6.

The inoculated production medium is transferred to 14 L fermentor and fermented at about 30 ° C. for about 66 hours. The fermentation broth is stirred at about 600 RPM using a conventional stirrer and sterilized air is blown so that the dissolved oxygen level at atmospheric pressure exceeds 30% of air saturation.

B. Deacylation of A-21978C

a. Fermentation of utahensis is carried out as described in section A by incubating the production medium for about 67 hours using slope medium A and production medium I. Crude A-21978C complex (100 g), prepared as described in US Pat. No. 4,208,403, is added to the fermentation medium.

A-21978C complex deacylation is monitored by the assay for Micrococcus luteus. Fermentation is M. Continue for about 24 hours until complete loss of deacylation with loss of activity against Luteus.

C. A-21978C Separation of Nuclei

The whole fermented broth (20 L) obtained as described in section B is filtered through a filter aid (Hyflo Super-cel, Johns Manville Corp.) and the mycelium cake is discarded. The filtrate thus obtained is passed through a column containing 1.5 L of HP-20 resin (DIAION High Porous Polymer, HP Series, Mitsubishi Chemical Industries Limited, Tokyo, Japan), and the passing liquid is discarded. The column is washed with deionized water (10 L) and then the remaining filtration broth is removed. Discard the wash water. The column is then eluted with a water: acetonitrile mixture (10 L, 95: 5, 9: 1 and 4: 1, respectively) to collect 1 L fraction.

Elution is monitored by paper chromatography using n-butanol: pyridine: acetic acid: water (15: 10: 3: 12) solvent system and detecting the compound by UV fluorescence. In this system, A 21978C factor represents a R _f value of about 0.56, A-21978C nucleus represents a R _f value of about 0.32. The product may also be tested by analytical HPLC using silica gel / C ₁₈ and water: methanol (3: 1) containing 0.1% ammonium acetate in the solvent system and detecting nuclei with a UV monitor at 254 nm.

The fractions containing the nuclei were combined and concentrated in vacuo to remove acetonitrile and lyophilized to yield 40.6 g of semi-clean A-21978C nuclei.

D.A-21978C Purification of Nuclei

Semi-purified A-21978C nuclei (15 g) obtained as described in Section C are dissolved in 75 ml of water: methanol: acetonitrile (82: 10: 8) containing 0.2% acetic acid and 0.8% pyridine. This solution is applied on a 4.7 × 192 cm column containing 3.33 L of silica gel / C ₁₈ (Quantum LP-1, Quantum Industries, 341 KaplanDrive, Fairfield, NJ 07006). The column is developed in the same solvent system. Collect 350 ml volume fractions. The separation process is tracked with a UV monitor at 280 mm. The fractions containing the nuclei were combined and concentrated in vacuo to remove the solvent and lyophilized to yield 5.2 g of purified A-21978C nuclei.

E.A-21978Cnuclear characteristics

The A-21978C nucleus has the following characteristics:

a) Form: white amorphous solid that fluoresces under shortwave UV

b) Experimental formula: C ₆₂ H ₈₀ N ₁₇ O ₁₅

c) molecular weight: 1465

d) Solubility: Water Soluble

e) The infrared absorption spectrum (KBr) shows the absorption maximum at the following frequencies (cm ^-1 ): 3300 (broad), 3042 (weak), 2909 (weak), 1655 (strong), 1530 (strong), 1451 (About), among 1399 (medium), 1222 (about), 1165 (about), 1063 (about) and 758 (medium to about)

f) The UV absorption spectrum in methanol shows an absorption maximum at 223 nm (ε41, 482) and 260 (ε8, 687).

g) As a result of electrostatic determination in 66% aqueous dimethylformamide, four titratable groups with pKa values of about 5.2, 6.7, 8.5 and 11.1, respectively, were identified (initial pH6.12).

Production Example 2

A-21978COther Manufacturing Methods

A-21978C nuclei are prepared according to the method of Preparation Example 1, with the exception of some variations of paragraph B. a. Utahensis cultures are initially incubated for about 48 hours, substrate (50 g of semi-refined A-2978C complex) is added and then incubated for about 16 hours. The broth filtrate is passed through a column containing 3.1 L of HP-20 resin, and the column is washed with 10 times the amount of water and then eluted with water: acetonitrile (95: 5). Elution is monitored as in Production Example 1. After collecting 24 liters, the elution solvent was changed to water: acetonitrile (9: 1). Fractions containing nuclei are eluted with this solvent. These fractions are combined, concentrated in vacuo to remove acetonitrile and then lyophilized to yield 24.3 g of semi-purified A-21978C nuclei.

This semi-purified A-21978C nucleus (24.3 g) is dissolved in water (400 ml). The solution was prepared on a 4.7 × 192 cm steel column containing 3.33 L of silica gel (Quantum LP-1 / C ₁₈ ) prepared in water: methanol: acetonitrile (8: 1: 1) containing 0.2% acetic acid and 0.8% pyridine. Applies to The column is run with the same solvent under a pressure of about 2000 psi to collect 350 ml fractions. Elution was monitored by UV at 280 to collect the nucleated fractions, concentrated in vacuo to remove solvent, and lyophilized to yield 14 g of highly purified A-21978C nuclei.

Preparation Example 3

Preparation of N _orn- t-BOC-A-21978 Factors C ₂ and C ₃

A mixture of A-21978C factors C ₂ and C ₃ (10 g), prepared as described in US Pat. No. 4,208,403, is dissolved in water (50 ml) with high frequency decomposition (200 mg / ml). The pH of the solution is adjusted from 4.05 to 9.5 using 5N sodium hydroxide (3.6 ml). Di-tert-butyl dicarbonate (3.0 ml) is added and the reaction mixture is stirred at room temperature for 2 hours. The pH of the reaction solution was maintained at 9.5 by the slow addition of 5N sodium hydroxide (6.5 ml added over 2 hours).

The reaction is periodically monitored by TLC on silica gel using a CH ₃ CN: H ₂ O (7: 3 and 8: 2) solvent system and detected by UV.

After about 10 minutes, the reaction solution is rapidly turbid and the consumption of base is increased. After 30 minutes, the rate of increase in turbidity and the rate of consumption of base are reduced, suggesting completion of the reaction. However, the reaction is continued for an additional 90 minutes to ensure completion of the reaction. End-stage reaction over the second time be immediately lyophilized and the reaction material was to afford _{N orn -t-BOC-A-} 21978 factor C ₂ and C ₃ 12.7g.

Using a similar process, the reaction is carried out twice with 10 g of reactant and once with 30 g of reactant. In each case, the reaction time is only 40 minutes. Two experiments using 10 g of the reaction yield 11.9 g and 12.1 g of product, respectively. In the reaction using 30 g, the product was obtained 35.4.

Production Example 4

A-21978C Preparation of N _orn- t-BOC Nuclei

A. A. Fermentation of Utah

a. Fermentation of utahensis is carried out using the slope medium A and the production medium I, and the production medium is incubated for about 66 hours as described in Preparation Examples 1, A.

B. Deacylation of N _orn- t-BOC Complex

The A-21978C N _orn -t-BOC complex (A-21978C early quality 1185g of a composite containing about 176g) and to the fermentation medium. Deacylation is performed as described in Preparation Example 1, B. After about 24 hours, completion of deacylation is confirmed by HPLC.

C. A-21978C Isolation of N _orn- t-BOC Nuclei

The fermentation broth (100 L) obtained as described in section B is filtered through a filter aid (Hyflo Super-cel). The filtrate is passed through a column containing 7.5 L of HP-20 resin (DIAION) and the column is washed with water (38 L). Elution is monitored by silica gel / C ₁₈ HPLC and UV detection at 254 nm.

A small amount of nuclei elutes when washed. The nuclei are eluted successively with the following water: acetonitrile mixture: (95: 5) -40 L; And (85: 15) -100 L. The combined fractions containing the nucleus is concentrated under vacuum and removal of the solvent obtained, then the freeze-dried to give the semi-purified A-21978C N _orn -t-BOC nucleus 298.5g.

D. A-21978C Purification of N _orn- t-BOC Nuclei

A semi-purified A-21978C N _orn -t-BOC nucleus (30g) obtained as described in section C containing 0.25% acetic acid and 0.8% pyridine (75㎖) Water: for: acetonitrile (1: 9) Dissolve. This solution is applied to a 4.7 × 192 cm steel column containing 3.33 L of silica gel (Quantum LP-1 / C ₁₈ ) equilibrated in the same solvent system. The column is developed under pressure with water: acetonitrile: methanol (80: 15: 5) containing 0.2% acetic acid and 0.8% pyridine, and fractions of 350 ml are collected and the product is detected by 280 nm UV.

The combined product-containing fractions were concentrated in vacuo and removal of the solvent to give the following, the lyophilized and purified A-21978C N _orn -t-BOC nucleus 8.4g.

A-21978 t-BOC nuclei have the following characteristics:

a) Form: white amorphous solid that fluoresces under shortwave UV

b) Experimental formula: C ₆₇ H ₉₁ N ₁₇ O ₂₇

c) molecular weight: 1565

d) Solubility: Water Soluble

e) The hostile absorption spectrum (KBr) shows the absorption maximum at the following frequency (cm ^-1 ):

3345 (broad), 3065 (weak), 2975 (weak), 2936 (weak), ~ 1710 (show rate), 1660 (strong), 1530 (strong), 1452 (strong), 1395 (medium), 1368 (weak ), 1341 (about), 1250 (medium), 1228 (medium), 1166 (medium to about), and 1063 (about)

f) The UV absorption spectrum in 90% ethanol shows the maximum absorption at 220 nm (ε 42,000) and 260 nm (ε 10,600).

g) HPLC retention time: ₂ ml / under UV detection using H ₂ O / CH ₃ CN / CH ₃ OH (80: 15: 5) solvent containing 0.2% ammonium acetate on a 4.6 × 300 mm silica gel C ₁₈ column. The residence time is 6 minutes when eluted at a flow rate of min.

Production Example 5

A-21978 Alternative Purification of N _orn- t-BOC Nuclei

Preparation Example 4, a half tablet obtained by the A-21978C N _orn -t-BOC was dissolved in water nuclear (10..8g) AMBERLITE IRA-68 as described in Section C (Rohm and Haas, Philadelphia, PA, acetate cycle) is applied to a column containing 80 ml. The column was washed with water and eluted with 0.5 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 to collect 120 mL fractions. On silica gel / C ₁₈ , analytical HPLC is performed using a water: acetonitrile: methanol (80: 15: 5) system containing 0.2% ammonium acetate, and the column is monitored by detecting the product at UV at 254 nm. The product containing fractions are collected and the pH of the solution is adjusted to 5.8 using pyridine, then the resulting solution is concentrated in vacuo to a volume of about 200 ml.

Water is added to the concentrate and the resulting solution is reconcentrated to remove pyridine. By freeze-drying the concentrate to obtain the A-21978C N _orn -t-BOC nucleus tablet 3.46g.

[Examples 1 to 16]

Representative methods for preparing the complexes of Formula (I) include the preparation of various alkanoyl and alkenoyl derivatives by acylation of Formula (3). The derivatives in Table I are prepared by the Shoten-Baumann reaction using acid chloride as the acylating agent (method A) or by the active ester method (method B) using 2, 4, 5-trichlorophenyl ester as the acylating agent. do.

The general process for carrying out the acylation reaction by Method A or Method B is as follows:

Method A (Modified Shoten-Bauman Reaction)

This process consists of the reaction of the nucleus of formula (3) with the alkanoic acid or alkenoic acid chloride corresponding to the desired acyl side chain.

The mixture of formula (3) (1.95 to 2.16 g, 1.33 to 1.47 mmol) or the mixture of formula (2) (409 mg, 0.25 mmol) was dissolved in 200 ml of pyridine / water (9: 1). Acylating agents (18-20 molar excess of acylchloride dissolved in 15 ml of acetone) are suitable over 1 to 3 hours and the reaction is stirred for an additional 2 to 3 hours at ambient temperature. The reaction mixture is concentrated to remove acetone. The remaining aqueous phase is diluted with water to about 200 mL volume. The pH of the solution is adjusted to pH 3.0 to 3.5 by adding glacial acetic acid. The solution is washed eight times with the same volume of diethyl ether and then lyophilized.

The crude acylated derivatives are purified by reverse phase HPLC as follows: Samples dissolved in water or eluent system (about 4 to 6.5 ml) are injected onto 33 × 5/8 inch stainless steel filled with LP ₁ / C ₁₈ support. . The column is eluted with a solvent system consisting of H ₂ O: MeOH: CH ₃ CN: pyridine: HOAC. The elution step is carried out using an LDC duplex pump (Milton Roy) at a flow rate of about 10-12 ml / min under pressure of about 1500-2000 psi.

The eluate is monitored by UV detection using an ISCO-UA-5 detector at 280 nm. The desired fractions are combined and evaporated to dryness in vacuo to give the desired alkanoyl derivatives. The purified product is analyzed by TLC using a reversed phase plate (Whatman KC ₁₈ ) and a H ₂ O: MeOH: CH ₃ CN: pyridine: HOAC (45: 15: 40: 2: 2) solvent system. The plate is observed under UV light to detect the product. The product is also tested by UV (absorption coefficient at 220 nm and 260 nm) and amino acid analysis. Purity is measured on a monitor at UV 280 nm by analytical reversed phase HPLC (C ₁₈ Microbondapak, Waters Co.) using a H ₂ O: MeOH: CH ₃ CN: pyridine: HOAC solvent system.

Method B (2, 4, 5-trichlorophenyl active ester method)

Nob-t-BOC A-21978C nucleus (1.0 g, 0.64 mmol), A-21978C nucleus (0.5 to 1.0 g, 0.34 to 0.68 mmol) or A-21978C (9.46 mg, 0.58 mmol) and 3.5 mol excess trichloro A solution of phenyl-acyl active ester is dissolved in DMF (100 mL) and stirred at about room temperature to about 50 ° C. for about 6 to about 20 hours. The reaction mixture is concentrated to oil under vacuum. The resulting oil is triturated with 50 ml of Et ₂ O: toluene (1: 1) and washed with Et ₂ O. Monoacylated and diacylated A-21978C nuclear derivatives, acyl A-21978C and acyl t-BOC-A-21978C nuclei are purified as described in Method A.

The acylated t-BOC-A-21978C nucleus was deblocked using 50 mg / ml of trifluoroacetic acid: anisole: triethylsilane (10: 1: 1) at about -10 ° for 3 to 5 minutes. The reaction mixture is concentrated in vacuo to afford an oil which is triturated twice with 20 mL Et ₂ O. The crude acyl product is purified by reverse phase HPLC and analyzed as described in Method A.

Particular compounds as examples summarized in Tables I-X described below are complexes of the following general formula (I):

TABLE 1

A-21978C Preparation of N _Trp -Monoacyl Derivatives of Cyclic Peptides

a: R ¹ , R ² = H,

b: N _Trp acyl-H _orn -tBOC,

c: H ₂ O: CH ₃ OH: CH ₃ CH (V: V: V) containing 0.2% pyridine and 0.2% HOAC

d: The reaction temperature is maintained at 5 ℃.

e: contains 1% pyridine and 1% HOAC.

TABLE II

A-21978C Properties of N _Trp -Monoacyl Derivatives of Cyclic Peptides

a: Reversed-phase silica gel TLC (Whatman KC ₁₈ containing fluorescence and indicator) using H ₂ O: CH ₃ OH: CH ₃ CN (45: 15: 40) containing 0.2% pyridine and 0.2% HOCA in the solvent system. R _f value by

b: H ₂ O: CH ₃ OH: CH ₃ CN (V: V: V) containing 0.2% pyridine and 0.2% HOCA.

c: containing 1% pyridine and 1% HOAC.

TABLE III

Preparation of N _Trp , N _orn -Diacyl-A-12978C Derivatives

a: H ₂ O: CH ₃ CH: CH ₃ CN containing 0.2% pyridine and 0.2% HOAC (V: V: V).

b: succinic anhydride, 90% pyridine.

c: The reaction temperature is kept at 5 ° C.

d: 1% pyridine and 1% HOAC.

e: (t-BOC) ₂ / H ₂ O.

Table IV

_{Preparation of} N _TrP , N _Orn -Diacyl-A-21978C Derivatives

a: Reversed-phase silica gel TLC (Whatman KC ₁₈ ) containing a fluorescent indicator using a H ₂ O: CH ₃ OH: CH ₃ CN (45: 15: 40) solvent system containing 0.2% pyridine and 0.2% HOAC R _f value by.

b: H ₂ O: CH ₃ OH: CH ₃ CN (V: V: V) containing 0.2% pyridine and 0.2% HOAC.

c: containing 1% pyridine and 1% HOAC.

d: NT not tested.

TABLE V

_{Preparation of} N _TrP and N _Kyn - _Diacyl Derivatives

a: see Example 28 below.

b: H ₂ O: CH ₃ OH: CH ₃ CN (V: V: V) containing 0.2% pyridine and 0.2% HOAC.

Table VI

_{Characteristics of} N _TrP and N _Kyn - _Diacyl Derivatives

a: reversed phase silica gel TLC (Whatman KC _{18 with} fluorescence indicator) using a H ₂ O: CH ₃ OH: CH ₃ CN (45: 15: 40) solvent system containing 0.2% pyridine and 0.2% HOAC R _f value by.

b: H ₂ O with 0.2% pyridine and 0.2% HOAC: CH ₃ OH: CH ₃ CN: NH ₄ OAC.

Example 27

N _TrP- (n-decanoyl) A-21978C Nucleus [Compound (4)]

The following process describes a large scale process for the preparation of compounds by the active ester method.

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

A solution of decanoyl chloride (Pfaltz and Bauer, 5.6 ml) and 2, 4, 5-trichlorophenol (5.6 g) in 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 silica gel column (Woelm) using toluene as eluent. Fractions are monitored by TLC using detection shortwave UV. The appropriate fractions are combined and dried in vacuo to yield 10.4 g of 2, 4, 5-trichlorophenyl n-decanoate.

B. Acylation of N _Orn- t-BOC-A-219784C Nuclei with 2, 4, 5-trichlorophenyl n-decanoate

A solution of N _Orn- t-BOC A-21978C nucleus (15.0 g) and 2,4,5-trichlorophenyl n-decanoate (15.0 g) in anhydrous DMF (500 ml) was 25 at N ₂ at ambient temperature. Stir for hours. The mixture is then stirred at 60 ° C. for 5 hours until complete reaction is confirmed by TLC. The reaction mixture is concentrated to about 200 ml in vacuo and stirred with 1.2 l of Et ₂ O / toluene (5: 1). The product was filtered, separated and washed with Et ₂ O and dried in vacuo to give N _TrP — (n-decanoyl) -N _Orn- t-BOC-A-21978 C nuclear intermediate [Formula (1); 15.05 g of R = n-decanoyl, R ¹ = H, R ² = t-BOC.

C. _{Purification of} N _TrP- (n-decanoyl) -N _Orn- t-BOC-A-21978C Nuclei

N _TrP- (n-decanoyl) -N _Orn- t-BOC-A-21978C The nuclear intermediate is purified by the following method: The crude formulation is dissolved in about 50 ml of an eluting solvent system and then filled with a reversed phase C ₁₈ silica gel adsorbent. Purification by HPLC using a Waters Prep / 500 system containing a prepared cartridge. The system is eluted with H ₂ O: MeOH: CH ₃ CN (50: 15: 35) containing 0.2% pyridine and 0.2% HOAC. Fractions are monitored by UV of 280 nm. The appropriate fractions are combined and dried in vacuo to yield _{8.56 g of} purified N _TrP- (n-decanoyl) -N _Orn -t-BOC-A-21978C nuclei.

D. Removal of N _Orn -t-BOC Group

The t-BOC group was used to _prepare N _TrP- (n-decanoyl) -N _Orn- t-BOC-A-21978C nuclei (1.47 g) in 15 ml of trifluoroacetic acid / 1, 2-ethanedithiol (10: 1) Remove by stirring for 3 minutes at ambient temperature. The reaction mixture is dried in vacuo and the residue is triturated with Et ₂ O (50 ml). After washing with 20 ml of Et ₂ O, the ground material was dried in vacuo to give crude N _TrP- (n-decanoyl) -A-21978C nucleus [Formula (1): R = n-decanoyl; 2.50 g of R ¹ and R ² = H] are obtained.

E. _{Purification of} N _TrP- (n-decanoyl) -A-21978C Nuclei

The crude N _TrP- (n-decanoyl) -A-21978C nuclei are purified by reverse phase HLPC in the following manner: H ₂ O: MeOH: CH ₃ CN: Pyridine: HOAC (50: 15: 35: 2: 2) A sample (2.59 g) dissolved in 4.0 ml is injected into a 33 × 1 inch stainless steel column filled with LP-1 / C ₁₈ adsorbent. The column is eluted with the same solvent system. The elution step is performed at 1200 to 1700 psi pressure using a LDC duplex pump (Milton-Roy) at a flow rate of 10 to 12 ml / min. Eluate is monitored at 280 nm using a UV detector (Isco Model UA-5, Instrument Specialist Co., 4700 Superior Avenue, Lincoln, NB 68504). Collect fractions (20-24 ml) every 2 minutes. The desired fractions identified as exhibiting antimicrobial activity are combined and dried in vacuo to yield 1.05 g of product.

Repeating the above purification process using 4.35 g, 4.25 g, 2.14 g, 2.00 g and 1.75 g of crude starting derivative yields a total of 5.58 g of purified N _TrP- (n-decanoyl) -A-21978C nuclei.

Example 28

N _TrP- (n-decanoyl) -N _Kyn- (n-decanoyl) -A-21978C [Compound (21)]

In _preparing the N _TrP- (n-decanoyl) derivative of Example 27, the main reaction product was N _TrP- (n-decanoyl) -N _Kyn- (n-decanoyl) -A-21978C nucleus. 1): R and R ¹ = n-decanoyl; R ² = H]. This is separated during the reversed phase HLPC purification described in section E above. The desired fractions were combined and dried in vacuo to yield 211 mg crude product.

The compound was purified by analytical HLPC (C ₁₈ microbondapak, Waters Co.) using H ₂ O: MeOH: CH ₃ CN: NH ₄ OH: HOAC (6: 23: 15: 0.5: 0.5) as the elution system (32 repetitions). , 500 _μg of each injection sample) to obtain 4.4 mg of N _TrP- (n-decanoyl) -N _Kyn- (n-decanoyl) -A-21978C nuclei, identified by 360 MHz proton NMR.

Example 29

N _TrP -Cbz-N _Orn -Lauroyl A-21978C Nucleus [Compound (20)]

This method protects tryptophan α-NH ₂ of the N _Orn- t-BOC-A-21978C nucleus with the Cbz group, then removes the t-BOC group and acylates with trichlorophenyl-lauroyl ester (ornithine Α-NH ₂ position).

A. _{Preparation of} N _TrP -Cbz-N _Orn -t-BOC-A-21978C Nuclei

N _Orn- t-BOC-A-21978C nuclei (1.0 g) are dissolved in DMF (150 ml) and warmed to 60 ° C. N, O-bis-trimethylsilyl acetamide (0.55 ml) is added followed by benzyl pentachlorophenyl carbonate (257 mg). After stirring for 20 hours, the reaction mixture is concentrated to a volume of about 20-25 ml. Water (100 ml) is added and 1N sodium hydroxide is added to adjust the pH of this solution to 6.0. The mixture is washed with Et ₂ O (6 times, 200 ml volume) and then lyophilized.

The crude derivative is purified by reverse phase HLPC as follows: H ₂ O: MeOH: CH ₃ CN: Pyridine: HOAC (55: 15: 30: 2: 2) Sample dissolved in about 6 ml LP-1 / C ₁₈ support Fill into a 33 × 1/2 inch stainless steel column filled with. The desired fractions are identified by UV absorption (280 nm) and antimicrobial activity. These fractions are then combined and lyophilized to _{yield 951} mg of N _TrP - _Cbz -N _Orn- t-BOC-A-21978C nuclear derivative.

B. Removal of the t-BOC Group

The t-BOC group is removed by dissolving the intermediate at 50 mg / ml in trifluoroacetic acid: anisole: triethylsilane (10: 1: 1) at -10 ° C for 3 minutes. The oil obtained by concentrating the reaction was ground twice with 25 ml of Et ₂ O each time to give 520 mg of crude N _TrP -Cbz-A-21978C nuclei.

_{Acylation of} C. N _TrP -Cbz-A-21978C Nuclei

N _TrP -Cbz-A-21978C nuclei are acylated by the trichlorophenyl active ester acylation method. N _TrP -Cbz-A-21978C (520 mg) is added to a solution of 1-hydroxybenzotriazole (7 mg) and lauroyltrichlorophenol active ester (123 mg) in pyridine (150 ml). After stirring at 60 ° C. for 20 hours, the residue obtained by concentrating the reaction mixture was _triturated with Et ₂ O (3 times, 25 ml volume) to give N _TrP -Cbz-N _{Orn -lauroyl} -A-21978C nuclei (550 mg). To obtain.

Example 30

Preparation of Schiff Bases and Reduced Schiff Bases

Several reactions are carried out as follows: A-21978C The nucleus (40 mg) is dissolved in water (2 ml) and the pH of the solution is adjusted from 4 to 9 by addition of 1N sodium hydroxide. An aliquot of this solution (0.5 ml) is then mixed with each of the following aldehydes (5 μl) in methanol: 1) heptaldehyde, 2) octyl aldehyde, 3) decyl aldehyde, 4) undecyl aldehyde reactant Stir overnight at. The Schiffbase formed is reduced using NaBH ₃ CN (2.5 mg per reaction) for 5 minutes at room temperature.

The reaction is confirmed by testing for silica gel TLC and Staphylococcus aureus using a CH ₃ CH: H ₂ O (7: 3) solvent system. In this test, the Schiff base is S. It does not show activity against Oreus because it is likely that the Schiff base is unstable under test conditions. These compounds are S. Since it shows activity against Oreus and similar mobility in TLC system, formation of the following general formula (1) compound is confirmed (in each case, R ¹ is hydrogen).

Preparation of Schiff Bases and Reduced Schiff Bases

^a The lack of Schiff base activity in this test seems to reflect instability under test conditions.

Example 31

_{Preparation of} N _TrP - _{lauradaldehyde} Schiff Base and N _TrP , N _Orn - _{dilaualdehyde} Schiff Base and Reduced Schiff Bases thereof [Compounds (22) and (23)]]

A-21978C The nucleus (1 g) is dissolved in water (50 ml) and a solution of dodecyl aldehyde (500 μl) in methanol (200 ml) is added. The reaction mixture is stirred overnight at room temperature. Then NaBH ₃ CN (291 mg) was added to the reaction to reduce for 50 minutes. The reaction mixture is filtered under vacuum using Whatman 1 filter paper to remove particles. The aqueous solution obtained by concentrating the supernatant was lyophilized to yield 1.256 g of product. The product is examined by analytical HPLC and purified little by preparative reverse phase HPLC. Each portion (350 mg) is dissolved in 50% aqueous methanol (6 ml) with high frequency decomposition and heating to dissolve the material.

The solution was then 1.5x80 cm LP ₁ -C ₁₈ eluted with CH ₃ OH: CH ₃ CN: H ₂ O (25: 40: 35) containing 0.2% pyridine and 0.2% acetic acid at a flow rate of about 7.5 ml / min. Pass the column. Collecting a total of 104mg N _TrP the fraction containing the desired product - (n- dodecyl) -A-21978C nucleus [Compound (22)] and N _TrP of 19.2mg - (n- dodecyl) -N _Orn - ( n-dodecyl) -A-21978C nucleus [compound (23)] is obtained.

Example 32

The antimicrobial activity of the compound of formula (1) can be demonstrated by in vitro tests. The antimicrobial test results of the representative compounds of the general formula (1) using the standard plate agar disk diffusion method are shown in Table VII.

In Table VII, the activity is measured by the size of the inhibitor zone (in mm) that is observed to inhibit microbial growth by the test compound.

[Table VII]

Antimicrobial Activity of the Compound of Formula (1) by Plate Agar Disc Diffusion

^a compound 1mg / ml in concentration of sikimyeo suspended in water, was immersed a 7mm disk to the suspension, and then placed on the agar surface and incubated 24-48 hours at 25 to 37 ℃.

^b Minimum nutrient agar growth

^c tr = smile

The antimicrobial test results of typical Formula (1) compounds by standard agar dilution are summarized in Table VIII. In Table VII, activity is expressed as minimum inhibitory concentration (MIC), the lowest concentration of test compound that inhibits the growth of microorganisms.

[Table VII]

A-21978C Antibiotic Activity of Cyclic Peptides

^a MIC (mcg / ml); Compound Nos. In Tables II, IV and VI and Example 31

^b penicillin resistant strains

^c methicillin resistant strains

^d Median from 3 tests

^e 2 test

The A-21978C cyclic peptide of formula (1) exhibits antimicrobial activity in vivo against laboratory bacterial infections. Two doses of test compound were subcutaneously or orally administered to mice of demonstrative infections, and their activity was adjusted to ED ₅₀ values [effective dose in mg / kg protecting 50% of test animals; See: Warren Wick, et al., J. Bacteriol. 81, 233-235 (1961). The ED ₅₀ values for A-21978C compounds of representative formula (1) are shown in Table VII.

[Table VII]

In vivo activity of A-21978C cyclic peptide

^a mg / kg x 2, ^b 2 tests ^{c not} tested ^d 3 tests

The results of the toxicity studies on the A-21978C cyclic peptides of several general formulas (1) are shown in Table VII.

[Table VII]

A-21978C Toxicity of Cyclic Peptides

^a intravenous dose ^b two trials

Claims (16)

(Correction) A-21978C cyclic peptide nucleus or derivative thereof of the following general formula (3) is reacted with an acylating agent or an active derivative thereof of the following general formula (5), and A- 21978C Method for preparing a cyclic peptide derivative.

In the above formula, R, R ¹ and R ² are independently hydrogen, C ₄ to C ₁₄ -alkyl, optionally substituted C ₂ to C ₁₉ -alkanoyl 'C ₅ to C ₁₉ -alkenoyl or amino protecting groups, R ³ , R ⁴ and R ⁵ are both hydrogen, or R ³ and R ¹ , R ⁴ and R, or R ⁵ and R ² together may represent a C ₄ to C ₁₄ -alkylidene group, and R ′ and R ⁰ are independently Hydrogen or an aminoprotecting group, R ⁶ is optionally substituted C ₁ to C ₁₈ -alkyl or C ₄ to C ₁₈ -alkenyl, provided that 1) at least one of R, R ¹ or R ² is hydrogen or amino protecting group 2) at least one of R ¹ or R ² must be a hydrogen or amino protecting group, 3) the R, R ¹ and R ² groups together contain at least 4 carbon atoms, 4) R ¹ and When R ² is all selected from hydrogen or amino protecting group, R is 8-methyldecanoyl, 10-methylundecanoyl, 10-methyldodecanoyl, A-21978C factor C _{It cannot be} a specific C ₁₀ -alkanoyl group of ₀ or a specific C ₁₂ -alkanoyl group of A-21978C factors C ₄ and C ₅ . The compound of claim 1, wherein R is a general formula

Wherein alkanoyl is n is an integer of 3 to 17. The compound of claim 1, wherein R is a general formula

Is an alkanoyl of n and m are each independently an integer of 0 to 14, provided that the sum of n and m is 1 or more and 15 or less, and if n is 0, m cannot be 8 and n is 1 m cannot be 6 or 8. The compound of claim 1, wherein R is a general formula

And cis or trans alkenyl, wherein n and m are each independently an integer of 0 to 14, provided that the sum of n and m is 1 or more and 15 or less. The compound of claim 1, wherein R is a general formula

Cis or trans alkenyl, n is an integer from 2 to 16. (delete) (delete) (delete) (delete) (New) The method according to claim 1, wherein R is n-decanoyl [CH _3- (CH ₂ ) ₈ -CO-]. (Newly) A-21978C cyclic peptide nucleus of the following general formula (3) or a derivative thereof is characterized by reacting with a carbonyl derivative of the following general formula (6) Methods of Making Peptide Derivatives.

Wherein R, R ¹ and R ² are independently hydrogen, C ₄ to C ₁₄ -alkyl, optionally substituted C ₂ to C ₁₉ -alkanoyl, C ₅ to C ₁₉ -alkenoyl or amino protecting group, R ³ , R ₄ and R ⁵ are all hydrogen, or R ³ and R ¹ , R ⁴ and R, or R ⁵ and R ² together may represent a C ₄ to C ₁₄ -alkylidene group, and R ′ and R ⁰ Are independently hydrogen or an amino protecting group, and R ⁷ R ⁸ C = group represents a C ₄ to C ₁₄ -alkylidene group, provided that at least one of R, R ¹ or R ² is hydrogen or an amino protecting group 2) at least one of R ¹ or R ² must be a hydrogen or amino protecting group, 3) the R, R ¹ and R ² groups together contain at least 4 carbon atoms, 4) R ¹ and R ^When both are selected from hydrogen or amino protecting groups, R is 8-methyldecanoyl, 10-methylundecanoyl, 10-methyldodecanoyl, A-21978C factor It cannot be a specific C ₁₀ -alkanoyl group of C ₀ or a specific C ₁₂ -alkanoyl group of A-21978C factors C ₄ and C ₅ . (New) The method according to claim 11, wherein R is C ₄ to C ₁₄ -alkylidedenyl. (New) The method according to claim 12, wherein R is dodecyldenyl. (Newly) A-21978C cyclic peptide nucleus of the following general formula (3) or a derivative thereof is reacted with a carbonyl derivative of the following general formula (6), and the resulting C ₄ to C ₁₄ -alkylidene product is reduced to A process for preparing the A-21978C cyclic peptide derivative of the following general formula (1), wherein R, R ¹ or R ² forms a compound of the general formula (1) wherein C ₄ to C ₁₄ -alkyl group.

Wherein R, R ¹ and R ² are independently hydrogen, C ₄ to C ₁₄ -alkyl, optionally substituted C ₂ to C ₁₉ -alkanoyl, C ₅ to C ₁₉ -alkenoyl or amino protecting group, R ³ R ⁴ and R ⁵ are all hydrogen, or R ³ and R ¹ , R ⁴ and R, or R ⁵ and R ² together may represent a C ₄ to C ₁₄ -alkylidene group, and R ′ and R ⁰ are Independently hydrogen or an aminoprotecting group, and R ⁷ R ⁸ C = group represents a C ₄ to C ₁₄ -alkylidene group, provided that 1) at least one of R, R ¹ or R ² is not a hydrogen or amino protecting group 2) at least one of R ¹ or R ² must be a hydrogen or amino protecting group, 3) the R, R ¹ and R ² groups together contain at least 4 carbon atoms, and 4) R ¹ and R ² Is both hydrogen or amino protecting group, R is 8-methyldecanoyl, 10-methylundecanoyl, 10-methyldodecanoyl, A-21978Cfactor C _{It cannot be} a specific C ₁₀ -alkanoyl group of ₀ or a specific C ₁₂ -alkanoyl group of A-21978C factors C ₄ and C ₅ . (New) The method according to claim 14, wherein R is C ₄ to C ₁₄ -alkyl. (New) The method according to claim 15, wherein R is dodecyl.