BE886576A - CYCLIC PEPTIDE CORE DERIVATIVES, THEIR PREPARATION AND THEIR USE AS ANTIFUNGAL AGENTS - Google Patents

Description

       

  The present invention relates to novel semi-synthetic antifungal compounds which are prepared by acylation.

  
of cyclic peptide nuclei obtained by enzymatic deacylation of the cyclic peptide antibiotic

  
corresponding.

  
The cyclic peptide antibiotic is an antifungal compound having the general formula:

  

  <EMI ID = 1.1>


  
  <EMI ID = 2.1>

  
Throughout this application, the formulas of the cyclic peptides,

  
like formula I, assume that the amino acids represented

  
are in L configuration.

  
Factors A, B, D and H of A-30912 are

  
  <EMI ID = 3.1>

  
  <EMI ID = 4.1>

  
  <EMI ID = 5.1>

  
R and R are both H and R and R are both OH.

  
  <EMI ID = 6.1>

  
  <EMI ID = 7.1>

  
The antibiotic S 31794 / F-1 is an antifungal cyclic peptide of formula 1 in which R is a group

  
  <EMI ID = 8.1>

  
Each factor is isolated from the A30912 complex which contains the other factors arbitrarily called factors

  
  <EMI ID = 9.1>

  
Individual Gs are described by M. Hoehn and K. Michel in

  
the U.S. patent N [deg.] 4,024,245. The factor A of antibiotic A-30912 is identical to antibiotic A-22802

  
  <EMI ID = 10.1>

  
Chim. Acta, 57, 2459 (1974) and Swiss patent N [deg.] 568,386] and

  
to the antibiotic SL 7810 / F [see C. Keller-Juslen et al.

  
  <EMI ID = 11.1>

  
The factor A of the antibiotic A-30912 is prepared by aerobic immersion fermentation using one of several different organisms, namely: (a) Aspergillus rugulosus NRRL 8113; (b) Aspergillus nidulans NRRL 8112;
(c) Aspergillus nidulans var. echinulatus A-32204, NRRL
3860; (d) Aspergillus rugulosus NRRL 8039; or (e) Aspergillus nidulans var. roseus NRRL 11440.

  
We also found that factor B is identical

  
antibiotic echinocandin C [see R. Traber et al., Helv.

  
  <EMI ID = 12.1>

  
[see Belgian patent N [deg.] 834,289].

  
The factor B of the antibiotic A-30912 is prepared by aerobic immersion fermentation using one of several different organisms, namely: (a) Aspergillus rugulosus NRRL 8113; (b) Aspergillus nidulans var. echinulatus A-32204, NRRL 3860; (c) Aspergillus rugulosus NRRL 8039; or
(d) Aspergillus niduians var. roseus NRRL 1440.

  
We also found that the factor D is identical

  
  <EMI ID = 13.1>

  
Helv. Chim. Acta, 62, 1252 (1979)] and the antibiotic SL
7810 / F-III [see Belgian patent N [deg.] 834,289].

  
We prepare factor D of antibiotic A-30912

  
  <EMI ID = 14.1> several different organisms, namely: (a) Aspergillus

  
  <EMI ID = 15.1>

  
echinulatus A-32204, NRRL 3860; (c) Aspergillus rugulosus NRRL 8039 (see Belgian patent N [deg.] 834,289); or (d) Aspergillus nidulans var. roseus NRRL 11440.

  
Factor H is a factor in the antibiotic A-30912

  
discovered later, and it is described in the application for

  
  <EMI ID = 16.1>

  
here by way of reference.

  
The factor H of A-30912 is prepared by fermentation using one of several different organisms, namely: (a) Aspergillus rugulosus NRRL 8113; or (b) Aspergillus nidulans var. roseus NRRL 11440.

  
A subculture of A. nidulans var. roseus has been deposited and is part of the collection of permanent crops of the Northern Regional Research Labotatorv,

  
U.S. Department of Agriculture, Agricultural Research Service,

  
  <EMI ID = 17.1>

  
under the number NRRL 11440.

  
When using a strain of A. nidulans var. roseus NRRL 11440 to produce any of the factors A-30912, we get a complex set of factors that

  
for convenience is called antibiotic complex A-42355. Factor A of A-30912 is the main factor in the antibiotic complex A-42355, while factors B, D and H are minor factors. The following preparations 2 to 7 illustrate the preparation of the A-42355 complex

  
  <EMI ID = 18.1>

  
A-30912.

  
  <EMI ID = 19.1>

  
  <EMI ID = 20.1>

  
cyclic on the α-amino grouping of the ornithine residue. It has surprisingly been found that the linoleoyl side chain can be cut from the nucleus by an enzyme without changing the chemical integrity of the nucleus. The enzyme used to carry out the deacylation reaction is produced by a microorganism

  
  <EMI ID = 21.1>

  
To deacylate, add the appropriate factor of antibiotic A-30912 to a culture of the microorganism

  
and the culture is allowed to incubate with the substrate until

  
  <EMI ID = 22.1>

  
cyclic thus obtained is separated from the fermentation broth by methods known in the art. Unlike factors A, B, D and H of antibiotic A-30912, the ring nucleus (lacking the linoleoyl side chain) is essentially free of antifungal activity.

  
The antibiotic S31794 / F-1, which is described in the German patent application published after examination N [deg.] 2,628,965

  
and in the U.S. patent N "4.173.629, is produced by Acrophialophora limonispora nov. Spec. Dreyfuss and Muller

  
  <EMI ID = 23.1>

  
  <EMI ID = 24.1>

  
CH3OH) or + 37 [deg.] (C 0.5, pvridine) (crystallized); maxima

  
  <EMI ID = 25.1>

  
  <EMI ID = 26.1>

  
carbon C in deuterom &#65533; thanol (190 mg in 1.5 ml of

  
  <EMI ID = 27.1>

  
internal standard) having the following characteristics (in the crystallized state):

  

  <EMI ID = 28.1>
 

  
an approximate elementary analysis (after drying the crystallized material for two hours under high vacuum at
100 [deg.] C) as follows: 55.5-56.5% carbon, 7.5-7.7%

  
  <EMI ID = 29.1>

  
is readily soluble in methanol, ethanol, pyridine, dimethyl sulfoxide and sparingly soluble in water, chloroform, ethyl acetate, diethyl ether, benzene and hexane; and has antifungal activity, especially against Candida albicans.

  
The antibiotic S31794 / F-1 is prepared by aerobic culture in immersion of Acrophialophora limonispora NRRL
8095 as described in preparations 8 and 9. This microorganism is part of the collection of permanent cultures 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 access number.

  
The antibiotic S31794 / F-1 has an antifungal activity, in particular against Candida strains such as Candida albicans. Thus, the production and isolation of the antibiotic can be controlled by bioautograph using a Candida species such as Candida albicans.

  
In the antibiotic molecule S31794 / F-1

  
  <EMI ID = 30.1>

  
hydroxy and R is a group -CO-NH-f the myristoyl side chain (R) is attached to the cyclic peptide nucleus on the a-amino group of the dihydroxyornithine residue. We found

  
  <EMI ID = 31.1>

  
be cut from the nucleus by an enzyme without harming the chemical integrity of the nucleus. The enzyme used to carry out the deacylation reaction is produced by a microorganism of the family Actinoplanaceae, preferably the microorganism Actinoplanes utahensis NRRL 12052 or one of its variants. To carry out the deacylation, the antibiotic S31794 / F-1 is added to a culture of the microorganism and the culture is left to incubate with the substrate until

  
  <EMI ID = 32.1> cyclic thus obtained is separated from the fermentation broth by methods known in the art. In contrast to the antibiotic S31794 / F-1, the cyclic nucleus (not having the myristoyl side chain) is essentially free of antifungal activity.

  
The cyclic peptide nuclei provided by the above-mentioned enzymatic deacylation reactions of the antibiotics of formula I are represented by the general formula II.

  

  <EMI ID = 33.1>

J

  
The compound of formula II where R, R and R are all hydroxy groups and R <2> is H is the nucleus of factor A of A-3C912 and, for convenience, will be referred to herein as "nucleus of A-30912A". The core of A-30912A has a formula

  
  <EMI ID = 34.1>

  
The compound of formula II where R and R are both

  
  <EMI ID = 35.1>

  
of A-30912 and, for convenience, will be referred to herein as "core of A-30912B". The core of A-30912B has a formula

  
  <EMI ID = 36.1>

  
  <EMI ID = 37.1>

  
The compound of formula II where R, R, R and R are all hydrogen atoms is the nucleus of factor D of A-30912 and will be called here for convenience "nucleus of A-30912D". The core of A-30912D has a formula

  
  <EMI ID = 38.1>

  
The compound of formula II where R and R 'are both OH, R2 is H and R <3> is CH30- is the nucleus of factor H of A-30912 and, for convenience, will be referred to herein as "nucleus of A-30912H".

  
The compound of formula II in which R, R and

  
  <EMI ID = 39.1>

  
a molecular weight of 840.87.

  
The removal of the side chain provides a

  
  <EMI ID = 40.1>

  
cyclic peptide. As will be appreciated by those skilled in the art, the nuclei can be obtained in the form of the free amine or in the form of an acid addition salt. Although any suitable acid addition salt can be used, those which are non-toxic and pharmaceutically acceptable are preferred.

  
The process for the preparation of each nucleus from the appropriate antibiotic using a fermentation using Actinoplanes utahensis NRRL 12052 is described in the patent application filed on the same day as the present

  
  <EMI ID = 41.1>

  
Cultures of representative species of Actinopnanaceae are made available to the public at the Northern Regional Research Laboratory under the following access numbers:

  

  <EMI ID = 42.1>
 

  
  <EMI ID = 43.1>

  
for carrying out the deacylation of this invention is determined by the following procedure. An appropriate culture medium is inoculated with the microorganism. We

  
  <EMI ID = 44.1>

  
days on a rotary shaker. Then one of the antibiotics used as a substrate is added to the culture. We maintain

  
the pH of the fermentation medium at about 6.5. The culture is checked for its activity using an analysis by Candida albicans. The loss of antibiotic activity is an indication that the microorganism has produced the enzyme necessary for deacylation. This should however be checked using one of the following methods: 1) analysis by high pressure liquid chromatography to determine the presence of the intact nucleus; or 2) reacylation with an appropriate side chain (eg linoleoyl, stearoyl, palmitoyl or myristoyl) to restore activity.

  
It is known that other antibiotic substances have the same nucleus as that of factor A of antibiotic A-30912. These antibiotics differ from factor A of antibiotic A-30912 in that different acyl groups are present in place of the linoleoyl group.
(R) of formula I. Such antibiotics are: (a)

  
  <EMI ID = 45.1>

  
tetrahydro êchinocandin B) described in the Belgian patent

  
  <EMI ID = 46.1>

  
  <EMI ID = 47.1>

  
is a stearoyl group; and (b) aculaecin A, which is a component of the aculaecin complex (prepared by

  
fermentation using Aspergillus aculeatus NRRL 8075) and is described in the U.S. Patent. N [deg.] 3,978,210. As described in Belgian patent N [deg.] 859,067, in aculaecin A, the palmitoyl side chain is present in place of the linoleoyl group. Factor A of tetrahydrogenated A-30912 can be prepared from factor A of A-30912 by

  
  <EMI ID = 48.1>

  
under positive pressure. The A-factor of A-30912 7

  
  <EMI ID = 49.1>

  
used as substrates for enzymatic deacylation using the procedures described here.

  
It is also known that another antibiotic substance has the same nucleus as factor B of the antibiotic A-30912. This substance, which differs from factor B of antibiotic A-30912 in that a different acyl group is present in place of the group

  
  <EMI ID = 50.1>

  
tetrahydrogenated (tetrahydro-SL 7810 / F-II; tetrahydro ëchinocandine C) described by R. Traber et al., Helv.

  
  <EMI ID = 51.1>

  
can be prepared from factor B of A-30912 by

  
  <EMI ID = 52.1>

  
under positive pressure. Factor B of tetrahydrogenated A-30912

  
  <EMI ID = 53.1>

  
Antibiotic A-30912 for enzymatic deacylation using the procedures described here.

  
It is also known that another antibiotic substance has the same nucleus as factor D of A-30912. This substance, which differs from factor D of the antibiotic

  
A-30912 in that a different acyl group is present in place of the linoleoyl group (R) of formula I, is factor D of A-30912 tetrahydrogenated (tetrahydro-SL
7810 / F-III; tetrahydro-ëchinocandine D) described by R.

  
  <EMI ID = 54.1>

  
of tetrahydrogenated A-30912 is described by formula I when R is a stearoyl group. Factor D of tetrahydrogenated A-30912 can be prepared from factor D of antibiotic A-30912 by catalytic hydrogenation to

  
  <EMI ID = 55.1>

  
tetrahydrogenated A-30912 factor D can be used as a substrate in place of the antibiotic A-30912 factor D for enzymatic deacylation using the procedures described herein.

  
  <EMI ID = 56.1> 5-hydroxy group present in the dihydroxyornithine residue of the peptide ring is methylated, while in factor A of antibiotic A-30912, the 5hydroxy group is not substituted. It will therefore be seen that factor H can be synthetically prepared by methylating factor A according to conventional methods for preparing an aliphatic ether. from an alcohol. It will also be seen that factor A can be alkylated with other lower alkyl groups to form homologs with alkyloxy groups of the factor H molecule. Homologues with alkyloxy groups of factor H which can be prepared by

  
  <EMI ID = 57.1>

  
of factor H type homologs of A-30912. Compounds of

  
  <EMI ID = 58.1>

  
formula II where R and R are both OH, R is H and R is a C2-C6 alkyloxy group are here called as "nuclei of type A-30912H".

  
We will also see that the linoleoyl side chain

  
  <EMI ID = 59.1>

  
alkyloxy groups (R is a stearoyl group). It is also possible to prepare the tetrahydrogenated derivatives by first hydrogenating the factor A of the antibiotic A-30912 to obtain the factor A of tetrahydrogenated A-30912 and then forming the desired alkyloxy derivative.

  
It is understood that the factor H of the antibiotic A-30912, the factor H of tetrahydrogen A-30912, a homolog with C2-C6 alkyloxy groups of factor H or a tetrahydrogen derivative of a homolog of factaur H &#65533; C2-C6 alkyloxy group can be used as substrate for enzymatic deacylation using

  
the procedures described here.

  
The invention which is the subject of the present patent application describes new compounds obtained by acylation of cyclic peptide nuclei of formula II. The compounds of the present invention have the chemical formula described by formula III:

  

  <EMI ID = 60.1>


  
in which R is H or OH and:

  
  <EMI ID = 61.1>

  
H or both OH,

  
and

  
  <EMI ID = 62.1>

  
  <EMI ID = 63.1>

  
  <EMI ID = 64.1>

  
0

  
  <EMI ID = 65.1>

  
W is a divalent aminoacyl radical of formula:

  
0
(a) -C-A-NH- <EMI ID = 66.1> <EMI ID = 67.1>

  
0 R7
(b) -C-CH-NH- <EMI ID = 68.1>

  
  <EMI ID = 69.1>

  
mercaptoethyl, methylthioethyl, 2-thienyl, 3-indole-methyl, phenyl, benzyl or substituted phenyl or substituted benzyl in which the benzene ring is substituted by a chloro radical,

  
  <EMI ID = 70.1>

  
C1-C3 alkylthio, carbamyl or alkyl-

  
  <EMI ID = 71.1>

  

  <EMI ID = 72.1>


  
in which X is a hydrogen, chlorine, bromine or iodine atom or a group

  
  <EMI ID = 73.1>

  
  <EMI ID = 74.1>

  
  <EMI ID = 75.1>

  

  <EMI ID = 76.1>


  
  <EMI ID = 77.1>

  
bromine or iodine;

  

  <EMI ID = 78.1>
 

  

  <EMI ID = 79.1>


  
where B is a divalent radical of formula <EMI ID = 80.1>
-CH = CH-CH2-; or 0

-CNHCH -

  
  <EMI ID = 81.1>

THIS?

  
As used herein, the terms alkylene, alkyl, alkoxy, alkylthio and alkenyl refer to branched and straight hydrocarbon chains. The term alkyl denotes a monovalent saturated hydrocarbon radical. The term alkenyl denotes a monovalent unsaturated hydrocarbon radical containing one, two or three double bonds, which may be in

  
  <EMI ID = 82.1>

  
divalent saturated hydrocarbon. Cycloalkylene denotes a divalent cyclic saturated hydrocarbon radical.

  
  <EMI ID = 83.1>

  
preferred for the purposes of this invention are:

  

  <EMI ID = 84.1>


  
  <EMI ID = 85.1>

  
C4 (i.e. methyl, ethyl, n-propyl, iso-propyl, n-

  
  <EMI ID = 86.1>

  
p is an integer from 1 to 8 and q is an integer from 0 to 7, provided that p + q is not greater than 8.

  
  <EMI ID = 87.1>

  
preferred for the purposes of the present invention are:
(a) CH3-
(b) - (CH2) nCH3 where n is an integer from 1 to 16; and

  
CH3 <EMI ID = 88.1> an integer from 0 to 14 provided that r + s is not greater than 14. <EMI ID = 89.1> independently an integer from 0 to 14 provided that t + u is not greater than 14. <EMI ID = 90.1> where v and z are independently an integer from 0 to 11 and y is an integer from 1 to 12 provided that v + y + z is not greater than 11.

  
The following examples of C1-C1 alkyl groups are particularly preferred. :

  

  <EMI ID = 91.1>


  
The following examples are particularly preferred:

  
  <EMI ID = 92.1>

  

  <EMI ID = 93.1>


  

  <EMI ID = 94.1>
 

  
0

  
he

  
those skilled in the art will see that the function -C- and the function

  
  <EMI ID = 95.1>

  
ortho, meta or para one with respect to the other. The substituent represented by X can be in any available position on the benzene ring. Preferred compounds are those in which X is a hydrogen atom

  
0

  
laughed

  
and the functions -C- and -NH- are placed in para configuration.

  
The expressions "substituted phenyl" and "benzylc

  
'7

  
substituted "as used for R 'in formula III, denote the substitution of a group on any of the available positions of the benzene ring, that is to say that the substituent can be in the ortho position,

  
  <EMI ID = 96.1>

  
defined by R ′ or X in formula III comprises the methyl, ethyl, n-propyl and iso-propyl groups.

  
The invention provides in particular a compound of formula:

  

  <EMI ID = 97.1>


  
where R is H or OH and 16

  
  <EMI ID = 98.1>

  
  <EMI ID = 99.1>

  
and

  
  <EMI ID = 100.1>

  
C 1-6 alkyloxy group, and R is OH, or R is a -CO-NH2 group and R and R are both OH;

  
  <EMI ID = 101.1>

  
0

  
formula -W-C-R where:

  
W is a divalent aminoacyl radical of formula:

  
0

  
  <EMI ID = 102.1>

  
CS-C6 cycloalkylene;

  
  <EMI ID = 103.1>
(b) -C-CH-NH- <EMI ID = 104.1>

  
hydroxyethyl, mercaptomethyl, mercaptoethyl, methylthioethyl, 2-thicnyl, 3-indole-methyl, phenyl, benzyl or substituted phenyl or

  
  <EMI ID = 105.1>

  
substituted by a chloro, bromo, iodo radical,

  
  <EMI ID = 106.1>

  

  <EMI ID = 107.1>


  
where X is a hydrogen, chloro, bromo radical,

  
  <EMI ID = 108.1>

  

  <EMI ID = 109.1>
 

  
where X is a chlorine, bromine or iodine atom;

  

  <EMI ID = 110.1>


  
where B is a divalent radical of formula.

  
  <EMI ID = 111.1>
-CH = CH-CH2; or <EMI ID = 112.1>

THIS?

  
Compounds of formula III inhibit growth

  
pathogenic fungi as shown in conventional bioassay procedures. The compounds can therefore be used to combat the growth of fungi on environmental surfaces (as antiseptics) or for the treatment of infections caused by fungi. The antifungal activity of the compounds has been demonstrated against Candida albicans in vitro in diffusion tests on agar discs and plates and in

  
  <EMI ID = 113.1>

  
in mice infected with C. albicans. The compounds are therefore particularly useful for treating infections caused by strains of C. albicans (candidiasis). The compounds of formula III also exhibit activity

  
in vitro in diffusion tests on discs and agar plates against Trichophyton mentagrophytes, a dermatophyte organism. Activation has also been found in diffusion assays on discs and agar plates in vitro against Saccharomyces pastorianus and Neurospora <EMI ID = 114.1>

  
Table 8), give significant blood levels after oral administration in mice.

  
When administered to a dog intravenously at 100 mg / kg per day for five

  
13 days, the compound of formula III in which R, R and R are all OH, R2 is H and R 5 is an n-dodecanoylep-aminobenzoyl group, shows no external signs of toxicity although levels are observed increased serum glutamo-pyruvic transaminase.

  
The compounds of formula III are prepared by acylating the appropriate nucleus on the α-amino group d: ornithine with the side chain N-alkanoyl-aminoacyl &#65533; or N-alkenoylamine-acyl suitable using standard methods 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 (formula IV) corresponding to the desired acyl side chain.

  

  <EMI ID = 115.1>


  
  <EMI ID = 116.1>

  
The expression "activated derivative" denotes a derivative which

  
  <EMI ID = 117.1>

  
coupling with the primary amino group to form the amide bond which links the acyl side chain to the nucleus. Appropriate activated derivatives, their preparation methods 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 (e.g. acid chloride), (b) an acid anhydride (e.g. an alkoxyformic acid anhydride or an aryloxyformic acid anhydride) or ( c) an activated ester (for example a

  
  <EMI ID = 118.1>

  
benzotri.azole or an ester of -hydroxysuccinimide). Other methods of activating the carboxy function include 19

  
  <EMI ID = 119.1>

  
  <EMI ID = 120.1>

  
diisopropylcarbodiimide) to obtain a reactive intermediate which, due to its instability, is not isolated, the reaction with the primary amine being carried out in situ.

  
A preferred method of preparing the compounds of formula III is the method using the active ester.

  
The use of the 2,4,5-trichlorophenyl ester of N-

  
  <EMI ID = 121.1>

  
formula IV as an acylating agent is much preferred.

  
In this process, an excess of the active ester is reacted with the nucleus at room temperature in a solvent.

  
  <EMI ID = 122.1>

  
Duration of the reaction is not critical, although it is preferred to last from about 15 to about 18 hours. At the end of the reaction, the solvent is removed and the residue is purified, as by column chromatography using gel.

  
  <EMI ID = 123.1>

  
ethyl and methanol as solvent system-

  
2,4,5- esters can be conveniently prepared

  
  <EMI ID = 124.1>

  
alkenoylamino acids, by treating the desired amino acid
(formula IV) with 2,4,5-trichlorophenol in the presence of a coupling agent, such as N, N'-dicyclohexylcarbodiimide. Other suitable methods for preparing amino acid esters will be apparent to those of skill in the art.

  
  <EMI ID = 125.1>

  
acids are known compounds or they can be prepared by acylation of the appropriate amino acid with the appropriate alkanoyl or alkenoyl group using conventional methods, such as those described above. A preferred way of preparing the N-alkanoylamino acids is

  
  <EMI ID = 126.1>

  
used in the preparation of the products of the invention

  
are known compounds or they can be prepared

  
by known methods or by modification of known methods, which will be obvious to those skilled in the art.

  
If a particular amino acid contains an acylable functional group other than the group

  
  <EMI ID = 127.1>

  
protected before the reaction of the amino acid with the reagent used to fix the alkanoyl or alkenoyl group. Suitable protecting groups can be any group known in the field as usable for the protection of a functional group of a side chain 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 known to those skilled in the art [see for example "Protective Groups In Organic

  
Chemistry ", M. McOmie, Edit, Plenum Press, N.Y., 1973].

  
It will be seen that certain amino acids used in the synthesis of the products of this invention can exist in optically active forms, and it is possible to use both the natural configuration (L configuration) and the other.

  
  <EMI ID = 128.1>

  
and there will be obtained products which are part of the present invention.

  
When used generally, the dose of the 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 will be started at low doses, the dose being increased until the desired antifungal effect is obtained. The compounds can be administered intravenously or intramuscularly in the form of a sterile aqueous solution or suspension to which can be added, if desired, various conventional preservatives, buffers, solubilizers or suspensions acceptable on the pharmaceutical plan. Other additives, such as salt 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 administration with chalk (see

  
Example 40, Table 8) and can be administered systemically orally. For oral use,

  
  <EMI ID = 129.1>

  
pharmaceutically acceptable carriers or excipients in the form of capsules, tablets or powders. The nature and proportion of these supports or excipients are well known to those skilled in the art.

  
When used to treat vaginal infections caused by Candida, the compounds of formula III can be administered in combination with conventional pharmaceutically acceptable excipients suitable for intravaginal use. The compositions designed for intravaginal administration are well known to those skilled in the art.

  
The preparation and use of the compounds of the present invention are illustrated in the following examples:

  
Preparation 1

  
Fermentation of Actinoplanes utahensis NRRL 12052

  
  <EMI ID = 130.1>

  
inclined agar. The medium used to prepare the tilted culture is chosen from one of the following:

  
Middle A

  

  <EMI ID = 131.1>


  

  <EMI ID = 132.1>


  
the pH before autoclaving is approximately 5.9; the pH is adjusted to 7.2 by addition of NaOH; after autoclaving, the pH is approximately 6.7.

  
  <EMI ID = 133.1>

  
Ingredient Quantity

  

  <EMI ID = 134.1>


  

  <EMI ID = 135.1>


  
Middle B
  <EMI ID = 136.1>
  <EMI ID = 137.1>
 Czapek mineral solution 2.0 ml Agar 20.0 g

  
Deionized water q.s.p. 1 liter N-Z-Amine A, Humko Sheffield Chemical, Lyndhurst, New

  
Jersey, U.S.A.

  
Inoculated culture is inoculated with Actinoplanes

  
utahensis NRRL 12052, and the inoculated culture is incubated at 30 [deg.] C for about 8-10 days. About half of the culture is used to inoculate 50 ml of a vegetative medium having the following composition:

  

  <EMI ID = 138.1>


  

  <EMI ID = 139.1>


  
adjust to pH 7.4 with NctOII; after autoclaving, the

  
  <EMI ID = 140.1>

  
New York, U.S.A.

  
The inoculated vegetative medium is incubated in a 250 ml Erlenmeyer flask with a wide neck at 30 [deg.] C

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

  
This directly incubated vegetative medium can be used to inoculate a second stage vegetative medium. Or, and preferably, it can be stored for later use by keeping the culture in the 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. Dailey and C.E. Higgens, "Preservation and Storage of Microorganisms in the Gas Phase of Liquid Nitrogen, Cryobiol 10,
364-367 (1973) for details]. The suspensions thus prepared are stored in the vapor phase of liquid nitrogen.

  
A conserved suspension (1 ml) thus prepared is used to inoculate 50 ml of a first stage vegetative medium (having the composition described above). The inoculated first stage vegetative medium is incubated as described above.

  
To obtain a larger volume of inoculum, 10 ml of the incubated first stage vegetative medium are used to inoculate 400 ml of a second stage vegetative medium having the same composition as the first stage vegetative medium. The second stage medium is incubated in a

  
  <EMI ID = 141.1>

  
approximately 48 hours on an agitator rotating with an arc of 5 cm in diameter at 250 revolutions per minute.

  
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:

  
Middle 1

  

  <EMI ID = 142.1>


  

  <EMI ID = 143.1>


  
Tap water q.s.p. 1 litre

  
The pH of the medium is approximately 6.9 after sterilization by autoclaving at 121 [deg.] C for 45 minutes under a pressure of approximately 1.1 to 1.24 bar.

  
Middle II

  

  <EMI ID = 144.1>


  

  <EMI ID = 145.1>
 

  
  <EMI ID = 146.1>

  
the pH is around 7.0.

  

  <EMI ID = 147.1>


  
  <EMI ID = 148.1>

  
The final pH is around 6.6.

  
The inoculated production medium is left to ferment in a 165 l fermentation tank at a temperature of about 30 [deg.] C for about 42 hours. The fermentation medium is stirred with conventional stirrers at about 200 revolutions per minute and is aerated with sterile air to

  
  <EMI ID = 149.1>

  
air saturation at atmospheric pressure.

  
Preparation 2

  
Preparation of the antibiotic mixture A-42355

  
A. Fermentation in shaken flask

  
A culture of Aspergillus nidulans var. roseus NRRL 11440 and it is maintained on an inclined agar of 18 x 150 ml, prepared with a medium having the following composition:

  

  <EMI ID = 150.1>
 

  
Deionized water q.s.p. 1 litre
(initial pH 6.1)

  
  <EMI ID = 151.1>

  
  <EMI ID = 152.1>

  
The tilted culture is inoculated with Aspergillus nidulans var. roseus NRRL 11440 then the culture is incubated at 25 [deg.] C for about seven days. Cover the ripe slanted crop with water and scrape it off with a handle

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

  
1 ml of the tilted suspension culture is used

  
  <EMI ID = 153.1>

  
250 ml. The vegetative medium has the following composition:

  
Ingredient Quantity

  

  <EMI ID = 154.1>


  
(initial pH 6.5-6.7)

  
  <EMI ID = 155.1>

  
The inoculated vegetative medium is incubated at 25 [deg.] C for 48 hours, at 250 revolutions per minute, on an agitator.

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

  
(Waring type), then incubate it again for the remaining 24 hours. Alternatively, the inoculated vegetative medium can be incubated for 48 hours and then homogenized

  
  <EMI ID = 156.1>

  
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, keeping the culture in the vapor phase of liquid nitrogen. The culture is prepared for this preservation in numerous small ampoules, as follows.

  
The volume / volume vegetative cultures are mixed with a suspension solution having the following composition:

  

  <EMI ID = 157.1>


  
The prepared suspensions are distributed in small sterile tubes with screw caps (4 ml per tube). These tubes are kept in the vapor phase of liquid nitrogen.

  
A preserved suspension thus prepared can be used to inoculate tilted cultures or liquid seed media. The tilted cultures are incubated at 25 [deg.] C in the light for 7 days.

  
B. Fermentation in tanks

  
To obtain a larger volume of inoculum, 10 ml of the first stage vegetative culture are used.

  
  <EMI ID = 158.1>

  
second stage vegetative having the same composition as that of the vegetative medium. The second stage medium is incubated

  
  <EMI ID = 159.1>

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

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

  
  <EMI ID = 160.1>
  <EMI ID = 161.1>
 
  <EMI ID = 162.1>
(initial pH 6.8-7.0)

  
  <EMI ID = 163.1>

  
  <EMI ID = 164.1>

  
:::::; N-Z-Amine A, Humko Sheffield Chemical, Lyndhurst, N.J.

USA.

  
:::::::: P2000, Dow Corning, Midland, Michigan, U.S.A.

  
Middle V

  

  <EMI ID = 165.1>


  
The inoculated production medium is left to ferment in a

  
  <EMI ID = 166.1>

  
for 7 days. The fermentation medium is aerated with

  
  <EMI ID = 167.1> greater than around 50% of air saturation.

  
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 larger tank. A preferred third stage vegetative medium has the following composition:

  

  <EMI ID = 168.1>
 

  

  <EMI ID = 169.1>


  
Preparation 3

  
Separation of the antibiotic mixture A-42355

  
The whole fermentation broth (4127 liters), obtained by the process described in Example 1, is carefully stirred using production medium II, with
4,280 liters of methanol for one hour, then filtered, using a filter aid (Hyflo Supercel, diatomaceous earth, Johns-Manville Products Corp.). The pH of the filtrate is adjusted to 4.0 by adding 5N HCl.

  
  <EMI ID = 170.1>

  
concentrated in vacuo to a volume of 20 liters. This concentrate is added to 200 liters of diethyl ether to precipitate the mixture A-42355. The precipitate is separated by filtration and 2775 g of mixture A-42355 are obtained in the form of a gray-white powder.

  
Separation and identification of factors of A-30912

  
Preparation 4

  
Separation of factor A from A-30912

  
  <EMI ID = 171.1>

  
prepared as described in Preparations 2 and 3, in 7 ml of a 7: 2: 1 mixture of methanol, water and acetonitrile. This solution is filtered and introduced into a glass column with an internal diameter of 3.7 cm and a length of 35 cm.

  
  <EMI ID = 172.1>

  
microns), which was prepared as described in Preparation 10, through a loop using a system

  
with valves. The column is packed in the 7: 2: 1 system with methanol, water and acetonitrile, using the suspension packing procedure described in Preparation 11.

  
An F.M.I. with piston design without valves
(maximum flow rate 19.5 ml / minute) is used to move the solvent in the column at a flow rate of 9 ml / minute at 7 bars, fractions being collected every minute. The elution of the antibiotic is controlled at 280 nm using a UV control device (ISCO Model UA-5, Instrument Specialist

  
Co., 4700 Superior Ave., Lincoln, Nebraska 68504) equipped with an optical unit (ISCO Type 6).

Preparation 5

  
  <EMI ID = 173.1>

  
The mixture A-42355 is separated as described in Preparation 3, but the concentrated chloroform extracts (285 l) are chromatographed on a column of silica gel (150 l of Grace silica gel, quality 62) # a flow rate of 2 1 / minute. The column is washed with 200 liters of

  
  <EMI ID = 174.1>

  
it is continuously eluted with a 98: 2 mixture of acetonitrile and water at a rate of 1 l / minute. We collect fractions

  
  <EMI ID = 175.1>

  
to determine their biological activity. The biological analysis is carried out by an analysis on paper disc on agar plate, seeded with Candida albicans. We join the fractions 77 &#65533; 103 (1365 liters) and we

  
concentrated in vacuo. The concentrated solution (4.5 liters) contains a precipitate which is removed by filtration and 119 g of mixture A-42355 enriched in factor B are obtained. The filtrate is concentrated to dryness. The residue obtained is redissolved in an appropriate volume of methanol. The methanolic solution is added to 10 volumes of diethyl ether to precipitate the antibiotic complex containing factor B. This precipitate is also separated by filtration and dried, and an additional 24 g of mixture A-42355 enriched with are obtained. factor B in the form of a gray powder.

  
The mixture A-42355 enriched in factor B thus obtained <EMI ID = 176.1>

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

  
  <EMI ID = 177.1>

  
Preparation 10, using the 7: 2: 1 mixture of methanol, water, and acetonitrile, as described in Preparation 11.

  
The solvent moves in the column at a flow rate of 10 ml / minute at approximately 7 bars, using an FMI piston pump without valves. A fraction is collected every minute. The elution of the antibiotic is monitored at 280 nm as in Preparation 15. The fractions 102-110 are combined and they are combined.

  
concentrated in vacuo to obtain an oil. The oil is dissolved in a small volume of t-butanol and lyophilized, giving 22 mg of factor B of A-30912.

  
Preparation 6

  
Isolation of factor D from A-30912

  
The concentrated chloroform extracts from two fermentation tests (3800 1 and 4007 1) obtained by the process described in Preparation 3 are combined and chromatographed on a column of silica gel (Grace, quality 62). The column is washed with chloroform and then eluted with acetonitrile and a 98: 2 mixture of acetonitrile and water. Fractions having a volume of about 200 l are collected and subjected to biological analysis on paper disc on agar

  
  <EMI ID = 178.1>

  
active (850 l) and concentrated in vacuo. The concentrated solution (0.7 l) is added to 10 volumes of diethyl ether to precipitate the factor-enriched mixture A-42355

  
D. This precipitate is removed by filtration and dried, which gives 32 g of mixture A-42355 enriched in factor D in the form of a gray powder.

  
The A-42355 mixture enriched in factor D thus obtained (1.0 g) is dissolved in 5 ml of a 7: 2: 1 mixture of methanol, water and acetonitrile. However, this solution is filtered and it is introduced onto a column of silica gel (Michel-Miller column, 3.7 cm internal diameter x 30 cm) via a loop using a valves.

  
The column was packed with reverse phase silica gel resin LP-1 / C (10-20 microns) prepared as described in Preparation 10. The packing was carried out in a mixture

  
  <EMI ID = 179.1>

  
packing operation of Preparation 11. The solvent moves in the column at a flow rate of 8 ml / minute at approximately 3.1 bars, using an FMI pump with piston design without valves. A fraction is collected every 2 minutes.

  
  <EMI ID = 180.1>

  
described in Preparation 4. The 96-108 fractions are combined and concentrated in vacuo 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--30912.

  
Preparation 7

  
Separation of factor H from A-30912

  
5.0 g of the antibiotic mixture A-42355, prepared as described in Preparations 2 and 3, are dissolved in 35 ml

  
  <EMI ID = 181.1>

  
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 = 182.1>

  
  <EMI ID = 183.1>

  
of water and acetonitrile as described in Preparation 11. The solvent moves through the column at a Cite crime 13 ml / minute at about 8.3 bar, using an FMI pump with piston design without valves, and l 'a fraction is collected every two minutes. The elution of the antibiotic is monitored at 280 nm as described in Preparation 19, paragraph C. Fractions 112-132 are combined with fractions 106-117 of a second similar purification. The combined fractions are concentrated in vacuo to an oil. The oil is dissolved in a small volume of t-butanol

  
  <EMI ID = 184.1>

  
A-30912.

  
  <EMI ID = 185.1>

  
in 8 ml of a 7: 2: 1 mixture of methanol, water and acetonitrile; the resulting solution is filtered and reintroduced on a glass column of 2.0 cm internal diameter x 32 cm, as described above. The solvent moves through the column at a flow rate of 8 ml / minute at about 5.5 bar, and a fraction is collected every 3 minutes. The elution of the antibiotic is monitored at 280 nm. Fractions 17 and 18 are combined and concentrated in vacuo to obtain an oil. The oil is dissolved in a small volume of t-butanol and lyophilized to obtain 29 mg of factor E of A-30912.

  
  <EMI ID = 186.1>

  
The different factors of A-30912 can be identified by thin layer chromatography (TLC). The Rf of factors A to G of A-3C912, using TLC on silica gel (Merck, Darmstadt), a 7: 3 solvent system of benzene and methanol and a bioautograph by Candida albicans are shown in Table VII.

TABLE VII

  

  <EMI ID = 187.1>


  
  <EMI ID = 188.1>

  
  <EMI ID = 189.1>

  
TLC on silica gel (plates of silica gel n [deg.] 60 from Merck Darmstadt, 20 x 20 cm) and a bioautographer by Candida albicans, are given in Table VIII.

  
  <EMI ID = 190.1>

  

  <EMI ID = 191.1>


  
Solvent systems

  
a: ethyl acetate: methanol (3: 2)

  
  <EMI ID = 192.1>

  
We can also identify factors A, B, D and

  
  <EMI ID = 193.1>

  
high analytical characteristics (CLPFCE), under the following conditions:

  

  <EMI ID = 194.1>
 

  
Injection loop injection

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

TABLE IX

  
A-30912 factor Retention time (s)

  

  <EMI ID = 195.1>

Preparation 8

  
Preparation of the antibiotic S31794 / F-1

  
The antibiotic S31794 / F-1 is produced by immersion culture of Acrophialophora limonispora NRRL 8095, with shaking, stirring and / or aerating at pH 3-8, preferably 5-7, and at 15-30 [deg.] C, preferably at 18-27 [deg.] C, for 48 to 360 hours, preferably for 120 to 288 hours.

  
  <EMI ID = 196.1>

  
  <EMI ID = 197.1>

  
ethyl acetate and isopropanol and homogenizing for 30 minutes at room temperature. The organic phase is separated and evaporated in vacuo at about 40 [deg.] C. The residue thus obtained is chromatographed on approximately ten times its quantity of silica gel, using mixtures of chloroform and methanol (95: 5 to 60:40). The fractions which have an antifungal activity are combined and they are chromatographed on 100 times their quantity of "Sephadex

  
  <EMI ID = 198.1>

  
  <EMI ID = 199.1>

  
combined and rechromatographed on 100 times their quantity of silica gel (0.05-0.2 mm) with a solvent system
71: 25: 4 of chloroform, methanol and water. The eluted fractions which have an antifungal activity are combined and evaporated under vacuum to obtain the crude antibiotic S31794 / F-1. We dissolve this product in small quantities

  
  <EMI ID = 200.1>

  
to obtain S31794 / F-1 in the form of an amorphous powder

  
  <EMI ID = 201.1> vacuum pushed to 25-30 [deg.] C. Crystallization from ten times its mixing volume 30: 12: 8 of ethyl acetate, methanol and water gives crystallized S31794 / F-1, m.p. 181-183 [deg.] C with

  
  <EMI ID = 202.1>

  
Preparation 9

  
Separation of antibiotic S31794 / F-1

  
The crude antibiotic S31794 / F-1, obtained as described in Preparation 8, is introduced after chromatography on Sephadex, on a column of silica gel (MichelMiller column) via a loop using a valve system. The slurry packing procedure described in Preparation 11 is used. The solvent is moved through the column using an FMI pump with a piston design without valves. We control the elutior. of the antibiotic using a UV monitor at

  
280 nm as in Preparation 22. The fractions having an antifungal activity are combined with gold: they are concentrated under

  
empty to obtain the antibiotic S31794 / F-1.

  
  <EMI ID = 203.1>

  
on a thin layer of silica gel (Merck, 0.25 mm):

  
Solvent system Rf

  

  <EMI ID = 204.1>


  
S31794 / F-1 can also be detected by iodine vapor.

  
Preparation 10

  
Preparation of reverse phase silica gel resin /

  
C18

  
Step 1: Hydrolysis

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

  
  <EMI ID = 205.1>

  
concentrated sulfuric and 1650 ml of concentrated nitric acid in a 5 l round bottom flask and stirred to obtain an appropriate suspension. The mixture is heated on a steam bath overnight (16 hours) with a water-cooled refrigerant attached to the flask.

  
The mixture is cooled in an ice bath and

  
  <EMI ID = 206.1>

  
sintered. The silica gel is washed with deionized water until neutral pH. Then, the silica gel is washed with 4 l of acetone and it is dried under vacuum at 100 ° 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) are added, and the reaction mixture is heated at reflux overnight (16 hours) with slow mechanical stirring at 60 [deg.] C. Care is taken to avoid the agitator being too close to the bottom of the flask, to avoid crushing of the particles

  
  <EMI ID = 207.1>

  
The mixture is allowed to cool. The silanized silica gel is collected, washed with 3 l of toluene and 3 l

  
acetone, then air dry overnight (16-20 hours). The dried silica gel is suspended in 3.5 l of a 1: 1 mixture of acetonitrile and water in a 5 l flask, stirred carefully at room temperature for two hours, filtered, wash with 3 l of acetone and air dry overnight.

  
Step 3: Second silylation

  
We repeat the procedure of the first

  
  <EMI ID = 208.1>

  
heats the suspension at reflux at 60 [deg.] C for two hours, while shaking carefully. We collect the final product

  
  <EMI ID = 209.1>

  
methanol, then dried under vacuum at 50 [deg.] C overnight
(16-20 hours).

  
Preparation 11

  
Suspension packing procedure for Michel-Miller columns General information

  
This procedure is used to garnish a

  
  <EMI ID = 210.1>

  
that prepared by the process of Preparation 10.

  
In general, a pressure of less than 14 bars and flow rates of between 5 and 40 ml / minute are necessary for this suspended 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 packing material, which would significantly reduce the efficiency of the column. It is therefore important to allow the pressure to slowly drop to zero each time the pump is stopped.

  
Approximate volume of columns (Ace glass columns, Category No., not filled): 5795-04, 12 ml;
5795-10, 110 ml; 5795-16, 300 ml; 5795-24, 635 ml; and 5796-
34, 34 ml.

  
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

  
  <EMI ID = 211.1>

  
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-

  
  <EMI ID = 212.1>

  
4. Shake well until all particles are wet, allow to sit overnight or longer to ensure complete impregnation of the particles with the solvent. Decant the supernatant liquor.

  
Dissolve the resin with enough solvent to fill the tank column. Quickly pour into the tank. The column: must be pre-filled with the same solvent and the reservoir column must be partially filled with the 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 <EMI ID = 213.1>

  
6. Close the tank with the Teflon plug below the column (see Figure 1 of the U.S. Patent.

  
N [deg.] 4,131,547, plug N [deg.] 3); connect to the pump; and immediately begin pumping solvent into the 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 adsorbent. The pressure must not exceed the maximum tolerance of the column during this operation

  
(14 bars for large columns and 21 bars for analytical columns). In most cases, pressures

  
below 14 bars will be sufficient.

  
  <EMI ID = 214.1>

  
the flow and pressure will decrease.

  
9. After filling the column with adsorbent, stop the pump; let the pressure drop to zero; disconnect the tank; replace the tank with a pre-column; fill the pre-column with solvent and a small amount of adsorbent, and pump at maximum pressure until the column is completely packed. For additional information, see the general procedure. Always allow the pressure to drop slowly after stopping the pump; this will prevent the formation of any preferential cracks or channels in the packing material.

  
  <EMI ID = 215.1>

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

  
  <EMI ID = 216.1> gently to the top of the packing material, and place a Teflon plug at the top of the column until

  
  <EMI ID = 217.1>

  
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 upholstery material should continue # this file (this can be the case with large columns), a certain dead space will appear or else, it will form preferential channels and step 9 must be repeated.

  
Preparation 12

  
Preparation of the core of A-30912A

  
  <EMI ID = 218.1>

  
Fermentation of A. utahensis is carried out as described in Preparation J, using the medium of

  
  <EMI ID = 219.1>

  
incubate the production medium for approximately 42 hours.

  
  <EMI ID = 220.1>

  
A of A-30912 (340 g of raw substrate which contains approximately
19.7 g of factor A of A-30912, dissolved in 1.5 l of ethanol). We control the deacylation of factor A of A-

  
  <EMI ID = 221.1>

  
fermentation continues until complete deacylation 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 nucleus from about 20 g of factor A of A-30912.

  
We reject the mycelial cake. The clear filtrate thus obtained (about 93 l) is passed through a column containing 4.5 l of HP 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. The column is then washed with up to eight volumes (column volume) of 6.5 to 7.5 fold deionized water 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 (85 l) at a flow rate of 200-300 ml / minute.

  
  <EMI ID = 222.1>

  
following procedure: two aliquots of each eluted fraction are taken. One of these parts is concentrated to a small volume and treated with an acid chloride such as myristoyl chloride. This product and the other untreated part are tested to determine their activity against Candida 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 nucleus of

  
  <EMI ID = 223.1>

  
liquid on reverse hase

  
25 g of the core of crude A-30912A, obtained, are dissolved

  
  <EMI ID = 224.1>

  
96: 2: 1: 1 of water, acetonitrile, acetic acid and pyridine. This solution is chromatographed on a 4 l stainless steel column (8 cm x 80 cm) filled 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 Canal, 91160 Longjumeau, France). The column is operated at a pressure of 6.2-6.9 bars, giving a flow rate of about 60 ml / minute, using the same solvent. The separation is monitored at 280 nm using a UV monitor (ISCO Absorption Monitor Model UA-5, Instrumention Specialties Co., 4700 Superior Ave. Lincoln, Nebraska 68504) with an optical unit (ISCO Type 6). We collect per minute

  
  <EMI ID = 225.1>

  
Based on the absorption at 280 nm, the fractions containing the core of A-30912A are combined, evaporated in vacuo and lyophilized, which gives 2.6 g of the nucleus. The amount of solvent required to complete this chromatographic separation procedure ranges from 7 to # 1. D. Characteristics of the core of A-30912A <EMI ID = 226.1> (b) molecular weight: 797.33.
(c) white amorphous solid, soluble in water, dimethylformamide, dimethyl sulfoxide and methanol; insoluble in chloroform,

  
  <EMI ID = 227.1>

  
(d) Infrared absorption spectrum (pellet of

  
KBr)

  
presents absorption iraximas to:

  
3340 broad (OH, hydrogen bond); 2970, 2930 and 289C

  
  <EMI ID = 228.1>

  
1660 and 1625 (several carbonyl groups C = O); 1510-
1550; 1430-1450 (deformation of CH "wag"), 1310-1340; 1230-

  
  <EMI ID = 229.1>

  
(e) the electrometric titration in 66% aqueous dimethylformamide indicates the presence <EMI ID = 230.1>

  
7.35 (initial pH 7.32).

  
(f) retention time in liquid chromatography <EMI ID = 231.1>

  
the following conditions:

  
Column: 4 x 300 mm

  
Filling: silica gel / C18

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

  
  <EMI ID = 232.1>

  
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.

  
Preparation 13

  
The core of A-30912A is prepared and purified by the process of Preparation 12, but uses it as a substrate

  
  <EMI ID = 233.1>

  
Preparation 14

  
The nucleus of A-30912A is prepared and purified

  
  <EMI ID = 234.1>

  
substrate aculeacin A.

  
Preparation 15

  
Preparation of the core of A-30912B

  
A. Deacylation of factor B of antibiotic A-30912

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

  
We control the deacylation of factor B of A -
30912 by paper disk test against Candida albicans or Neurospora crassa. The fermentation is allowed to continue until &#65533; that deacylation is complete as shown by the disappearance of the activity.

  
  <EMI ID = 235.1>

  
The fermentation broth thus obtained is filtered as described in paragraph A. The mycelial cake is rejected. Passing the clear filtrate thus obtained

  
  <EMI ID = 236.1>

  
porous DIAION, HP-Series, Mitsubishi Chemical Industries Limited, Tokyo, Japan). The effluent thus obtained is rejected.

  
The column is then 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 according to the following procedure: two aliquots are taken from each eluted fraction. One of the aliquots is concentrated? up to a small volume and it is treated with a chloride decides like chloride

  
of myristoyl. This product and the other untreated part are titrated to determine their activity vis-à-vis Candida albicans. If the untreated part has no activity and the acylated aliquot part has activity, the fraction contains

  
  <EMI ID = 237.1>

  
core of A-30912B under vacuum to a small volume and lyophilized to obtain the raw core.

  
  <EMI ID = 238.1>

  
reverse phase liquid

  
The core of crude A-30912B, obtained as described in paragraph C, 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 Canal, Longjumeau, France). The column is operated at a pressure of 6.2-5.9 bars, giving a flow rate of about 60 ml per minute, using the same solvent.

  
The separation is monitored at 280 nm using a UV monitor (ISCO Absorption Monitor Mode! UA-5, Instrumentation Specialties Co., 4700 Superior Ave., Lincoln, Nebraska 68504) with an optical unit (ISCO Type 6).

  
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.

  
Preparation 16

  
The novau of A-30912B is prepared and purified by the process of Preparation 15, but a tetrahydrogenated substrate A-30912B is used.

  
Preparation 17

  
A-30912D kernel preparation

  
A deacylation of factor D 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 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 test 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 1

  
The whole fermentation broth obtained is filtered.

  
  <EMI ID = 239.1> mycelial. The clear filtrate thus obtained is passed through a column containing HP-20 resin (very polymer

  
  <EMI ID = 240.1>

  
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 filtered broth

  
  <EMI ID = 241.1>

  
column with a 7: 3 solution of water and methanol. The elution is checked using the following procedure:

  
  <EMI ID = 242.1>

  
One of the aliquots is concentrated to a small volume and treated with an acid chloride such as myristoyl chloride. This product and the other untreated part are titrated to determine their activity with respect to Candida albicans. If the untreated part has no activity and the acylated aliquot part has activity, the fraction contains the core of A-30912D. The eluate containing the nucleus

  
of A-30912D is concentrated in vacuo to a small volume and lyophilized to obtain the raw core.

  
  <EMI ID = 243.1>

  
reverse phase liquid

  
The core of crude A-30912D obtained as described in section C is dissolved in a 96: 2: 1: 1 mixture of water, acetonitrile, acetic acid and pyridine. of 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 Canal, 91160 Longjumeau, France). The column is operated at a pressure of 6.2-6.9 bars, which gives a flow rate of about 60 ml / minute, using the

  
  <EMI ID = 244.1>

  
a UV control device (ISCO Absorption Monitor Model UA-5, Instrumentation Spécialités Co., 4700 Superior Ave., Lincoln, Nebraska 68504) with an optical unit (ISCO Type 6).

  
Based on the absorption at 280 nm, the fractions containing the core of A-30912D are combined and evaporated.

  
  <EMI ID = 245.1> A-30912D purified.

Preparation 18

  
The core of A-30912D is prepared and purified by the method of Preparation 17, but a tetrahydrogenated substrate A-30912D is used.

Preparation 19

  
Preparation of the core of A-30912H

  
A. Deacylation of factor H from antibiotic 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 H of A-30912 dissolved in a mixture is added to the fermentation medium. small amount of methanol.

  
The decacylation of factor H of A-30912 is checked by analysis on a paper disc against Candida albicans or Neurospora crassa. The fermentation is allowed to continue until deacylation

  
  <EMI ID = 246.1>

  
  <EMI ID = 247.1>

  
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 (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. one rejects this wash water. Then, the column is eluted with a 7: 3 solution of water and methanol. We

  
  <EMI ID = 248.1>

  
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

  
  <EMI ID = 249.1>

  
Candida albicans. If the untreated aliquot has not

  
  <EMI ID = 250.1> the fraction contains the nucleus of A-30912H. The eluate containing the core of A-30912H is concentrated under vacuum until

  
a small volume and lyophilized to obtain the raw core.

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

  
reverse phase liquid

  
Dissolve the core of crude A-30912H, obtained

  
as described in paragraph C, 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

  
  <EMI ID = 251.1>

  
16-18 rue du Canal 91160 Longjumeau, France). We operate

  
the column at a pressure of 6.2-6.9 bars, giving a flow rate of about 60 ml / minute, using the same solvent.

  
  <EMI ID = 252.1>

  
  <EMI ID = 253.1>

  
Instrumentation Specialties Co., 4700 Superior Ave., Lincoln, Nebraska 68504) equipped with an optical unit (ISCO Type 6).

  
Preparation 20

  
The core of A-30912H is prepared and purified by the method of Preparation 19 but a tetrahydrogenated substrate A-30912H is used.

  
Preparation 21

  
Preparation of the core S31794 / F-l

  
A. Deacylation of the antibiotic S31794 / F-1

  
Fermentation of A. utahensis is carried out as described in Preparation 1, using the medium of

  
  <EMI ID = 254.1>

  
about 48 hours, the antibiotic S31794 / F-1 dissolved in a small amount of methanol is added to the fermentation medium.

  
  <EMI ID = 255.1>

  
analysis with paper disc against Candida albicans.

  
  <EMI ID = 256.1>

  
deacylation is complete as shown by the disappearance of the activity.

  
B. Separation of the S31794 / F-1 nucleus

  
The whole fermentation broth obtained is filtered as described in paragraph A. The mycelial cake, 01 is discarded. The clear filtrate thus obtained is passed through a column containing HP-2G resin (polymer

  
very porous DIAION, HP-Series, Mitsubishi Chemical Industries Limited, Tokyo, Japan). The effluent thus obtained is rejected.

  
The column is then 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 we elect the

  
column 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 treated with an acid chloride such as myristoyl chloride. This product and the other untreated aliquot are titrated to determine their activity vis-à-vis Candida albicans. If the untreated aliquot part has no activity and the acylated aliquot part has activity, the fraction contains the core of S31794 / F-1. The eluate containing the nucleus of S31794 / F-1 is concentrated in vacuo to a small volume and lyophilized to obtain the crude nucleus.

  
C. Purification of the nucleus of S31794 / F-1 by chromatography

  
reverse phase liquid

  
It is dissolved in a mixture 96: 2: 1: 1 of water,

  
of acetonitrile, of acetic acid and of pyridine the nucleus of crude S31794 / F-1 obtained as described in paragraph B. This solution is chromatographed on a column packed with

  
  <EMI ID = 257.1>

  
Manufacturing Chemists, Inc. E / M, Cincinnati, OH). The column is part of a Chromatospac Prep 100 unit (Jobin Yvon,
16-18 iue du Canal 91160 Longjumeau, France). The column is operated at a pressure of 6.2-6.9 bars, giving a flow rate of about 60 ml / minute, using the same solvent. The separation is checked at 280 nm using an apparatus

  
UV control system (ISCO Absorption Monitor Model UA-5, Instrumentation Specialties Co., 4700 Superior Ave., Lincoln. Nebraska 68504) equipped with an optical unit (ISCO Type 6).

  
  <EMI ID = 258.1>

  
the fractions containing the nucleus of S31794 / F-1, they are evaporated in vacuo and lyophilized to obtain the nucleus of purified S31794 / F-1.

  
Preparation 22

  
Preparation of tetrahydrogenated A-30912A

  
Factor A of A-30912 is dissolved in ethanol.

  
Pt02 is reduced in absolute ethanol to form Pt which is

  
  <EMI ID = 259.1>

  
factor A of A-30912, by 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 tetrahydrogenated A-30912A.

  
Preparation 23

  
  <EMI ID = 260.1>

  
Factor B of A-30912 is dissolved in ethanol.

  
  <EMI ID = 261.1>

  
platinum which is used in turn to catalytically reduce factor B of A-30912 by 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 tetrahydrogenated A-30912B.

  
Preparation 24

  
  <EMI ID = 262.1>

  
Factor D of A-30912 is dissolved in ethanol. Pt02 is reduced in absolute ethanol to form Pt which is used in turn to catalytically reduce factor D of A-30912, by 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

  
  <EMI ID = 263.1>

  
Proxy 25

  
Preparation of tetrahydrogenated A-30912H

  
Factor H of A-30912 is dissolved in ethanol. Pt02 is reduced in absolute ethanol to form Pt which is used in turn to catalytically reduce the H factor of A-30912, by hydrogenation under positive pressure until the reaction is complete
(about 2 to 3 hours). The reaction mixture is filtered

  
and it is concentrated in vacuo. The residue is dissolved in a small amount of t-butanol and lyophilized to obtain tetrahydrogenated A-30912H.

  
Preparations 26-55

  
Table 1 below gives the preparation of various N-alkanoyl amino acids. The compounds described in Table 1 are prepared according to the following general procedure:

  
The appropriate alkanoic acid chloride is added dropwise to the appropriate amino acid (1: 1 molar ratio) dissolved in pyridine. The quantity of pyridine used must be such that a concentration of the reagents is obtained between 0.1 and 0.2 M. The solution is stirred at room temperature for approximately 3 to 6 hours, after which it is poured into a large volume of water. The product precipitates from the solution and is collected by filtration and crystallized from methanol.

  

  <EMI ID = 264.1>


  

  <EMI ID = 265.1>
 

  

  <EMI ID = 266.1>


  

  <EMI ID = 267.1>
 

  

  <EMI ID = 268.1>


  

  <EMI ID = 269.1>
 

  

  <EMI ID = 270.1>


  

  <EMI ID = 271.1>
 

  

  <EMI ID = 272.1>


  

  <EMI ID = 273.1>
 

  
Examples 56-85

  
Table 2 below gives the preparation of

  
  <EMI ID = 274.1>

  
acids described in Table 1, The compounds described in Table 2 are prepared according to the following general procedure:

  
We dissolve one mole of N-alkanoylamino acid,

  
  <EMI ID = 275.1>

  
carbodiimide in methylene chloride, ether or tetrahydrofuran. The solution is stirred at room temperature for about 16 to about 20 hours, after which it is filtered. The filtrate is brought to dryness and the product is crystallized from a mixture of acetonitrile and water or from a mixture of diethyl ether and petroleum ether.

  

  <EMI ID = 276.1>


  

  <EMI ID = 277.1>
 

  

  <EMI ID = 278.1>


  

  <EMI ID = 279.1>
 

  

  <EMI ID = 280.1>


  

  <EMI ID = 281.1>
 

  

  <EMI ID = 282.1>


  

  <EMI ID = 283.1>
 

  

  <EMI ID = 284.1>


  

  <EMI ID = 285.1>
 

  
EXAMPLES 1 to 30

  
Table 3 below gives the preparation of the derivatives of the nucleus A-30912A prepared from the esters of

  
  <EMI ID = 286.1>

  
indicated in Table 2. The compounds described in Table 3 are generally prepared according to the following procedure:

  
Core 30912A, dissolved in dimethylformamide
(DMF) the 2,4,5-trichlorophenyl ester of Nalcanoyl-amino acid is added. The reaction mixture is stirred

  
  <EMI ID = 287.1>

  
get a residue. The residue is washed (twice each) with ethyl ether and methylene chloride. Washing liquids are rejected. The remaining residue is dissolved in a 3: 2 by volume mixture of ethyl acetate and methanol and

  
it is chromatographed on a column of silica gel

  
(&#65533; '&#65533; elm 99-210 microns) using the above mentioned solvent system as eluent. The fractions from the chromatograph are checked by TLC on silica gel (Merck) using a 3: 2 volume / volume mixture of ethyl acetate and methanol as solvent system. The fractions containing the desired product are combined and the solvent is removed

  
  <EMI ID = 288.1>

  
analyze the product by reverse phase high pressure liquid chromatography as follows: the dissolved sample is injected into a 1: 2: 2 volume / volume mixture of water, methanol and acetonitrile (1 mg / ml) in a steel column

  
  <EMI ID = 289.1>

  
Bondapak (Waters Associates, Inc., Milford, Massachussets, U.S.A.) and the column is eluted with a solvent system comprising a 1: 2: 2 by volume mixture of water, methanol

  
and acetonitrile. The elution is carried out at a pressure of
103.5 bars with: a flow rate of 3 ml / minute using a Waters 600A pump (Waters Associates, Inc.) and a speed: paper of 0.5 cm / minute. The eluent is checked with a Varian Vari-Chrom UV detection device at 230 nm.

  
The products can also be analyzed by field desorption mass spectrometry (FDMS).

  

  <EMI ID = 290.1>


  

  <EMI ID = 291.1>
 

  

  <EMI ID = 292.1>


  

  <EMI ID = 293.1>
 

  

  <EMI ID = 294.1>


  

  <EMI ID = 295.1>
 

  

  <EMI ID = 296.1>


  

  <EMI ID = 297.1>
 

  

  <EMI ID = 298.1>


  

  <EMI ID = 299.1>
 

  

  <EMI ID = 300.1>


  

  <EMI ID = 301.1>
 

  
EXAMPLES 31-40

  
Examples 31-40 illustrate the preparation on a larger scale of the compounds of formula III. The particular compounds prepared by the procedures indicated above

  
  <EMI ID = 302.1>

  
  <EMI ID = 303.1>

  
8.74 g (40 mmol) of

  
  <EMI ID = 304.1>

  
benzoic (5.5 g, 40 mmol) dissolved in 100 ml of pyridine.

  
The mixture is stirred for three hours and poured into 3 l of water. The precipitate which forms is filtered and dried

  
  <EMI ID = 305.1>

  
benzoic (11.01 g).

  
  <EMI ID = 306.1>

  
mmoles) of tricyclohexylcarbodiimide. The mixture is stirred at room temperature for 3.5 hours and then filtered. The filtrate is evaporated under vacuum to obtain a residue which is crystallized from a mixture of acetonitrile and water

  
  <EMI ID = 307.1>

  
p-aminobenzoic acid. The solution 3 is stirred at room temperature for 15 hours. The solvent is removed in vacuo

  
  <EMI ID = 308.1>

  
  <EMI ID = 309.1>

  
residue washed in 50 ml of methanol and purified by reverse phase CLPE using a "Prep LC / System 500" unit
(Waters Associates, Inc., Milford, Mass.) Using a

  
  <EMI ID = 310.1> <EMI ID = 311.1>

  
a 25:65:10 mixture by volume of water, methanol and acetonitrile at 34.5 bars. Fractions are analyzed by CCM

  
using silica plates and the same solvent system.

  
The fractions containing the desired product are combined and lyophilized to obtain the derivative with an N- group (n-

  
  <EMI ID = 312.1>

EXAMPLE 32

  
Acylatic-: from the core of A-30912B

  
10.2 ml of the core of A-30912B and 10.2 mmol of the ester are dissolved in 100 ml of dimethylformamide

  
  <EMI ID = 313.1>

  
aminobenzoic acid (prepared in Steps A and B of the Example

  
31). 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 ethyl ether. Washing products 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, Inc., Milford, Massachussets) using a Prep column

  
  <EMI ID = 314.1>

  
The fractions are analyzed by TLC using silica gel plates and the same solvent system. The fractions containing the desired product are combined and lyophilized to obtain the N- (n-dodecanoyl) p-aminobenzoyl group derivative of the core of A-30912B.

  
EXAMPLE 33

  
The method described in the Example can be used
32, with minor modifications, to synthesize other derivatives of the core of A-30912B. The substitution of the appropriate acyl chloride and the appropriate ar'no acid in Step A, the substitution of the appropriate N-alkanoyl-amino acid (more

  
  <EMI ID = 315.1>

  
and substitution of the appropriate 2,4,5-trichlorophenyl ester in Example 33 can give the core derivatives of A-30912B shown below:

  
  <EMI ID = 316.1>

  

  <EMI ID = 317.1>
 

  

  <EMI ID = 318.1>
 

  

  <EMI ID = 319.1>
 

  

  <EMI ID = 320.1>
 

EXAMPLE 34

  
Core acylation of A-30912D

  
Dissolve in 100 ml of dimethylformamide, 10.2

  
  <EMI ID = 321.1>

  
benzoic (prepared in Steps A and B of Example 31).

  
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 products 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, Inc., Milford, Massachussets) using a Prep column

  
  <EMI ID = 322.1>

  
the column isocratically with a 25:65:10 mixture in

  
volume of water, methanol and acetonitrile at 34.5 bars. The fractions are analyzed by TLC using silica gel plates and the same solvent system. The fractions containing the desired product are combined and lyophilized to obtain the derivative containing an N- (n-dodecanoyl) -p-amino- group.

  
  <EMI ID = 323.1>

EXAMPLE 35

  
The method described in the Example can be used
34, with slight modifications, to synthesize other derivatives of the nucleus of A-30912D. The use of the appropriate acyl chloride and amino acid in Step A, the use of the appropriate N-alkanoyl amino acid (more

  
  <EMI ID = 324.1>

  
  <EMI ID = 325.1>

  
the use of the appropriate 2,4,5-trichlorophenyl ester

  
  <EMI ID = 326.1>

  
  <EMI ID = 327.1>

  
Example 33.

  
N-alkanoylamino acid derivatives of the core of A-30912D

  

  <EMI ID = 328.1>


  
  <EMI ID = 329.1>

  
Acylation of A-30912H nucleus

  
It is dissolved in 100 ml of dimethylformamide

  
  <EMI ID = 330.1>

  
  <EMI ID = 331.1>

  
aminobenzoic acid (prepared in Steps A and B of the Example

  
31). 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. We

  
  <EMI ID = 332.1>

  
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 using silica gel plates and the same solvent system. The fractions containing the desired product are combined and lyophilized to obtain the derivative <EMI ID = 333.1>

  
A-30912H.

  
EXAMPLE 37

  
  <EMI ID = 334.1>

  
  <EMI ID = 335.1>

  
other derivatives of the core of A-30912H. Use of appropriate acyl chloride and amino acid in Step

  
  <EMI ID = 336.1>

  
N-alkanoyl monochloro-aminobenzoic acids) in Step B, and the use of the appropriate 2,4,5-trichlorophenyl ester in Example 36 makes it possible to obtain the derivatives of the core of A-30912H indicated above. below where R is as defined in Example 33.

  
N-alkanoylamino acid derivatives of the A-30912H nucleus

  

  <EMI ID = 337.1>


  
EXAMPLE 38

  
The following procedure illustrates the preparation

  
  <EMI ID = 338.1>

  
A-30912H from the core of A-3C912A.

  
  <EMI ID = 339.1>

  
  <EMI ID = 340.1> the procedure of Example 36. The derivative is methylated

  
  <EMI ID = 341.1>

  
  <EMI ID = 342.1>

  
let the solution stand with stirring for 16 hours, after which the solvent is removed under reduced pressure and

  
a residue is obtained. The residue is purified by phase CLPE

  
  <EMI ID = 343.1>

EXAMPLE 39

  
S31794 / F-1 nucleus acylation

  
It is dissolved in 100 ml of dimethylformamide 10.2

  
  <EMI ID = 344.1>

  
aminobenzoic acid (prepared in Steps A and B of the Example

  
31). 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 Associates, Inc.), using a Prep Pak-500 column. /

  
  <EMI ID = 345.1>

  
column isocratically with a mixture 25:65:10 in

  
  <EMI ID = 346.1>

  
fractions by TLC using silica gel plates and the same solvent system. The fractions containing the desired product are combined and lyophilized to obtain

  
  <EMI ID = 347.1>

  
core of S31794 / F-1.

  
EXAMPLE 40

  
The method described in the Example can be used
39 with small modifications to synthesize other derivatives of the nucleus of S31794 / F-1. Use of appropriate acyl chloride and amino acid in Step A, use of appropriate N-alkanoylamino acid (plus use of tetrahydrofuran as solvent for N-alkanoyl-monochloro acids aminobenzoic acid) in the Step

  
  <EMI ID = 348.1>

  
in example 39 allow to obtain the derivatives of the nucleus <EMI ID = 349.1>

  
in Example 33.

  
  <EMI ID = 350.1>

  

  <EMI ID = 351.1>


  
EXAMPLE 41

  
The antifungal activity of the compounds of formula III can be demonstrated in vitro in disk diffusion and dilution tests on conventional agar and in conventional in vivo tests on mice which demonstrate the efficacy with respect to a systemic fungal infection. The results of the antifungal tests of representative compounds of formula V (Examples 1 to 30) are shown in Tables 4, 5, 6 and 7.

  
Tables 4 and 5 give the results of the in vitro tests of the compounds of Examples 1 to 21 by the methods of diffusion on disc on agar plate. In Table 4, activity is measured by size
(diameter in mm) of the zone of inhibition of the microorganism observed produced by the test compound - In Table 5, the activity is measured by the inhibitory concentration

  
  <EMI ID = 352.1>

  
inhibit the growth of the test organism. Table 6 ts

  
gives the results of the in vitro test of the derivative to

  
  <EMI ID = 353.1>

  
albicans, by the dilution method on agar. In the

  
  <EMI ID = 354.1>

  
substance (pg / ml) needed to inhiberate the test organism.

  
In vivo test results to determine

  
the efficacy of the compounds of Examples 1 to 21, 26 and 28 against an infection caused by Candida albicans A-26

  
in mice are given in Table 7, where the activity is measured by the ED 50 (dose in mg / kg necessary to cure
50% of test animals). When ED is not observed, -0 / activity is indicated by the lowest dose at which

  
there is a significant antifungal effect. In this test, groups of male albino mice (free of specific pathogens) weighing 18 to 20 g 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 eight minutes (total dose

  
400) to reduce immunological responses to

  
of the infectious organism. At 0, 4 and 24 hours after infection, each group of mice is given progressive subcutaneous doses of the test compound as a

  
  <EMI ID = 355.1>

  
of water. The day of death is noted for each animal. A statistical comparison of Student's mean day of death test "t" was made between each group of treated and infected animals and a group of ten infected and untreated animals to determine whether the treatment significantly extended survival time.

  
Table 8 gives the results of the test of the compounds for absorption after oral administration. In this trial, mice are force-fed at a dose of
416 mg / kg of the test compound suspended in a mixture with

  
  <EMI ID = 356.1>

  
take blood samples from the orbital sinus and determine their antibiotic activity as follows: <EMI ID = 357.1>

  
seeded with Aspergillus montevidensis A35137. After 40 hours of incubation at 30 [deg.] C, the zones of inhibition from the blood samples are compared to a standard obtained from the test compound, and the amount of compound in the blood sample is calculated. .

  

  <EMI ID = 358.1>


  

  <EMI ID = 359.1>
 

  

  <EMI ID = 360.1>


  

  <EMI ID = 361.1>
 

  

  <EMI ID = 362.1>


  

  <EMI ID = 363.1>
 

  

  <EMI ID = 364.1>


  

  <EMI ID = 365.1>
 

  

  <EMI ID = 366.1>


  

  <EMI ID = 367.1>
 

  

  <EMI ID = 368.1>


  

  <EMI ID = 369.1>
 

  

  <EMI ID = 370.1>


  

  <EMI ID = 371.1>
 

  

  <EMI ID = 372.1>


  

  <EMI ID = 373.1>
 

  
TABLE 6

  
In vitro activity of N- group derivatives (n-

  
  <EMI ID = 374.1>

  
N- (n-dodecanoyl) -5-amino-n-pentanoyl (Example 2) from the core of A-30912A against five strains of Candida albicans by the agar dilution test.

CIM (q q / ml)

  

  <EMI ID = 375.1>
 

  

  <EMI ID = 376.1>


  

  <EMI ID = 377.1>
 

  

  <EMI ID = 378.1>


  

  <EMI ID = 379.1>
 

  

  <EMI ID = 380.1>


  

  <EMI ID = 381.1>
 

  

  <EMI ID = 382.1>


  

  <EMI ID = 383.1>


  

  <EMI ID = 384.1>
 

  

  <EMI ID = 385.1>


  

  <EMI ID = 386.1>
 

  

  <EMI ID = 387.1>


  

  <EMI ID = 388.1>
 

  

  <EMI ID = 389.1>


  

  <EMI ID = 390.1>
 

  

  <EMI ID = 391.1>


  

  <EMI ID = 392.1>


  

  <EMI ID = 393.1>



    

Claims (1)

1. Compound of formula  <EMI ID = 394.1> in which R is H or OH and when R is H, R2 is H and R <3> and R are both H or both OH, and 123 when R is OH, R is H, R is OH or a group <EMI ID = 395.1> <EMI ID = 396.1>  <EMI ID = 397.1> 6 formula -W-C-R in which:  <EMI ID = 398.1> 0 (a) -C-A-NH-  <EMI ID = 399.1>  <EMI ID = 400.1> (b) -C-CH-NH- <EMI ID = 401.1> <EMI ID = 402.1> phenyl, benzyl, substituted phenyl or substituted benzyl whose benzene ring is substituted by a chloro, bromo, iodo, nitro radical,  <EMI ID = 403.1>  <EMI ID = 404.1> where X is a hydrogen, chlorine, bromine or iodine atom or a nitro group,  <EMI ID = 405.1>  <EMI ID = 406.1> where X is a chlorine, bromine or  <EMI ID = 407.1>  <EMI ID = 408.1> where B is a divalent radical of formula: <EMI ID = 409.1> -CH = CH; -CH = CH-CH2-; or 0 -CNHCH2- 1 <EMI ID = 410.1> c17 * 2. Compound according to claim 1, characterized 0 0  <EMI ID = 411.1> in that R is a group -C-A-NH-CR where A. is a  <EMI ID = 412.1> 3. Compound according to claim 2, characterized in that R5 is an N- (n-dodecanoyl) -5-amino-n- group  <EMI ID = 413.1> (n-heptanoyl) -li-amino-n-hendecanoyl. 4. Compound according to claim 1, characterized in that R5 flood is  <EMI ID = 414.1>  <EMI ID = 415.1> in this one? straight chain. 5. Compound according to claim 4, characterized  <EMI ID = 416.1> 6. Compound according to claim 1, characterized  <EMI ID = 417.1>  <EMI ID = 418.1> in which X is a chloro, bromo, iodo, nitro radical,  <EMI ID = 419.1> <EMI ID = 420.1> alanyl. 9. Process for the preparation of a compound of formula III, characterized in that it consists in acylating with a Nalcanoylamino-acid, a cyclic peptide nucleus of formula:  <EMI ID = 421.1> in which R is H or OH and  <EMI ID = 422.1> H or both OH, and 123 when R is OH, R is H, R is OH or a  <EMI ID = 423.1> 0 <EMI ID = 424.1> <EMI ID = 425.1> in that R5 is an N-alkanoyl-amino-acyl group, derived from the corresponding acid, of formula 0 -W-C-R where W and R6 are as defined in <EMI ID = 426.1> 11. Method according to claim 9, characterized in that R is 0 0 <EMI ID = 427.1> a straight chain C 1 -C 4 alkyl group.  <EMI ID = 428.1>  <EMI ID = 429.1> 13. Method according to claim 9, characterized  <EMI ID = 430.1>  <EMI ID = 431.1>  <EMI ID = 432.1>  <EMI ID = 433.1> 14. Method according to claim 13, characterized  <EMI ID = 434.1>  <EMI ID = 435.1> 15. Method according to claim 9, characterized in that R is  <EMI ID = 436.1> in which X is a chloro, bromo, ioda, nitro radical, <EMI ID = 437.1> 17. The method of claim 10, characterized in that R is an N- (n-dodecanoyl) -3-amino-2,5- group  <EMI ID = 438.1> <EMI ID = 439.1> dodecanoyl) -2-aminonicotinyl or N- (n-dedecanoyl) -phenylalanyl. 18. Antifungal composition containing as active ingredient a compound according to any one of claims from 1 to 8.