HU185021B - Process for the preparation of cyclic hexapeptides - Google Patents

Abstract

The present invention relates to a process for the preparation of cyclic hexapeptide derivatives of the formula I and their acid addition salts, wherein R1 is hydrogen or hydroxy, and when R1 is hydrogen, R2 is hydrogen and R3 and R4 are hydrogen or hydroxy and R1 is hydroxy, R2 is hydrogen, R3 is hydroxy. and R4 is hydroxy or R2 is carbamoyl and R3 and R4 are hydroxy. The novel antibiotics of the present invention have antifungal activity. -1-

Description

The present invention relates to a process for the preparation of novel cyclic hexapeptide derivatives of the general formula (1). The compounds of the present invention are useful as intermediates in the preparation of semi-synthetic antifungal compounds.

In preparing these compounds are prepared by acilhasítunk to a cyclic peptide antibiotic of formula II, wherein in formula R is saturated or unsaturated fatty acid, and R ^1, R ^2, R ³ and R ⁴ are as defined below meanings.

It has been found that the fatty acid residue R can be enzymatically cleaved from the compound to give the cyclic hexapeptide parent compound. According to the method of the present invention, the antibiotic is exposed to an enzyme produced by a microorganism of the Actionoplanaceae family in an aqueous medium until acyl cleavage occurs.

In a preferred embodiment of the method of the invention, the enzyme biosynthesized by Actionplanes utahensis NRL 12052 is used to cleave the fatty acid side chain. Generally, acyl cleavage is achieved by adding the appropriate antibiotic to the culture of A. utahensis and incubating the culture until the acyl group is cleaved. The parent compound thus obtained is isolated from the culture broth by known methods. These parent compounds can be used to acylate again to produce new antibiotics.

More particularly, the present invention relates to a process for the preparation of cyclic hexapeptide parent compounds of formula I wherein:

R ^{1 is} hydrogen or hydroxy and when R ^{1 is} hydrogen, R ^{2 is} hydrogen, R ³ and R ⁴ are either hydroxy and when R ^{1 is} hydroxy, R ^{2 is} hydrogen, R ^{3 is} hydroxy or methoxy and R ^{4 is} hydroxy or R ^{2 is} carbamoyl and R ³ and R ⁴ are each hydroxy.

One of the parent compounds used in the process of the present invention is wherein R *, R ³ and R ^{4 are} hydroxy and R ^{2 is} hydrogen. This parent compound is known as A-30912 A parent compound. The parent compound A-30912 A is prepared by acylating the compound of formula II wherein R is a linoleyl group, a stearoyl group or a palmitoyl group and R ¹ , R ² , R ³ and R ^{4 are the} same as the corresponding groups of the parent compound A-30912 A.

When the cyclic peptide of formula II is R-linoleoyl, the antibiotic is referred to as antibiotic A-30912 A, while if R is stearoyl, this antibiotic is the tetrahydro-A-30912 component A, while R is palitoyl. Aculeacin A.

Component A-30912 A is a member of the A-30912 blend, which also contains components B, C, D, E, F and G. Mixture A-30912 and component A to component G are known from Hoehn and Michel, see U.S. Patent 4,024,245. Component A-30912 A is identical to the antibiotic A-22082 described by Higgens and Michel in U.S. Patent 4,024,246.

No. 4,024,245. and 4.24.246. Since the disclosure of U.S. Patents Nos. 3,690,940 and 5,630,600, it has been found that component A-30912 A is identical to Echinocandin B (see Benz et al., Helv. Chim. Acta, 57;

2459, 1974, and 568,386. Swiss Patent Specification No. Antibiotic SL 7810 / F is also identical to echinocandin B (see Keller-Juslen et al., Tetrahedron Letters, 46, 4147-4150, 1976) and Belgian Patent 823.289,

Keller-Juslen et al. Describe the structure of component A-30912 with a compound of formula II wherein R is a linoleoyl group, R ¹ , R ³ and R ^{4 are} hydroxy groups, and wherein R ^{2 is} hydrogen.

Tetrahydro-A-30912 Component A (Tetrahydro-SL 7810 / F, Tetrhydro-Echinocandin B) is disclosed in U.S. Patent 834,289. Belgian et al., Helv. Chim. Acta, 57, 2459-2477 (1974). The structure of the compound is described by a compound of formula II wherein R is stearoyl, R ¹ , R ³ and R ^{4 are} hydroxy and R ^{2 is} hydrogen. For simplicity, this compound is referred to herein as tetrahydro-A-30912A.

Aculeacin A is one of the components of the Aculeacin blend that is found in the highest amounts among the six minor components (Aculeacin B, C, D, E, F, and G). The aculeacin components are described in Mizuno et al., U.S. Patent 3,978,210. U.S. Pat. As described in 859.067. In the Belgian patent no. 808, aculeacin A is likely to have a similar cyclic peptide structure to tetrahydro-A-30912A except that the stearoyl side chain is replaced by a palmitoyl side chain.

The novel cyclic peptide parent compound of the invention, namely Compound A-30912 A (echinocandin B, SL 7810 / F), Compound A tetrahydro-A-30912 and aculeacin A, are compounds of Formula I wherein R ¹ , R ³ and R ^{4 is} hydroxy, while R ^{2 is} hydrogen.

The base parent compound (i.e. parent compound A-30912A) is a white, amorphous material, soluble in water, dimethylformamide, dimethylsulfoxide and methanol, but not in chloroform, toluene and diethyl ether, for example.

Experimental formula for parent compound A-30912 A 'C ₃ 4 H _s j N ₇ O! ₅ and a molecular weight of 797.83,

The infrared absorption spectrum of parent compound A-30912 A in a potassium bromide tablet is shown in Figure 1. The absorption maxima shown below are -33340, broad (hydroxy, hydrogen bonds), 2970, 2930, 2890 (CH, aliphatic CH ₃ , CH), 1660 and 1625 (more carbonyl), 1510-1550, 1430-1450 (CH). ), 1310-1340 1230-1260, 1080,835, 650 wide and 550 wide, cm ⁴ .

An electrometric titration of parent compound A-30912 A in 66% aqueous dimethylformamide showed a titratable group having a pK _a of 7.35 (original pH 7.32).

The parent compound A 30912 A was isolated by high performance liquid chromatography. Approximate retention time (K *) of parent compound A-30912 A: 11.52 minutes when separated by high performance liquid chromatography under the following conditions: column: 4,300 mm packing: silica gel / Cg • solvent: ammonium acetate: acetonitrile / water 1: 2: 97 mixture flow rate: 3 ml / min pressure: 10 atm detector: variable ultraviolet wavelength,

230 nm sensitivity. 0-04 A.U.F.S.

The preparation of the parent compound A-30912 A is described below.

Preparation of the substrate

The parent compound of the present invention, A-30912 A, is prepared from component A-30912 A, tetrahydro-30912 A, or aculeacin A. These materials may be used as pure compounds, but this is not essential. For example, the A-30912 A mixture in which A-30912 A is present as a major component may be used as a substrate for preparing the parent compound A-30912 A.

Component A-30912 A is prepared by culturing immersed aerated fermentation conditions of the following strains: a) Aspergillus rugulosus NRRL 8113, b) Aspergillus nidulans NRRL 8112, c) Aspergillus nidulans var. echinulatus A32204, NRRL 3860, d) Aspergillus rugulosus NRRL 8039, or e) Aspergillus nidulans var. roseus NRRL 11440.

Using the microorganism A. nidulans var, roseus NRLL 11440 for the preparation of component A-30912 A results in a mixture of components, referred to simply as the A-42355 antibiotic mixture. Major component of the A-42355 antibiotic mixture is component A-30912. Minor components of the A-42355 mixture are q, A-30912 components B, D and H. The A-30912 A-tetrahydro derivative is prepared from component A-30912 A by known hydrogenation procedures, whereby the reduction is effected until both double chemical bonds of the linoleoyl side chain are reduced.

For the production of aculeacin A, the microorganism Aspergillus aculeatus NRRL 8075 is disclosed in U.S. Patent 3,978,210. fermented according to U.S. Pat.

In another embodiment of the process of the present invention there is provided a parent compound wherein R ¹ and R ^{2 are} hydrogen and R ^{3 and} R ⁴ are hydroxy. Such a parent compound of the invention is referred to as A-30912 B parent compound. The parent compound A-30912 B is prepared by acylating a cyclic peptide antibiotic of formula II wherein R is a linoleoyl group or a stearoyl group, and R ¹ , R ² , R ³ and R ^{4 are} as described in parent compound A-30912 B.

In the case where R is a linoleyl group in Formula II, the cyclic peptide antibiotic is referred to as Component A-30912, whereas when R is a stearoyl group, this antibiotic is referred to as tetrahydro-A-30912 Component B.

Component A-30912 B is a member of the A-30912 blend, and also includes A, C, D, E, F, and G. A mixture of A-30912 is described by Hoehn and Micnel in 4,024,245. U.S. Pat.

Component A-30912 B has been shown to be identical to echinocandin C (see Traber et al., Helv. Chim Aeta, 62, 1252, 1979) and antibiotic SL 7810 / F-II (see Belgian Patent No. 834,289). description).

According to Traber et al., The structure of the echinocandin C antibiotic is provided by a compound of formula II wherein R * and R ^{2 are} hydrogen atoms,

R and R ^{4 are} hydroxy and R is linoleoyl. The structure of tetrahydro-echinocandin C, according to Traber et al., Is a compound of formula II wherein R * and R ^{2 are} hydrogen, R ³ and R ^{4 are} hydroxy and R is stearyl.

Tetrahydro-A-30912B (Tetrahydro-SL 7810 / F-II, Tetrahydro-echinocandin C) is described in Belgian Patent 834,289, and Traber et al., Helv, Chim. Aeta, 62, 1979, Compound II has the same structure as the compound of formula II wherein R 1 and R ^{2 are} hydrogen, R ³ and R ^{4 are} hydroxy and R is stearoyl. For purposes of simplicity, this compound is referred to herein as tetrahydro-A-30912B.

The structure of the novel cyclic hexapeptide parent compound of the invention, the parent compound A-30912 B and the tetrahydro-A-30912 B parent compound, is represented by a compound of formula I wherein R ¹ and R ^{2 are} hydrogen and R ³ and R ^{4 are} hydroxy.

The parent compound of A-30912 B has an empirical formula C ₃ 4 H _s ] N ₇ O 14 and a molecular weight of 781.83.

The preparation of parent compound A-30912 B is described below.

Preparation of the substrate

The parent compound A-30912 B is prepared from component A-30912 B or tetrahydro-A-30912 B. Because Component A-30912 is not a major component of the antibiotic blend, it must be purified to some extent before being used as a substrate to remove the other components that are formed.

Component A-30912 B is cultured under (a) immersed aerated fermentation conditions: a) Aspergillus rugulosus NRRL 8113, b) Apergillus nidulans var. echinulatus A-32204, NRRL 3860, c) Aspergillus rugulosus NRRL 8039, or d) Aspergillus nidulans var. roseus NRRL 11440.

If A.nidulans var. roseus NRRL 11440 is used to produce component A-30912 B, so for the sake of simplicity we get a mixture called antibiotic mixture A-42355? The major component of the A-42355 antibiotic mixture is component A-30912. Minor components of the mixture are A-30912 B, D and H.

Tetrahydro-A-30912 B is prepared from component A-30912 B by known hydrogenation techniques. The reduction is performed until the double chemical bonds of the linoleoyl side chain are reduced.

In another embodiment of the process of the invention, compounds are prepared wherein R ¹ , R ² , R ³ , and R ⁴ are each hydrogen. This compound is referred to as the parent compound A-30912 D. This compound is prepared by deacylating a cyclic hexapeptide antibiotic of formula II wherein R is a linoleoyl or stearoyl group and R ¹ , R ² , R ³ and R ^{4 are} as defined in parent compound A-30912 D.

When the cyclic hexapeptide of formula II is an R-linoleoyl group, the antibiotic is referred to as Component A-30912, whereas when the R is a stearoyl group, the compound is designated as tetrahydro-A-30912 Component D.

Component A-30912 D is a component of the A-30912 blend, and also includes A, B, C, D, E, FesG. The A-30912 blend is described by Hoehn and Michel in 4,024,245. U.S. Pat.

Component D is identical to antibiotic echnoncandin D (see Traber et al., Helv. Chim. Acta, 62, 1252, 1979) and antibiotic SL-7810 / F-II (see Belgian Patent No. 834,289). |

Traber et al. Propose a compound of formula II for the echinocandin D antibiotic structure wherein R ¹ , R ² , R ³ and R ⁴ are each hydrogen and R is a linoleoyl group. The structure of the antibiotic tetrahydro-echinocand D is, according to Traber et al., Identical to that of a compound of the formula ΓΙ wherein R ¹ , R ² , R ³ and R ^{4 are} hydrogen atoms and R is a stearoyl group.

The above compound is referred to as tetrahydro-A-30912-D for simplicity (identical to tetrahydro-SL 7810 / F-III, tetrahydro-echinocandin D).

The novel cyclic peptide parent compound of the present invention, that is, the parent compound of Component A-30912 D (echinocandin D, SL 7810 / F-IIII) and tetrahydro-A-30912 Dt can be characterized by the general formula ¹ wherein R ¹ , R ² , R ³ and R ⁴ are each hydrogen.

Experimental formula for parent compound A-30912 D C3 4 H _s i N ₇ 0! ₂ and a molecular weight of 749.83.

The preparation of parent compound A-30912 D is described below.

Preparation of the substrate

The parent compound A-30912 D is prepared from component A-30912 D or tetrahydro-A-30912 D. Since Component A-30912 is a minor component of the antibiotic mixture, it needs to be purified to some extent before being used as a substrate to remove other co-producing components.

The antibiotic component A-30912 D is prepared by culturing the following microorganisms under immersed, aerated fermentation conditions: a) Aspergillus rugulosus NRRL 8113, b) Aspergillus nidulans var. echinulatus A-32204, NRRL 3860, c) Aspergillus rugulosus NRRL 8039 (Belgian Patent 834289), or d) Aspergillus nidulans var, roseus NRRL 11440.

If A. nidulans var. roseus NRRL 11440 is used to obtain a mixture of components, referred to simply as the A4-2355 antibiotic mixture, for simplicity, the major component of which is component A-30912 A. Components B, F and H of A-30912 are minor components of A-42355.

Tetrahydro-A-30912 D is hydrogenated from component A-30912 D! methods. The reduction is carried out until the double bonds of the linoleol side chain are reduced.

The invention according to a further embodiment variant is prepared by a basic compound, wherein ^R1 and ^R4 are hydroxyl groups, R ² hydrogen and R ³ 1-6 methoxy. This parent compound is referred to as the parent compound A-30912 H. The A-30912 H-type parent compound is prepared by acylating the cyclic hexapeptide antibiotic of formula II wherein R is a linoleoyl or stearoyl group, and R *, R ² , R ³ and R ⁴ are as defined in the A-30912 H-type parent compound.

When R is a linoleoyl group and R ^{3 is a} methoxy group in the cyclic hexapeptide of formula II, this compound is referred to as Component A-30912 H, whereas when R is a stearoyl group and R ^{3 is} methoxy, the antibiotic is called tetrahydro-A-30912 Component H.

Component A-30912 H is a component of the A-30912 blend, further comprising A, C, D, E, D, and G. Mixture A-30912 is described by Hoehn and Michel in U.S. Patent 4,024,245. Component A-30912 H is a later discovered antibiotic component A-30912 and is described in Michel Antibiotic A-309J2 Factor H, 117,739. filed February 1, 980, which is part of U.S. Patent No. 46,875, filed June 8, 1979. are incorporated herein by reference.

Following the discovery of the structure of component A-30912 H, it became apparent that the lower alkyloxy homologues of component A-30912 H would be useful compounds. Prior to this time, the usefulness of the alkyloxy derivative prepared was not recognized, but was only made for structure determination. Short-chain 2-6-alkoxy homologs are prepared from component A-30912A.

The antibiotic component A-30912 A is prepared by culturing the following microorganisms under submerged aeration fermentation conditions: a) Aspergillus rugulosus NRRL 8113, b) Aspergillus nidulans NRRL 8112, c) Apergillus nidulans var. echinulatus A-32204, NRRL 3860, d) Aspergillus rugulosos NRRL 8039, or e) Aspergillus nidulans var. roseus NRRL 11440.

Because parent compound A-30912 H contains an amino group, the compound may exist as a salt. These salts can be used as intermediates and for purification. Pharmaceutically acceptable salts of parent compound A-30912 H can be used particularly advantageously since the final products are easier to purify. Pharmaceutically acceptable salts are salts that do not increase their toxicity in comparison to the free base in warm-blooded animals.

The preparation of the parent compound A-30912 H is described below.

Preparation of the substrate

The A-30912 H parent compounds, i.e. the compound of formula I, wherein in formula R ¹ and R ⁴ OH, R ² is hydrogen and R ³ methoxy, A-30 912 H-component or tetrahydro-A-30912 H from. Since Component A-30912 H is a minor component of the antibiotic mixture, it is necessary to purify to some extent to remove the remaining antibiotic components before use as a substrate.

The antibiotic component A-30912 H is prepared by fermentation of the following microorganisms: a) Aspergillus rugulosus NRRL 8113, or b) Aspergillus nidulans var. roseus NRRL 11440.

If A. nidulans var. roseus strain NRRL 11440 to obtain a mixture of components as described in FIG

This is called a -42355 antibiotic mixture. The mixture is a major component of A-30912 component A, while B, D and H are minor components of A-42355 mixture.

The process of the present invention provides a further parent compound wherein R *, R ³ and R ^{4 are} hydroxy and R ^{2 is a} carboxylic acid amide. The above parent compound of the invention is referred to as the parent compound S 31794 / F1. S 31794 / Fl parent compound prepared is the II cyclic peptide antibiotic acilhasításával general, where in the formula the meanings given R myristoyl and R ^1, R ^2, R ³ and R ⁴ S-31794 / Fl basic compound represents.

To produce the parent compound of the present invention, S31794 / F-1, the cyclic hexapeptide antibiotic S 31794 / F-1 is deacylated.

Anti-fungal antibiotic S 31794 / Fl is disclosed in U.S. Patent No. 2,662,965. German Patent No. 4,673,629; U.S. Pat. No. 4,198,123 to Acrophialophora limonsipora nov. spec. Dreyfuss et Muller NRRL 8095 was cultured. Compound S 31794 / F1 has the following characteristics: At 178 ° C to 180 ° C, the amorphous powder melts at decomposition and the crystalline at 1 ° C to 183 ° C also decomposes: (at ² p ^ 4: [ α-λθθ: -24 ° (c = 0.5 in methanol) or ,37 ° (c = 0.5 in pyridine) (crystalline material): ultraviolet absorption spectrum in methanol: 194 nm (ε 1%, cm: 807), 225 nm (shoulder), (E1%, cm: 132), 276 nm (E1%, cm: 12.8), 284 nm (shoulder), E1%, cm: 10, 5), the C 13 nuclear magnetic resonance spectrum (crystalline material) of deuteromethanol (190 mg, 1.5 ml deuteromethane, internal standard tetramethylsilane) is as follows:

PPM PPM PPM 176.2 75.5 51.2 175.0 74.0 39.7 173.7 71.0 38.8 172.6 70.5 36.6 172.0 69.7 34.8 171.8 68.0 32.8 171.7 62.2 30.6 168.6 58.3 · 26.7 157.7 57.0 23.5 132.5 56.2 19.7 129.0 55.4 14.3 115.9 52.9 11.1 76.6

The crystalline material is dried for 2 hours at 100 [deg.] C. under a high vacuum to give the following elemental analysis: 55.5-56.5% carbon, 7.5-7.7% hydrogen, 10.5-10.8% nitrogen and 25.5) 26.0% oxygen, the compound is highly soluble in methanol in ethanol, pyridine, dimethylsulfoxide, slightly soluble in water, chloroform, ethyl acetate, diethyl ether, benzene and hexane, the compound has antifungal activity, especially Candida albicans. against a microorganism.

The structure of S 31794 / Fl antibiotic enter a compound of Formula II wherein R myristoyl, R, R ³ and R ⁴ OH and R ^2-carboxylic acid amide.

The novel cyclic peptide parent compound of the present invention, the parent compound of antibiotic S 31794 / F1, has the following structure: A compound of formula I wherein R ¹ , R ³ and R ^{4 are} hydroxy, R? and carbonsa is a v-amide group.

The parent compound of S 31794 / Fl has an empirical formula C ₃ s H _{s 2} N ₈ O ₁ β and a molecular weight of 840.87.

The preparation of the parent compound S 31794 / F-1 is described below.

Preparation of the substrate

The parent compound S 31294 / F-1 is prepared from the antibiotic S 31794 / F-1.

The antibiotic S 31794 / F-1 is prepared by culturing the microorganism Acrophialophora limonispora limonspora NRRL 89595 under submerged aeration. The microorganism was deposited with the Norther Region Research Center, U.S.A. Department of Agriculture, Agricultural Research Cultures College, North Central Region, Peoria, Illinois 61604, here the strain is given the NRRL 8095.

Final purification of the individual antibiotics preferably reversed-phase high-resolution, low-pressure liquid chromatography using silica gel / C _l8 adsorbent. The process involves dissolving the crude antibiotic mixture in a solvent and placing the solution on a column equilibrated with a similar solvent. The column is then eluted with the solvent. Fractions were collected and analyzed by Candida albieans bioautography and / or ultraviolet light (based on relative retention time). Fractions containing each antibiotic component were collected. Occasionally, additional chromatographic separation is required to isolate the pure form of each component.

For example, the crude antibiotic mixture for A-30912 or A-42355 is prepared by extraction of the whole culture broth or mycelium with methanol and chloroform.

Preferably, a 7: 2: 1 mixture of methanol, water, and acetonitrile is used for chromatographic separation of the antibiotics.

The crude antibiotic S 31794 / F-1 was prepared by extracting the whole fermentation broth with ethyl acetate: isopropanol (4: 1) and then chromatographing the extracts.

Each antibiotic component is identified by thin layer chromatography. The adsorbent is preferably silica gel.

The R / P values of the A-30912 A-G components on a silica gel thin layer with a 7: 3 mixture of benzene and methanol are given in Table I.

The studies were performed by Candida albicans bioautography.

Table I

Component 30912 has a value on it

A 0.35

B 0.45

C, 0.54

D, 0.59

E, 0.27

F, 0.18

G, 0.13

III. spreadsheet

Component 30912 is the Retention Time in seconds

792

B 870

H 990

D 1140

Components A-30912 A, B, C, D, and H have Ri values obtained using bioautography of Candida albicans with various solvents on silica gel (Merck-Darmstadt silica gel No. 60, 20 x 20 cm).

H, Table

The Rp values for component 30912 are in the solvent mixture

Component A 0.20 0.14 0.28, 0.43

Component B 0.39 0.21 0.42 0.47

Component C 0.46 0.31 0.51 0.58

Component D 0.50 0.38 0.57 0.61

H component 0.42 0.27 0.36 0.53

Solvent tickets a: ethyl acetate: methanol 3: 2, b: ethyl acetate: methanol 7: 3 c: acetonitrile: water 95: 5 d: ethyl acetate, ethanol: acetic acid 40:60 : 0.25.

The A-30912 A, B, D and H components can also be identified high-resolution, low-pressure liquid chromatography under the following conditions: Column: glass, 0.8 x 15.0 cm packing: Nucleosil ^C-1 lÖ _je (Mechery-Nagel and Company), fill as follows Solvent: methanol: water: acetonitrile 7: 2: 1 Sample volume: 8 μί Sample weight: 8 pg Column temperature: Room temperature Flow rate: 18, ml / min Pressure: 9 , 8 atm detector: UV detection at 222 nm (ISCO Model 1800, variable wavelength, UV sensor monitor) Pump: LDC Duplex Minipump injection: needle injection.

In III. Table A shows the retention times for components A-30912 A, B, D and H.

Preparation of the enzyme

1. Producing micro-organism

The enzyme used to deacylate the antibiotics of the invention is produced by certain microorganisms of the Actinolanaceae family, preferably strain Actinoplanes utahensis NRRL 12052.

The enzyme may be similar to that used for the deacylation of penicillins, this work is described in Kleinschmid, Wright, Kvanagh and Stark 3,150,059. U.S. Pat. Although Example 1 describes a preferred method of culturing A. utahensis strain NRRL 12052, other methods will obviously be apparent to those skilled in the art.

The Actinoplanaceae is a relatively new family of the Actinomycetales, first described by Coueh in 1955 (J. Elisha-Mitchell, Sci. Soc. 71, 148, 1955). Family and gender characteristics can be found in Bergey's Manual of Determinative Bacteriology, 8tn ed ,, R.E. Ruchanan and N. E. Gibbons, Eds., Th Williams Wilkins Co., Baltimore, Md., 1974, 7Ű6-723. side. So far, 10 genders have been distinguished:

I. Actinoplanes (the type and the most common,

II. Spirillospora, Ilii. Streptosporangium, IV. Amorphosporangurn, V. Ampullariella, VI. Pilimelia, VII. Planomonospora, VTII. Planobispora, IX. Datylospprangium and X. Kitasatoa,

For example, isolated and characterized species and variants include Actinoplanes philippinensis, Actinoplanes armenlacus, Actinoplanes utahensis and Actinoplanes missurensens, Spirillospora albida, Streptosporiangium roseum, Streptosporangium vulgare, Streptosporangium roseum var. Hollandensis, Streptospirangium album, Streptosporangium viridialbum, Amorphosporangium auranticolor, Ampullariella regular, Ampullariella campanulata, Ampullariella lobata, Ampullariella digitata, Pilimelia terevasa, Pilimelia anulata, Planomonospora parontospora, Planomonospora, Planomonospora

A preferred source of the enzyme used in the process of the invention is not Actinoplanes. Within the genus Actinoplanes, the species Actinoplanes utahensis is particularly advantageous.

Cultures of the species used in the method of the present invention can be ordered from the Northern Region Research Laboratory strain collection under the following code number:

Actinoplanes utahensis NRRL 12052

Actinoplanes missouriensis' NRRL 12053

185 021

Actlnoplanes sp. Actinoplanes sp. Actinoplanes sp.

NRRL 8122 NRRL 12065. NRRL 42064

A. utahensis strain NRRL 12052 is from the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md .. 20852 (A. utahensis ATCC 14539). A. utahensis strain ATCC 14539 may also be used as an enzyme source.

A. mossourinesis strain NRRL 12053 is also from the ATCC strain collection (A. missouriensis ATCC 14538) and is also an enzyme source.

The potency of particular strains of the Actinoplanaceae family of microorganisms in the acyl cleavage reaction of the present invention is determined by the following procedure. The microorganism was inoculated with a medium of preferred composition. The culture was cultivated on a rotary shaker at 28 ° C for 2 or 3 days. One of the substrate antibiotics is then added to the culture. The pH of the broth is maintained at 6.5. During cultivation, activity is determined by measuring with Candida albicans. A decrease in antibiotic activity indicates that the microorganism produces an enzyme that catalyses the acyl cleavage reaction. However, this must be demonstrated by one of the following methods:

1) the axle? performing high performance liquid chromatography (HPLC) to confirm the presence of a skeleton, or

2) re-acylating the compound with a suitable side chain, such as a linoleoyl, stearoyl, palmitoyl or myrostoyl side chain, to restore activity.

2. Enzyme production conditions

Enzyme production is carried out under conditions that are advantageous to the growth of Actionplanaceae, i.e., at 25 ° C to 30 ° C, at a pH of 5.0 to 8.0, with stirring and aeration. The culture medium must contain: a) a useful carbon source such as sucrose, glucose, glycerol or others, b) a nitrogen source such as peptone, urea, ammonium sulfate or others, c) a phosphate source such as soluble phosphate salt, and d) inorganic salts, which generally increase the growth of microorganisms.

An effective amount of the enzyme is 40-60 after the growth phase. hours, when a certain amount of time has passed after the rapid growth. The amount of enzyme produced will depend on the species used and the different growth conditions.

As will be apparent to one of ordinary skill in the art, microorganisms such as Actinoplanes utahensis NRRL 12052, i.e., the enzyme producing microorganism, may also vary. For example, artificial variants and mutants can be removed from these strains using various known mutagens, such as ultraviolet radiation, X-rays, high-frequency waves, radioactive radiation and chemicals. All natural and artificial variants and mutants of the above strains which produce the enzyme can be used in the process of the present invention,

Conditions for acyl cleavage

Preferably, the substrate is added to the culture of Actinoplanaceae after incubation of the strain for at least 48 hours. The concentration of the substrate in the transforming medium may vary within wide limits. However, for maximum utilization of the enzyme, and for virtually complete deacylation within 24 hours, substrate concentrations of 0.5 mg to 1.0 mg per ml are generally used. However, less substrate may be added, but the enzyme is not fully utilized, and at higher substrate concentrations, the deacylation is incomplete without prolongation of the fermentation time.

The fastest conversion of substrate antibiotics to the corresponding parent compound of the present invention occurs when the pH of the fermentation broth is maintained between 6.0 and 7.0. At pH = 6 or lower, deacylation is slow as the pH rises above 6.0, and both the substrate and the parent compound formed are less and less stable. Maximum stability is eaten at pH 6.0, but at pH 6.0 deacylation occurs more slowly over a period of about 30 to 36 hours. For faster deacylation (24 hours), we ferment at pH 6.5, with no major loss. The pH in the mixed fermentors can be controlled by sensors. Where this is impractical, for example in a flask fermenter, the pH is resolved by adding 0.1 M phosphate buffer, which is added to the medium prior to the addition of the substrate.

Fermentation must continue for 24 hours or more after the substrate is added. The purity of the substrate influences the degree of deacylation. For example, when a substrate with a purity greater than 50% is used, it is deacylated in an amount of 0.8 to 1.2 mg / ml of antibiotic per 24 hours. If the substrate is less pure, the deacylation proceeds more slowly.

The substrate may be dosed several times. For example, 0.30.5 mg of antibiotic is added to small fermentors at least five times per 12 hour intervals. Larger fermenters receive 0.5 mg / ml twice per ml.

The deacylation reaction may be carried out over a wide range of temperatures, for example between 20 ° C and 45 ° C. However, it is preferred that the deacylation temperature is adjusted to 25 ° C and 35 ° C, more preferably 26 ° C, in which case the deacylation is optimal and the substrate and parent compound are most stable.

4. Purified antibiotics are preferably used as substrates. Subcutaneous antibiotics have antifungal activity but no antibacterial activity. Thus, the substrate, by carrying bacterial cells or spores, can infect the fermentation broth during the deacylation reaction. These infections may affect the deacylation reaction or the stability of the parent antibiotics and parent compounds obtained as a product. Therefore, it is important that the substrates are sterile administered. Since most substrate antibiotics are degraded during autoclaving, the compositions are preferably sterilized with ethylene oxide in a pressurized system.

5. Follow-up of the deacylation reaction

The starting materials have antifungal activity, in particular against the microorganism Candida albicans. Thus, the amount of substrate present is determined by measuring against C. albicans. The resulting parent compound is water soluble but biologically inactive. Thus, the reduction in biological activity is rapid and provides a useful means of assaying for deacylation. We also need to measure the fermentation broths and alcoholic extracts of the fermentation broth as the substrates are only slightly soluble in the broth.

Use of the parent compound

The parent compounds and their acid addition salts can be used as intermediates in the preparation of synthetic antifungal compounds. Useful antifungal compounds prepared from these parent compounds are disclosed in Abbott and Fukusa (Docket No. X-5396) and in Docket No. X-5595 and X-5564, derivatives of Cyclic Feptide Base Compounds.

The term alkyl as used hereinbefore denotes a monovalent, saturated, straight or branched hydrocarbon group. Alkenyl means a monovalent, unsaturated, straight or branched chain hydrocarbon group having up to three double bonds. The double bonds in the unsaturated hydrocarbon chain may be in the cis or trans configuration. C 6 -C 24 is a straight-chain or branched hydrocarbon group having from 6 to 24 carbon atoms.

Compounds of formula III are prepared by acylating the corresponding parent compound on the α-amino group of the ornithine group of the parent compound with the corresponding acyl side chain. The method used for acylation is known and is used to form the amide bond. Generally speaking, the acylation reaction is carried out by reacting the parent compound with an activated derivative of the acid corresponding to the corresponding acyl side chain.

By an activated derivative is meant a derivative which is capable of attaching the carboxyl group of the acylating agent to a primary anino group when the resulting amide group attaches the acyl side chain to the parent compound. Preferred activated derivatives and methods for their preparation and use are known to those skilled in the art. Preferred activated derivatives include:

(a) an acid halide (for example (acid chloride),

(b) acid anhydride (for example, alkoxy formic anhydride or aryloxy formic anhydride); or

c) an activated ester (e.g., 2,4,5-trichlorophenyl ester, N-hydroxybenzotriazole ester, or N-hydroxy succinimide ester).

Alternatively, the carboxylic acid can be activated by reacting the carboxylic acid with carbonyl diimide, such as N, N-dicyclohexylcarbodiimide or N, N-diisopropylcarbodiimide, without isolating the resulting reactive intermediate due to its instability. , but is reacted with the primary amine in the reaction mixture itself.

It will be apparent to those skilled in the art that, if a particular amino acid contains an acylable functional group other than the amino group, it is protected before the reaction of the amino acid with the alkanoyl or alkenoyl group coupling agent to the amino acid. need. A preferred protecting group may be any group which may be used to protect functional side chains during peptide synthesis. These groups are well known, and the protecting groups and the manner in which they are attached to the compound are not difficult to those skilled in the art (see, for example, Protective Groups In Organic Chemistry, dcOmie, Editor, Plenum Press, N.Y. 1973).

The compounds of formula III inhibit the growth of fungal pathogens. The compounds of the invention can thus be used to inhibit fungi growing on surfaces in our environment (all antiseptic) or to treat fungal infections with the compounds. The antifungal activity of the compounds is demonstrated by in vitro agar diffusion assays and agar dilution assays, or by in vivo assays in mice infected with Candida albicans fungi. The compounds of the formula III are also active against Trichophyton against the micro-organism.

Certain compounds, as described below, provide significant blood levels in mice upon oral administration.

When administered to dogs by intravenous injection at a dose of 100 mg / kg body weight per day for five days, the compound of formula III wherein ar-p- (n-octyloxy) -benzoyl is non-toxic! signals, although an increase in serum glutainate pyruvate transaminase activity is observed.

For systemic use, the dosage of the compounds of Formula III will depend on the compound employed, the severity and nature of the infection, and the physical condition of the subject. Treatment should be initiated at low dosages and subsequently increased until adequate antifungal activity is achieved. The compounds may be administered intravenously or intramuscularly by injection, in which case a sterile aqueous solution or suspension may be added, optionally together with various known pharmaceutically acceptable preservatives, buffering agents, solubilizers or suspending agents. The compositions may also contain other additives, such as salt or glucose, which are added to render the solutions isotonic. The proportions and quality of the additives are known to those skilled in the art. '

Certain compounds of Formula II, after oral administration, provide significant blood levels, as shown in Example 58, Example VIII. table. The compound may be systemically administered orally. For oral administration, these compounds are used in combination with pharmaceutically acceptable carriers or diluents, as capsules, tablets or powder formulations. The nature and ratio of the carriers or diluents will be known to those skilled in the art.

When used to treat vaginal Candida infection, the compound of Formula III is administered in conjunction with a pharmaceutically acceptable diluent known for use in vaginal use. The preparation of compositions for vaginal administration is known to those skilled in the art.

The following examples illustrate the process of the invention.

Example 1

Fermentation of Actinoplanes utahensis NRRL 12052

Cultures of Actinoplanes utahensis NRRL 12052 were prepared and maintained on inclined agar. For the preparation of inclined agar, the following feed-81 was used

We use one of 185,021 species:

The medium ingredient Quantity baby oatmeal 60.0 g yeast 2.5 g dipotassium hydrogen phosphate 1.0 g

Czapek's Mineral Saline * 5.0 ml ugar 25.0 g

Pre-ionized water 11 filled

The pH of the medium before autoclaving was adjusted to 5.9 and adjusted to 7.2 with sodium hydroxide. The pH after autoclaving is 6.7.

^x = The components of the mineral salt solution of Czapek are as follows:

ingredient quantity

FeSO ₄ .7H ₃ O (dissolved in 2 ml concentrated hydrochloric acid) 2 g potassium chloride 100 g magnesium sulfate.7 water 100 g deionized water 11

Medium B component potato dextrin yeast extract enzymatically hydrolyzed casein ^x couscous extract dextrose corn starch meat peptone beet molasses magnesium sulfate.7 water calcium carbonate Czapek mineral salt agar

Deionized water ^x 'NZ-amine A, Humko Sheffield Chemical, Lyndhurst, NJ

Inclined agar was inoculated with Actionplanes utahensis strain NRRL 12052 and incubated at 30 ° C for 8-10 days. Half of the slant agar culture was inoculated with 50 ml of vegetative medium of the following composition:

Quantity 5.0 g 0.5 g 3.0 g 0.5 g 12.5 g 5.0 g 5.0 g 2.5 g 0.25 g '1.0 g 2.0 ml 20.0 g 11- filled ingredient pre-cooked oatmeal sucrose yeast distillation residue ^x dipotassium hydrogen phosphate mineral salt of Czapek deionized water '' Quantity 20.0 g. 20.0 g

2.5 g 5.0 g 1.0 g

Make up to 5.0 ml with 11.

The pH of the medium was adjusted to 7.4 with sodium hydroxide, and the pH of the medium after autoclaving was 6.8. ^x = National Distillers Products Co., 99 Park Ave.,

New York, N.Y.

The inoculated vegetative medium is incubated in a 250 mL wide-mouth Erlenmeyer lornbik at 30 ° C for 72 hours on a rotary shaker rotating at 250 rpm and circling about 5 cm in diameter.

This vegetative culture can be used directly to inoculate a second vegetative medium. In another embodiment, and preferably, it is stored for later use in the vapor phase of liquid nitrogen. The culture is subdivided into small vials for this storage as follows: each vial contains 2 ml of an overgrown culture of vegeta50 and 2 ml of glycerol-lactose solution (see Hailey and Higgens, Preservation and Storage of Gas Phase of Liquid Nitrogen, Cryobi). 10, 364-367 (1973). The prepared suspensions are stored in the vapor phase above liquid nitrogen.

ml of the suspension thus stored was inoculated with 50 ml of the first phase vegetative medium having the same composition as above. The inoculated vegetative medium is cultured as above.

To produce a larger volume of inoculum, 10 ml of overgrown first-stage vegetative medium was inoculated with 400 ml of second vegetative medium having the same composition as the first vegetative medium. This second inoculum was cultured in a two-liter, wide-mouth Erlenmeyer lornbik at 30 ° C for 48 hours on a rotary shaker rotating at 250 rpm at ca. 5 cm in diameter.

80 µl of the second vegetative culture grown as above was inoculated with 1001 sterile production medium. The production medium is selected from the following:

Medium I Component Quantity (g / l) Peanut Flour 10.0 g Meat Meat Peptone 5.0 Sucrose 20.0 Potassium Dihydrogen Phosphate 0.5 Dipotassium Hydrogen Phosphate 1.2 Magnesium Sulfate. 7 water. 0.25 tap water to 1 liter

PH of the utánθ medium after sterilization is 6, 9, and the gylation is carried out at 121 ° C for 45 minutes,

II. broth

Quantity (g / l) 30,000 5,000. 1,000 0.500 0.002 11 filled ingredient sucrose peptone dipotassium hydrogen phosphate potassium chloride magnesium sulfate. 7 water deionized water

The pH of the medium was adjusted to 7.0 with hydrochloric acid, and the pH of the medium after sterilization was 7.0.

III. medium ingredient glucose ammonium chloride sodium sulfate zinc chloride magnesium chloride. 6 water iron trichloride. 6 water manganese dichloride. 4 Water Copper Chloride. 2 water calcium carbonate potassium dihydrogen phosphate ^x tap water.

^x = Sterilize separately and dispense sterile.

The pH of the medium after sterilization is 6.6.

The inoculated production medium is fermented in a 165 L total volume fermentor at 30 ° C for 42 hours. The fermentation broth is mixed with a known mixer, the mixer turning at 200 rpm. Sterile air is bubbled through the medium so that the concentration of dissolved oxygen is at atmospheric Quantity (g / l) 20,000 3,000 2,000 0.019 0.304 0.062 0.035 0.005 6.000 0.670 11 Filled with dipotassium hydrogen phosphate 2 g tap water 1000 ml (original pH 6, 1).

* = N-Z-Case,

Example 3

Isolation of the A-42355 antibiotic complex

4127 1 of the whole fermentation broth prepared in Example 2 production medium V was thoroughly stirred with 4280 L of methanol for one hour and then filtered with Hyflo Super (diatomaceous earth, Johns-Manville 'roducts Corp.) in the presence of a filter aid. . The filtrate was adjusted to pH 4.0 with 5N hydrochloric acid. The acidic filtrate was extracted twice with an equal volume of chloroform. Combine the chloroform extracts and concentrate to 201 under reduced pressure. To the concentrate was added 200 L of diethyl ether to precipitate the A-42355 complex. The precipitate was collected by filtration to give 2775 g of A-42355 as an off-white powder.

Example 4

Isolation of Component A-30912 A 1 g of the antibiotic A-42355 complex prepared as in Example 3 was dissolved in 7 ml of a 7: 2: 1 mixture of methanol, water and acetonitrile. The solution was filtered and a glass column packed with 3.7 cm internal diameter 35 cm long LP-1 / Cjg silica gel (reverse phase, 10-20 μm particle size), Michel-Miller High Performance Low Pressure Chromatography column, Ace Glass Invorporated, Vineland, NJ. 08360) is poured onto the filtrate. The chromatographic column was prepared as described in Example 10 and the solution was applied to the top of the column using a valve system. The column was prepared using a slurry filling method using methanol: water: acetonitrile (7: 2: 1) as described in Example 11. The solvent was F.M.I. with a valveless pump (maximum flow rate 19.5 ml / min), through the column, flow rate about 9 ml / min, pressure applied 0.45 atm. A fraction is separated every minute. Elution of the antibiotic was monitored with a UV monitor operating at 280 nm (1SCO Model UA-5, Instrument Specialist Co., 4700 Superioir Ave., Lincoln, Nebraska, 68504) using ISCO Type 6 as an optical unit.

112-140. fraction was combined and added to 20 ml of water. The solution was extracted twice with an equal volume of chloroform, and the chloroform extracts were combined and concentrated under reduced pressure to an oily distillation residue. This oil was dissolved in tert-butanol and freeze dried to give 524 mg of component A-42355 A (component A-30912 A, A-22082).

Example 5

Isolation of Component A-30912 B

The A-42355 complex was isolated as described in Example 3, except that the 285 L concentrated chloroform extract was chromatographed on a column of silica gel (150 L Grace silica gel, grade 62) at an elution rate of 2 L / min. The column was washed with 200 L of chloroform, eluting with 500 L of acetonitrile and continuing with acetonitrile / water 98: 2 at 1 L / min. Fractions of about 200 L were collected and analyzed individually for biological activity. Bioassay on agar plates was performed using the paper disk method using Candida albicans. 77-103. Fractions (1365 L) were combined and concentrated under reduced pressure. The 4.5 L concentrate contains a precipitate which is collected by filtration to give 119 g of Component B rich A-42355. The filtrate was distilled to dryness and the residue was dissolved in a sufficient amount of methanol. The methanolic solution is added to 10 times diethyl ether to precipitate the antibiotic complex containing component B. The precipitate was collected by filtration and dried to give A-42355 complex as an additional 24 g of Compound B as a gray powder.

1.0 g of the resulting Component B-rich A-42355 complex is dissolved in 8 ml of a 7: 2: 1 mixture of methanol, water and acetonitrile. The solution was filtered and applied through a valve system to a top of a 3.7 cm diameter and 33 cm high Michel-Miller column filled with silica gel. The column was methanol: water: acetonitrile 7 as described in Example 10 is prepared consisting of 10-20 μ diameter particles made by reverse phase 1 mixture of LP-1 / _C18 silica gel: 29th The suspension was charged as described in Example 11. The solvent was flushed at a rate of 0.45 atm at a flow rate of 10 mL / min using an FMI pump. One fraction is collected every minute. The elution of the antibiotic was monitored using a UV monitor at 280 nm as in Example 15. 102-110. fractions were combined and distilled under reduced pressure to an oil-distillation residue. Dissolve the oil in a small amount of tert-butanol and freeze-dry to give 22 mg of Compound A-30912.

Example 6

Isolation of Component A-30912 D was prepared using concentrated chloroform extracts from 3800 L and 4007 L fermentations as in Example 3, and the concentrates were combined and chromatographed on a silica gel chromatography column (Grace, grade 62). Wash the column with chloroform and elute with acetonitrile and 98: 2 acetonitrile / water. Fractions of about 200 L were collected and assayed for biological activity using Candida albicans micro paper on an agar plate. The fractions containing the activity were combined and the 850 L solution was distilled under reduced pressure. The 0.7 L concentrate was added to 10 times the volume of diethyl ether to precipitate the component D rich A-42355 complex. The precipitate was collected by filtration and dried to give 32 g of Compound D as a gray powder A42355.

1.0 g of the Compound D rich A42355 complex thus prepared is dissolved in 5 ml of a 7: 2: 1 mixture of methanol, water and acetonitrile. The solution was filtered and applied through a valve system to a top of silica gel having an internal diameter of 3.7 cm and a length of 30 cm in a Michel-Miller column with a wind chill. 10-20 μ particle size reversed phase LP-1 / C The column was prepared as described in Example 10 from ₈ made of silica. Charging all. The suspension was carried out as described in Example 1, using a slurry filling method using methanol: water: acetonitrile (7: 2: 1). The solvent was injected at a rate of 8 ml / min11

Move the column at -101 at 0.2 atm, F.M.I. pump. A fraction was separated every second minute. Elution of the antibiotic is monitored with a UV monitor operating at 280 nm (1SC0 Model UA-5) with an ISCO Type 6 optical unit. 96-108. fractions were combined and distilled under reduced pressure to an oil-distillation residue. The oil was dissolved in a small amount of tert-butanol and freeze-dried to give 89 mg of Compound A-30912.

Example 7

Isolation of component A-30912 H

5.0 g of the A-42355 antibiotic complex prepared as described in Examples 2 and 3 are dissolved in 35 ml of a 7: 2: 1 mixture of methanol: water: acetonitrile. The solution was filtered and loaded onto the top of a Michel-Miller chromatographic column, 3.7 cm internal diameter and 42 cm high, using a column system. The column was methanol: water: acetonitrile, 7: 2: 1 mixture of filling is made as described in Example 10, a LP-1 / Cl ₈ reverse phase, particle size 10-20 μ silica gel by the procedure described in Example 11. The solvent was flushed at 0.5 atm at 13 mL / min using an FMI valveless pump. A fraction is separated every two minutes. Elution of the antibiotic was monitored by a 280 nm UV photometer as in Example 19

C. 112-132. fractions were collected and pooled for a second purification in a similar fashion. fraction thereof. The combined fractions were evaporated under reduced pressure to an oil-distillation residue. The oily substance was dissolved in a small amount of tert-butanol and freeze-dried to give 173 mg of component A-30912H.

Dissolve 150 mg of crude Compound A-30912 H in 8 ml of a 7: 2: 1 mixture of methanol, water and acetonitrile, filter, and proceed as above on a silica column of 2.0 cm internal diameter and 32 cm high on a glass column. top it. The solvent was flushed at 0.3 atm at 8 mL / min, separating each fraction every 3 minutes. The elution of the antibiotic is monitored at 280 nm. Fractions 17 and 18 were combined and distilled under reduced pressure to give an oily residue. This material was dissolved in a little tert-butanol and freeze dried to give 29 mg of Compound A-30912H.

Example 8

Preparation of antibiotic S 31794 / F-1

The antibiotic S 31794 / F1 is prepared by submerged culturing of the Acrophialophora limonispora NRRL 8095 under agitation, agitation and / or aeration at pH 3 to 8, preferably 5 to 7, 15 to 30, preferably 18 to 27. ° C. The culturing is carried out for 48 hours to 360 hours, preferably 120 hours to 288 hours.

To isolate antibiotic S 31794 / F-1, 90 L of culture broth was treated with 90 L of ethyl acetate: isopropanol (4: 1) and homogenized for 30 minutes at room temperature. The organic phase was separated and distilled under reduced pressure at 40 ° C. The distillation residue was chromatographed on a column of silica gel (10x) eluting with methanol: chloroform 5:95;

40:60. Fractions with antifungal activity were collected and chromatographed on 100x Sephadex LH-20 resin, eluting with methanol. Fractions from the column having antifungal activity were collected and re-chromatographed on 100 column silica gel (0.05-0.2 mm) eluting with chloroform: methanol: water (71: 25: 4). Among the eluting fractions, those having antifungal activity were collected and distilled under reduced pressure to give the crude antibiotic S 31794 / F-1 as a distillation residue. This product was dissolved in a small amount of methanol and precipitated with diethyl ether to give antibiotic S 31794 / F-2 as a white amorphous powder, m.p. 178-180 ° C. It is dried at 25 ° C to 30 ° C under reduced pressure. The product was crystallized from 10 times ethyl acetate: methanol: water (80: 12: 8) and then dried under reduced pressure at 20 ° C to give crystalline S 31794 / Fl, melting at 181-183 ° C. antibiotic.

Example 9

Isolation of antibiotic S 31794 / F-1

The crude antibiotic S 31794 / F-1, prepared as described in Example 8, is chromatographed on a Michel-Miller column using a valve system after chromatography on Sphadex resin. The column was prepared from LP-1 / Cjg reverse phase silica gel 10-20 μg, prepared as in Example 10, and suspended in chloroform / methanol / water (71 + 25: 4). All. The column was filled in as described in Example 1-1. The solvent on the column was F.M.I. with a valveless pump. Elution of the antibiotic is monitored with a UV monitor operating at 280 nm. Fractions with antifungal activity were pooled and distilled under reduced pressure to yield antibiotic S 31794 / F-1 as the distillation residue.

Example 10

Preparation of the silica gel / C _and reverse phase resin

Step 1: Hydrolysis

LP-1 silica gel (1000 g, Quantum Corp., now Whatman) was added to a mixture of 1650 mL of concentrated sulfuric acid and 1650 mL of concentrated nitric acid, placed in a 5 L round bottomed flask and shaken for 16 hours to obtain a suitable suspension. a condenser tube with a water cooler is connected to the flask.

Cool the mixture in an ice bath and carefully filter through a glass filter. Wash the silica gel with deionized water until the pH is neutral. It is then washed with 4 L of acetone and dried in a vacuum at 100 ° C for 2 days.

Step 2: First silylation

The dry silica gel obtained in the first step was filled into a round-bottomed flask and suspended in 3.5 L of toluene. The suspension is heated in a steam bath for 2 hours for azeotropic distillation of the remaining water. 321 ml of octadexyl trichlorosilane (aldirch Chemival Company) was added and the reaction was stirred at 60 ° C under gentle stirring for 16 hours12

-111 here. Make sure that the mixer does not touch the bottom of the ball joints. This prevents the assembly of silica gel particles.

Allow the suspension to cool. The silylated silica gel is then collected, washed with 3 L of toluene, then with 3 L of acetone and air-dried for 16 to 20 hours. The dry silica gel was suspended in 3.5 L acetonitrile / water (1: 1), stirred at room temperature for 2 hours, filtered, washed with 3 L acetone and air-dried overnight.

Step 3: Second silylation

Repeat the procedure described for the first silylation with 200 ml of oxadecyl trichlorosilane. The suspension was heated at 60 ° C under reflux for 2 hours while stirring gently. The final product was collected by filtration, washed with 3 L of toluene, then 6 L of methanol, and dried at 50 ° C for 16 to 20 hours under reduced pressure.

Example 11

Suspension of Meichel-Miller columns

General Information

A. Analytical or preparative columns may be filled using the following procedure.

B. Use silica gel or silica gel reversed phase filler (e.g. Quantum LP-1.10-20 μ particle size, LiChroprep RP-8 and RP-18, particle size 25-40 μ).

However, other silica gels, such as Shandons ODS Hypersil, particle size 5 μ, may be used, but also others. '

C. The suspension is generally filled at a pressure of less than 0.8 atmospheres and with a flow rate of between 5 ml and 40 ml / min, the flow rate depending on the size of the column. The pressure applied during filling should be greater than the pressure used during actual separation, with an excess pressure of 0.1-0.2 atm. The additional pressure ensures that the adsorbent is no longer a bulk charge during chromatography.

D. Rapid drop in pressure can create fractures or channels in the filler, greatly reducing the efficiency of the column. Therefore, it is important that the pressure is slowly reduced to 0, even if the pump is turned off.

E. Columns of suitable size are as follows (Ace Glass Cat. No., Unfilled), No. 5795-04, 12 ml. No. 5795-10, 110 ml, No. 5796-16, 300 ml, No. 5795-24,635 ml, and nO. 5796-34.34 ml.

D. The time it takes to kill a glass column can vary from minutes to several hours, depending on the size of the column and how the researcher is practicing.

Here's how to upload a column:

1. The glass column is connected to a reservoir column, the latter having twice the volume of the former. Both columns are set upright with the reservoir column on top.

2. Weigh the filler (about 100 g for a 200 ml column).

3. Approximately five volumes of solvent are added to the filler using 70-80% methanol and 20-30% water.

4. Shake the slurry until the particles are wet, then leave overnight to allow the particles to completely soak. The supernatant is then decanted.

5. The resin is suspended in a suitable solvent when the reservoir column is filled. The reservoir is filled, but partially filled with solvent. Larger volumes of slurry can give better results, but in this case a larger reservoir tank or a multi-step fill must be used.

6. Seal the reservoir tank with a Teflon plug (cf. Item 3 of Figure 1 of U.S. Patent 4,331,547), connect it to the pump, and, in the case of an Ace 13265-25 pump or similar, solvent transfer using a system, immediately starting the solvent flow at maximum speed (about 20 ml / min).

7. Continue the flow until the column is completely filled with the adsorbent. The filling pressure should not change during the operation (about 0.8 atm for larger columns and 1.2 atm for analytical columns. In most cases, less than 0.8 atm will suffice).

8. When the maximum pressure is reached, the flow rate is reduced and the pressure is reduced.

9. When the column is filled with adsorbent, the pump is turned off. The pressure is allowed to drop to 0, the reservoir tank is shut off, and it is replaced by a pre-column. The pre-column is filled with solvent and a small amount of adsorbent and maintained at maximum pressure until the column is fully charged. For more information, study the general presentation. In all cases, the pressure should be reduced slowly after the pump has been switched off, thus avoiding breakage of the filler material or the formation of channels in it.

10. Decrease the pressure and carefully disconnect the pre-column from the column. Using a spatula, remove 2-4 mm filler from the top of the column and place one or two filters on the column. Lightly press the top of the filler and place a Teflon plug on top. Attach the pump to the column, providing pressure, usually less than 0.8 atm, while observing through the column glass wall whether additional resin is applied to the top of the column. If additional filler is applied to the column - this is the case when larger columns are used - so-called "columns". dead spaces or battles are created. In this case, you need to repeat step 9.

Example 12

Preparation of tetrahydro-A-30912A

Component A-30912 A is dissolved in ethanol. Platinum oxide in absolute ethanol is reduced to produce platinum, which is used for the catalytic reduction of component A-30912 A with hydrogen and under positive pressure. The reaction is carried out for 2-3 hours, during which time it is complete. The reaction mixture was filtered and distilled under reduced pressure. The distillation residue was dissolved in a small amount of tert-butanol and freeze-dried to give tetrahydro-A-30912A.

Example 13

Preparation of tetrahydro-A-30912B

Component A-30912 B is dissolved in ethanol. Platinum oxide in absolute ethanol is reduced to produce platinum, which is used for the catalytic reduction of Component A-30912 B with hydrogen and positively.

-121 under pressure. The reaction is carried out for 2-3 hours, during which time it is complete. The reaction mixture was filtered and distilled under reduced pressure. The distillation residue was dissolved in a small amount of tert-butanol and freeze-dried to give tetrahydro-A-30912B.

Example 14

Preparation of tetrahydro-A-30912 D

Component A-30912 D is dissolved in ethanol. Platinum oxide in absolute ethanol is reduced to produce platinum, which is used for the catalytic reduction of component A-30912 with hydrogen and under positive pressure. The reaction is carried out for 2-3 hours, during which time it is complete. The reaction mixture was filtered and distilled under reduced pressure. The distillation residue was dissolved in a small amount of tert-butanol and freeze-dried to give tetrahydro-A-30912 D.

Example 15

Preparation of tetrahydro-A-30912 H

Component A-30912 H is dissolved in ethanol. Platinum oxide in absolute ethanol is reduced to produce platinum, which is used for the catalytic reduction of component A-30912 H with hydrogen and under positive pressure. The reaction is complete for 2-3 hours, during which time the reaction is complete. The reaction mixture was filtered and distilled under reduced pressure. The distillation residue was dissolved in a small amount of tert-butanol and freeze-dried to give tetrahydro-A-30912H.

Example 16

A. Acyl cleavage of the A-30912 A antibiotic component

As in Example 1, it was fermented with A. utahensis. Medium A is used as slanted agar and medium I is used as production medium. The production medium was incubated for 42 hours. To the fermentation broth is added 340 g of crude substrate containing 19.7 g of component A-30912 A in 1.5 l of ethanol.

Deacylation of component A-30912 A is monitored by biological measurement with Candida albicans. The fermentation is continued until the acyl cleavage is complete, which is indicated by the loss of activity against C. albicans.

B, Isolation of parent compound A-30912 A

1001 whole fermentation broths prepared as described in A containing approximately 20 g of the parent compound prepared from component A-30912 A are filtered. The mycelium is discarded. The clear filtrate (ca. 93 L) was chromatographed on a packed column of 4.5 L HP-20 resin (Diaion High Pore Polymer, HP-Series, Mutsubishi Chemical Industries Limited, Tokyo, Japan) at 200 mL / min. The resulting effluent is discarded. The column was washed with 8 column volumes of deionized water, pH 6.5 to 75, to remove contaminants. The wash was discarded. It is then eluted with 85 liters of a 7: 3 mixture of water and methanol at an elution rate of 200-300 ml / min.

Elution is followed by the following procedure: 2 aliquots of each eluate are collected, one is concentrated to a small volume and treated with an acid chloride such as myristoyl chloride. This and the other untreated sample were evaluated for Candida albicans. If the untreated sample is inactive and the acylated sample is active, fraction A-30912 A contains the parent compound. The eluates containing the parent compound A-30912 A were concentrated by distillation under reduced pressure and freeze-dried to give 97 g of crude parent compound.

C. Purification of the parent compound A-30912 A by reverse-phase liquid chromatography g of the crude parent compound A-30912 A prepared in the preceding paragraph was dissolved in a mixture of water, acetonitrile, acetic acid and pyridine (96: 2: 1: 1). The solution was chromatographed on a column of Lochroprep RP-18 resin (8 cm x 80 cm, 4 L) made of acid-proof steel (MC / B Manufacturing Chemists, Inc. E / M, Cincinati, Ph). The column is part of the Chromatospec Prep 100 unit (Jobin Yvon, 16-18 Rue du Canal 91160 Longjumeau, France). The column operates at a pressure of 0.36-0.45 atm with a flow rate of 60 ml / min. The solvent used is the above solvent. The material was monitored on a 280nm UV monitor (ISCO Absorption Monitor Model UA-5, 4700 Superior Ave., Lincoln, Nebraska 68504) equipped with an ISCO Type 6 optical unit. Fractions of 500 ml volume were collected every minute.

On the basis of the absorbance at 280 nm, the fractions containing the parent compound A-30912 A were combined, distilled under reduced pressure, and the residue was freeze-dried to yield 2.6 g of the parent compound. The chromatographic separation process requires 7-8 L of solvent.

D. Characteristics of A-30912 A parent compound

a) Experiment: C ₃ 4 H ₅₁ N ₅ .

b) Molecular Weight: 797.83.

c) The substance is soluble in white, amorphous, solid water, dimethylformamide, dimethylsulfoxide, methanol, insoluble in chloroform, toluene and diethyl ether.

d) The infrared absorption spectrum studied in the potassium bromide tablet exhibits absorption maxima at 3303, broad (hydroxyl group, H-bonds), 2970, 2930, and 2890 (CH, aliphatic methylpropyl group, methylene group, CH group), 1660 and 1625 (more carbonyl groups), 1510-1550, 1430-1450 (CH-), 1310-1340, 1230-1260, 1080, 835, 650, broad, and 550, broad, cm ⁴ .

e) electrometric titration in 66% aqueous dimethylformamide contains a titratable moiety having a characteristic of 7.35 pK _a (original pH 7.32).

(f) Retention time (K *) of HPLC: 11.52 minutes. This was measured using the following system:

column: 4 x 300 mm filler: silica gel / C, _8: ammonium acetate: acetonitrile, water: 1: 2: 97 flow rate: 3 ml / min pressure: 100 atm detector: variable wavelength ultraviolet detector, 230 mm sensitivity: 0-0.4 AUFS

Example 17

In the same manner as in Example 16, except that tetrahydro-A-30912 A is used as a substrate, the parent compound A-30912 A is prepared.

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Example 18

Proceeding as in Example 16, except that aculeacin A is used as a substrate, the parent compound A-30912A is prepared and purified.

Example 19

A. Deacylation of the parent compound component A-30912B

Following the procedure of Example 1, fermentation of A. utahensis was carried out using production medium I. The culture was incubated for 48 hours and then methanol-dissolved A-30912B was added to the culture.

Deacylation of Component A-30912 B was followed by paper disk measurement with Candida albicans or Neurospora crassa. Fermentation is continued until the deacylation is complete, which is indicated by the loss of biological activity.

B. Isolation of parent compound A-30912B

Filter the entire fermentation broth prepared as described in A above. The mycelium is discarded. The clear filtrate was chromatographed on a column of HP-20 resin (Diaion High Porous Polymer, Series-Series, Mitsubishi Chemical Industries Limited, Tokyo, Japan). The effluent is discarded. The column is then washed with 8 column volumes of deionized water of pH 6.5-7.5 to remove the filtered fermentation broth. The wash was discarded. The column was then eluted with water / methanol (7: 3). The elution is followed as follows: two aliquots of each fraction are taken. One sample is concentrated to a small volume and acid chloride, such as myristyl chloride, is added. This sample and the other, i.e. untreated, are weighed against Candida albicans. If the untreated sample is inactive and acylated, the fraction contains parent compound A-30912B. The eluates containing the parent compound A-30912 B were distilled under reduced pressure and the low volume concentrate was freeze-dried to give the crude parent compound.

C. Purification of parent compound A-30912 B by reverse phase liquid chromatography

The crude base compound A-30912B obtained in B. is dissolved in a mixture of water, acetonitrile and acetic acid and pyridine 96: 1: 1: 1. The solution was chromatographed on a column packed with Lichroprep RP-18 resin. The resin has a particle size of 25-40 g (MC / B Manufacturing Chemists, Inc. E / M, Cincinnati, OH). The column is part of a unit of a Chromatospec Prep 100 (Jobin Yvon, 16-18 Rue du Canal 91160 Longjumeau, France). The column is operated at a pressure of 3.5-0.45 atm, with a flow rate of 60 mL / min. The solvent mixture used is the above solvent mixture. Compound isolation was monitored by a UV monitor operating at 280 nm (ISCO Absorption Monitor Model UA-5, 4700 Superior Ave., Lincoln, Nebraska 68504) equipped with an ISCO Type 6 optical unit.

On the basis of the absorbance at 280 nm, the fractions containing the parent compound A-30912 B were combined, the mixture was distilled under reduced pressure and the distillation residue was freeze-dried to give the purified parent compound A 30912 B.

Example 20

In the same manner as in Example 19, the parent compound A-30912 B was prepared and purified except that the substrate was tetrahydro-A-30912 B.

Example 21

A. Deacylation of Component A-30912 D

Proceeding as in Example 1, using the production medium 1, the A. utahensis microorganism is fermented. The culture is incubated for 48 hours and then a small amount of methanol-dissolved A-30912 component D is added to the culture.

Deacylation of Component A-30912 was monitored by paper disk measurement using Candida albicans or Neurospora crassa. Fermentation is continued until the deacylation is complete, which is indicated by the loss of biological activity.

B. Isolation of parent compound A-30912 D

The whole fermentation broth prepared as described in A above is obtained. The mycelium is discarded. The clear filtrate was chromatographed on a column of HP-20 resin (Diaion High Ptous Polymer, HP-Seties, Mitsubishi Chemical Industries Limited, Tokyo, Japan). The effluent is discarded. The column is then washed with 8 column volumes of deionized water of pH 6.5-7.5 to remove the filtered fermentation broth. The wash was discarded. The column is then eluted with a 73% water / methanol mixture. The elution is followed as follows: two aliquots of each fraction are taken. One sample is concentrated to a small volume and acid chloride such as myristoyl chloride is added. This sample and the other, i.e. untreated, are weighed against Candida albicans. If the untreated sample has no activity and the acylated one, the faction contains parent compound A-30912 D. The eluates containing the parent compound A-30912 D were distilled under reduced pressure and the low volume concentrate was freeze-dried to give the crude parent compound.

C. Purification of parent compound A-30912 D by reverse-phase liquid chromatography

The parent compound A-30912 D, obtained in crude form B, was purified as described in Example 19.

On the basis of the absorbance at 280 nm, the fractions containing the parent compound A-30912 D were selected, distilled under reduced pressure and freeze-dried to yield pure A-30912 D parent compound.

Example 22

By proceeding as in Example 21, except that tetrahydro-A-30912 B is used as a substrate, the parent compound A-30912 D is prepared and purified.

Example 23

A. Deacylation of the A-30912 H antibiotic component

Using the same procedure as in Example 1, fermentation of A. utahensis, using production medium I, was carried out. The culture is incubated for 48 hours and then a small amount of methanolic A-30912 H component is added to the culture.

D-acylation of component H of A-30912 by paphr disks, Candida albicans or Neurospora

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185,021 with crassa microorganism. Fermentation is continued until the deacylation is complete, which is indicated by the loss of biological activity.

B. Isolation of parent compound A-30912 H

Filter the entire fermentation broth obtained in (A) above. The mycelium is discarded. The clear filtrate was chromatographed on a column of HP-20 resin (Diaion High Porous Polymer, HP-Series, Mitsubishi Chemical Industries Limited, Tokyo, Japan). The effluent is discarded. The column is then washed with 8 column volumes of deionized water of pH 6.5-7.5 to remove the filtered fermentation broth. The wash was discarded. Then elute the column with a mixture of water and methanol (7 □). The elution is followed as follows: two aliquots of each fraction are taken. One sample is concentrated to a small volume and acid chloride such as myristoyl chloride is added. This sample, and the other, untreated, was evaluated against Candida albicans. If the untreated sample has no activity and the acylated one, the fraction contains parent compound A-30912 H. The eluates containing the parent compound A-30912 H were distilled under reduced pressure and the low volume concentrate was freeze-dried to give the crude parent compound.

C. Purification of parent compound A-30912 H by reverse-phase liquid chromatography

The crude parent compound A-30912 H obtained in (B) was purified as described in Example 19 (C).

On the basis of the absorbance at 280 nm, the fractions containing the parent compound A-30912 H were combined, distilled under reduced pressure and freeze-dried to yield pure A-30912 H parent compound.

Example 24

In the same manner as in Example 23, except that tetrahydro-A-30912 H was used as a substrate, the parent compound A-30912 H was prepared and purified.

Example 25

The title compound A-30912 H was prepared and purified as in Example 23, except that component A-30912 H was prepared from component A-30912 H by the following procedure:

Dissolve 19.6 mg of antibiotic component A-30912 A in 1 ml of dimethylformamide. To the solution was added 0.06 ml of a 3% solution of hydrogen chloride in methanol. The resulting solution was stirred overnight at room temperature and then evaporated to dryness under reduced pressure. The resulting distillation residue was purified by high performance liquid chromatography as described in Example 7, purified by reverse phase silica gel (LP-1 / Cu, as in Example 10) and methanol: water: acetonitrile (7: 2: 1). . Thus 1.4 mg of A-30912 H is obtained (vegvület of the compound of formula II wherein R ¹ and R ⁴ OH, R ³ is hydrogen, R ³ and methoxy, and R linoleoilcsoport).

Example 26

A. Deacylation of antibiotic S 31794 / F-1

As described in Example 1, the microorganism A. utahensis is fermented using the working medium I. ter10. The culture is incubated for 48 hours and a small amount of S 31794 / F-1 in methanol is added.

Deacylation of the S-31794 / F-1 component was followed by paper disk measurement with Candida albicans or Neurospora crassa. Fermentation is continued until the deacylation is complete, which is indicated by the loss of biological activity.

B. Isolation of parent compound S 31794 / F-l

Filter the entire fermentation broth prepared as described in A above. The mycelium is discarded. The clear filtrate was chromatographed on a column of HP-20 resin (Diaion High Porous Polymer, HP-Series, Mitsubishi Chemical Industries Limited, Tokyo, Japan). The efflient is discarded. The column is then washed with 8 column volumes of deionized water of pH 6.5-7.5 to remove the filtered fermentation broth. The wash was discarded. The column is then eluted with a 7: 3 mixture of water and methanol. The elution is followed as follows: two aliquots of each fraction are taken. One sample is concentrated to a small volume and acid chloride such as myristoyl chloride is added. This sample, and the other, untreated, was evaluated against Candida albicans. If the untreated sample has no activity and the acylated one, the fraction contains the parent compound S 31794 / F-1. The fractions containing the parent compound S 31794 / F1 are distilled under reduced pressure and the low volume concentrate is freeze-dried to give the crude parent compound.

C. Purification of parent compound S 31794 / F-1 by reverse-phase liquid chromatography

The crude parent compound S 31794 / F-1 obtained in B. was purified as described in Example 19 (C).

On the basis of the absorbance at 280 nm, the fractions containing the parent compound S 31794 / F-1 are combined, distilled under reduced pressure and freeze-dried to give pure S 31794 / F-1 parent compound.

16-26. The NMR spectra of the parent compounds prepared in Examples 1 to 4 are given below. The peaks for each amino acid component are reported separately for each parent compound. The measurement was performed in DMSO-dj solvent at 360 MHz; expressed in.

A-30912 Compound A ppmmethylhydroxyproline (1) sub-formula dihydroxy-ornithine (2) sub-formula threonine / 1 (3) sub-formula threonine / 2 (4) sub-formula dihydroxy-homothyrosine (5) sub-formula a = 4.17, (3 = 4.01.7 = 2.33, CH ₃ = 0.95, δ = 3.82 and 3.17.

a = 3.22, (3 = 1.69 and 1.42, γ = 3.79, δ = 5.20, ΝΗ = 7.89.

7.24, a = 4.64, β '

NH 3.88, CH ₃ <

1.06.

NH = 8.03, α = 4.76, β 459, CHj = 1.06.

NH = 7.39.0 = 3.94, (3 =

3.90, γ = 4.18, ortho = 6.99, Meta = 6.66.

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185.021 Hydroxyproline (6) Sub-Formula A-30912 B Base Methyl Hydroxyproline (1) Sub-Dihydroxy-Ornithine (2) Sub-Thiononyl / 1 (3) Sub-Form Trconone / 2 (4) Hydroxy-Homothir ) sub-formula hydroxyproline (6) formula A-30912 parent compound methyl-hydroxyproline (1) sub-formula ornithine (8) sub-formula threonine / 1 (3) sub-formula trconin / 2 (4) sub-formula Wdroxy-homothyrosine (7) -proline (6) sub-formula A-30912 H parent compound methyl hydroxyproline (1) sub-formula methoxy-hydroxy-ornithine (9) sub-formula threonine / 1 (3) sub-formula trconine / 2 (4) sub-formula hydroxy-homothyrosine (5) hydroxyproline (6)

The parent compound S 31794 / F-1 is the methyl hydroxyproline (1) sub-formula dihydroxy-ornithine = 4.29.0 = 2.20 and 1.84, 7 = 4.41.0 = 3.79 and 3.69.

4,15,0 3,98,7 c = 2.35, _CH3 = 0.96, and δ = 3.83 '3.22.

a = 3.14.3 = 1.69 and 1.41, 7 = 3.85, 5 = 5.24, NH =

7.98.

NH = 7.30.0 = 4.65.0 = 3.87,

CH ₃ = 1.05.

NH = 8.18, a = 4.75.0 = 4.46,

CH ₃ = 0.99.

NH = 7.82.0 = 4.17.0 =

3.99, = 4.36, δ = 3.96, and 3.57. a = 4.28, 0 = 2.18 and 1.90, 7 = 4.36, o = 3.96 and 3.57, = 4.14.0 = 3.98,7 = 2.29, CH ₃ = 0.94, δ = 3.80 and 3.22.

= 3.16.0 = 1.04 and 1.79, 7 = 1.60, δ = 3.33 and 2.87, NH = 7.70,

NH = 7.44.0 = 4.69.0 = 4.06,

CH ₃ = 1.03.

NH = 7.85.0 = 4.66.0 = 4.42,

CH ₃ = 1.04.

NH = 8.04.0 = 4.12.0 = 4.06, = 2.57 and 2.48, ortho = 6.92, Meta = 6.63. 0 = 4.34, 0 = 2.19 and 1.93, 7 = 4.40, 3.98 and 3.64.

0 = 4,21,0 = 4,03,7 =

2.31, CH ₃ = 0.96, δ = 3.85 and 3.22. 0 = 3,12,0 = 1,69 and 1,42, 7 = 3,97, δ = 5,03,0 CH ₃ = 3,20,

NH = 7.95,

NH = 7.15, α = 4.61, O = 3.83,

CH ₃ = 1.08.

NH = 8.24.0 = 4.79.0 = 4.51,

CH ₃ l = 03.

NH = 7.46.0 = 3.94.0 = 3.77.7 = 4.27, Ortho = 6.95, Meta = 6.65. o = 4.25.0 = 2.19 and 1.86, 7 = 4.42, = 3.87 and 3.67.

4,23,0 3,98,7 = = = 2,32, _CH3 = 0.94, δ = 4.05 and 3.12. aNH = 8.18.0 = 4.22.0 =

1.75 and 1.75, (2) sub-formula 0-hydroxyglutamine (10) sub-formula threonine (4) sub-formula dihydroxy-homothyrosine (5) sub-formula = 3.73, δ = 4.95, NH = 8.05.

NH = 7.19, a = 4.8-5.0 = 4,16,7 = 2.45 and 2.25, -CO-NH ₂ = 6 78 7,29és

NH = 8.23, α = 4.75, β = 4.34 CH ₃ = 1.06.

NH = 7.40, a = 3.97, 0 =

3.97,7.7 = 4.13, Ortho = 7.00, Meta = 6.66, Phenol 9.29.

hydroxyproline O = 4.33, O = 1.85 and 2.22, 4.40, (6) sub-formula δ 3.72 and 3.72.

The following describes the preparation of the compounds of the general formula III using the so-called. by the active ester method. The process provides a compound of Formula III wherein R ^s is CH + CH 3) 4, and wherein R 1, R ² , R ³ and R ^{4 are} as defined in the parent compound to be prepared. 27-47. The methods described in Examples 1 to 4 are not within the scope of the invention.

Example 27

Preparation of 2,4,5-trichlorophenyltridecanoate

In 650 ml of methylene chloride was dissolved 12.5 g of n-tridecanoic acid (Sigma Chemical CO.), 11.5 g of 2,4,5-trichlorophenol and 12.0 g of Ν, il'-dicyclohexylcarbodiimide. The reaction mixture was stirred at room temperature for 16 hours. It was then filtered and distilled to dryness under reduced pressure to give 22 g of 2,4,5-trichlorophenyltridecalate as a distillation residue. The product was purified by column chromatography on silica gel (Woelm) and toluene as eluent. The fractions were examined by thin layer chromatography and detected under shortwave ultraviolet light. The pure product fractions were collected and distilled to dryness under reduced pressure.

Example 28

Acylation of parent compound A-30912 A with 2,4,5-trichlorophenyl-n-tridecanoate

Dimethylformamide (600 ml) was dissolved in 2,4,5-trichlorophenyltridecanoate (6.0 g) and parent compound A-30912A (4.5 g), and the solution was stirred at room temperature for 16 hours. Distillation under reduced pressure gave 12 g of product as a distillation residue. This is suspended in 500 ml of methylene chloride and after 45 minutes the suspension is filtered. The filtrate is discarded. The resulting solid was extracted with 500 mL of methanol, filtered through methanol extract and distilled under reduced pressure to give 5.0 g of crude product.

The crude product was purified by reversed phase high performance liquid chromatography as follows: Dissolve 1 g of the crude product in 5 ml of methanol and inject 2 and 1/2 times the LP-1 / C 1 _S resin in a 65 cm acid resistant column. and Examples 11). The column was eluted with a 4: 4 mixture of water, methanol and acetonitrile. Elution is carried out at a pressure of 40-60 atm, with a flow rate of 11-12 ml / min provided by an LDC pump (Milton-Roy). The effluent was examined at 280 nm with an ultraviolet detector (ISCO UA-5). A fraction (21-24 mL) was collected every other minute. The fractions containing the product to be prepared were collected and reduced

-161 centrifuges are distilled to dryness under pressure. 550 mg of product are obtained. The above chromatographic purification was repeated four times to give four more products: 620 mg, 520 mg, 670 mg and 490 mg. their total weight is 2.8 g.

Following the above procedure, 40 g of the parent compound A-30912 A is reacted with 2,4,5-trichlorophenyl n-tridecanoate to give 2.6 g of pure product. The preparations from both experiments were combined to give 5.4 g of product. Field Mass Desorption Mass Spectrometry (M + Na ⁺ ): 1016. (Theoretical: 1016). Analytical HPLC (C ₁₈ Micro Bondapak, Waters Co.) by (eluted with water: methanol: acetonitrile, 2: 1: 2 mixture of a) the product shows a single peak.

Example 27

Acylation of parent compound A-30912 B with 2,4,5-trichlorophenyl-n-tridecanoate

3.3 mmol of 2,4,5-trichlorophenyl-n-triadecanoate and 1 mmol of parent compound A-30912 B are dissolved in 200 ml of dimethylformamide. The reaction mixture was stirred at room temperature for 16 hours. The solvent was removed by distillation under reduced pressure, the residue was suspended in 300 ml of methylene chloride and filtered after 45 minutes. The filtrate was discarded. The solid product was extracted with methanol (300 mL), the methanolic extract was filtered and distilled under reduced pressure to give the crude product as a distillation residue.

The crude product was purified by high performance liquid chromatography as follows:

The product is dissolved in 5 ml of methanol and injected onto the top of an LP-1 / C18 resin in a 2.5 x 65 cm column made of stainless steel (see _Examples 10 and 11). The column was eluted with 33: 4 water: methanol: acetonitrile. Elution was carried out at 40 to 60 atm, with a flow rate of 11-12 ml / min provided by an LDC pump (Milton-Roy). The effleun was examined with an ultraviolet detector (ISCO UA-5) at 280 nm. A fraction (21-24 mL) was collected every second minute. The fractions containing the product to be prepared are collected and distilled under reduced pressure to give the product to be obtained. The pure product was analyzed by thin layer chromatography on reversed-phase _Ig plates (Whatman KCl), eluted with water, methanol: acetonitrile (1: 2: 2). After run, the plates were examined under ultraviolet light to induce the product.

Example 30

Acylation of parent compound A-30912 D with 2,4,5-trichlorophenyl-n-tridecanoate

In dimethylformamide (200 mL) was dissolved 2,3 mmol of 2,4,5-trichlorophenyl n-tridecanoate and 1 mmol of parent compound A-30912 D, and the solution was stirred at room temperature for 16 hours. The solvent was removed by distillation under reduced pressure and the residue was suspended in 300 ml of methylene chloride. After 45 minutes, the suspension is filtered. The filtrate was discarded. The residual solid was extracted with methanol (300 mL), filtered and distilled under reduced pressure to give the crude product.

The crude product was purified by reversed-phase high performance liquid chromatography as described in Example 29.

Example 31

Acylation of parent compound A-30912 H with 2,4,5-trichlorophenyl-n-tridecanoate

In dimethylformamide (200 mL) was dissolved 2,3 mmol of 2,4,5-trichlorophenyl n-tridecanoate and 1 mmol of parent compound A-30912 H and the solution was stirred at room temperature for 16 hours. The solvent was removed by distillation under reduced pressure and the residue was suspended in 300 ml of methylene chloride. After 45 minutes, the suspension is filtered. The filtrate was discarded. The residual solid was extracted with methanol (300 mL), filtered and distilled under reduced pressure to give the crude product.

The crude product was purified by reversed phase high performance liquid chromatography as described in Example 37.

Example 32

Acylation of parent compound S 31794 / F-1 with 2,4,5-trichlorophenyl-n-tridecanoate

3.3 mmol of 2,4,5-trichlorophenyl n-tridecanoate and 1 mmol of parent compound S 31794 / F-1 are dissolved in 200 ml of dimethylformamide and the solution is stirred for 16 hours at room temperature. The solvent was removed by distillation under reduced pressure and the residue was suspended in 300 ml of methylene chloride. After 45 minutes, the suspension is filtered. The filtrate was discarded. The residual solid was extracted with methanol (300 mL), filtered and distilled under reduced pressure to give the crude product.

The crude product was purified by reversed phase high performance liquid chromatography as described in Example 29.

Preparation of Debono Ι derivatives

The preparation of compounds of formula III wherein R ^{s is} a group of formula IV is described below. This illustrates a method for preparing Debono I compounds of formula III.

Example 33

Preparation of N- (n-dodecanol) p-aminobenzoic acid

5.5 g (40 mmol) of p-aminobenzoic acid are dissolved in 100 ml of pyridine and 8.74 g (40 mmol) of n-dodecanoyl chloride are added dropwise. After stirring for 3 hours, the reaction mixture was poured into 3 L of water. The precipitate was collected by filtration and dried under reduced pressure to give 11.01 g of N, N -dodecanoyl) -p-aminobenzoic acid.

Example 34

Preparation of 2,4,5-trichlorophenyl ester of N- (n-dodecanoyl) -p-aminobenzoic acid

In 250 mL of methylene chloride, 11.01 g (34.5 mmol)

N- (n-dodecanoyl) -p-aminobenzoic acid was dissolved in 7.5 g (38 mmol) of 2,4,5-trichlorophenol and 6.94 g (34.5 mmol) of N, N-dicyclohexylcarbodiimide. The reaction mixture was stirred at room temperature for 3.5 hours and then filtered. The filtrate was distilled under reduced pressure, and the residue was crystallized from acetonitrile / water to give 12.84 g of 2,4,5-trichlorophenyl ester for N- (n-dodecanoyl) -p-aminobenzoic acid.

Example 35

Acylation of A-30912 A parent compound

In 100 ml of dimethylformamide, 8.16 g (10.2 mmol) of the parent compound A-30912 A and 4.72 g (10.2 mmol) of N18

-171 • (n -do decanoyl) -p-amino-benzoic acid 2,4,5-trichlorophenyl ester is dissolved and the reaction mixture is stirred at room temperature for 15 hours. The solvent was removed by distillation under reduced pressure, and the residue was washed twice with diethyl ether. The wash was discarded. The washed product was dissolved in 50 ml of methanol and purified by high performance reverse phase liquid chromatography on the Prep LC / System 500 unit (Waters Associates Inc., Milford, Mass.) Prop Pak-500 / C ₁₈ column (Waters Associates, Inc.) as the stationary phase. The column was eluted with water / methanol / acetonitrile (2565: 10 v / v) at 2 atm. The fractions were monitored by thin layer chromatography on silica gel plates, eluting with water, methanol and acetonitrile (25:65:10 v / v). The product-containing fractions were combined and freeze-dried to give 3.5 g of the parent compound A-30912A N- (n-dodecanoyl) -p-aminobenzoyl.

Example 36

Acylation of parent compound A-30912 B in 100 µl dimethylformamide 10.2 mmol A-30912

Basic compound B and 10.2 mmol of N- (n-dodecanoyl) -p-aminobenzoic acid 2,4,5-trichlorophenyl ester are dissolved. The reaction mixture was stirred at room temperature for 15 hours. The solvent was removed by distillation under reduced pressure, and the residue was washed twice with diethyl ether. The wash was discarded. The washed product was dissolved in 50 ml of methanol and Prep LC / System 500 units and Prep Pak-500 / Cl ₈ column (Waters Associates, Inc., Milford, Mass.) Was purified using high pressure as the stationary phase for reverse phase chromatography. The column was eluted with water, methanol: acetonitrile (2565: 10) at 2 atm and fractions were analyzed by silica gel plates, eluting with water: methanol: acetonitrile (25:65:10). The fractions containing the product to be prepared were combined and then freeze-dried to give the N- (n-dodecanoyl) -p-aminobenzoyl derivative of parent compound A-30912B.

Example 37

Acylation of parent compound A-30912 D

In 100 ml of dimethylformamide was dissolved 10.2 mmol of the parent compound A-30912 D and 10.2 mmol of N- (n-dodecanoyl) aminobenzoic acid 2,4,5-trichlorophenyl ester. The reaction mixture was stirred at room temperature for 15 hours. The solvent was removed by distillation under reduced pressure, and the residue was washed twice with diethyl ether. The washings were discarded. The washed product was dissolved in methanol (50 mL) and purified by reverse phase high performance liquid chromatography on a Prep LC / System 500 (Waters Associates, Inc., Milford, Mass.) Having a Prep Pak-500 / Ci _e column (Water Associates Inc.). , as a stationary phase. The column was eluted with water / methanol / acetonitrile (26:65:10, v / v) at 2 atm. The fractions were analyzed by thin layer chromatography using silica gel plates and a 25:65:10 by volume mixture of water, methanol and acetonitrile. The product-containing fractions were combined and freeze-dried to give the N-n-dodecanoyl-β-aminobenzoyl derivative of parent compound A-30912 D.

Example 38

Acylation of parent compound A-30912 H

A-30912 in 100 ml of dimethylformamide was 10.2 mmol

The parent compound H and 10.2 mmol of N- (n-dodecanoyl) -p-aminobenzoic acid were dissolved. The solution was stirred at room temperature for 15 hours. The solvent was removed by distillation under reduced pressure, and the residue was washed twice with diethyl ether. The washings were discarded. The washed product was dissolved in 50 ml of methanol and purified using a Prep LC / System 500 unit (Waters Associates Inc ,, Milford, Mass.) Using reverse phase high performance liquid chromatography, which is mounted as a stationary phase Prep Pak-500) C _& columns. The column was eluted with water / methanol / acetonitrile (25:65:10) at 2 atm. The fractions were analyzed by thin layer chromatography on silica gel plates using a 25:65:10 by volume mixture of water, methanol and acetonitrile as solvent. The product-containing fractions were combined and freeze-dried to give the N-(n-dodecanoyl) -p-aminobenzoyl derivative of parent compound A-30912 H

Example 39

Acylation of parent compound S 31794-F / l

In 100 ml of dimethylformamide, 10.2 mmol of S 31794 /

The parent compound F1 and 10.2 mmol of N- (n-dodecanoyl) -p-aminobenzoic acid 2,4,5-trichlorophenyl ester were dissolved and the reaction mixture was stirred at room temperature for 15 hours. The solvent was removed by distillation under reduced pressure, and the resulting distillation residue was washed twice with diethyl ether. The washings were discarded. The washed product was dissolved in 50 ml of methanol and purified using a Prep LC / System 500 units of reverse phase high pressure liquid chromatography (Waters Associates Inc., Milford, Mass.), Which is Prep Pak-500 / C _I8 column (Waters Associates Inc.) equipped as a stationary phase. The column was eluted with 65:10 (v / v) water, methanol and acetonitrile at 2 atm. The fractions were analyzed by thin layer chromatography on silica gel plates using a 65:10 by volume mixture of water, methanol and acetonitrile as solvent. The product-containing fractions were combined and freeze-dried to give the N- (dodecanoyl) -p-aminobenzoyl derivative of the parent compound S 31794 / F1.

Preparation of Debono II Derivatives The preparation of compounds of formula III wherein R ^{5 is} a group V is described below. This illustrates the preparation of Debono type II compounds of Formula III.

Example 40

Preparation of Pj (n-octyloxy) benzoic acid

Dissolve 19.2 g (150 mmol) of p-hydroxybenzoic acid in 120 ml of 10% aqueous sodium hydroxide and add to 480 ml of dimethylsulfoxide, previously heated to 80 ° C. 28.95 g (150 mmol) of n-octyl bromide are added dropwise. After stirring at room temperature for 4 hours, the reaction mixture was poured into 1200 ml of ice water. Concentrated hydrochloric acid (30 ml) was added and the mixture was allowed to stand until the precipitate was complete. The precipitate was collected, dried and crystallized from acetonitrile / water. The product was obtained

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Melting point 97-99 ° C.

Elemental analysis for Ci _s H ₂₂ O ₃ :

Found: C, 71.97; H, 8.86; 71.72, H: 9.10%.

Example 41

Preparation of 2,4,5-trichlorophenyl ester of p- (n-octyloxy) benzoic acid

In 200 mL of methylene chloride, 6.18 g (24.7 mmol) of p- (n-octyloxy) benzoic acid, 5.39 g (27.2 mmol) of 2,4,5-trichlorophenol and 4.94 g of ,7, Ν-Dicyclohexylcarbodiimide (24.7 mmol) was dissolved. The reaction mixture was stirred at room temperature for 18 hours and then filtered. The filtrate was distilled off, and the oil obtained by distillation was crystallized from a mixture of acetonitrile and water to give p- (n-octyloxy) -benzoic acid 2,4,5-trichlorophenyl ester.

Nuclear Magnetic Resonance Spectrum of the prepared compound is 4.02 (2H, t, J = 3Hz), 7.0 (1H, d, .MHz), 7.23 (1H, s), 7.3 (1H, s) ), 8.08 (1H, d, J = 4Hz).

Example 42

Acylation of A-30912 A parent compound

In 150 ml of dimethylformamide, 14.2 g (17.8 mmol) of the parent compound A-30912 A and 15.32 g (35.1 mmol) of p- (n-octyloxy) benzoic acid 2,4,5-trichlorophenyl are added. ester. The reaction mixture is stirred for 16-20 hours at room temperature. The solvent was removed by distillation under reduced pressure, and the residue was washed twice with diethyl ether and twice with methylene chloride. The washings were discarded. The washed residue was dissolved in 80 mL of ethyl acetate: methanol (1: 3) and purified by silica gel chromatography using a Prep LC / System 500 as the stationary phase. The column was eluted step by step with a mixture of methanol and ethyl acetate (1 A and 23, respectively). Fractions were analyzed by silica gel (Merck) thin layer chromatography (ethyl acetate: methanol = 3: 2, v / v). Fractions containing no parent compound A-30912 A were combined and freeze-dried to give 7.13 g of the parent compound A-30912 A, p-(n-octyloxy) -benzoyl.

♦ 23.1052 (by field desorption mass spectrometry).

Example 43

Acylation of parent compound A-30912 B

17.8 mmol of the parent compound A-30912 B and 35.6 mmol of p- (n-octyloxy) benzoic acid 2,4,5-trichlorophenyl ester are dissolved in 150 ml of dimethylformamide. The solution is stirred for 16-20 hours at room temperature and then the solvent is distilled off under reduced pressure. The distillation residue was washed twice with diethyl ether and twice with methylene chloride. The washings were discarded. The washed residue was dissolved in 80 mL of a 1: 3 mixture of ethyl acetate and methanol and purified by high performance liquid chromatography on silica gel using a Prep LC / System 500 stationary phase. The column was eluted stepwise with methanol: ethyl acetate (1: 4 and 23, respectively). The fractions were run on a 3: 2 by volume mixture of ethyl acetate and methanol. Fractions containing no parent compound A-30912 B were collected and freeze-dried to give the p-(n-octyloxy) benzene derivative of parent compound A-30912 B.

Example 44

Acylation of parent compound A-30912 D

17.8 mmol of A-30912 in 150 ml of dimethylformamide

Basic compound D and 35.7 mmol of p- (n-octyloxyl) -benzoic acid 2,4,5-trichlorophenyl ester were dissolved. The solution was stirred for 16-20 hours at room temperature and then distilled under reduced pressure to remove the solvent. The distillation residue was washed twice with diethyl ether and twice with methylene chloride. The washings were discarded. The washed residue was dissolved in 80 mL of a 13% ethyl acetate / methanol mixture and purified by high performance liquid chromatography (silica gel, Prep LC / System 500 as stationary phase). The column was eluted stepwise with methanol / ethyl acetate 1: 4 and 2: 3, respectively. Fractions were analyzed by silica gel (Merck) thin layer chromatography (3: 2 ethyl acetate: methanol). Fractions containing no parent compound A-30912 D were collected and freeze-dried to give the p-(n-octyloxy) benzoyl derivative of parent compound A-30912 D.

Example 45

Acylation of parent compound A-30912 H

17.8 mmol of A-30912 in 150 ml of dimethylformamide

The parent compound H and 35.7 mmol of p- (n-octyloxy) -benzoic acid 2,4,5-trichlorophenyl ester were dissolved. The solution is stirred for 16 to 20 hours at room temperature and then the solvent is distilled off under reduced pressure. The distillation residue was washed twice with diethyl ether and twice with methylene chloride. The washings were discarded. The washed residue was dissolved in 80 mL of a 13% ethyl acetate / methanol mixture and purified by high performance liquid chromatography using silica gel using a Prep LC / System 500 as the stationary phase. The column was eluted stepwise with methanol: ethyl acetate (1: 4 and 23, respectively). The fractions were analyzed by silica gel (Merck) thin layer chromatography (3: 2 ethyl acetate: methanol). Fractions containing no parent compound A-30912 H were collected and freeze-dried to give the p-(n-octyloxy) benzoyl derivative of parent compound A-30912 H.

Example 46

Acylation of parent compound S 31794 / F-l

17.8 mmol of S 31794 in 150 ml of dimethylformamide /

The parent compound F-1 and 35.7 mmol p (n-octyloxy) benzoic acid 2,4,5-trichlorophenyl ester were dissolved. The solution is stirred for 16 to 20 hours at room temperature and then the solvent is distilled off under reduced pressure. The distillation residue was washed twice with diethyl ether and twice with methylene chloride. The washings were discarded. The washed residue was dissolved in 80 mL of a 13% ethyl acetate / methanol mixture and purified by high performance liquid chromatography using silica gel using a Prep LC / System 500 as the stationary phase. The column was eluted stepwise with methanol: ethyl acetate (1: 4 and 23, respectively). Fractions were analyzed by silica gel (Merck) thin layer chromatography (3: 2 ethyl acetate: methanol). Fractions containing no parent compound S 31794 / F-1 were collected and freeze-dried.

-191

185.021 gives p-n-octyloxy) -benzoyl] -hydrous salt of parent compound S 31794 / E-1.

Example 47 5

In III. The antifungal activity of the compounds of Formula I is detected in vitro by standard disk diffusion method and agar dilution method, and by elimination of in vivo standard systemic fungal infections in mice. The following sections 4-8. Tables III. some compounds of formula (I) are tested for antifungal activity.

Tables IV and V show in vivo results of the compounds described in Examples 36, 44 and 52 on agar plates by disk diffusion.

The IV. The data presented in Table II are obtained by plotting the magnitude of the zones of inhibition caused by the test compound by inhibiting the growth of the microorganism in mm. In Table V, activity is determined by the lowest growth inhibitory concentration (Gug / disk), which is the concentration required to inhibit the growth of the test microorganism.

VI. The data in Table III are the results of in vitro assays of p- (n-octyloxy) -benzoyl-A-30912 A parent compound against five microorganisms of Candida albicans. The assays were performed using the agar dilution method. VI. The activities of Table II are determined at the lowest growth inhibitory concentration (pg / ml) required to inhibit the growth of the test microorganism.

VII. Table I shows the in vivo efficacy of the compounds of the invention in mice infected with Candida albicans A-26, where the activity is expressed as EDjn / kg amount of material required to recover 50% of the animals. ). Where no ED ^ value is obtained, the activity is given at the lowest dose θθ necessary to produce a significant antifungal activity. Male albino mice, which are pathogen-free animals and weigh between 18 g and 20 g, were used in the study. Infection with Candida albicans Α-26 was performed intravenously. Twenty-four hours prior to infection, the animals were exposed to approximately 50 X-rays for 8 minutes (400 X total) to reduce the immune response to the infectious organism. 0, 4 after infection. and at 24 hours, each group was dosed with an increasing dose of the test compound in a 33% polyethylene glycol / water suspension subcutaneously. The deaths of each animal were recorded. Student t-statistic comparisons were made for mean daily mortality at a given dose level for each infected and treated animal group compared to 10 infected and untreated animals to determine whether treatment significantly increases survival?

The Vili. Table IV shows the absorption observed upon oral administration of the compounds of the invention. In this study, mice were treated with doses of 416 mg / kg, the test compounds were suspended in 33% polyethylene glycol 400, and water. At intervals, blood samples were taken from the orbital sinus and the samples were determined for antibiotic activity. This was done as follows: 20 µl of whole blood disc of 7 ml diameter was placed on agar previously infected with Aspergillus montevidensis A35137. Following a 40 hour incubation at 30 ° C, the inhibition zone from the blood sample was determined and compared to the inhibition zones obtained by the test compound standard. The drug content of the blood sample was then calculated.

ARC. Spreadsheet

Agar plate disc diffusion assay for parent compound A-30912 A ^- ~ antifungal activity

The size of the inhibition zone in mm ®

Example The formula -------------—-- of formula III

number compound wherein R ³ = Saccharomyces pastorianis X-52. Neurospora crassa 846 Trichophyton mentagroahytes A-23 Candida albicans A-26 36th CH ₃ (CH ₂ ) ₁₁ - 21,, 32 ^60x 30 44th ARC 21 33 ^x 55 ^x 23 17 35 ^x 56 ^x 19 52nd V 18 23 ^x - 28

^x = A measurable and well-defined inhibition zone, the microorganism growing again around the disk.

The assays were carried out by immersing discs of 7 mm diameter in a methanol suspension of 1 mg / ml and placing them on the agar surface.

Incubation time: 24-48 hours at 25-37 ° C.

-201

185 021

V, Table

Antifungal activity of derivatives of parent compound A-30912 A as determined by agar plate diffusion method

Minimum growth inhibitory concentration (mg / kroon) ^x

Example song The compound of general formula III wherein R ^b Candida albicans A-26 Trichophyton mentagroahytes 6 36th CH ₃ (CH ₂ ) _n - 0,625 0,039 44th ARC 0, and 25 1.25 > 0.039 0.678 52nd V 0.16> 0.302 (0.312) ^xx

^x = The compounds are suspended in 0.01 M sodium borate, pH 7.5. Compounds are assayed to reach peak concentrations of 20 mg / disc and then double-folded until the end point is reached. Incubation time: 24 hours at 30 ° C.

^xx = 5 isolate.

Table VI

The parent compound A-30912 A, wherein R ⁵ is the in vitro activity of a group of Formula V against Candida albicans

Minimum growth inhibitory concentration (µg / ml) A26 SBH 16 SBH 31 SBH 28 SBH 29 0.312 0.312 0.312 0.312 0.312

VII. Spreadsheet

Therapeutic activity of derivatives of parent compound A-30912 A against C. albicans in mouse

Example number Dosage plan of formula III is ^X where R ⁵ = ^ED 50 (mg / kg) Lowest active dose (mg / kg) 36th ch ₃ (ch ₂ ) _u - b 34.0 20.0 44th IV A 15.0 10.0 15.0 > 5.0 52nd A A 13.0 10.0 22.2 > 12.5

^x = Dose plan: 40, 20, 15 and 10 mg / kg, B = 80, 40, 20, and 10 mg / kg. Doses were administered as suspensions in 30% polyethylene glycol-water at 0, 4 and 24 hours post injection. Test compounds were administered to each group of 6 mice at each dose level. Control (10 untreated mice per group).

^xx = Increase in Survival Time of Treated Animals versus Control as determined by Reed and Muench (Amer. J. Hygiene, 27,493, 1938).

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185 021

Vili, table

Blood levels of A-30912 A parent compound in mice following oral administration

Example number The formula III is halohane where R - Blood level ^x (mg / ml) 36th CH ₃ (CH _? ), R 0.10 44th ARC 0.83 52nd V 23.0 ^x = 4 hours after test compound administration

the dose was 416 mg / kg, the compound was 33% polyethylene glycol; 400 and water. The amount of the compound was determined by biological measurement with Aspergillus montevidensis strain Λ-35137.

Claims (6)

A process for the preparation of the cyclic hexapeptide derivatives of the formula I wherein: R ^{1 is} hydrogen or hydroxy, and when R ^{1 is} hydrogen, R ^{2 is} hydrogen, and R ⁴ and R ^{4 are} hydrogen or hydroxy, and R is hydroxy, R ^{2 is} hydrogen, R ^{4 is} hydroxy or R ^{4 is} carbamoyl and R ⁴ and R ^{4 are} hydroxy groups, characterized in that a compound of formula II wherein R ^{1 is} hydrogen or hydroxy and when R ^{1 is} hydrogen, R ^{2 is} hydrogen and R ⁴ and R ^{4 are} hydrogen or hydroxy and R is stearoyl is a linoleic group and when R ^{1 is} hydroxy, R ^{2 is} hydrogen and R ^{4 is} hydroxy, R ^{4 is} hydroxy and R a stearoyl group, a linoleoyl group or a palmitoyl group, or a R ⁴ methoxy group and a R stearoyl group or a linoleoyl group, and when R 1, R ⁴ R ⁴ hydroxyl group and R ² carbamoyl group, R myristoyl group, Actinoplanes sp. NRRL 8122, NRRL 12064, NRRL 12065, Actinoplanes utahensis NRRL 12052 or Actinoplanes missouriensis NRRL 12 053 or a mutant thereof Second embodiment of the method according to claim 1, the I A-30912 of formula basic compound - wherein R, R ⁴ and R ⁴ is hydroxy and R ² is hydrogen preparation, characterized in that starting with a compound of formula II wherein R ^1, R ⁴ and R ⁴ OH, R ² and R hidrogén25 linoleoílcsoport, stearoyl or palmatoilcsoport. 3. The process of claim 1, wherein the parent compound A-30912 B has the formula: wherein R ¹ and R ^{2 are} hydrogen and R and R are hydroxy, characterized in that starting from a compound of formula II wherein R 1 and R ^{2 are} hydrogen, R ⁴ and R ^{4 are} hydroxy and R is stearoyl or linoleoyl. 4. The method of claim 1 embodiment are of the general kéjüetű A-30 912 D parent compound qc - wherein R, R ^2, R ⁴ and R ⁴ a hydrogen atom the preparation of ^v, characterized in that starting with a compound of formula II, álról ^R1, R, R ⁴ and R ⁴ is H and R is stearoyl or Iinoleoilcsoport. The method of claim 1, which is an embodiment Method 4Q is the parent compound A-3O9J2 H of Formula 1 - "wherein R ¹ and R ^{4 are} hydroxy, R ^{2 is} hydrogen and R ^{4 is} methoxy, characterized in that starting from a compound of formula II wherein R and R ^{4 are} hydroxy, R ^{2 is} hydrogen, R ^{4 is} methoxy and R is a stearoyl group. or a linoleic group. 6. The method of claim 1 embodiment, the ζ I S 31794 / Fl aiapvegyület - wherein R, R ⁴ and R ⁴ is hydroxy and R ² carbamoyl - preparing, characterized made to compounds of formula II indu50 about wherein R, R ⁴ and R ^{4 are} hydroxy, R ^{2 is} carbamoyl and R is myristoyl.