BE886577A - CYCLIC PEPTIDE CORES AND THEIR PREPARATION - Google Patents

Description

       

  This invention relates to cyclic peptide nuclei of the general formula:

  

  <EMI ID = 1.1>

I

  
and their acid addition salts. Kernels and their salts

  
can be used as intermediates in the preparation

  
semi-synthetic antifungal agents.

  
The nuclei are prepared by deacylating an antibiotic

  
cyclic peptide of general formula:

  

  <EMI ID = 2.1>
 

  
in which R is a side chain of saturated fatty acid

  
or unsaturated and R1, R, R and R are substituents defined below.

  
A process for the enzymatic removal of

  
the fatty acid side chain R, to obtain the cyclic peptide nucleus. The method involves bringing the antibiotic into an aqueous medium in contact with an enzyme

  
produced by a microorganism of the Actinoplanaceae family until substantial deacylation.

  
A preferred method according to this invention is to use an enzyme produced by the microorganism

  
Actinoplanes utahensis NRRL 12052 for cutting the chain

  
lateral fatty acid. Deacylation is generally

  
performed by adding the appropriate antibiotic to a culture

  
ae A. utahensis and incubating the culture until

  
that we get deacylation. The cores thus obtained

  
are separated from the fermentation broth by processes

  
known in the art. These kernels are useful in that

  
can reacylate them to get new antibiotic substances.

  
In particular, the invention provides a cyclic peptide nucleus of formula

  

  <EMI ID = 3.1>


  
i

  
wherein R is H or OH and;

  
  <EMI ID = 4.1> <EMI ID = 5.1>

  
and

  
  <EMI ID = 6.1>

O

  
  <EMI ID = 7.1>

  
group -C-NH2 and R and R are both OH, and its acid addition salts.

  
In one embodiment of the cores of this invention

  
  <EMI ID = 8.1>

  
embodiment is known as the core of A-30912A. The core of A-30912A is prepared by deacylating a cyclic peptide antibiotic of formula II where R is a linoleoyl group,

  
  <EMI ID = 9.1>

  
in the nucleus of A-30912A.

  
When R in the cyclic peptide antibiotic of formula II is a linoleoyl group, the antibiotic is known as factor A of A-30912, when R is a stearoyl group, the antibiotic is known as factor A of A-30912- tetrahydrogen &#65533; and when R is a group

  
  <EMI ID = 10.1>

  
Factor A of A-30912

  
The factor A of A-30912 is a factor of the mixture A-30912 which also contains the factors B, C, D, E, F and G. The mixture A-30912 and the various factors A to G are described in the patent of USA N [deg.] 4,024,245. The A-factor of A-30912 is identical to the antibiotic A-22082 which is described in the U.S. patent. N [deg.] 4,024,246.

  
  <EMI ID = 11.1>

  
and 4,024,246, factor A of A-30912 was found to be identical to the antibiotic echinocandin B [See F. Benz et al., Helv. Chim. Acta 57, 2459-2477 (1974) and Swiss patent N [deg.] 568.386J. The antibiotic SL 7810 / F has also been identified as echinocandin B [C. KellerJuslen, et al., Tetrahedron Letters 1976 (46), 4147-4150, and Belgian patent N [deg.] 834,289].

  
Keller-Juslen, et al. proposed the structure of

  
  <EMI ID = 12.1>

  
formula II where R is a linoleoyl group, R, R and R are OH and R <2> is H for factor A of antibiotic A-30912.

  
  <EMI ID = 13.1>

  
The factor A of tetrahydrogenated A-30912 (SL 7810 / F tetrahydrogenated, tetrahydroechinocandin B), which is described in Belgian patent N [deg.] 834.289 and by F. Benz et al., Helv.

  
Chim. Acta 57, 2459-2477 (1974), at formula II in

  
13 4 which R is a stearoyl group, R, R and R are OH and R <2> is H. For convenience, this substance will be referred to herein as tetrahydro-A-30912A.

  
Aculeacin A

  
Aculeacin A is a component of the aculeacin mixture, which consists of one main component (aculeacin A) and six minor components (aculeacins B, C, D, E, F and G). The components of aculeacin are described in the U.S. Patent. N [deg.] 3,978,210. As described in Belgian patent N [deg.] 859,067, aculeacin A probably has the same cyclic peptide structure as tetrahydro-A-30912A but the stearoyl side chain is replaced by a palmitoyl chain.

  
A-30912A core

  
The novel cyclic peptide nucleus of this embodiment, i.e., the nucleus of factor A of A-30912 (echinocandin B, SL 7810 / F), factor A of tetrahydrogenated A-30912, and aculeacin A, has the formula I in which

  
  <EMI ID = 14.1>

  
This core (core of A-30912A) is a white amorphous material which is soluble in solvents such as water, dimethylformamide, dimethylsulfoxide and 'methanol and which is insoluble in solvents such as chloroform, toluene and diethyl ether.

  
The core of A-30912A has a crude formula of C34H51N7015 and a molecular weight of 797.83.

  
The infrared absorption spectrum of the core of A-30912A in KBr pellets is shown in Figure I. The following absorption maxima are observed: 3340 broad (OH, hydrogen bond), 2970, 2930 and 2890 (CH stretching) , aliphatic in the groups CH3, CH2, CH), 1660 and 1625 (several

  
  <EMI ID = 15.1>

  
"wagging" from CH), 1310-1340, 1230-1260, 1080, 835, 650 large

  
  <EMI ID = 16.1>

  
  <EMI ID = 17.1>

  
66% aqueous dimethylformamide indicates the presence of a

  
  <EMI ID = 18.1>

  
7.32).

  
The core of A-30912A can be separated by high pressure liquid chromatography (CLPE). The core of A-30912A has an approximate retention time (k ') of 11.52 minutes when measured by CLPE using the following conditions:

  

  <EMI ID = 19.1>


  
Preparation of the core of A-30912A

  
Substrate preparation

  
The core of A-30912A according to this invention can be prepared from factor A of A-30912, factor A of tetrahydrogenated A-30912 or aculeacin A. These substrates can be used in the form of purified materials but it does not it is not essential that it be purified. Thus, for example, the mixture A-30912 of which the factor A of A-30912 is the main component, can be used as a substrate for preparing the core of A-30912A.

  
Factor A of A-30912

  
Factor A of A-30912 can be obtained by fermenta-

  
  <EMI ID = 20.1>

  
1) a strain of Aspergillus rugulosus NRRL 8113;

  
2) a strain of Aspergillus nidulans NRRL 8112;

  
3) a strain of Aspergillus nidulans var. echinulatus

  
A-32204, NRRL 3860;

  
4) a strain of Aspergillus rugulosus NRRL 8039; or

  
5) a strain of Aspergillus nidulans var. roseus,

  
NRRL 11440.

  
When using a strain of A. nidulans var. roseus NRRL 11440 to produce factor A of A-30912, we obtain <EMI ID = 21.1>

  
is the main factor in the antibiotic mixture A-42355.

  
  <EMI ID = 22.1>

  
of mixture A-42355.

  
Tetrahydro-A-30912A

  
Tetrahydro-A-30912A is prepared from factor A of A-30912 by standard hydrogenation techniques, performing the reduction until the two double bonds of the linoleoyl side chain have been reduced.

  
Aculeacin A

  
Aculeacin A is prepared by fermentation of a strain of Aspergillus aculeatus NRRL 8075 as described in US patent N [deg.] 3,978,210 which is incorporated herein by reference.

  
According to another embodiment of the core of this

  
  <EMI ID = 23.1>

  
two OH. The core of this embodiment is known as the core of A-30912B. The core of A-30912B is prepared by

  
  <EMI ID = 24.1>

  
in which R is a linoleoyl or stearoyl group

  
123

  
  <EMI ID = 25.1>

  
When R in the cyclic peptide antibiotic of formula II is a linoleoyl group, the antibiotic is known as factor B of A-30912 and when R is a stearoyl group, the antibiotic is known as factor

  
B of tetrahydrogenated A-30912.

  
Factor B of A-30912

  
The factor B of A-30912 is a factor of the mixture A-30912 which also contains the factors A, C, D, E, F and G. The mixture A-30912 is described in the patent of E.U.A. N [deg.] 4,024,245.

  
Factor B of A-30912 has been found to be identical

  
antibiotic echinocandin C [See R. Traber et al.,

  
  <EMI ID = 26.1>

  
SL 7810 / F-ll (Belgian patent N [deg.] 834,289).

  
Traber et al. proposed the structure of formula II

  
12 3 in which R and R are both H, R and R are all

  
  <EMI ID = 27.1> <EMI ID = 28.1>

  
antibiotic, Traber et al. offer formula II in

  
  <EMI ID = 29.1>

  
OH and R is a stearoyl group.

  
Factor B of tetrahydrogenated A-30912

  
Factor B of tetrahydrogenated A-30912 (tetrahydro-SL
7810 / F-II; tetrahydroechinocandin C), which is described in Belgian patent N [deg.] 834,289 and by R. Traber et al., Helv.

  
Chim. Acta 62, 1252-1267 (1979), has the formula II in

  
  <EMI ID = 30.1>

  
OH and R is a stearoyl group. For convenience, this material will be called tetrahydro-A-30912B.

A-30912B core

  
The. new cyclic peptide nucleus of this embodiment, i.e. the factor B nucleus of A-30912 (echinocandin C, SL 7810 / F-II) and tetrahydro-A-30912B, has the structure represented by formula I, in which

  
  <EMI ID = 31.1>

  
and a molecular weight of 781.83.

Preparation of the core of A-30912B

  
Substrate preparation

  
The core of A-30912B can be prepared from factor B of A-30912 or tetrahydro-A-30912B. As this factor B is not the main component of the antibiotic mixtures in which it is produced, it must be purified to remove the other co-produced factors before being used as a substrate.

  
Factor B of A-30912

  
This factor can be obtained by aerobic fermentation of:

  
1) a strain of Aspergillus rugulosus NRRL 8113;

  
2) a strain of Aspergillus nidulans var. echinulatus

  
A-32204, NRRL 3860;

  
3) a strain of Aspergillus rugulosus NRRL 8039; or

  
4) a strain of Aspergillus nidulans var. roseus,

  
NRRL 11440.

  
When using a strain of A. nidulans var. roseus NRRL 11440 to produce factor B of A-30912, a mixture of factors is obtained which, for reasons of convenience, is called antibiotic mixture A-42355. The A-factor of A-30912 is the main factor in the A-42355 antibiotic mixture. Factors B, D and H of A-30912 are minor factors in the A-42355 mixture.

  
Tetrahydro-A-30912B

  
Tetrahydro-A-30912B is prepared from factor B of A-30912 by standard hydrogenation techniques, carrying out the reduction until the two double bonds of the linoleoyl side chain have been reduced.

  
In another embodiment of the nuclei of this

  
1 2

  
invention, R, R, R and R are all hydrogen atoms. The core of this embodiment is called the core of A-30912D. The core of A-30912D is prepared by deacylating a cyclic peptide antibiotic of formula II where R is a linoleoyl or stearoyl group and R, 1 R, R and R are as in the core of A-30912D.

  
When R in the cyclic peptide antibiotic of formula II is a linoleoyl group, the anti.biotic is called factor D of A-30912 and when R is a stearoyl group, the antibiotic is called factor D of A-30912 tetrahydrogen.

  
Factor D of A-30912

  
The factor D of A-30912 is a factor of the mixture A-30912 which also contains the factors A, B, C, E, F and G. The mixture A-30912 is described in the US patent. N [deg.] 4,024,245.

  
Factor D of A-30512 has been found to be identical to the antibiotic echinocandin D [See R. Traber et al., Helv. Chim. Acta 62, 1252-1267 (1979)] and the antibiotic SL 7810 / F-III (Belgian patent N [deg.] 834,289).

  
Traber et al. proposed the structure of the formula

  
123

  
  <EMI ID = 32.1>

  
linoleoyl group for the antibiotic echinocandin D For tetrahydroechinocandin D, Traber et al. have proposed

  
1 2 the structure of formula II in which R, R, R and R are each H and R is a stearoyl group.

  
For convenience, the factor D of tetrahydro-hydrogenated A-30912 (tetrahydro-SL 7810 / F-III; tetrahydro-echinocandin D) will be called tetrahydro-A-30912D.

A-30912D core

  
The new cyclic peptide nucleus of this embodiment, i.e. the factor D nucleus of A-30912 (echinocandin D, SL 7810 / F-III) and tetrahydro-A-30912D, has the structure shown in formula I in which
12

  
R, R, R and R are hydrogen atoms.

  
  <EMI ID = 33.1>

  
and a molecular weight of 749.83.

  
Preparation of the core of A-30912D Preparation of the substrate

  
We can prepare the core of A-30912D from

  
  <EMI ID = 34.1>

  
factor D of A-30912 is not the main component of the antibiotic mixtures in which it is produced, it must be purified to remove the factors produced simultaneously before using it as a substrate.

  
Factor D of A-30912

  
Factor D of A-30912 can be produced by aerobic fermentation by immersion of:

  
1) a strain of Aspergillus rugulosus NRRL 8113;

  
2) a strain of Aspergillus nidulans var. echinulatus

  
A 32204, NRRL 3860;

  
3) a strain of Aspergillus rugulosus NRRL 8039; or

  
4) a strain of Aspergillus nidulans var. roseus,

  
NRRL 11440.

  
When using a strain of A. nidulans var. roseus NRRL 11440 to obtain factor D of A-30912, a mixture of factors is obtained which is called for reasons of convenience antibiotic mixture A-42355. The A-factor of A-30912 is the main factor in the A-42355 antibiotic mixture. Factors B, D and H of A-30912 are minor factors in the A-42355 mixture.

  
Tetrahydro-A-30912D

  
Tetrahydro-A-30912D is prepared from factor D of A-30912 by standard hydrogenation techniques, performing the reduction until the two double bonds of the linoleoyl side chain have been reduced.

  
In yet another embodiment of the nuclei of this invention, R and R are both OH, R is H and R

  
  <EMI ID = 35.1>

  
of realization are called type A-30912H cores. The nuclei of type A-30912H are prepared by deacylating a cyclic peptide antibiotic of formula II in which R is a linoleoyl or stearol group and R, R, R and R 4 are as in the nuclei of type A-30912H.

  
When R in the cyclic peptide antibiotic of formula II is a linoleoyl group and R is a methoxy group, the antibiotic is called factor H of A-30912 and, when R is a stearoyl group and R is a methoxy group, the antibiotic is called A-30912 tetrahydrogenated factor H.

  
When R, in the cyclic peptide antibiotic of formula II, is a linoleoyl group and R is a C2-C6 alkyloxy group, the antibiotics are called homologs with a lower alkyloxy group of factor H of A-30912 and when R is a group stearoyl and R is a C2-C6 alkyloxy group, the antibiotics are called homologs with a lower alkyloxy group of factor H of A-30912 tetrahydrogenated.

  
H-factor of A-30912

  
The H-factor of A-30912 is a factor of the A-30912 mixture which also contains the factors A, B, C, D, E, F and G. The A-30912 mixture is described in the U.S. Patent. N [deg.] 4,024,245. The H-factor of A-30912 is a factor of A-30912 discovered later. The H-factor of A-30912

  
  <EMI ID = 36.1>

  
A-30912 factor H counterparts

  
After discovering the structure of factor H of A-30912, it was emphasized that the lower alkyloxy homologs of factor H of A-30912 would be useful products. Prior to this time, the alkyloxy derivatives which had been prepared had not been recognized as having utility and were prepared only as tools for determining the structure. The homologs with C2-C6 alkyloxy grouping of factor H of A-30912 are prepared from factor A of A-30912.

  
Factor H from A-30912 can be produced by fermen - ion of 1) a strain of Asperqillus rugulosus NRRL 8113;

  
  <EMI ID = 37.1>

  
A-32204, NRRL 3860;

  
4) a strain of Aspergillus rugulosus NRRL 8039 as

  
described in Belgian patent N [deg.] 834,289; or

  
5) a strain of Aspergillus nidulans var. roseus,

  
NRRL 11440.

  
As each nucleus of A-30912H contains an amino part, they can exist in the form of salts. These .sels can also be used as intermediates and for purification purposes. The pharmaceutically acceptable salts of A-30912H nuclei are particularly useful because they will minimize the purification of the end products. "Pharmaceutically acceptable" salts refer to salts in which the toxicity of the product as a whole to warm-blooded animals is not increased.

  
The acid addition salts of the nuclei of A-30912H can be prepared by standard reaction procedures with inorganic or organic acid. Examples of mineral and organic acids include hydrochloric, hydrobromic, hydroiodic, sulfuric, phosphoric, acetic, benzolic, sulfamic, tartaric, citric, maleic, succinic, ascorbic, glycolic acids; lactic, fumaric, palmitic, cholic, pamolic, mucic,

  
  <EMI ID = 38.1>

  
stearic, salicylic - methanesulfonic, benzenesulfonic, sorbic, picric, cinnamic and other suitable acids.

Preparation of A-30912H nuclei

  
Substrate preparation

  
The core of A-30912H can be prepared, i.e. the compound of formula I in which R1 and R4 are both

  
  <EMI ID = 39.1>

  
factor H of A-30912 or tetrahydro-A-30912H. Since factor H of A-30912 is not a main component of the antibiotic mixtures in which it is produced, it must be purified to remove other antibiotic factors <EMI ID = 40.1>

  
a C2-C6 alkyloxy group (homologs of A-30912H) by reaction of factor A of A-30912 or of tetrahydro-A-
30912A with an alcohol suitable for preparing the derivative with a corresponding C2-C6 alkyloxy group. Since factor A of A-30912 is the main component of the antibiotic mixtures in which it is produced, this process is also a preferred way of preparing factor H of A-30912 and tetrahydro-A-309l2H.

  
H-factor of A-30912

  
Factor H can be obtained from A-30912 by aerobic fermentation by immersion of a strain of Aspergillus rugulosus NRRL 8113 or of a strain of Aspergillus nidulans var. roseus NRRL 11440.

  
When using a strain of A. nidulans var. roseus NRRL 1140 to produce factor A-30912, a mixture of factors is obtained which is called for reasons of convenience antibiotic mixture A-42355. The A-factor of A-30912 is the main factor in the A-42355 antibiotic mixture. Factors B, D and H of A-30912 are the minor factors in mixture A-42355.

  
Counterparts of A-30912H

  
Homologues of A-30912H are prepared by reacting factor A of A-30912 or tetrahydro-A-30912A with the appropriate alcohol to form the derivative to

  
  <EMI ID = 41.1>

  
R4 are both OH, R <2> is H and R <3> is a C2-C6 alkyloxy group. This is a preferred process for the preparation of factor H from A-30912 and tetrahydro-A-30912H. The homologues of A-30912H of formula II can be prepared in which R is a tearoyl group and R is an alkyloxy group in

  
  <EMI ID = 42.1>

  
  <EMI ID = 43.1>

  
alkyloxy group, or b) reacting factor A of A-30912 with the appropriate alcohol to form the derivative with alkyloxy group and then reducing the double bonds <EMI ID = 44.1>

  
We prepare tetrahydro-A-30912A, tetrahydro-A-.30912H, and the compounds of formula II where R is a Coxy alkyl group; -C &#65533; and R a stearoyl group from the factors A and H of A-30912 and from the compounds of

  
  <EMI ID = 45.1>

  
is a linoleoyl group, by conventional hydrogenation techniques, carrying out the reduction until the two double bonds of the linoleoyl side chain have been reduced.

  
In yet another embodiment of the nuclei of

  
  <EMI ID = 46.1>

  
carboxamide. The core of this embodiment is known as the core of S 31794 / F-1. We prepare the nucleus of

  
S 31794 / F-1 by deacylating a cyclic peptide antibiotic of formula II in which R is a group

  
1 2

  
myristoyl and R, R, R and R are as in the nucleus of S 31794 / F-1.

  
  <EMI ID = 47.1>

  
The nucleus of S 31794 / F-1 of this invention is obtained by deacylating the cyclic peptide antibiotic S 31794 / F-1.

  
The antibiotic S 31794 / F-1, which is described in the German patent application published after examination

  
N [deg.] 2,628,965 and the U.S. Patent N [deg.] 4,173,629, is an antifungal compound produced by Acrophialophora limonispora nov. Spec. Dreyfuss and Muller NRRL 8095. S 31794 / F-l a

  
  <EMI ID = 48.1>

  
(amorphous) or 181-183 [deg.] C. (decomposition) (crystallized);

  
  <EMI ID = 49.1>

  
in the crystallized state); maximum UV absorption in the

  
  <EMI ID = 50.1>

  
deuteromethanol (190 mg in 1.5 ml of deuteromethanol), tetramethylsilane being used as internal standard) with the following characteristics (in the crystallized state):

  

  <EMI ID = 51.1>


  
an approximate elementary analysis (after drying the crystalline material for two hours under high vacuum at

  
  <EMI ID = 52.1>

  
hydrogen, 10.5 - 10.8% nitrogen and 25.5 - 26.0% oxygen. S 31794 / F-l is easily soluble in

  
  <EMI ID = 53.1>

  
is sparingly soluble in water, chloroform, ethyl acetate, diethyl ether, benzene and hexane and has antifungal activity, in particular vis-à-vis Candida Albicans.

  
The antibiotic S 31794 / F-1 is believed to have the formula II in which R is a myristoyl group, R, R and R 4 are OH and R <2> is a carboxamide group.

  
Core of S 31794 / F-l

  
It is believed that the new cyclic peptide nucleus of this invention, i.e. the antibiotic nucleus

  
S 31794 / F-l, has the structure represented by the formula 1

  
134

  
  <EMI ID = 54.1>

  
carboxamide.

  
The core of S 31794 / F-l has a crude formula of

  
  <EMI ID = 55.1>

  
Preparation of the core of S 31794 / F-l, Preparation of the substrate

  
The nucleus of S 31794 / F-1 of this invention is prepared from the antibiotic S 31794 / F-1.

  
The antibiotic S 31794 / F-1 is prepared by aerobic culture in immersion of Acrophialophora limonispora NRRL
8095. This microorganism is part of the permanent culture collection of the Northern Regional Research Center, U.S. Department of Agriculture, Agricultural Research Culture Collection, North Central Region, Peoria, Illinois
61604, where it is available to the public under the indicated NRRL number.

  
As the nuclei each contain an amino group, they can exist in the form of salts. These salts can also be used as intermediates and for purification. The pharmaceutically acceptable salts of the nuclei are particularly useful because the purification of the final products is minimized. The "pharmaceutically acceptable" salts designate the salts in which the toxicity of the product as a whole with respect to warm-blooded animals is not increased.

  
The acid addition salts of the nuclei can be formed by standard reaction procedures with a mineral or organic acid. Typical mineral and organic acids include hydrochloric, hydrobromic, hydroiodic, sulfuric, phosphoric, acetic, benzoic, sulfamic, tartaric, citric, maleic, succinic, ascorbic, glycolic, lactic, fumaric, palmitic, cholic, pamolic, mucic, D -glutamic, d-camphoric, glutaric, phthalic, lauric, stearic, salicylic, methanesulfonic, benzenesulfonic, sorbic, picric, cinnamic, and other suitable acids.

  
High performance low pressure liquid chromatography (CLFPCE) reversed phase using

  
  <EMI ID = 56.1>

  
the final purification of the various antibiotics. In this process, the crude antibiotic mixture dissolved in a solvent is placed on a column equilibrated with the same solvent. Then, the column is eluted with the solvent. The fractions collected are controlled by bioautograph by Candida albicans and / or by UV (based on the relative retention times). The fractions containing identical antibiotic factors are pooled. It is sometimes necessary to carry out an additional chromatographic separation in order to obtain the various factors in purified form.

  
  <EMI ID = 57.1>

  
or A-42355, for example, by extracting whole broth or mycelium with methanol or chloroform. The 7: 2: 1 mixture of methanol, water and acetonitrile is a preferred solvent system for the chromatography of these antibiotics.

  
The crude antibiotic S 31794 / F-1 is obtained by extraction of the whole broth with a 4: 1 mixture of ethyl acetate and isopropanol and chromatography of the extracts.

  
The different factors can be identified by thin layer chromatography (TLC). Silica gel is a preferred adsorbent.

  
The Rf of factors A to G of A-30912, using a TLC on silica gel with a high carbon content, a 7: 3 solvent system of benzene and methanol and a bioautograph Candida albicans, are given in Table I

  
  <EMI ID = 58.1>

  

  <EMI ID = 59.1>


  
The approximate Rf of factors A, B, C, D and H of A-30912 in different solvent systems, using TLC on carbon rich silica gel and bioautograph by. Candida albicans, are given in Table II

TABLE II

  

  <EMI ID = 60.1>


  
Solvent systems:

  
a: ethyl acetate: methanol (3: 2) b: ethyl acetate: methanol (7: 3) c: acetonitrile: water (95: 5)

  
  <EMI ID = 61.1>

  
Factors A, B, D and H of A-30912 can also be identified by analytical CLFPCE using the following conditions:

  

  <EMI ID = 62.1>


  
The approximate retention times for factors A, B, D and H of A-30912 under these conditions are summarized in Table III.

TABLE III

  

  <EMI ID = 63.1>


  
Preparation of the enzyme

  
A. Producing microorganism

  
The enzyme which can be used for deacylation of the antibiotics of this invention is produced by certain microorganisms of the family Actinoplanaceae, preferably

  
the microorganism Actinoplanes utahensis NRRL 12052.

  
The enzyme can be the same enzyme as the one

  
used to deacylate penicillins. This work has

  
has been described in U.S. Patent No. 3,150,059. Good

  
that a preferred method of culturing A. utahensis NRRL 12052 for the production of this enzyme is described in Preparation 1, those skilled in the art will see that other methods can be used.

  
Actinoplanaceae are a relatively family

  
recent microorganisms of the Actinomycetales order.

  
Described for the first time by Dr. John N. Couch, this family was created in 1955 [J. Elisha Mitchell Sci. Soc. 71,
148-155 (1955)]. Family characteristics and many distinct genres can be found in: "Bergey's Manual

  
of Determinative Bacteriology ", 8th Ed., R.E. Buchanan and

  
N.E. Gibbons, Eds., The Williams & Wilkins Co., Baltimore,

  
Md., 1974, pages 706-723. Ten genera have thus been distinguished since:

  
I. Actinoplanes (i.e. the type genus and, in fact

  
by far the most common kind);

  
II. Spirillospora;

  
III. Streptosporangium;

  
IV. Amorphosporangium;

  
V. Ampullariella;

  
VI. Pilimelia VII. Planomonospora; VIII. Planobispora;

  
  <EMI ID = 64.1>

  
X. Kitasatoa.

  
Some of the species and varieties that have been isolated and characterized so far are: Actinoplanes

  
  <EMI ID = 65.1>

  
utahensis, and Actinoplanes missouriensis; Spirillospora albida; Streptosporangium roseum, Streptosporangium vulgare, Streptosporangium roseum var. hollandensis, Streptosporangium album, Streptosporangium viridialbum, Amorphosporangium auranticolor, Ampullariella regularis, Ampullariella campanulata, Ampullariella lobata, Ampullariella digitata, Pilimelia terevasa, Pilimelia anulata, Planomonospora parontospora, Planomonospa venezora, Planomonisora planoraisora planoraisora planoraisora planoraisora planoraisora planoraisora planoraisora planorospor

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

  
Cultures of representative species are available to the public at the Northern Regional Research Center, see above, under the following access numbers:

  

  <EMI ID = 66.1>


  
A. utahensis NRRL 12052 was obtained from a mother culture which has been deposited with the American - Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville,

  
Md. 20852 (A. utahensis ATCC 14539). The culture of

  
A. utaherisis ATCC 14539 can also be used as the source of the enzyme.

  
A. missouriensis NRRL 12053 was obtained from a culture which is also deposited with ATCC (A. missouriehsis ATCC 14538) and which is another source of the enzyme.

  
The effectiveness of any given strain of microorganism belonging to the Actinoplariaceae family for carrying out the deacylation of this invention is determined by the following procedure. An appropriate culture medium is inoculated with the microorganism. The culture is incubated at about 28 [deg.] C for 2 or 3 days on a rotary shaker. Then one of the antibiotics used as a substrate is added to the culture. The pH of the fermentation medium is maintained at approximately pH 6.5. The activity of the culture is monitored using a Candida albicans assay. This procedure is described in section E. The loss of antibiotic activity is an indication that the microorganism is producing the enzyme necessary for deacylation. However, this should be verified using one of the following methods:

  
1) Analysis by CLPE to determine the presence of the intact nucleus; or

  
2) Reacylation with an appropriate side chain to restore activity.

  
B. Conditions for production of the enzyme

  
The production of the enzyme takes place under conditions allowing the growth of Actinoplanaceae, that is to say at a temperature between about 25 and about
30 [deg.] C and at a pH between about 5.0 and about 8.0, with agitation and aeration. The culture medium must contain:
a) a source of assimilable carbon such as sucrose, glucose, glycerol, etc.; b) a source of nitrogen such as peptone, urea, <EMI ID = 67.1> c) a source of phosphate such as soluble phosphate; and d) mineral salts which are generally found to be effective in enhancing the growth of microorganisms.

  
We generally obtain an effective quantity of

  
  <EMI ID = 68.1>

  
growth cycle and it persists for some time after actual growth has been reached. The quantity of enzyme produced varies from one species of organism to another and according to different culture conditions.

  
As one skilled in the art will see, microorganisms,

  
  <EMI ID = 69.1>

  
the enzyme is subject to variations. For example, artificial variants and mutants of these strains can be obtained by treatment with various known mutagens such as ultraviolet rays, X-rays, high frequency waves, radioactive rays, and chemicals. All natural and artificial variants and mutants which are obtained from Actinoplanaceae and which produce the enzyme can be used in this invention.

  
C. Deacylation conditions

  
The substrate is preferably added to the culture of Actinoplanaceae after the culture has incubated for at least about 48 hours. The concentration of the substrate in the transformation medium can vary significantly. However, for maximum use of the enzyme and for practically complete deacylation in 24 hours, the concentration of the substrate will generally be between approximately 0.5 and approximately 1.0 mg / ml. Lower concentrations can be used but they do not allow maximum use of the enzyme; higher concentrations can also be used, but the substrate cannot be completely deacylated if the fermentation time is not extended.

  
The transformation of antibiotic substrates into corresponding nuclei of this invention is done for the best when the pH of the fermentation medium is maintained

  
  <EMI ID = 70.1>

  
deacylation is done slowly; when the pH rises above 6.0, the substrate and the nucleus that forms are less and less stable. For maximum stability, a pH of 6.0 is preferred, but at this pH, deacylation will take place less quickly (approximately 30 to approximately 36 hours). For a faster deacylation (approximately 24 hours) without significant losses, a pH of approximately 6.5 is preferred. In stirred fermenters, the pH can be adjusted by detection and control devices. When this is not practical, <EMI ID = 71.1>

  
adding 0.1 M phosphate buffer to the medium before adding the substrate.

  
After addition of the substrate, incubation of the culture should continue for approximately 24 hours or more. The purity of the substrate will affect the deacylation rate.

  
For example, a substrate having a purity greater than 50% is deacylated at a rate of about 0.8 about 1.2 mg / ml of antibiotic in 24 hours. When substrates of lower purity are used, deacylation takes place more slowly.

  
The substrate can be introduced several times. For example, in small reservoirs, 0.3 to 0.5 mg / ml of antibiotic can be introduced at 12-hour intervals for at least five additions, and in larger reservoirs, two times 0 can be added, 7 mg / ml.

  
The deacylation can be carried out over a wide range of temperatures, for example from about 20 to about 45 [deg.] C. It is however preferable to carry out the

  
  <EMI ID = 72.1>

  
  <EMI ID = 73.1>

  
for optimal deacylation and stability of the substrate and the nucleus.

  
D. The substrate

  
It is preferable to use purified antibiotics as substrates. Antibiotic substrates have antifungal but non-antibacterial activity. Thus, the substrates can harbor bacterial cells or spores which could develop in the deacylation fermentation medium. Such impurities can harm

  
  <EMI ID = 74.1>

  
starting biotics or obtained nuclei. It is therefore important that the substrates are sterile. As the autoclave destroys most of the antibiotic substrates, it is preferable to sterilize the preparations by treatment with ethylene oxide in a pressurized system.

  
E. Control of deacylation

  
The starting substances are anti-fungal antibiotics which are particularly active against Candida albicans. For this reason, an analysis using C. albicans is preferable to determine the amounts of substrates present. The nuclei that form are soluble in water but are inactive biologically. A decrease in biological activity is therefore a rapid presumptive deacylation test. Both broth samples and alcoholic extracts from the fermentation solids should be analyzed as the substrates are only slightly soluble in the broth.

  
F. Use of remaining cells

  
Another deacylation method consists in collecting the cells of Actinoplanaceae from the culture medium, in resuspending the cells in a buffer solution and in carrying out the deacylation as described in paragraph C. When this method is used, the myceliums enzyme active ingredients can be reused. For example, the myceliums of A. utahensis NRRL 12052 retain deacylase activity after storage for a month or more under refrigeration (4 - 8 [deg.] C) or in the frozen state (-20 [deg.] C) . A preferred buffer solution is 0.1 M phosphate buffer.

  
G. Immobilized enzymes

  
Another method of carrying out the deacylation consists in immobilizing the enzyme by methods known in the field. (See, for example, "Biomedical Applications of Immobilized Enzymes and Proteins", Thomas Ming Swi Chang, Ed., Plenum Press, New York, 1977; Vol. 1). The immobilized enzyme can then be used in a column (or any other suitable type of reactor) to carry out the deacylation.

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

Usefulness of the cores

  
The nuclei and their acid addition salts are intermediates which can be used for the preparation of synthetic antifungal compounds. The usable antifungal compounds prepared from these nuclei are described in patent applications N [deg.] Filed on the same day as the present patent application.

  
These compounds are represented by the following formula.

  
  <EMI ID = 75.1>

  
  <EMI ID = 76.1>

  
corresponding requests mentioned on page 23, line 39 above.

  

  <EMI ID = 77.1>


  
The compounds of formula III are prepared by acylating the appropriate nucleus on the α-amino group of the ornithine part of the nucleus by the appropriate acyl side chain using methods known in the art to form an amide bond. The acylation is generally carried out by reacting the nucleus with an activated derivative of the acid corresponding to the desired acyl side chain.

  
The term "activated derivative" designates a derivative which makes the carboxy function of the acylating agent reactive to coupling with the primary amino group to form the amide bond which links the acyl side chain to the appropriate nucleus. Suitable activated derivatives, their methods of preparation and their methods of use as acylating agents of a primary amine are known to those skilled in the art. The preferred activated derivatives are:
(a) an acid halide (for example an acid chloride), (b) an acid anhydride (for example an anhydride <EMI ID = 78.1>
(c) an activated ester (for example a 2,4,5-trichlorophenyl ester).

  
Other methods of activating the carboxy function include reacting the carboxylic acid with a carbonyldiimide (e.g. N, N'-dicyclohexylcarbodiimide or N, N'-diisopropylcarbodiimide) to obtain a reactive intermediate which, due of its instability, is not isolated, the reaction with the primary amine then being carried out in situ.

  
If a particular amino acid contains a group

  
  <EMI ID = 79.1>

  
understood for the skilled person that such a group must be protected before the reaction of the amino acid with the reagent used to fix the N-alkanoyl or N-alkenoyl group. Suitable protecting groups can be any group known in the field as usable for the protection of a functional chain group.

  
lateral in the synthesis of peptides. Such groups are well known and the choice of a particular protective group and its method of use will be easily appreciated by those skilled in the art [See, for example, "Protective

  
  <EMI ID = 80.1>

  
Press, N.Y., 1973].

  
The compounds of formula III inhibit the growth of pathogenic fungi and are therefore useful for combating the growth of fungi on environmental surfaces (as an antiseptic) or in the treatment of infections caused by fungi. The compounds are, in particular, active against Candida albicans and are therefore particularly useful for treating candidiasis. The activity of the compounds can be determined by conventional microbiological test procedures, such as in vitro by diffusion tests on discs on agar plate or diffusion tests on agar, or

  
  <EMI ID = 81.1>

  
albicans. The compounds are also active against trichophyton mentagrophytes (dermatophyte organism), Saccharomyces pastorianus and Neurospora crassa.

  
Certain compounds (As indicated in Reference Example 19, Table VIII) give significant levels in the blood after oral administration in mice.

  
When administered to a dog intravenously at a dose of 100 mg / kg per day for 5 days, the compound

  
  <EMI ID = 82.1>

  
Benzoyl, there is no outward sign of toxicity although there are temporarily increased levels of serum glutamo-pyruvic transaminase.

  
When used systematically, the dose of compounds of formula III will vary depending on the particular compound being used, the severity and nature of the infection, and the physical condition of the subject being treated. Therapy should be started at low doses, and the dose should be increased until the desired antifungal effect is obtained. The compounds can be administered intravenously or intramuscularly by injection in the form of a sterile aqueous solution or suspension to which

  
conventional pharmaceutically acceptable agents such as preservatives, buffering agents, solubilizing agents or the like may be added, if desired.

  
suspension. Other additives, such as saline or glucose, can be added to make the solutions isotonic. The proportions and the nature of these additives will be obvious to those skilled in the art.

  
Certain compounds of formula III give significant levels in the blood after oral administration
(See Reference Example 19, Table VIII) and can be routinely administered orally. For oral use, such compounds can be administered in combination with pharmaceutically acceptable carriers or excipients in the form of capsules, tablets or powders. The nature and proportion of these supports or excipients are known to those skilled in the art.

  
When used to treat vaginal infections due to Candida, the compounds of formula III can be administered in combination with excipients <EMI ID = 83.1>

  
the following examples are given.

  
Preparation 1

  
  <EMI ID = 84.1>

  
inclined is chosen from one of the following:

  
Middle A

  

  <EMI ID = 85.1>


  
the fold before autoclaving is about 5.9; we adjust

  
  <EMI ID = 86.1>

  
  <EMI ID = 87.1>

  
  <EMI ID = 88.1>

  

  <EMI ID = 89.1>


  
Middle B
  <EMI ID = 90.1>
 
  <EMI ID = 91.1>
  <EMI ID = 92.1>

  
Jersey, U.S.A.

  
Inoculated culture is inoculated with Actinoplanes

  
  <EMI ID = 93.1>

  
  <EMI ID = 94.1>

  
vegetative with the following composition:

  

  <EMI ID = 95.1>


  
  <EMI ID = 96.1>

  
New York, U.S.A.

  
The inoculated vegetative medium is incubated in

  
  <EMI ID = 97.1>

  
for approximately 72 hours on an agitator rotating on an arc 5 cm in diameter at 250 revolutions per minute.

  
  <EMI ID = 98.1>

  
  <EMI ID = 99.1>

  
subsequent one now cultivation in Here vapor phase of liquid nitrogen. The culture is prepared for such conservation in numerous small vials as follows:
2 ml of incubated vegetative medium and 2 ml of a glycerol and lactose solution are placed in each ampoule [see W.A.

  
  <EMI ID = 100.1>

  
prepared :: are kept in the vapor phase of nitrogen <EMI ID = 101.1>

  
stage (negating the composition described above). incubating as described above the inoculated first stage vegetative medium.

  
To obtain a larger volume of inoculum, we

  
  <EMI ID = 102.1>

  
to inoculate 400 ml of a second stage vegetative medium

  
  <EMI ID = 103.1>

  
Stadium. The second stage medium is incubated in a

  
  <EMI ID = 104.1>

  
about 48 hours on a stirrer rotating with a 5 cm arc

  
  <EMI ID = 105.1>

  
The incubated second stage vegetative medium (800 ml), prepared as described above, is used to inoculate
100 1 of a sterile production medium chosen from one

  
of the following:

  
  <EMI ID = 106.1>

  

  <EMI ID = 107.1>


  
  <EMI ID = 108.1>

  
  <EMI ID = 109.1>

  
  <EMI ID = 110.1>

  
Middle II
  <EMI ID = 111.1>
 
  <EMI ID = 112.1>
  <EMI ID = 113.1>

  
  <EMI ID = 114.1>

  
Middle III

  

  <EMI ID = 115.1>


  
  <EMI ID = 116.1>

  
  <EMI ID = 117.1>

  
The inoculated production medium is left to ferment

  
  <EMI ID = 118.1>

  
  <EMI ID = 119.1>

  
  <EMI ID = 120.1>

  
revolutions per minute and air it with sterile air to

  
  <EMI ID = 121.1>

  
air saturation at atmospheric pressure.

Preparation 2

  
  <EMI ID = 122.1>

  
  <EMI ID = 123.1>

  
next :

  

  <EMI ID = 124.1>
 

  

  <EMI ID = 125.1>


  
  <EMI ID = 126.1>

  
  <EMI ID = 127.1>

  
the ripe tilted crop and it is scratched with a handle

  
sterile to release spores. The resulting suspension is then suspended in 10 ml of sterile deionized water.

  
  <EMI ID = 128.1>

  
to inoculate 55 ml of vegetative medium in a bottle of

  
  <EMI ID = 129.1>

  

  <EMI ID = 130.1>


  
  <EMI ID = 131.1>

  
rotary type. After 24 hours, the medium is homogenized for one minute at low speed in a mixer

  
  <EMI ID = 132.1>

  
  <EMI ID = 133.1>

  
vegetative medium inoculated for 48 hours then homogenize it for 15 seconds at low speed.

  
This incubated vegetative medium can be used to inoculate a fermentation culture medium in a shaken flask or to inoculate a second stage vegetative medium. It can also be stored for later use, by '

  
  <EMI ID = 134.1> many small bulbs, as follows:

  
Mix the volume / volume vegetative crops

  
  <EMI ID = 135.1>

  

  <EMI ID = 136.1>


  
  <EMI ID = 137.1>

  
these tubes in the vapor phase of liquid nitrogen.

  
A stored suspension thus prepared can be used to inoculate tilted cultures or media. <EMI ID = 138.1> inclined at 25 [deg.] C in the light for 7 days. B. Fermentation in tanks

  
  <EMI ID = 139.1>

  
  <EMI ID = 140.1>

  
incubated to inoculate 400 ml of growth medium

  
  <EMI ID = 141.1>

  
25 [deg.] C for 24 hours, on a stirrer rotating with an arc 5 cm in diameter at 250 revolutions per minute.

  
  <EMI ID = 142.1>

  
as described above, is used to inoculate 100 liters of a sterile production site chosen from one of the following:

  
Middle IV
  <EMI ID = 143.1>
 
  <EMI ID = 144.1>
(initial fold 6.8-7.0)

  
  <EMI ID = 145.1>

  
  <EMI ID = 146.1>

  
Middle V

  

  <EMI ID = 147.1>


  
  <EMI ID = 148.1>

  
The inoculated production medium is left to ferment in a 165 l fermentation tank at a temperature of 25 [deg.] C for: 7 days. We air the middle of Lion with

  
  <EMI ID = 149.1>

  
C. Third stage vegetative medium

  
If the fermentation is carried out in larger tanks than those used for fermentation by
100 liters, it is recommended to use a third stage vegetative culture to seed the tank

  
  <EMI ID = 150.1>

  
has the following composition:

  

  <EMI ID = 151.1>
 

  

  <EMI ID = 152.1>


  
Preparation 3

  
  <EMI ID = 153.1>

  
  <EMI ID = 154.1>

  
whole (4,127 liters), obtained by the process described in Example 1 using production medium II, with
4280 liters of methanol for one hour, then filtered,

  
  <EMI ID = 155.1>

  
Diatomaceous Earth, Johns-Manville Products Corp.). The fold of the filtrate is adjusted to 4.0 by addition of 5N IICI.

  
  <EMI ID = 156.1>

  
chloroform. The chloroform extracts are combined and concentrated in vacuo to a volume of 20 liters. We add

  
  <EMI ID = 157.1>

  
precipitate mixture A-42355. The precipitate is separated by

  
  <EMI ID = 158.1>

  
form of a gray white powder.

  
  <EMI ID = 159.1>

  
Preparation 4

  
  <EMI ID = 160.1>

  
  <EMI ID = 161.1>

  
  <EMI ID = 162.1>

  
  <EMI ID = 163.1>

  
filter this solution and put it in a column in

  
  <EMI ID = 164.1>

  
  <EMI ID = 165.1>

  
  <EMI ID = 166.1> <EMI ID = 167.1>

  
suspension packing described in Preparation 11.

  
  <EMI ID = 168.1>

  
(maximum flow 19.5 ml / minute) is used to move the

  
  <EMI ID = 169.1>

  
from fractions being collected every minute. Elution

  
  <EMI ID = 170.1>

  
UV control device (ISCO Model UA-5, Instrument Specialist Co., 4700 Supcrior Ave., Lincoln, Nebraska 68504) equipped

  
of an optical unit (ISCO Type 6).

  
Preparation 5

  
  <EMI ID = 171.1>

  
One separates the mixture A-42355 as described in Preparation 3, but the extracts are chromatographed

  
  <EMI ID = 172.1>

  
  <EMI ID = 173.1>

  
to determine their biological activity. Biological analysis is performed by analysis on paper disc

  
  <EMI ID = 174.1> <EMI ID = 175.1> concentrated under vacuum. The concentrated solution (4.5 liters)

  
  <EMI ID = 176.1>

  
concentrates the filtrate to dryness. The residue obtained is redissolved in an appropriate volume of methanol. We add the solution

  
  <EMI ID = 177.1>

  
precipitate the antibiotic complex containing factor B. This precipitate is also separated by filtration and is

  
  <EMI ID = 178.1> <EMI ID = 179.1>

  
and it is introduced on a column of silica gel (Michel-Miller column of 3.7 cm internal diameter x 33 cm) via a loop using a valve system. The column is packed with a reverse-phase gel resin.

  
  <EMI ID = 180.1>

  
Preparation 15. The fractions 102-110 are combined and concentrated under vacuum to obtain an oil. The oil is dissolved in a small volume of t-butanol and lyophilized, which

  
  <EMI ID = 181.1>

  
Preparation 6

  
Isolation, of factor U of A-30912

  
  <EMI ID = 182.1>

  
silica gel (Grace, grade 62) the concentrated chloroform extracts from two fermentation tests (3800 l and 4007 l) obtained by the process described in Preparation 3. The column is washed with chloroform and then with ac6to-

  
  <EMI ID = 183.1>

  
to precipitate the mixture A-42355 enriched in factor D .. This precipitate is removed by filtration and dried, this

  
  <EMI ID = 184.1>

  
form of a gray powder.

  
  <EMI ID = 185.1>

  
introduced it onto a silica gel column (Michel column <EMI ID = 186.1>

  
Prepared Lion 10. The filling was carried out in a 7: 2: 1 mixture of methanol, water and acetoniLrilc,!; According to the mode

  
  <EMI ID = 187.1>

  
  <EMI ID = 188.1>

  
without valves. A fraction is collected every 2 minutes.

  
  <EMI ID = 189.1>

  
described in Preparation 4. The fractions 96-108 are combined and concentrated under vacuum to obtain an oil. This oil is dissolved in a small volume of t-butanol and lyophilized to obtain 89 mg of factor D of A-30912.

  
Preparation 7

  
Separated Lion from factor Il of A-30912

  
  <EMI ID = 190.1>

  
prepare as described in Preparations 2 and 3, in 35 ml

  
  <EMI ID = 191.1>

  
filters the resulting solution and introduces it onto a glass column 3.7 cm inside diameter x 42 cm
(Michel-Miller column) via a loop using a valve system. The column is lined with

  
  <EMI ID = 192.1>

  
piston design without valves, and a fraction is collected every two minutes. The elution of the antibiotic is monitored at 280 nm as described in Preparation 19, paragraph C. The fractions 112-132 are combined with the

  
  <EMI ID = 193.1>

  
  <EMI ID = 194.1>

  
oil. The oil is dissolved in a small volume of t-butanol

  
  <EMI ID = 195.1> <EMI ID = 196.1>

  
  <EMI ID = 197.1>

  
inside x 32 cm, as described above. The solvent

  
  <EMI ID = 198.1>

  
and methanol and a bioautograph by Candida albicans are shown in Table VII.

  
  <EMI ID = 199.1>

  

  <EMI ID = 200.1>


  
  <EMI ID = 201.1>

  
  <EMI ID = 202.1>

  
  <EMI ID = 203.1>

  
are given in Table VIII.

  
  <EMI ID = 204.1>

  

  <EMI ID = 205.1>


  
  <EMI ID = 206.1>

  
following conditions:

  

  <EMI ID = 207.1>
 

  
Injection injection on goat: the

  
Duration: approximate tick retention for factors A,

  
  <EMI ID = 208.1>

  
  <EMI ID = 209.1>

TABLE IX

  

  <EMI ID = 210.1>


  
  <EMI ID = 211.1>

  
  <EMI ID = 212.1>

  
stirring, stirring and / or aerating at pH 3-8, preferably 5-7,

  
  <EMI ID = 213.1>

  
fractions that have antifungal activity and they are

  
  <EMI ID = 214.1>

  
LII-20 "with methanol. The fractions from the

  
  <EMI ID = 215.1>

  
  <EMI ID = 216.1>

  
71: 25: 4 of chloroform, methanol and water. Elected fractions which have an antifungal activity are combined and evaporated under vacuum to obtain the crude antibiotic

  
  <EMI ID = 217.1> <EMI ID = 218.1>

  
decomposition after drying under high vacuum at 20 [deg.] C.

Preparation 9

  
Separation of the antibiotic S31794 / F-J

  
  <EMI ID = 219.1>

  
as described in Preparation 8 after chromatography on Sephadex, on a silica gel column (MichelMiller column) via a J'aide loop of a winnowing system. The filling procedure is used in

  
  <EMI ID = 220.1>

  
  <EMI ID = 221.1>

  
the antibiotic using a UV tester at
280 nm as in Preparation 22. The fractions are combined

  
  <EMI ID = 222.1>

  

  <EMI ID = 223.1>


  
  <EMI ID = 224.1>

  
diode.

  
Preparation 10

  
  <EMI ID = 225.1>

  
CI8

  
Step 1 Hydrolysis

  
1000 g of silica gel LP-1 (Quantum) are added

  
  <EMI ID = 226.1>

  
concentrated sulfuric and 1650 ml of concentrated nitric acid

  
  <EMI ID = 227.1> 5 water cooling fixed on the tank.

  
Or "cools the mixture in an ice bath and

  
  <EMI ID = 228.1>

  
frit. The silica gel is washed with deionized water until neutral. Then, the silica gel is washed with 4 l of acetone and dried under vacuum at 100 [deg.] C for two days.

  
Step 2: First silylation

  
The dry silica gel from Step 1 is transferred to a round bottom flask and suspended in 3.5 1 of toluene. The flask is heated in a steam bath for two hours to remove all the residual water by azeotropic distillation. 321 ml of octadecyltrichlorosilane (Aldrich Chemical Company) is added, and the reaction mixture is heated at reflux overnight (16 hours)

  
  <EMI ID = 229.1>

  
avoid the agitator being too close to the bottom of the flask, to avoid crushing of the silica gel particles.

  
The mixture is allowed to cool. The silanized silica gel is collected, washed with 3 l of toluene and 3 l of acetone, then air-dried overnight (16-20

  
  <EMI ID = 230.1>

  
  <EMI ID = 231.1>

  
ambient for two hours, filter, wash with 3 1
-acetone and air dried overnight.

  
Step 3: Second silylation

  
We repeat the procedure of the first

  
  <EMI ID = 232.1>

  
heats the suspension at reflux at 60 [deg.] C for two hours, while shaking carefully. The final product is collected by filtration, washed with 3 1 of toluene and 6 1 of

  
  <EMI ID = 233.1>

  
(16-20 hours).

  
  <EMI ID = 234.1>

  
  <EMI ID = 235.1> <EMI ID = 236.1>

  
that prepared by the process of Preparation 10.

  
  <EMI ID = 237.1>

  
flow rates between 5 and 40 ml / minute are necessary for this suspension packing technique; this depends on the volume and dimensions of the column. The packing pressure must be higher than the pressure used during the actual separation by 2 to 3.5 bars; this ensures that no subsequent packing of the adsorbent occurs during the separations.

  
A sudden decrease in pressure can cause cracks or channels in the material

  
  <EMI ID = 238.1>

  
of the column. It is therefore important to leave the. pressure slowly descend to zero each time the pump is stopped.

  
Approximate volume of columns (Ace glass columns, Category No., not filled): 5795-04, 12 ml;

  
  <EMI ID = 239.1>

  
  <EMI ID = 240.1>

  
The time required to garnish a glass column can vary from a few minutes to a few hours, depending on the size of the column and the operator's experience. Steps :

  
1. Connect the glass column to a tank column by a coupling (the volume of the tank column must be

  
twice that of the column). Place the two columns vertically with the tank column above.

  
2. Weigh the packing material (100 g for 200 ml of column).

  
3. Add approximately 5 volumes of solvent to the packing material, use a mixture of 70-80% methanol and 20-

  
30 5 of water.

  
  <EMI ID = 241.1> <EMI ID = 242.1>

  
fill the tank column. Pour quickly into the

  
  <EMI ID = 243.1>

  
solvent and the tank column must be partially filled with solvent before pouring the suspension. The use of larger suspension volumes can also give good results; however, this requires
(a) a larger tank or (b) more than one filling of the tank during the filling operation.

  
  <EMI ID = 244.1>

  
devices at maximum flow if using an Ace Cat pump. No. 13625-25, or a similar solvent delivery system (20 ml / minute).

  
7. Continue until the column is completely filled with the adsorbent. The pressure must not exceed the

  
  <EMI ID = 245.1>

  
below 14 bars will be sufficient.

  
8. If the pressure exceeds the maximum values, reduce

  
  <EMI ID = 246.1>

  
  <EMI ID = 247.1>

  
fill the pre-column with solvent and a small amount

  
  <EMI ID = 248.1>

  
the column is completely packed. For additional information, see the operating mode. Always

  
  <EMI ID = 249.1>

  
pump ; this will prevent the formation of any preferential cracks or channels in the packing material.

  
  <EMI ID = 250.1>

  
pre-column. With a small spatula, remove a few millimeters (2-4) of packing at the top of the column:

  
  <EMI ID = 251.1>

  
  <EMI ID = 252.1>

  
introduce the pressure (generally less than 14 bars) and observe through the glass wall at the top of the column if the resin settles more. If the packing material must continue to deposit (this can be the case with large columns), a certain dead space will appear or else, preferential channels will form and step 9 must be repeated.

  
Preparation 12

  
  <EMI ID = 253.1>

  
Factor A of A-30912 is dissolved in ethanol.

  
PtO2 is reduced in absolute ethanol to form Pt which is used in turn to reduce the A factor of A-30912 catalytically, using hydrogenation under positive pressure until the reaction is complete
(about 2 to 3 hours). The reaction mixture is filtered and concentrated in vacuo. The residue is dissolved in a small amount of t-butanol and lyophilized to obtain tetrahydro-A-309l2A.

  
Preparation 13

  
Preparation of tetrahydro-A-30912B

  
Factor B of A-30912 is dissolved in ethanol.

  
  <EMI ID = 254.1>

  
this in turn is used to reduce the B factor of A-30912 catalytically, using hydrogenation under positive pressure until the reaction is complete
(about 2 to 3 hours). The reaction mixture is filtered and concentrated in vacuo. The residue is dissolved in a small amount of t-butanol and lyophilized to obtain tetrahydro-A-30912B.

  
Preparation 14

  
  <EMI ID = 255.1>

  
Factor D of A-30912 is dissolved in ethanol. Pt02 is reduced in absolute ethanol to form Pt which is used in turn to reduce the D factor of A-30912 catalytically, using hydrogenation under positive pressure until the reaction is complete
(about 2 to 3 hours). The reaction mixture is filtered and concentrated in vacuo. The residue is dissolved in a small amount of t-butanol and lyophilized to obtain tetrahydro-A-30912D.

  
Preparation 15

  
Preparation of tetrahydro-A-30912H

  
Factor H of A-30912 is dissolved in ethanol. Pt02 is reduced in absolute ethanol to form Pt that

  
  <EMI ID = 256.1>

  
catalytically, using hydrogenation under

  
ftA.-positive pressure until the reaction is complete
(about 2 to 3 hours). The reaction mixture is filtered and concentrated in vacuo. The residue is dissolved in a small amount of t-butanol and lyophilized to obtain tetrahydro-A-30912H.

Example 1

  
A. Deacylation of factor A from A-30912

  
Fermentation of A. utahensis is carried out as described in Preparation 1, using inclined culture medium A and production medium I and incubating the production medium for about 42 hours.

  
Factor A of A-30912 is added to the fermentation medium
(340 g of raw substrate which contains approximately 19.7 g of factor A of A-30912, dissolved in 1.5 l of ethanol).

  
The deacylation of factor A of A-30912 is checked by analysis with Candida albicans. The fermentation is allowed to continue until the deacylation is complete as shown by the disappearance of the activity vis-à-vis C. albicans.

  
B. Separation of the core from A-30912A

  
The whole fermentation broth (100 liters) is filtered, obtained as described in paragraph A and containing the core of approximately 20 g of factor A of A-30912. We reject the mycelial cake. The clear filtrate thus obtained (about 93 liters) is passed through a column containing 4.5 liters of HP-20 resin (very porous polymer DIAION, HP-Series, Mitsubishi Chemical Industries Limited, Tokyo, Japan) at a flow rate of 200 ml / minute. The effluent thus obtained is rejected. Then the column is washed with up to eight volumes of deionized water at pH 6.5 - 7.5 to remove the residual filtered broth. We reject this &rsquo; washing water. Then the column is eluted with a 7: 3 solution of water and methanol (85 liters) at a flow rate of 200-300 ml / minute.

  
The elution is checked using the following procedure: Two aliquots are taken from each eluted fraction. One of the aliquots is concentrated to a small volume and treated with an acid chloride such as myristoyl chloride, using a procedure such as that described in Reference Example 1. The activity of this product and of the other aliquot part (untreated) vis-à-vis Candica albicans. If the untreated aliquot has no activity and the acylated aliquot has activity, the fraction contains the nucleus of A-30912A. The eluate containing the core of A-30912A is concentrated in vacuo to a small volume and lyophilized to obtain approximately 97 grams of raw core.

  
C. Purification of the nucleus of A-30912A by chromatography

  
reverse phase liquid

  
  <EMI ID = 257.1>

  
obtained as described in paragraph B, in 300 ml of a 96: 2: 1: 1 mixture of water, acetonitrile, acetic acid and pyridine. This solution is chromatographed on a 4 liter stainless steel column (8 cm x 80 cm) packed with Lichroprep RP-18, particle size 25-40 microns
(MC / B Manufacturing Chemists, Inc. E / M, Cincinnati, OH). The column is part of a Prep 100 chromatospac unit
(Jobin Yvon, 16-18 rue du Canal 91160 Lor.gjumeau, France). The column operates at a pressure of 6.2 - 6.9 bars, giving a flow rate of approximately 60 ml / minute, using the same solvent. The separation is monitored at 280 nm using a UV control device (ISCO Absorption Monitor Model UA-5, Instrumentation Specialties Co., 4700 Superior Av., Lincoln, Nebraska 68504) equipped with an optical unit.
(ISCO Type 6).

   Fractions having a volume of about 500 ml are collected per minute.

  
Based on the absorption at 280 nm, the fractions containing the core of A-30912A are pooled, evaporated in vacuo and lyophilized to obtain 2.6 grams of the core. The quantity of solvent necessary to complete this chromatographic separation varies from 7 to 8 liters.

  
D. Characteristics of the core of A-30912A <EMI ID = 258.1>
(b) Molecular weight: 797.83.
(c) White amorphous solid, soluble in water, dimethylformamide, dimethyl sulfoxide and methanol insoluble in chloroform,

  
tolèune and diethyl ether.

  
(d) Infrared absorption spectrum (KBr pellet).

  
Presents absorption maxima at:

  
3340 broad (OH, hydrogen bond); 2970, 2930 and 2890 (stretching of CH, aliphatic groups CH3, CH2, CH),
1660 and 1624 (several carbonyl groups C = O); 1510-
1550; 1430-1450 ("wagging" distortion); 1310-1340;

  
  <EMI ID = 259.1>
(e) The electrometric titration in 66% aqueous dimethylformamide indicates the presence of a titratable group with a pK a of approximately 7.35
(initial pH 7.32). <EMI ID = 260.1> under the following conditions:
Column: 4 x 300 mm

  
  <EMI ID = 261.1>

  
Solvent: 1: 2: 97 mixture of ammonium acetate,

  
acetonitrile and water

  
Flow rate: 3 ml / minute

  
Pressure: 172.5 bars

  
Detector: UV with variable wavelength at 230 nm Sensitivity: 0-0.4 A.U.F.S.

Example 2

  
The core of A-30912A is prepared and purified by the method of Example 1, but tetrahydro-A-30912A is used as the substrate.

Example 3

  
The core of A-30912A is prepared and purified by the method of Example 1, but aculeacin A is used as the substrate.

Example 4

  
A. Deacylation of factor B from A-30912

  
Fermentation of A. utahensis is carried out as described in Preparation 1, using production medium I. After having incubated the culture for approximately 48 hours, factor B of A-30912 dissolved in a mixture is added to the fermentation medium. small amount of methanol.

  
We control the deacylation of factor B of A-30912 <EMI ID = 262.1>

  
as the disappearance of the activity shows.

  
B. Separation of the core from A-30912B

  
The whole fermentation broth obtained is filtered as described in paragraph A. The mycelial cake is rejected. The clear filtrate thus obtained is passed through a column containing HP 20 resin (DIAION very porous polymer, HP-Series, Mitsubishi Chemical Industries Limited, Tokyo, Japan). The effluent thus obtained is rejected.

  
Then the column is washed with up to eight volumes of deionized water at pH 6.5 - 7.5 to remove the residual filtered broth. This washing water is rejected. Then the column is eluted with a 7: 3 solution of water and methanol. The elution is checked as follows: two aliquots are taken from each eluted fraction. One of the aliquots is concentrated to a small volume and treated with an acid chloride such as <EMI ID = 263.1> albicans of this product and of the other aliquot (untreated). If the untreated aliquot has no activity and the acylated aliquot has activity, the fraction contains the nucleus of A-30912B. The eluate containing the core of A-30912B is concentrated in vacuo to a small volume and lyophilized to obtain the crude core. C. Purification of the nucleus of A-30912B by chromatography

  
reverse phase liquid

  
The core of crude A-30912B, obtained as described in paragraph B, is dissolved in a 96: 2: 1: 1 mixture of water, acetonitrile, acetic acid and pyridine. This solution is chromatographed on a column packed with Lichroprep RP-18, particle size 25-40 microns (MC / B Manufacturing Chemists, Inc. E / M, Cincinnati, OH). The column is part of a Chromatospac Prep 100 unit (Jobin Yvon, 16-18 Rue du

  
  <EMI ID = 264.1>

  
column at a pressure of 6.2 - 6.9 bar, giving a flow rate of approximately 60 ml / minute, using the same solvent. The separation is checked at 280 nm using a <EMI ID = 265.1>

  
Based on the absorption at 280 nm, the fractions containing the core of A-30912B are pooled, evaporated in vacuo and lyophilized to obtain the core of purified A-30912B.

Example 5

  
The core of A-30912B is prepared and purified by the method of Example 4, but tetrahydro-A-30912B is used as the substrate.

Example 6

  
A deacylation of factor D from A-30912

  
Fermentation of A. utahensis is carried out as described in preparation I, using production medium I. After having incubated the culture for approximately 48 hours, factor D of A-30912 dissolved in a mixture is added to the fermentation medium. small amount of methanol.

  
The deacylation of factor D of A-30912 is checked by analysis on a paper disc against Candida albicans or Neurospora crassa. The fermentation is allowed to continue until the deacylation is complete, as shown by the disappearance of the activity.

  
B. Separation of the core from A-30912D

  
The whole fermentation broth obtained is filtered as described in paragraph A. The mycelial cake is rejected. The clear filtrate thus obtained is passed through a column containing HP-20 resin (DIAION very porous polymer, HP-Series, Mitsubishi Chemical Industries Limited, Tokyo, Japan). The effluent thus obtained is rejected. Then the column is washed with up to eight volumes of deionized water at pH 6.5 - 7.5 to remove the residual filtered broth. This washing water is rejected. The column is then eluted with a 7: 3 solution of water and methanol. The elution is checked using the following procedure:
Two aliquots of each eluted fraction are prepared. One of the aliquots is concentrated to a small volume and treated with an acid chloride such as <EMI ID = 266.1>

  
Candida albicans from this product and the other aliquot (untreated). If the untreated aliquot part

  
  <EMI ID = 267.1>

  
activity, the fraction contains the nucleus of A-30912D. The eluate containing the core of A-30912D is concentrated in vacuo to a small volume and lyophilized to obtain the crude core.

  
C. Purification of the nucleus of A-30912D by chromatography

  
reverse phase liquid

  
The core of crude A-30912D obtained is purified as described in paragraph B, using the procedure in paragraph C of Example 4.

  
Based on the absorption at 280 nm, the fractions containing the core of A-30912D are pooled, evaporated in vacuo and lyophilized to obtain the core of purified A-30912D.

Example 7

  
The core of A-30912D is prepared and purified by the method of Example 6, but tetrahydro-A-30912D is used as the substrate.

Example 8

  
A. Deacylation of factor H from A-30912

  
Fermentation of A. utahensis is carried out as described in Preparation I, using production medium I. After incubating the culture for 48 hours, factor H of A-30912 dissolved in a small amount is added to the fermentation broth. methanol.

  
The deacylation of factor H of A-30912 is checked by analysis on a paper disc against Candida albicans or N eurospora crassa. The fermentation is allowed to continue until the deacylation is complete as indicated by the disappearance of the activity.

  
B. Separation of the core from A-30912H

  
The whole fermentation broth obtained is filtered as described in paragraph A. The mycelial cake is rejected. The clear filtrate thus obtained is passed through a column containing HP-20 resin (DIAION very porous polymer, HP-Series, Mitsubishi Chemical Industries <EMI ID = 268.1>

  
  <EMI ID = 269.1>

  
  <EMI ID = 270.1>

  
control the elution using the following procedure:
Two aliquots of each eluted fraction are taken.

  
One of the aliquots is concentrated to a small volume and treated with an acid chloride such as myristoyl chloride. The activity of this product and of the other aliquot (untreated) part against candida albicans is determined. If the untreated aliquot part has no activity and the acylated aliquot part has activity, the fraction contains the core of A-30912H. The eluate containing the core of A-30912H is concentrated under vacuum to a small volume and lyophilized to obtain the crude core.

  
C. Purification of the nucleus of A-30912H by chromatography

  
reverse phase liquid

  
The core of crude A-30912H, obtained as described in paragraph B, is purified according to the method of paragraph C of Example 4.

  
Based on the absorption at 280 nm, the fractions containing the A-30912H nucleus are combined, evaporated in vacuo and lyophilized to obtain the purified A-30912H nucleus.

Example 9

  
The core of A-30912H is prepared and purified by the method of Example 8, but used as substrate

  
  <EMI ID = 271.1>

Example 10

  
The nucleus of A-30912H is prepared and purified by the method of Example 8, but the factor H of A-30912 is prepared from the factor A of A-30912 according to the following procedure:

  
19.6 mg of factor A of the antibiotic A-30912 is dissolved in 1 ml of dimethylformamide. Acid methanol (3% HCl, 0.06 ml) is added to this solution. The resulting solution is stirred at room temperature overnight and then evaporated to dryness in vacuo. The residue obtained is chromatographed by CLPECE as described in Preparation 7, using reverse phase silica gel

  
  <EMI ID = 272.1>

  
elution solvent and 1.4 mg of factor H of A-30912 are obtained (the compound of formula II where R and R 4 are both OH, R2 is hydrogen, R3 is a methoxy group and R is a linoleoyl group).

Example 11

  
The ring of type A-30912H of formula 1 in which R is an ethoxy group is prepared and purified by the method of Example 8, but the compound of formula II is used as substrate where R is a methoxy group and R is a linoleoyl group. The substrate is prepared by the procedure used in Example 10.

Example 12

  
Preparing and purifying by the method of Example 8

  
  <EMI ID = 273.1>

  
n-propoxy group, but the compound of formula II is used as substrate where R is an n-propoxy group and R is a linoleoyl group. The substrate is prepared as described in Example 10.

Example 13

  
The nucleus of A-30912H having the formula I where R is prepared and purified by the method of Example 8 <3> is an isobutoxy group, but the compound of formula II is used as substrate where R is an isobutoxy group and R is a linoleoyl group.

Example 14

  
The nucleus of A-30912H of formula I in which R is an n-pentyloxy group is prepared and purified by the process of Example 8, but the compound of formula II in which R is an n-pentyloxy group is used as substrate and R a linoleoyl group.

Example 15

  
The nucleus of type A-30912H of formula I in which R is an n-hexyloxy group is prepared and purified by the method of Example 8, but the compound of formula II in which R is an n-hexyloxy group is used as substrate and R a linoleoyl group.

Example 16

  
Preparing and purifying by the method of Example 8

  
  <EMI ID = 274.1>

  
ethoxy, but the compound of

  
  <EMI ID = 275.1>

  
stearoyl.

Example 17

  
Preparing and purifying by the method of Example 8

  
  <EMI ID = 276.1>

  
2-ethyl-1-butoxy, but the substrate used is

  
  <EMI ID = 277.1>

  
butoxy and R a linoleoyl group.

Example 18

  
The nucleus of type A-30912H having the formula I where R is a 3-methyl-1-butoxy group is prepared and purified by the method of Example 8, but the compound of formula II where R is used as substrate a 3-methyl-lbutoxy group and R a linoleoyl group.

Example 19

  
A. Deacylation of the antibiotic S 31794 / F-1

  
Fermentation of A. utahensis is carried out as described in Preparation 1, using production medium I. After having incubated the culture for approximately 48 hours, the antibiotic S 31794 / Fl, dissolved in a small amount of methanol.

  
The deacylation of S 31794 / F-1 is checked by analysis on a paper disc against Candida albicans. The fermentation is allowed to continue until the deacylation is complete as shown by the disappearance of the activity. B. Separation of the core of S 31794 / F-l

  
The whole fermentation broth obtained is filtered as described in paragraph A. The cake is discarded

  
  <EMI ID = 278.1>

  
column containing an HP-20 resin (very porous polymer DIAION, HP-Series, Mitsubishi Chemical Industries Limited, Tokyo, Japan). The effluent thus obtained is rejected. Then the column is washed with up to eight volumes of deionized water

  
at pH 6.5 - 7.5 to remove the residual filtered broth. This washing water is rejected. Then the column is eluted with a 7: 3 solution of water and methanol. The elution is checked using the following procedure: Two aliquots are taken from each eluted fraction. One of the aliquots is concentrated to a small volume and is

  
  <EMI ID = 279.1>

  
myristoyl. This product and the other untreated aliquot part are analyzed for their activity with respect to Candida albicans. If the untreated aliquot has no activity and the acylated aliquot has activity, the fraction contains the nucleus of S-31704 / F-1. The eluate containing the nucleus of

  
S 31794 / F-1 and lyophilized to obtain the raw nucleus. C. Purification of the nucleus of S 31794 / F-1 by chromatography

  
reverse phase liquid

  
The core of crude S 31704 / F-1 obtained as described in paragraph B above is purified according to the procedure of paragraph C of Example 4.

  
Based on the absorption at 280 nm, the fractions containing the nucleus of S 31794 / F-1 are combined, they are evaporated in vacuo and lyophilized to obtain the nucleus of

  
S 31794 / F-l purified.

  
Preparation of a first group of derivatives

  
The following procedure illustrates the preparation of compounds of formula III by the method using an "active ester". The particular compounds prepared by this mode

  
  <EMI ID = 280.1>

  
  <EMI ID = 281.1>

  
  <EMI ID = 282.1>

  
defined in the corresponding kernels.

  
Reference Preparation 1

  
Preparation of 2,4,5-trichlorophenyl tridecanoate

  
A solution of 12.5 g of n-tridecanoic acid (Sigma Chemical Co), 11.5 g of 2,4,5-trichlorophenol and 12.0 g of N is stirred at ambient temperature for 16 hours. N'-dicyclohexylcarbodiimide in 650 ml of methylene chloride. Then the reaction mixture is filtered and dried under vacuum to obtain 22 g of 2,4,5-trichlorophenyl tridecanoate: This material is purified by chromatography on a column of silica gel (Woelm) using toluene as eluent. Fractions are monitored by TLC using short wavelength ultraviolet light for detection. The fractions containing the purified product are combined and concentrated in vacuo to dryness.

  
Reference example 1

  
Acylation of the nucleus of A-30912A by n-tridecanoate

  
  <EMI ID = 283.1>

  
A solution of 6.0 g of 2,4,5-trichlorophenyl tridecanoate and 4.5 g of A-30912A core in 600 ml of dimethylformamide (DMF) is stirred at room temperature for 16 hours. Removal of the solvent in vacuo leaves a residue (12 g). We dilute the residue with

  
500 ml of methylene chloride for 45 minutes and the mixture is filtered. We reject the filtrate. The resulting solids are extracted with 500 ml of methanol. The methanolic extract is filtered and concentrated in vacuo to obtain a crude product (5.0 g).

  
The crude product is purified by reverse phase CIP E as follows:

  
A sample of the crude product (1 g), dissolved in 5 ml of methanol, is injected into a 2.5 x 80 cm stainless steel column packed with LP-1 / C18 resin (See Preparations 10 and 11). The column is eluted with a solvent system comprising a 3: 3: 3 mixture of water, methanol and acetonitrile. The elution is carried out at a pressure of

  
69 to 103.5 bars with a flow rate of 11 - 12 ml / minute using a LDC duplex pump (Milton-Roy). The effluent is monitored with an ultraviolet detector (ISCO-UA-5) at
280 nm. We collect fractions every two minutes
(21 - 24 ml). The fractions containing the desired product are combined and dried under vacuum. 550 mg of product are obtained. The above chromatography is repeated four times to obtain additional purified samples of the product as follows: 620 mg, 520 mg, 670 mg and 490 mg, for a total weight of 2.8 g.

  
Following the previous procedure, we react <EMI ID = 284.1>

  
trichlorophenyl and 2.6 g of the purified title product are obtained. The substances of the two preparations are combined

  
  <EMI ID = 285.1>

  
  <EMI ID = 286.1>

  
(C18 Micro Bondapak, Waters Co.) using the 2: 1: 2 system of water, methanol and acetonitrile as the eluent, indicates only one peak.

  
Reference example 2

  
Acylation of the core of A-30912B by n-tridecanoate

  
  <EMI ID = 287.1>

  
A solution of 3.3 mmol of 2,4,5-trichlorophenyl n-tridecanoate and 1 mmol of nucleus of A-30912B is stirred at room temperature for 16 hours.
200 ml of DMF. Removal of the solvent in vacuo provides a residue. The residue is diluted with 300 ml of methylene chloride for 45 minutes and the mixture is filtered. We reject the filtrate. The remaining solids are extracted with
300 ml of methanol and the methanolic extract is filtered and concentrated in vacuo to obtain a crude product.

  
The crude product is purified by reverse phase CLPE

  
as following :

  
A sample of the crude product (1 g) dissolved in 5 ml of methanol is injected into a stainless steel column

  
  <EMI ID = 288.1>

  
10 and 11). The column is eluted with a solvent system consisting of a 3: 3: 4 mixture of water, methanol and acetonitrile. The elution is carried out at a pressure of 69-103.5.

  
  <EMI ID = 289.1>

  
  <EMI ID = 290.1>

  
containing the desired product and they are dried under vacuum to obtain the title product. We analyze the product

  
  <EMI ID = 291.1>

  
  <EMI ID = 292.1>

  
1: 2: 2 by volume of water, methanol and acetonitrile. After development, the plates are observed under UV light to detect the product.

  
  <EMI ID = 293.1>

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

  
  <EMI ID = 294.1>

  
a solution of .3.3 mmol of 2,4,5-trichlorophenyl n-tridecanoate and of 1 mmol of A-30912D core in
200 ml of DMF. Removal of the solvent in vacuo gives a residue. The residue is diluted with 300 ml of methylene chloride for 45 minutes and the mixture is filtered. We reject the filtrate. The remaining solids are extracted with
300 ml of methanol, the methanolic extract is filtered and concentrated in vacuo to obtain a crude product.

  
The crude product is purified by reverse phase CLPE as described in Reference Example 2.

Reference example 4

  
Acylation of the nucleus of A-30912H by n-tridecanoate

  
  <EMI ID = 295.1>

  
A solution of 3.3 mmol of 2,4,5- n-tridecanoate is stirred at room temperature for 16 hours.

  
  <EMI ID = 296.1>

  
200 ml of DMF. Removal of the solvent in vacuo gives a residue. The residue is diluted with 300 ml of methylene chloride for 45 minutes and the mixture is filtered. We reject the filtrate. The remaining solids are extracted with
300 ml of methanol, the extract is filtered and concentrated in vacuo to obtain a crude product.

  
The crude product is purified by reverse phase CLPE as described in Reference Example 2.

  
  <EMI ID = 297.1>

  
according to the procedure of Reference Example 4, but using as starting material the compound of formula I where R is an n-propoxy group (prepared by the method of Example 12).

  
Reference example 6

  
  <EMI ID = 298.1>

  
2,4,5-trichlorophênyle

  
Stir at room temperature for 16 hours <EMI ID = 299.1>

  
200 ml of DMF. Removal of the solvent in vacuo gives a residue. The residue is diluted with 300 ml of methylene chloride for 45 minutes, and the mixture is filtered. We reject the filtrate. The resulting solids are extracted with
300 ml of methanol, the methanolic extract is filtered and concentrated in vacuo to obtain a crude product.

  
The crude product is purified by reverse phase CLPE as described in Reference Example 2.

  
Preparation of a second group of derivatives

  
The following procedure which gives the preparation

  
  <EMI ID = 300.1>

  
illustrates the process for the preparation of a second group of compounds of formula III.

  
  <EMI ID = 301.1>

  
  <EMI ID = 302.1>

  
n-dodecanoyl chloride to a solution of 5.5 g (40 mmol) of p-aminobenzoic acid dissolved in 100 ml of pyridine. The mixture is stirred for 3 hours and poured into 3 liters of water. The precipitate which forms is filtered and dried under vacuum and 11.01 g of N- (n-

  
  <EMI ID = 303.1>

  
Reference preparation 3

  
Preparation of 2,4,5-trichlorophenyl acid ester

  
  <EMI ID = 304.1>

  
mix at room temperature for 3.5 hours, then filter. The filtrate is evaporated under vacuum to obtain a residue which is crystallized from an acetonitrile / water mixture and 12.84 g of the 2,4,5- ester are obtained.

  
  <EMI ID = 305.1>

  
Reference Example 7

  
  <EMI ID = 306.1>

  
8.16 g (10.2 mmol) of A-30912A core and 4.72 g (10.2 mmol) of the 2,4,5-trichlorophenyl ester of N- (n-dodecanoyl acid) are dissolved. ) -p-aminobenzoic acid in 100 ml of DMF. The solution is stirred at room temperature for
15 hours. The solvent is removed in vacuo to obtain a residue which is washed twice with diethyl ether. Washing liquids are rejected. The washed residue is dissolved in 50 ml of methanol and purified by reverse phase CLPE using a "Prep LC / System 500" unit (Waters

  
  <EMI ID = 307.1>

  
(Waters Associates, Ine.) As a fixed phase. The column is eluted isocratically with a 25:65:10 mixture by volume of water, methanol and acetonitrile at 34.5 bars. The fractions are analyzed by TLC using silica gel plates and a 25:65:10 volume mixture of water, methanol and acetonitrile as the solvent system. We reunite

  
the fractions containing the desired product and they are lyophilized to obtain the N- (n-dodecanoyl) -p-aminobenzoyl derivative of the core of A-30912 &#65533; (3.5 g).

Reference Example 8

  
Acylation of A-30912B nucleus

  
Dissolve in 100 ml of dimethylformamide (10.2 mmol) of the core of A-30912B and 10.2 mmol of 2,4,5-trichlorophenyl ester of N- (n-dodecanoyl) -p-aminobenzolque acid. The solution is stirred at asbestos temperature for 15 hours. The solvent is removed in vacuo to obtain a residue which is washed twice with ether <EMI ID = 308.1> the residue washed in 50 ml of methanol and purified by reverse phase CLPE using a "Prep LC / System" unit
500 "unit (Waters Associates, Inc.), using a column

  
  <EMI ID = 309.1>

  
The column is eluted isocratically with a mixture
25:65:10 by volume of water, methanol and acetonitrile to
34.5 bars. The fractions are analyzed by TLC on silica gel plates and using a 25:65:10 by volume mixture of water, methanol and aceto- <EMI ID = 310.1>

  
  <EMI ID = 311.1>

  
dodecanoyl) -p-aminobenzoyl from the core of A-30912B.

  
Reference Example 9

  
Acylation of the nucleus of A-30912D

  
Dissolve in 100 ml of dimethylformamide (10.2 mmol) of the core of A-30912D and 10.2 mmol of 2,4,5-trichlorophenyl ester of N- (n-dodecanoyl) -p-aminobenzoic acid. The solution is stirred at room temperature for 15 hours. The solvent is removed in vacuo to obtain a residue which is washed twice with diethyl ether. Washing liquids are rejected. The washed residue is dissolved in 50 ml of methanol and purified by reverse phase CLPE using a "Prep LC / System" unit.
500 'unit (Waters Associates, Inc.), using a column

  
  <EMI ID = 312.1>

  
The column is eluted isocratically with a mixture

  
25:65:10 by volume of water, methanol and acetonitrile to
34.5 bars. The fractions are analyzed by TLC on silica gel plates and using as solvent system a

  
  <EMI ID = 313.1>

  
nitrile. The fractions containing the desired product are combined and lyophilized to obtain the N- (ndodecanoyl) -p-aminobenzyl derivative of the core of A-30912D.

Reference Example 10

  
Acylation of A-30912H nucleus

  
10.2 mmol of the core of A-30912H and 10.2 mmol of 2,4,5-trichloroester are dissolved in 100 ml of dimethylformamide

  
  <EMI ID = 314.1>

  
stir the solution at room temperature for 15 hours. The solvent is removed in vacuo to obtain a residue which is washed twice with diethyl ether. Washing liquids are rejected. The washed residue is dissolved in 50 ml of methanol and purified by reverse phase CLPE using a "Prep LC / System 500" unit (Waters Associates,

  
  <EMI ID = 315.1>

  
(Water Associates, Inc.), as a fixed phase. The column is eluted isocratically with a 25:65:10 mixture by volume of water, methanol and acetonitrile at 34.5 bars. The fractions are analyzed by TLC on silica gel plates and using a 25:65:10 mixture as the solvent system.

  
by volume of water, methanol and acetonitrile. The fractions containing the desired product are combined and lyophilized to obtain the N- (n-dodecanoyl) -p-aminobenzoyl derivative of the A-30912H nucleus.

  
Reference Example 11

  
  <EMI ID = 316.1>

  
  <EMI ID = 317.1>

  
according to the procedure of Reference Example 9, but using as starting material the compound of formula I where R <3> is an isobutoxy group (prepared by the method of Example 13).

Reference Example 12

  
Acylation of the nucleus of S 31794 / F-l

  
  <EMI ID = 318.1>

  
from the core of S 31794 / F-1 and 10.2 mmoles of 2,4,5-trichlorophenyl ester of N- (n-dodecanoyl) -p-aminobenzoic acid. The solution is stirred at room temperature for 15 hours. The solvent is removed in vacuo to obtain a residue which is washed twice with diethyl ether. Washing liquids are rejected. The washed residue is dissolved in 50 ml of methanol and purified by reverse phase CLPE using a "Prep LC / System" unit.
500 "unit (Waters Associates, Inc.) as a fixed phase. The column is eluted isocratically with a mixture of
25:65:10 by volume of water, methanol and acetonitrile to
34.5 bars. The fractions are analyzed by TLC on silica gel plates and using as a solvent system 25:65:10 by volume of water, methanol and acetonitrile.

   The fractions containing the desired product are combined and lyophilized to obtain the N- (n-dodecanoyl) p-aminobenzoyl derivative of the nucleus of S 31794 / F-1.

  
Preparation of a third group of derivatives

  
The following procedure which gives the preparation <EMI ID = 319.1>

  
illustrates the process for the preparation of a third group of compounds of formula III.

  
Reference preparation 4

  
  <EMI ID = 320.1>

  
A solution of 19.2 g (150 mmol) of p-hydroxybenzoic acid in 120 ml of 10% aqueous sodium hydroxide is added to 480 ml of dimethyl sulfoxide (DMSO) previously heated to 80 [deg.] C. It is added dropwise to the solution
28.95 g (150 mmol) of n-octyl bromide. The reaction mixture is stirred for 4 hours at room temperature, after which it is poured into 1200 ml of ice water. 30 ml of concentrated hydrochloric acid are added and the mixture is allowed to stand until precipitation is complete. The precipitate is collected, dried and crystallized from an acetonitrile / water mixture, m.p. 97-99 [deg.] C.

  

  <EMI ID = 321.1>


  
Reference preparation 5

  
Preparation of 2,4,5-trichlorophenyl acid ester

  
  <EMI ID = 322.1>

  
of N, N'-dicyclohexylcarbodiimide. The mixture is stirred at room temperature for 18 hours and then filtered. The filtrate is evaporated to obtain an oil, which is crystallized from a mixture of acetonitrile and water to obtain the ester.

  
  <EMI ID = 323.1>

  
7.23 (s, 1H), 7.3 (s, 1H), 8.08 (d, 1H, J = 4 Hz).

  
Reference Example 13

  
Acylation of the nucleus of A-30912A i

  
Dissolve in 150 ml of dimethylformamide 14.2 g
(17.8 mmol) of A-30912A core and 15.32 g (35.7 mmol)

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

  
benzoic. The solution is stirred at room temperature for 16-20 hours. The solvent is removed in vacuo and the residue is washed twice with diethyl ether and twice with methylene chloride. Washing products are rejected. The washed residue is dissolved in 80 ml of a 1: 2 mixture of ethyl acetate and methanol and it is purified by CLPE using a "Prep LC / System 500" unit using silica gel as fixed phase. . The column is eluted gradually with solvent systems of 1: 4 to 2: 3 of methanol and ethyl acetate. We analyze the

  
  <EMI ID = 324.1>

  
as a solvent system, a 3: 2 by volume mixture of ethyl acetate and methanol. We collect the exempt fractions

  
  <EMI ID = 325.1>

  
benzoylated p- (n-octyloxy) derivative of the core of A-30912A;

  
  <EMI ID = 326.1>

Reference Example 14

  
Acylation of A-30912B nucleus

  
17.8 mmol of A-30912B core and 35.7 mmol of 2,4,5-trichlorophenyl ester of p- (n-octyloxy) benzolic acid are dissolved in 150 ml of DMF. The solution is stirred at room temperature for 16-20 hours. The solvent is removed in vacuo and the residue is washed twice with diethyl ether and twice with methylene chloride. Washing liquids are rejected. The washed residue is dissolved in 80 ml of a 1: 3 mixture of ethyl acetate and methanol and it is purified by CLPE using a "Prep LC / System 500" unit and silica gel as fixed phase. The column is eluted in stages with solvent systems 1: 4 to 2: 3 of methanol and ethyl acetate. The fractions are analyzed by TLC using silica gel
(Merck) and a 3: 2 by volume mixture of ethyl acetate and methanol as the solvent system.

   The A-30912B nucleus-free fractions are pooled and lyophilized to obtain the benzoylated p- (n-octyloxy) derivative of the A-30912B nucleus.

  
Reference Example 15

  
Core acylation of A-30912D

  
17.8 mmol of A-30912D core and 35.7 mmol of the 2,4,5-trichlorophenyl ester of p- (n-octyloxy) benzolic acid are dissolved in 150 ml of dimethylformamide. We <EMI ID = 327.1>

  
hours. The solvent is removed in vacuo and the residue is washed twice with diethyl ether and twice with methylene chloride. Washing liquids are rejected.

  
The washed residue is dissolved in 80 ml of a 1: 3 mixture of ethyl acetate and methanol and it is purified by CLPE using a "Prep LC / System 500" unit and silica gel as fixed phase. The column is eluted gradually with solvent systems 1: 4 to 2: 3 of methanol and ethyl acetate. The fractions are analyzed by TLC using silica gel (Merck) and a 3: 2 by volume mixture of ethyl acetate and methanol as the solvent system. The nucleus-free fractions of A-30912D are combined and lyophilized to obtain the benzoylated p- (n-octyloxy) derivative of the nucleus of

  
A-30912D.

Reference Example 16

  
Acylation of A-30912H nucleus

  
17.8 mmol of A-30912H core and 35.7 mmol of the 2,4,5- ester are dissolved in 150 ml of dimethylformamide

  
  <EMI ID = 328.1>

  
stir the solution at room temperature for 16 - 20 hours. The solvent is removed in vacuo and the residue is washed twice with diethyl ether and twice with methylene chloride. Washing liquids are rejected.

  
The washed residue is dissolved in 80 ml of a 1: 3 mixture of ethyl acetate and methanol and it is purified by CLPE using a "Prep LC / System 500" unit and silica gel as fixed phase. The column is eluted gradually with solvent systems 1: 4 to 2: 3 of methanol and ethyl acetate. The fractions are analyzed by TLC using silica gel (Merck) and a 3: 2 by volume mixture of ethyl acetate and methanol as the solvent system. The A-30912H nucleus-free fractions are pooled and lyophilized to obtain the benzoylated p- (n-octyloxy) derivative of the A-30912H nucleus.

  
Reference example 17

  
The compound of formula III is prepared where R is a

  
  <EMI ID = 329.1> using the operating mode of Reference Example 16,

  
  <EMI ID = 330.1>

  
  <EMI ID = 331.1>

  
the method of Example 15).

Reference Example 18

  
Acylation of the nucleus of S 31794 / F-1

  
17.8 mmol of S 31794 / F-1 core and 35.7 mmol of the 2,4,5-trichlorophenyl ester of p- (n-octyloxy) benzolque acid are dissolved in 150 ml of dimethylformamide. The solution is stirred at room temperature for 16-20 hours. The solvent is removed in vacuo and the residue is washed twice with diethyl ether and twice with methylene chloride. Washing liquids are rejected. The washed residue is dissolved in 80 ml of a 1: 3 mixture of ethyl acetate and methanol and it is purified by CLPE using a "Prep LC / System 500" unit and silica gel as fixed phase. The column is eluted gradually with solvent systems 1: 4 to 2: 3 of methanol and ethyl acetate. The fractions are analyzed by TLC using silica gel (Merck) and a 3: 2 by volume mixture of ethyl acetate and methanol as the solvent system.

   The S 31794 / F-1 nucleus-free fractions are pooled and lyophilized to obtain the benzoylated p- (n-octyloxy) derivative of the S 31794 / F-1 nucleus.

Reference Example 19

  
The antifungal activity of the compounds of formula III can be demonstrated in vitro by disk diffusion tests and dilution tests on agar and in vivo by conventional tests on mice which show the efficacy vis-à-vis systemic fungal infection. The results of the antifungal tests of the standard compounds of formula III are given in Tables IV to VIII.

  
Tables IV and V give the results of the in vitro test of the compounds of Reference Examples 1, 7 and 13 by methods of diffusion of discs on agar plates. The activity in Table IV is measured by the dimension (diameter in mm) of the zone of inhibition of the microorganism observed that produced the test compound. In Table V, the activity is measured by the concentration <EMI ID = 332.1>

  
five strains of Candida albicans by the agar dilution process. In Table VI, the activity is measured by the ICD of the substance (mg / ml) necessary to inhibit the test organism.

  
The results of in vivo tests to determine the efficacy of the derivatives against an infection caused by Candida albicans A-26 in mice are given in Table VII. In these tests, the activity is measured by

  
  <EMI ID = 333.1>

  
50% of test animals). When DE $ 0 is not obtained, activity is indicated by the lowest dose at which a significant antifungal effect is observed. In these trials, groups of male albino mice (free of specific pathogens) weighing 18 to 20 grams are infected intravenously with Candida albicans A-26. Animals are treated with X-rays 24 hours before infection at approximately 50 roentgens per minute for minutes (dose

  
  <EMI ID = 334.1>

  
tion, each group of mice is given progressive doses subcutaneously of the test compound in the form of a suspension in 33% of polyethylene glycol (PEG)

  
in water. The day of death of each animal is noted. A statistical comparison by Student's t-test of the average day of death is carried out between each group of infected-treated animals at a particular dose and a group of 10 infected-untreated animals to determine whether the treatment prolongs significantly longer survival time.

  
Table VIII gives the results of the test of the compounds of Reference Examples 1, 7 and 13 to determine their absorption after oral administration. In these tests, mice were fed with a dose of 416 mg / kg of test compound suspended in a mixture of 33% of

  
PEG 400 and water. At time intervals, we take <EMI ID = 335.1>

  
their antibiotic activity as follows: a disc of 7 not containing 20 ml of whole blood is placed on agar

  
  <EMI ID = 336.1>

  
40 hours of incubation at 30 [deg.] C, the zones of inhibition of the blood samples are compared to a control obtained from the test compound, and the amount of the compound in the blood sample is calculated.

  

  <EMI ID = 337.1>


  

  <EMI ID = 338.1>


  

  <EMI ID = 339.1>
 

  

  <EMI ID = 340.1>


  

  <EMI ID = 341.1>


  

  <EMI ID = 342.1>
 

TABLE VI

  
In-vitro activity of the A-30912A nucleus derivative

  
  <EMI ID = 343.1>

  
from Candida albicans

  

  <EMI ID = 344.1>
 

  

  <EMI ID = 345.1>


  

  <EMI ID = 346.1>


  

  <EMI ID = 347.1>
 

  

  <EMI ID = 348.1>


  

  <EMI ID = 349.1>


  

  <EMI ID = 350.1>
 

CLAIMS

  
1. Cyclic peptide nucleus of formula:

  

  <EMI ID = 351.1>


  
wherein R is H or OH and;

  
  <EMI ID = 352.1>

  
or both OH,

  
and

  
when R is OH, R is H, R is OH or a group

  
  <EMI ID = 353.1>

  
0

  
  <EMI ID = 354.1>
-C-NH2 and R and R are both OH, and its acid addition salts.


    

Claims (1)

2. A-30912A core according to Claim 1, charac- <EMI ID = 355.1> 3. A-30912B core according to Claim 1, charac-  <EMI ID = 356.1> H and R <3> and R 4 are both OH. 4. A-30912D core according to Claim 1, charac-  <EMI ID = 357.1>  <EMI ID = 358.1> all H. 5. A-30912H core according to Claim 1, charac-  <EMI ID = 359.1> <EMI ID = 360.1> isopropoxy, n-butoxy, isobutoxy, t-butoxy, n-pentyloxy, n-hexyloxy, 2-ethyl-1-butoxy or 3-methyl-1-butoxy. 8. Core of S 31704 / F-1 according to Claim 1 ,. 13 characterized in that, in formula I, R, R and R are 0  <EMI ID = 361.1> both OH and R es &#65533; -C-NH2. 9. Addition salts of nucleic acids according to any one of Claims 2 to 8. 10. Process for the preparation of a peptide nucleus  <EMI ID = 362.1> characterized in that it consists in bringing into contact in an aqueous medium a cyclic peptide antibiotic of formula  <EMI ID = 363.1> II  <EMI ID = 364.1>  <EMI ID = 365.1> linoleoyl, and  <EMI ID = 366.1> <EMI ID = 367.1> is a stearoyl or linoleoyl group, and 0  <EMI ID = 368.1> myristoyl group, with a deacylation enzyme produced by a microorganism of the Actinoplanaceae family. 11. Method according to Claim 10 for preparing the core of A-30912A, characterized in that it consists in putting  <EMI ID = 369.1> is H and R is a linoleoyl, stearoyl or palmitoyl group, in contact with a deacylation enzyme produced by a microorganism of the family of Actinoplanaceae. 12. Method according to Claim 10 for preparing the core of A-30912B, characterized in that it consists in putting the antibiotic of formula II where R <1> and R <2> are both H and R <3> and R 4 are both OH and R is a stearoyl or linoleoyl group, in contact with a deacylation enzyme produced by a microorganism of the family of Actinoplanaceae. 13. Method according to Claim 10 for preparing the core of A-30912D, characterized in that it consists in putting  <EMI ID = 370.1> R is a stearoyl or linoleoyl group, in contact with a deacylation enzyme produced by a microorganism of the family of Actinoplanaceae. 14. The method of claim 10 for preparing the  <EMI ID = 371.1>  <EMI ID = 372.1> in contact with a deacylation enzyme produced by a microorganism of the Actinoplanaceae family. 15. The method of claim 10 for preparing a core type A-30912H, characterized in that it consists of <EMI ID = 373.1> is H, R is a C2-C6 alkyloxy group and R is a  <EMI ID = 374.1> deacylation enzyme produced by a microorganism of the Actinoplanaceae family. 16. A method according to claim 10 for preparing the core of S 31794 / F-1, characterized in that it consists in putting the antibiotic of formula II in which R, R and O 4 2 " R are OH, R is -C-NH2 and R is a myristoyl group, in contact with a deacylation enzyme produced by the microorganism of the family of Actinoplanaceae. 17. Method according to any one of the Claims 10 to 16, characterized in that the microorganism of the family of Actinoplanaceae is a member of the genus Actinoplanes. 18. Method according to any one of the Claims 10 to 17, characterized in that the microorganism is Actinoplanes utahensis. 19. Method according to Claim 18, characterized in that the microorganism is A. utahensis NRRL 12052 or one of its mutants producing the enzyme. 20. Method according to Claim 19, characterized in that the microorganism is A. utahensis NRRL 12052. 21. Method according to Claim 10, characterized in that the microorganism is Streptosporangium roseum var. hollandensis NRRL 12064, or one of its enzyme producing mutants. 22. Method according to Claim 21, characterized in that the microorganism is Streptosporangium roseum var. hollandensis NRRL 12064. 23. Method according to Claim 17, characterized in that the microorganism is Actinoplanes missouriensis NRRL 12053 or one of its enzyme producing mutants. 24. Method according to Claim 23, characterized in that the microorganism is Actinoplanes missouriensis NRRL 12053. 25. Method according to Claim 17, characterized in that the microorganism is Actinoplanes sp. NRRL 12065 or one of its enzyme producing mutants. 26. Method according to claim 25, characterized in that the microorganism is Actinoplanes sp. NRRL 12065. 27. Method according to Claim 17, characterized in that the microorganism is Actinoplanes sp. NRRL 8122 or one of its enzyme producing mutants. 28. Method according to Claim 27, characterized in that the microorganism is Actinoplanes sp. NRRL 8122. 29. Method according to any one of the Claims  <EMI ID = 375.1> in a culture of the producer Actinoplanaceae microorganism. 30. Method according to any one of the Claims 10 to 29, characterized in that it comprises the additional step of separation of the cyclic peptide nucleus or of one of its salts, from the fermentation medium. 31. Method according to any one of the Claims 10 to 30, characterized in that it comprises the additional step of purification of the cyclic peptide nucleus, or of a salt thereof. 27. Method according to Claim 17, characterized in  <EMI ID = 376.1> one of its enzyme producing mutants. 28. Method according to Claim 17, characterized in that the microorganism is Actinoplanes sp. NRRL 8122. 29. Method according to any one of the Claims 10 or 17 to 28, characterized in that the enzyme is present  <EMI ID = 377.1> producer. 30. Method according to any one of the Claims 10 to 29, characterized in that it comprises the additional step of separation of the cyclic peptide nucleus or of one of its salts, from the fermentation medium. 31. Method according to any one of the Claims 10 to 30, characterized in that it comprises the additional step of purification of the cyclic peptide nucleus, or of a salt thereof. 32. Cyclic peptide nucleus of formula I and its acid addition salts, in substance as described above with particular reference to Examples 1 to 19. 33. A-30912A nucleus of formula I where R <1>, R <3> and R4 are all OH and R2 is H, in substance as described above with particular reference to Examples 1 to 3.  <EMI ID = 378.1> both OH and R3 and R 4 are both OH, in substance as described here with particular reference to Examples 4 and 5.  <EMI ID = 379.1> are all H, in substance as described above with particular reference to Examples 6 and 7.  <EMI ID = 380.1>  <EMI ID = 381.1> described above with particular reference to Examples 8 to 10.  <EMI ID = 382.1>  <EMI ID = 383.1> in C2-C6, in substance as described above with reference  <EMI ID = 384.1> <EMI ID = 385.1> 0 " are all OH and R is -C-NH2, in substance as described above with particular reference to Example 19. 39. Process for the preparation of a peptide nucleus  <EMI ID = 386.1> substance as described above with particular reference to Examples 1 to 19. 40. Process for preparing the core of A-30912A from 134  <EMI ID = 387.1> substance as described above with particular reference to Examples 1 to 3. 41. Process for the preparation of the core of A-30912B from  <EMI ID = 388.1> two OH, in substance as described above with particular reference to Examples 4 and 5. 42. Process for the preparation of the core of A-30912B of formula I where R, R, R and R are H, in substance as described above with particular reference to Examples 6 and 7. 43. Process for preparing the core of A-30912H from  <EMI ID = 389.1> CH30-, in substance as described above with particular reference to Examples 8 to 10. 44. Process for the preparation of type A-30912H cores  <EMI ID = 390.1> described above with particular reference to Examples 11 to 18. 45. Process for preparing the core of S 31704 / F-1 from 0 '  <EMI ID = 391.1> substance as described above with particular reference to Example 19.