BE886578A - 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 deacylation

  
cyclic peptide antibiotic enzyme

  
corresponding.

  
The cyclic peptide antibiotic is a compound

  
  <EMI ID = 1.1>

  

  <EMI ID = 2.1>


  
  <EMI ID = 3.1>

  
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 Il of A-30912 are

  
  <EMI ID = 4.1>

  
  <EMI ID = 5.1>

  
Antibiotic S 31794 / F-1 is a cyclic peptide

  
  <EMI ID = 6.1>

  
  <EMI ID = 7.1>

  
Each factor is isolated from the complex A30912 which contains the other factors arbitrarily called factors B, C, D, E, F and G. The complex A-30912 and the factors A to

  
  <EMI ID = 8.1>

  
Antibiotic A-30912 is identical to Antibiotic A-22802 which is described in the U.S. patent. N [deg.] 4,024,246. He has. also found that factor A is identical to the antibiotic echinocandin B [see F. Benz et al., Helv.

  
  <EMI ID = 9.1>

  
to the antibiotic SL 7810 / F [see C. Keller-Juslen et al. Tetrahedron Letters, 4147 (1976) and Belgian patent N [deg.] 834,289].

  
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; <EMI ID = 10.1>
3860; (d) Aspergillus rugulosus NRRL 8039; or (e) Aspergillus nidulans var. roseus NRRL 11440.

  
Factor B has also been found to be identical to the antibiotic echinocandin C [see R. Traber et al., Helv. Chim. Acta, 62, 1252 (1979)] and the antibiotic SL 7810 / F-II [see Belgian patent N [deg.] 834,289].

  
The factor B of antibiotic A-30912 is prepared by aerobic immersion fermentation using one of several different organisms, namely: (a) Aspergillus

  
  <EMI ID = 11.1>
(d) Aspergillus nidulans var. roseus NRRL 1440.

  
Factor D has also been found to be identical to the antibiotic echinocandin D [see R. Traber et al.,

  
  <EMI ID = 12.1>

  
7810 / F-III See Belgian patent N [deg.] 834.2891.

  
The factor D of the antibiotic A-30912 is prepared by aerobic immersion fermentation using one of several different organisms, namely: (a) Aspergilius

  
  <EMI ID = 13.1>

  
NRRL 8039 (see Belgian patent N [deg.] 834,289); or (d) Aspergillus nidulans var. roseus NRRL 11440.

  
Factor H is a factor of the antibiotic A-30912 discovered later, and it is described in the application for

  
  <EMI ID = 14.1>

  
here by way of reference.

  
  <EMI ID = 15.1>

  
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 was deposited and is part of the collection of permanent cultures of the Northern Regional Research Laboratory,

  
U.S. Departmcnt of Agriculture, Agricultural Research Service, Peoria, Illinois 61604, where it is available to the public

  
  <EMI ID = 16.1>

  
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 and the isolation and purification of the various factors of A-30912.

  
  <EMI ID = 17.1>

  
Linoleoyl side chain (R) is attached to the cyclic peptide ring on the α-amino group 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 of the family Actinoplanaceae, preferably the microorganism

  
  <EMI ID = 18.1>

  
  <EMI ID = 19.1>

  
  <EMI ID = 20.1>

  
cyclic thus obtained is separated from the fermentation broth by methods known in the art. Unlike the factors A, B, D and H of the antibiotic A-30912, the cyclic nucleus (not having 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 US patent. N [deg.] 4.173.629, is produced by

  
  <EMI ID = 21.1>

  
NRRL 8095. S31794 / F-1 has the following characteristics:
m.p. 178-180 [deg.] C (with decomposition) (amorphous) or 181-183 [deg.] C
(with decomposition) (crystallized); The] 20 _ 24 [deg.] (C 0.5,

  
  <EMI ID = 22.1>

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

  

  <EMI ID = 23.1>
 

  
  <EMI ID = 24.1>

  
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 permanent culture collection of the Northern Regional Research Center, US Department of Agriculture, Agricultural Research Culture Collection, North Central Region, Peoria, Illinois 61604, where it is available at public under the NRRL access number indicated.

  
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 = 25.1>

  
surprisingly, the myristoyl side chain can 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 Actinoplanaceae family, 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 allowed to incubate with the substrate until

  
  <EMI ID = 26.1> <EMI ID = 27.1>

  
antifungal activity.

  
The cyclic peptide nuclei provided by the above-mentioned enzymatic deacylation reactions of the antibiotics of formula 1 are represented by the general formula II.

  

  <EMI ID = 28.1>


  
  <EMI ID = 29.1>

  
hydroxy groups and R is H is the nucleus of factor A of A-30912 and, for convenience, will be referred to herein as "nucleus of A-30912A". The core of A-30912A has a formula

  
  <EMI ID = 30.1>

  
  <EMI ID = 31.1>

  
H and R3 and R 4 are both OH is the nucleus of factor B of A-309L2 and, for convenience, will be called

  
  <EMI ID = 32.1>

  
]

  
  <EMI ID = 33.1>

  
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 = 34.1>

  
The compound of formula II in which R, R and

  
  <EMI ID = 35.1>

  
  <EMI ID = 36.1>

  
a molecular weight of 840.87.

  
Removal of the side chain provides a free primary u-amino group in the ornithine residue of the 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 preparing each nucleus from the appropriate antibiotic using a fermentation using Actinoplanes utahensis NRRL 12052 is described in the patent application filed the same day as the present patent application under N [deg. ]

  
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 = 37.1>
 

  
The effectiveness of any given strain of microorganisms belonging to the family of Actinoplanaceae 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 = 38.1>

  
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 around 6.5. We control

  
culture for its activity using 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) re-acylation 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 = 39.1>

  
2459 (1974), a compound which is described by formula 1 when R is a stearoyl group; and (b) aculaecin A, which is a component of the aculaecin complex (prepared by fermentation using Aspergillus aculcatus 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 = 40.1>

  
  <EMI ID = 41.1> <EMI ID = 42.1>

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

  
  <EMI ID = 43.1>

  
antibiotic has the same nucleus as factor B of antibiotic A-30912. This substance, which differs from factor B of antibiotic A-30912 in that a different aayle group is present in place of the linoleoyl group R of formula I, is factor B of tetrahydrogenated A-30912 (tetrahydro-SL 7810 / F-II; tetrahydro

  
  <EMI ID = 44.1>

  
stearoyl group. Factor B of tetrahydrogenated A-30912

  
  <EMI ID = 45.1>

  
catalytic hydrogenation using Pt02 in ethanol under positive pressure. Factor B of tetrahydrogenated A-30912 can be used as a substrate in place of factor B of antibiotic A-30912 for enzymatic deacylation using the procedures described herein.

  
We also know that another antibiotic substance

  
  <EMI ID = 46.1>

  
substance, which differs from factor D of antibiotic A-30912 in that a different acyl group is present in place of the linoleoyl (R) group of formula I, is

  
  <EMI ID = 47.1>

  
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 = 48.1>

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

  
In factor II of antibiotic A-30912, the 5-hydroxy group present in the dihydroxy residue

  
  <EMI ID = 49.1>

  
factor A of antibiotic A-30912, the 5hydroxy group is not substituted. So we will see that we can

  
  <EMI ID = 50.1>

  
factor A according to conventional methods for preparing an aliphatic ether from an alcohol. It will also be seen that the factor A can be alkylated with other lower alkyl groups to form homologs with alkyloxy groups of the factor H molecule. The homologs with alkyloxy groups of factor H which can be synthesized from factor A, are known as factor H type homologs of A-30912. Compounds of

  
  <EMI ID = 51.1>

  
a C2-C6 alkyloxy group are here called as

  
  <EMI ID = 52.1>

  
It will also be seen that the linoleoyl side chain of factor Il of A-30912 or of factor H type homologs of A-30912 can be hydrogenated according to conventional techniques to form the H factor of A-30912 tetrahydrogen or the corresponding tetrahydrogen derivative of homologs at

  
  <EMI ID = 53.1>

  
first the factor A of the antibiotic A-30912 to obtain the factor A of tetrahydrogenated A-30912 then by forming the desired alkyloxy derivative.

  
  <EMI ID = 54.1>

  
A-30912, the H factor of tetrahydrogenated A-30912, a

  
  <EMI ID = 55.1>

  
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 comprises new compounds obtained by acylation of a nucleus of formula II. The compounds of the

  
  <EMI ID = 56.1>

  
the general formula III

  

  <EMI ID = 57.1>


  
in which :

  
  <EMI ID = 58.1>

  
  <EMI ID = 59.1>

  
not be an n-tridecyl, n-tetradecyl, n-penta- group

  
  <EMI ID = 60.1>

  
  <EMI ID = 61.1>

  
heptadecyl and, when R is an alkenyl group, R

  
  <EMI ID = 62.1>

  
  <EMI ID = 63.1>

  
provided that, when R is an alkyl group, R cannot <EMI ID = 64.1>

  
when R <1> and R are both OH, R <2> is H and R is ur. C -Cg alkyloxy group (compound of type A-30912H), R is a C6 -C24 alkyl or alkenyl group

  
  <EMI ID = 65.1>

  
that, when R is an alkyl group and R a methoxy group, R cannot be an n-heptadecyl group;

  
  <EMI ID = 66.1>

  
(S31794 / F-1), R 5 is a C6-C24 alkyl or C6-C24 alkenyl group, provided that, when R is a group

  
  <EMI ID = 67.1>

  
The expression "alkyl" denotes a hydrocarbon radical with a straight or branched chain, saturated and monovalent. The expression "alkenyl" denotes a hydrocarbon radical with a straight or branched chain, unsaturated and monovalent, containing not more than three double bonds. The double bonds of the unsaturated hydrocarbon chain

  
  <EMI ID = 68.1>

  
a hydrocarbon (straight or branched chain) containing from 6 to 24 carbon atoms is designated.

  
In secondary aspects, taking into account the preceding restrictions, the invention contemplates the following preferred compounds of formula III: <EMI ID = 69.1> <EMI ID = 70.1>

  
integer from 5 to 23.

  
  <EMI ID = 71.1> alkyl of formula CH3 (CH2) n- 'where n is an integer from 10 to 20.
(c) Compounds where R is an alkyl group <EMI ID = 72.1>

  
each independently an integer from 0 to 21 provided that the sum n + m is not

  
  <EMI ID = 73.1> <EMI ID = 74.1> alkenyl containing a cis or trans double bond.
(e) The compounds in which R is a cis or trans alkenyl group of formula

  
  <EMI ID = 75.1>

  
where n and m are independently an integer from 0 to 21, provided that the sum n + m is not less than 3 and not more than 21.

  
(f) Compounds in which R is an alkenyl group containing two cis or trans double bonds.

  
(g) Compounds where R5 is an alkenyl group

  
cis or trans of formula

  
  <EMI ID = 76.1>

  
where n and p are independently an integer from 0 to 18 and m is an integer from 1 to 19, provided that the sum m + n + p is not less than 1 and not more than 19 and that RS cannot be a linoleoyl group.
(h) The compounds in which R is:

  
  <EMI ID = 77.1> <EMI ID = 78.1>

  
t-butoxy, n-pentyloxy, n-hexyloxy, etc.

  
In particular, the invention provides a cyclic peptide compound of formula:

  

  <EMI ID = 79.1>


  
in which R is H or OH and

  
  <EMI ID = 80.1>

  
or both OH,

  
and

  
  <EMI ID = 81.1>

  
C -Cg alkyloxy group and R 4 is OH, or R <2>

  
  <EMI ID = 82.1>

  
and

  
  <EMI ID = 83.1>

  
cannot be an n-tridecyl group ,, <EMI ID = 84.1>

  
123

  
two OH, or when R, R, R and R are all H, or

  
  <EMI ID = 85.1>

  
  <EMI ID = 86.1>

  
alkyl, R cannot be an n- group

  
  <EMI ID = 87.1>

  
  <EMI ID = 88.1>

  
  <EMI ID = 89.1>
-CO-NH2 and R an alkyl group, R cannot be an n-tridecyl group.

  
The compounds of formula III inhibit the growth of pathogenic fungi such as. highlight standard biological test procedures. The compounds are therefore useful for controlling the growth of fungi on environmental surfaces (as an antiseptic) or in the treatment of infections caused by fungi. The antifungal activity of the compounds has been demonstrated against Candida albicans in vitro in disk diffusion tests on agar plate and dilution tests on agar, or in in vivo tests on mice infected with C albicans. The compounds are thus particularly useful for the treatment of infections caused by strains of C. albicans
(candidiasis).

   The compounds of formula III also exhibit in vitro activity in disk diffusion tests on agar plate with respect to Tricophyton mentagrophytes, a dermatophyte organism. Activity has also been found in disk diffusion tests on in vitro agar plates against Saccharomyces pastorianus and Neurospora crassa. Certain compounds (as shown in Example 27, Table V) give significant blood levels after oral administration in mice.

  
When a dog is administered intravenously 100 mg / kg daily for five days of the compound

  
  <EMI ID = 90.1>

  
H and R5 is an n-dodecyl group (that is to say the n-tridecanoyl derivative of the nucleus of A-30912A), the dog shows no external signs of toxicity, although we observe ni. increased serum glutamo-pyruvic transaminase and some hemolysis.

  
The compounds of formula III are prepared by acylating the appropriate nucleus on the α-amino group of ornithine by the appropriate alkanoyl or alkenoyl 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 alkanoic acid

  
  <EMI ID = 91.1>

  
desired acyl (R 5 CO-). The expression “activated derivative” denotes a derivative which makes the carboxy function of the acylating agent reactive to coupling with the amino group.

  
primary 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 (for example an acid chloride); (b) an acid anhydride (for example an alkoxyformic acid anhydride or an aryloxyformic acid anhydride) or (c) an activated ester (a 2,4,5-trichlorophenyl ester). Others

  
  <EMI ID = 92.1>

  
reaction of the carboxylic acid with a carbonyl-diimide

  
(for example N, N-dicyclohexylcarbodiimide or N, N'diisopropylcarbodiimide) to form 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 active ester method. The use of the 2,4,5-trichlorophenyl ester of the desired alkanoic or alkenol acid as the acylating agent is much preferred. In this process, an excess is reacted

  
  <EMI ID = 93.1>

  
in a non-reactive organic solvent such as dimethylformamide (DMF). The reaction time is not critical, although a time of about 6 to about 10 days is preferred.

  
20 hours. At the end of the reaction, the solvent is removed and

  
the residue is purified as by liquid chromatography under high pressure (CLPE) in reverse phase using LP-1 / C.g as fixed phase and a mixture of water, methanol and acetonitrile as solvent system.

  
A preferred acylation process is a modified Schotten-Baumann procedure in which the nucleus is treated with the acid chloride of the desired alkanol or alkenol acid.

  
at an alkaline pH. In this process, a

  
excess of acid chloride in a non organic solvent

  
reagent (like acetone) to a solution of the nucleus in a

  
  <EMI ID = 94.1>

  
crude reaction product of the reaction medium by extraction in an immiscible organic solvent (chloroform and

  
ethyl acetate). Final purification is done by CLPE

  
reverse phase, as described above.

  
A process for the preparation of type A-30912H derivatives of formula III is as follows: (a) deacylation of factor A of the antibiotic A-30912 enzymatically using Actinoplanes utahensis NRRL 12052 as described in patent application N [ deg.] filed on the same day as the present patent application, the description of which is incorporated herein by reference, (b) acylation of the core of A-30912A thus produced with the acyl group constituting the appropriate side chain using the process described above for acylation of the core of A-30912H, and (c) alkylation of the appropriate acyl derivative of the core of A-30912A to form the desired alkyloxy derivative. The alkylation (Step C) can be carried out using methods which are conventional for the preparation of an aliphatic ether from an alcohol.

   For example, the appropriate acyl derivative of the core of A-30912A can be methylated by treating a solution of the derivative in an organic solvent <EMI ID = 95.1>

  
in the presence of an acid (for example 3% HCl in methanol). The product can be isolated by conventional methods and purified by reverse phase CLPE.

  
  <EMI ID = 96.1>

  
as starting materials for the acylation reaction and their activated derivatives (in particular acid chlorides and 2,4,5-trichlorophenyl esters) are known compounds or can be prepared from known compounds by methods known. 2,4,5-trichlorophenyl esters are conveniently prepared by treating the

  
  <EMI ID = 97.1>

  
trichlorophenol in the presence of pyridine or by treating free alkanol or alkenol acid with 2,4,5-trichlorophenol

  
  <EMI ID = 98.1>

  
copulation agent. The 2,4,5-trichlorophenyl ester derivative can be purified by chromatography on a column of silica gel in toluene.

  
When used systemically, 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 should be started at low doses, the dose being 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 can be added, if desired, various conventional pharmaceutically acceptable agents such as preservatives, buffers, solubilization or 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 blood levels after oral administration (see Example 27) and can be administered systemically orally. For oral use, such compounds can be administered in combination with pharmaceutical carriers or excipients

  
  <EMI ID = 99.1>

  
The nature and proportion of these supports or excipients will be 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 standard pharmaceutically acceptable excipients

  
suitable for intravaginal use. Compositions designed for intravaginal administration are known to those of skill in the art.

  
  <EMI ID = 100.1>

  
present in vent ion are illustrated in the following examples:

  
Preparation 1

  
Fermentation of Actinoplanes utahensis NRRL 12052

  
We are preparing a mother culture of Actinoplanes

  
utahensis NRRL 12052 and maintained on an inclined agar culture. The medium used to prepare the tilted culture is chosen from one of the following:

  
Middle A

  

  <EMI ID = 101.1>


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

  
  <EMI ID = 102.1>

  

  <EMI ID = 103.1>


  
Middle B
  <EMI ID = 104.1>
 
  <EMI ID = 105.1>
  <EMI ID = 106.1>

  
Jersey, U.S.A.

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

  

  <EMI ID = 107.1>


  
adjust to pH 7.4 with NaOH; after autoclaving. the pH is around 6.8.

  
  <EMI ID = 108.1>

  
New York, U.S.A.

  
The inoculated vegetative medium is incubated in

  
  <EMI ID = 109.1>

  
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.

  
  <EMI ID = 110.1>

  
by maintaining the culture in the vapor phase of liquid nitrogen. The culture is prepared for such storage in shady small bulbs 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 = 111.1> .organisms 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: for: inoculating 50 ml of a first vegetative medium

  
  <EMI ID = 112.1>

  
incubate as previously described the first stage inoculated vegetative medium.

  
To obtain a larger volume of inoculum, we

  
  <EMI ID = 113.1>

  
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 = 114.1>

  
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 = 115.1>


  
The pH of the medium is approximately 6.9 after sterilization

  
  <EMI ID = 116.1>

  
pressure of approximately 1.1 to 1.24 bar.

  
Middle II
  <EMI ID = 117.1>
 Deionized water q.s.p. 1 litre

  
  <EMI ID = 118.1>

  
  <EMI ID = 119.1>

  
Middle III

  

  <EMI ID = 120.1>


  
  <EMI ID = 121.1>

  
The final pH is around 6.6.

  
The inoculated production medium is left to ferment <1> in a 165 1 fermentation tank at a temperature of approximately 30 [deg.] C for approximately 42 hours. The fermentation medium is stirred with conventional stirrers at about 200 revolutions per minute and it is aerated with sterile air to maintain the dissolved oxygen level above 30% at 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 = 122.1>
 

  

  <EMI ID = 123.1>


  
(initial pH 6.1)

  
  <EMI ID = 124.1>

  
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 to inoculate 55 ml of vegetative medium in a vial of

  
250 ml. The vegetative medium has the following composition:

  

  <EMI ID = 125.1>


  
  <EMI ID = 126.1>

  
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 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, keeping the culture in the vapor phase of nitrogen

  
liquid. The culture is prepared for this conservation in numerous small ampoules, as follows:

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

  

  <EMI ID = 127.1>


  
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 incubated first stage vegetative culture are used to inoculate 400 ml of a second stage vegetative growth medium having the same composition as that of the vegetative medium. The second stage medium is incubated in a two-liter wide-mouth Erlenmeyer flask, at
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:

  
Middle IV
  <EMI ID = 128.1>
 
  <EMI ID = 129.1>
(initial pH 6.8-7.0)

  
  <EMI ID = 130.1>

  
  <EMI ID = 131.1>

  
Middle V

  

  <EMI ID = 132.1>


  
The inoculated production medium is left to ferment in a 165 l fermentation tank at a temperature of 25 [deg.] C for 7 days. The fermentation medium is aerated with sterile air, keeping the dissolved oxygen level above about 50% of the 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 environment

  
has the following composition:

  

  <EMI ID = 133.1>
 

  

  <EMI ID = 134.1>


  
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. The acidified filtrate is extracted twice with equal volumes of chloroform. The chloroform extracts are combined and concentrated in vacuo to a volume of 20 liters. This concentrate is added 3,200 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

  
1 g of the antibiotic mixture A-423S5, prepared as described in Preparations 2 and 3, is dissolved in 7 ml of a 7: 2: 1 mixture of methanol, water and acetonitrile. This solution is filtered and introduced into a glass column 3.7 cm in internal diameter and 35 cm in length [CLFPCE Chromatography Column :: (low pressure liquid chromatography with high characteristics) MichelMiller, Ace Glass Incorporated , Vineland, NJ 08360] topped with

  
  <EMI ID = 135.1>

  
microns), which was prepared as described in Preparation <EMI ID = 136.1>

  
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

  
Separation of factor B from A-30912

  
The mixture A-42355 is separated as described in Preparation 3, but the concentrated chloroform extracts (285 1) are chromatographed on a column of silica gel (150 1 of Grace silica gel, quality 62) at a flow rate of 2 1 /minute. The column is washed with 200 liters of chloroform, eluted with 500 liters of acetonitrile, then

  
it is continuously eluted with a 98: 2 mixture of acetonitrile and water at a rate of 1 l / minute. Fractions having a volume of 200 liters are collected and analyzed separately to determine their biological activity. Analysis. biological is carried out by an analysis on paper disc on agar plate, seeded with Candida albicans. Fractions 77 to 103 (1365 liters) are combined and 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 is obtained

  
A-42355 enriched with factor B in the form of a gray powder.

  
The mixture A-42355 enriched in factor B thus obtained <EMI ID = 137.1>

  
methanol, water and acetonitrile. This solution is filtered and introduced on a column of silica gel (Michel-Miller column of 3.7 cm internal diameter x 33 cm) by; through a loop using a valve system. The column is packed with a reverse phase resin of silica gel LP-1 / C18 (10-20 microns) prepared as described in Preparation 10, using the 7: 2: 1 mixture of methanol, water and acetonitrile, as described in Preparation 11.! The solvent moves through the column at a flow rate of 10 ml /. minute at about 7 bar, 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 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 aceto-

  
  <EMI ID = 138.1>

  
collects fractions having a volume of approximately 200 l and they are subjected to a biological analysis on paper disc on agar seeded with Candida albicans. We join the fractions

  
  <EMI ID = 139.1>

  
concentrated solution (0.7 1) containing 10 volumes of diethyl ether to precipitate the mixture A-42355 enriched in factor 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. This solution is filtered and introduced onto a column of silica gel (Michel column <EMI ID = 140.1>

  
  <EMI ID = 141.1>

  
The column was packed with reverse phase gel resin.

  
  <EMI ID = 142.1>

  
Preparation 10. The packing was carried out in a 7: 2: 1 mixture of methanol, water and acetonitrile, according to the packing procedure of Preparation 11. The solvent moves through the column at a flow rate of 8 ml / minute at approximately 3.1 bar, using an FMI pump with piston design

  
  <EMI ID = 143.1>

  
The elution of the antibiotic is controlled at 280 nm as described in Preparation 4. The fractions 96-108 are combined and concentrated in vacuo to obtain an oil. We dissolve this oil in a small volume of t-butanol and

  
  <EMI ID = 144.1>

  
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 of the 7: 2: 1 mixture of methanol, water and acetonitrile, the resulting solution is filtered and it is introduced on a glass column of 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 = 145.1>

  
microns) (Preparation 10) in a 7: 2: 1 mixture of methanol, water and acetonitrile as described in Preparation 11. The solvent moves through the column at a flow rate of 13 ml / minute at approximately 8.3 bars , using an FMI pump with 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. 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 = 146.1>

  
A-30912.

  
  <EMI ID = 147.1>

  
in 8 ml of a 7: 2: 1 mixture of methanol, water and

  
  <EMI ID = 148.1>

  
reintroduced onto 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 11 of A-30912. Identification of A-30912 factors

  
Can the different factors of A-30912 be identified? by thin layer chromatography (TLC). The Rf

  
  <EMI ID = 149.1>

  
silica (Merck, Darmstadt), a 7: 3 3 solvent system of benzene and methanol and a bioautographer by Candida albicans are shown in Table VII.

TABLE VII

  

  <EMI ID = 150.1>


  
  <EMI ID = 151.1>

  
A-30912 in different solvent systems, using a TLC on silica gel (silica gel plates n [deg.] 60 from Merck Darmstadt, 20 x 20 cm) and a bioautograph by Candida albicans, are given in Table VIII .

  
  <EMI ID = 152.1>

  

  <EMI ID = 153.1>


  
Solvent systems

  
a ethyl acetate: methanol (3: 2)

  
b: ethyl acetate: methanol (7: 3)

  
c acetonitrile: water (95: 5)

  
  <EMI ID = 154.1>

  
  <EMI ID = 155.1>

  
following conditions:

  

  <EMI ID = 156.1>
 

  
Injection loop injection

  
The approximate retention times for factors A,

  
  <EMI ID = 157.1>

  
Table IX.

TABLE IX

  

  <EMI ID = 158.1>

Preparation 8

  
Preparation of the antibiotic S31794 / F-1

  
  <EMI ID = 159.1>

  
by immersion of Acrophialophora limonispora NRRL 8095, stirring, stirring and / or aerating at pH 3-8, preferably 5-7,

  
  <EMI ID = 160.1>

  
hours, preferably 120 to 288 hours.

  
The antibiotic S31794 / F-1 is isolated by treating the culture broth (90 L) with a 4: 1 mixture (90 L) of ethyl acetate and isopropanol and homogenizing

  
  <EMI ID = 161.1>

  
organic phase and evaporated under vacuum 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 LH-20" with methanol. The fractions coming from the Sephadex column and which have an antifungal activity are 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

  
  <EMI ID = 162.1>

  
methanol and precipitated with diethyl ether to obtain S3J794 / F-1 in the form of an amorphous powder

  
  <EMI ID = 163.1> vacuum pushes 3 25-30 [deg.] C. Crystallization from ten times its 80: 12: 8 mixture volume of ethyl acetate, methanol and water gives crystallized S31794 / F-1, m.p. 181-183 [deg.] C with decomposition after drying under high vacuum at 20 [deg.] C.

  
Preparation 9

  
Separation of antibiotic S31794 / F-1

  
  <EMI ID = 164.1>

  
as described in Preparation 8 after chromatography on Sephadex, on a silica gel column (Michel column

  
  <EMI ID = 165.1>

  
with valves. 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. The elution of the antibiotic is monitored using a UV monitor at

  
280 nm as in Preparation 22. The fractions having an antifungal activity are combined and concentrated in vacuo to obtain the antibiotic S31794 / F-1.

  
S31794 / F-1 has the following Rf by chromatography on a thin layer of silica gel (Merck, 0.25 mm):

  

  <EMI ID = 166.1>


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

  
Preparation 10

  
Preparation of reverse silica gel resin /

  
C18

  
Step 1: Hydrolysis

  
'1000 g of LP-1 silica gel (Quantum Corp., now Whatman) is added to a mixture of 1650 ml of concentrated sulfuric acid and 1650 ml of concentrated nitric acid in a 5 l round bottom flask and the mixture is 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 carefully filtered using a sintered glass funnel. Wash the silica gel with deionized water

  
  <EMI ID = 167.1>

  
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 = 168.1>

  
prevent the agitator from 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 1 of toluene and 3 1 of acetone, then air-dried 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

  
The procedure for the first silylation is repeated using 200 ml of octadecyltrichlorosilane. We

  
  <EMI ID = 169.1>

  
while shaking carefully. The final product is collected by filtration, washed with 3 1 of toluene and 6 1 of

  
  <EMI ID = 170.1>

  
(16-20 hours).

  
Preparation 11

  
Suspension packing procedure for Michel-Miller columns General information

  
This procedure is used to garnish a

  
  <EMI ID = 171.1>

  
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 = 172.1>

  
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-
30% water.

  
4 .. Shake well until all the particles are wet, leave to stand overnight or longer to ensure complete impregnation of the particles by the solvent. Decant the supernatant liquor.

  
5. Dissolve the resin with sufficient 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 of the tank during the filling operation.

  
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 bar will be sufficient.

  
8. If the pressure exceeds the maximum values, reduce the flow and the 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; <EMI ID = 173.1> 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.

  
10. Remove the pressure and carefully disconnect the pre-column. With a small spatula, remove a few millimeters (2-4) of packing at the top of the column; place 1 or 2 filters at the top of the column; press gently to the top of the mating material, and place a Teflon plug at the top of the column until the seal is confirmed. Connect the column to the pump, 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

  
Preparation of the core of A-30912A

  
A. Deacylation of factor A of antibiotic A-30912

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

  
  <EMI ID = 174.1>

  
incubate the production medium for approximately 42 hours. A quantity of 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). We control the deacylation of factor A of A-
30912 by titration against Candida albicans. The fermentation is allowed to continue 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

  
  <EMI ID = 175.1>

  
clear thus obtained (approximately 93 1) on a column containing 4.5 1 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. Then the column is washed with up to eight volumes (volume of

  
  <EMI ID = 176.1>

  
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.

  
The elution is checked using the following procedure: or. take two aliquots of each eluted fraction. 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 aliquot part

  
  <EMI ID = 177.1>

  
acylated has an activity, the fraction contains the core of A-30912A. The eluate containing the nucleus of

  
  <EMI ID = 178.1>

  
to obtain approximately 97 g of the raw core.

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

  
liquid on reverse phase

  
25 g of the core of crude A-30912A, obtained as described in paragraph C, are dissolved in 300 ml of a mixture
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

  
  <EMI ID = 179.1>

  
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). 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 combined, evaporated in vacuo and lyophilized, which gives 2.6 g of the nucleus. The quantity of solvent necessary to complete this chromatographic separation procedure varies from 7 to 8 1.

  
  <EMI ID = 180.1> <EMI ID = 181.1>
(c) white amorphous solid, soluble in water, dimethylformamide, dimethylsulfoxide and methanol; insoluble in chloroform, toluene and diethyl ether.
(d) Infrared absorption spectrum (pellet of

  
KBr)

  
has absorption maxima at:

  
3340 wide (OR, hydrogen bonding); 2970, 2930 and 2890

  
  <EMI ID = 182.1>

  
(c) the electrometric titration in 66% aqueous dimethylformamide indicates the presence of a titratable group with a pK of approximately 7.35 (initial pH 7.32).
(f) retention time in high pressure liquid chromatography (K '): 11.52 minutes under the following conditions:

  
Column: 4 x 300 mm

  
  <EMI ID = 183.1>

  
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.

  
Preparation 13

  
The core of A-30912A is prepared and purified by the process of Preparation 12, but a tetrahydrogenated substrate A-30912A is used.

  
Preparation 14

  
The nucleus of A-30912A is prepared and purified

  
by the method of Preparation 12 but using aculeacin A as a substrate.

  
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. We leave the fermentation. continue until deacylation is complete as shown by the disappearance of the activity.

  
B. Separation of the core from A-30912B

  
The fermentation broth thus 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. 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 of each eluted fraction are taken. One of the aliquots is concentrated to a small volume and treated with an acid chloride such as 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 the core of A-30912B. The eluate containing the

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

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

  
reverse phase liquid

  
Dissolve the core of crude A-30912B, obtained as described in paragraph C, in a 96: 2: 1: 1 mixture of water, acetonitrile, acetic acid and pyridine. We

  
  <EMI ID = 184.1>

  
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 6.2-6.9 bars, giving a flow rate of approximately GO ml per minute, using the same solvent.

  
The separation is checked at 280 nm using an apparatus

  
  <EMI ID = 185.1>

  
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 core of A-30912B is prepared and purified by the process of Preparation 15, but used as

  
  <EMI ID = 186.1>

  
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.

  
  <EMI ID = 187.1>

  
The whole fermentation broth obtained is filtered as described in paragraph A. The cake is discarded <EMI ID = 188.1>

  
on a column containing IIP-20 resin (very porous polymer DIAION, HP-Series, Mitsubishi Chemical Industries Limited, Tokyo, Japan). The effluent thus obtained is rejected.

  
Then wash the column with up to eight volumes of water

  
  <EMI ID = 189.1>

  
residual. 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 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. 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

  
  <EMI ID = 190.1>

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

  
reverse phase liquid

  
The nucleus of crude A-30912D obtained as described in paragraph 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

  
  <EMI ID = 191.1>

  
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 bar, which gives a flow rate of about 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 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, evaporated in vacuo and lyophilized to obtain the core of 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 of 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 II of A-30912 dissolved in a small amount of methanol.

  
The desaçylation of factor H of A-30912 is checked by analysis on a paper disc against

  
  <EMI ID = 192.1>

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

  
  <EMI ID = 193.1>

  
residual. 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 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 untreated aliquot part with respect to Candida albicans is determined. If the untreated aliquot part has no activity and the acylated aliquot part has activity, <EMI ID = 194.1> the eluate containing the core of A-30912H under vacuum until

  
  <EMI ID = 195.1>

  
gross.

  
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 is part of a Chromatospac Prep 100 unit (Jobin Yvon,

  
16-18 rue du Canal 91160 Longjumeau, France). We make it work

  
the column 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).

  
Preparation 20

  
The nucleus of A-30912H is prepared and purified

  
by the process of Preparation 19, but a tetrahydrogenated substrate A-30912H is used.

  
Preparation 21

  
S31794 / F-1 kernel preparation

  
A. Deacylation of the antibiotic S31794 / F-1

  
Fermentation of A. utahensis is carried out as

  
  <EMI ID = 196.1>

  
production I. After having incubated the culture for approximately 48 hours, the antibiotic S31794 / F-1 dissolved in a small amount of methanol is added to the fermentation medium.

  
The deacylation of S31794 / F-1 is checked by analysis with a paper disc against Candida albicans. The fermentation is allowed to continue until the deacylation is conspicuous as shown by the disappearance <EMI ID = 197.1>

  
Lincoln, Nebraska 68504) equipped with an optical unit (ISCO

  
  <EMI ID = 198.1>

  
  <EMI ID = 199.1>

  
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.

  
  <EMI ID = 200.1>

  
in turn uses to catalytically reduce 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 = 201.1>

  
Factor B of A-30912 is dissolved in ethanol.

  
  <EMI ID = 202.1>

  
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

  
  <EMI ID = 203.1>

Preparation 24

  
Preparation of tetrahydrogenated A-30912D

  
Factor D is dissolved in ethanol

  
  <EMI ID = 204.1>

  
Pt which is used at its turn to catalytically reduce the factor D of A-30912, by hydrogenation under positive pressure until the reaction is complete

  
  <EMI ID = 205.1> of the activity.

  
  <EMI ID = 206.1>

  
The whole fermentation broth obtained is filtered as described in paragraph A. The mycelial cake is discarded, the clear filtrate thus obtained is passed through a column containing HP-20 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 water

  
  <EMI ID = 207.1>

  
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. We

  
  <EMI ID = 208.1>

  
to a small volume, and lyophilized to obtain the raw core.

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

  
reverse phase liquid

  
The core of crude S31794 / F-1 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

  
  <EMI ID = 209.1>

  
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 checked at 280 nm using an apparatus

  
  <EMI ID = 210.1>

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

Preparation 25

  
  <EMI ID = 211.1>

  
The factor il of A-30912 is dissolved in

  
  <EMI ID = 212.1>

  
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

  
  <EMI ID = 213.1>

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

EXAMPLES

  
Various derivatives containing alkanoyl and alkenoyl groups are prepared by acylation of the appropriate nucleus, either by the modified Schotten-Bauman reaction using an acid chloride as acylating agent.
(Method A) or by the aax active esters method using 2,4,5-trichlorophenyl ester as acylating agent
(Method B). The general procedures for carrying out the acylation reactions by Method A or Method B are indicated below:

  
Method A (Modified Schotten-Bauman reaction).

  
This process involves reacting the appropriate nucleus with the alkanol or alkenol acid chloride which corresponds to the desired acyl side chain.

  
The nucleus is dissolved in a mixture of 0.1 M KHPO4 buffer, pH 7.5 (5 to 10 ml) and acetone or methanol
(5 to 10 ml). To the core solution, cooled by an ice bath or at room temperature, is added slowly

  
  <EMI ID = 214.1>

  
  <EMI ID = 215.1>

  
If desired, the pH of the reaction mixture can be adjusted to 7.5-8.0 after each addition of the acid chloride; however, this step is not essential. The reaction mixture is stirred at ice bath temperature or at room temperature for 1.5 to 3.0 hours. As the reaction proceeds, a precipitate forms which is composed

  
  <EMI ID = 216.1>

  
at the end of the reaction period, the reaction mixture is centrifuged to remove the precipitate. The precipitate collected and the supernatant liquor are then treated according to one of the following purification procedures.

  
. Procedure (a) The precipitate is washed one, two or three times with 2 to 3 volumes of methanol or a 1: 1 mixture of methanol and water. The washing products are centrifuged with methanol or methanol-water and the methanolic supernatant liquors are combined with the supernatant liquor obtained after the initial centrifugation of the reaction mixture. The concentrated supernatant liquors are then concentrated under vacuum to remove the organic solvent. The aqueous concentrate is then combined with an equal volume of methanol and the resulting solution is extracted successively with chloroform and ethyl acetate as described above in Process (a).

  
Procedure (b) The precipitate is immediately discarded and the supernatant liquor is extracted

  
pH 5.0-7.0 once or twice with diethyl ether (equal volume) to remove alkanol or unreacted alkenol. The diethyl ether extract is discarded. The supernatant liquor extracted is concentrated in vacuo to remove the organic solvent. Then the aqueous concentrate is combined with an equal volume of methanol and the resulting solution is extracted successively with chloroform and ethyl acetate as described previously in Method ta).

  
After the extraction step with chloroform followed by ethyl acetate in the preceding procedure (a) or (b), all the phases of solvent extract are combined and concentrated to dryness to obtain the derivative with an alkanoyl or crude alkenoyl group of the core of A-30912A.

  
The crude derivative is purified by reverse phase CLPE as follows: the sample, dissolved in 5 to 6 ml of methanol, is injected into a stainless steel column

  
  <EMI ID = 217.1>

  
10) and the column is eluted with a solvent system comprising water, methanol and acetonitrile. We perform

  
  <EMI ID = 218.1>

  
10 to 12 ml per hour using an LD-C duplex pump
(Milton-Roy). The effluent is checked with an ISCOUA-5 detector at 280 nm. The fractions containing the desired product are combined and concentrated to dryness under vacuum to obtain the purified alkanoyl or alkyoyl derivative of the corresponding nucleus.

  
The purified product is analyzed by thin layer chromatography (TLC) using reverse phase C18 plates

  
  <EMI ID = 219.1>

  
development, the plates are observed under UV light to detect the product. The products are also analyzed by field desorption mass spectrometry.

  
Method B (Active ester method)

  
  <EMI ID = 220.1>

  
hours a solution of the appropriate nucleus and the appropriate 2,4,5-trichlorophenyl alkanoate or alkenoate in dimethylformamide (DMF) (10-20 ml). The reaction mixture is concentrated to dryness in vacuo to obtain the crude alkanoyl or alkenoyl derivative of the corresponding core. The crude product is purified by reverse phase CLPE as described above in method A. The purified product is also analyzed by the methods used in method A.

  
  <EMI ID = 221.1>

  
The preparation of various alkanoylated and alkenoylated derivatives, obtained by acylation of the core of A-30912A, representative of the compounds of formula III, is given in Table 1 below. The derivatives of Table 1 are prepared either by the modified Schotten-Bauman reaction using an acid chloride as acylating agent
(Method A) or by the active ester method using 2,4,5-trichlorophenyl ester as acylating agent
(Method B) described above.

  

  <EMI ID = 222.1>


  

  <EMI ID = 223.1>
 

  

  <EMI ID = 224.1>


  

  <EMI ID = 225.1>
 

  

  <EMI ID = 226.1>


  

  <EMI ID = 227.1>
 

  

  <EMI ID = 228.1>


  

  <EMI ID = 229.1>
 

EXAMPLE 17

  
N-tridecanoylated derivative of the core of A-30912A

  
The following procedure illustrates the preparation on a larger scale of the compounds of formula III by the active ester process. The particular compound prepared by the procedure given below is the compound of

  
  <EMI ID = 230.1>

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

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

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

  
2,4,5-trichlorophenyl

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

  
500 ml of methylene chloride for 45 minutes then the mixture is filtered. We reject the filtrate. The remaining solids are extracted with 500 ml of methanol and 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 CLPE as follows:

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

  
  <EMI ID = 231.1>

  
Preparation 10). The column is eluted with a solvent system comprising a 3: 3: 4 mixture of water, methanol and acetonitrile. The elution is carried out at a pressure of
69-103.5 bar with a flow rate of 11-12 ml per minute using a LDC duplex pump (Milton-Roy). The effluent is monitored with an ultra-violet detector (ISCO-UA-5) at
280.nm. The fractions containing the desired product are combined and dried under vacuum. 550 mg of product are obtained. The chromatography described above is repeated four times with samples of 1 g of crude product to obtain other purified samples as follows:
620 mg, 520 mg, 670 mg and 490 mg. Total weight of purified title product: 2.8 g.

   According to the preceding procedures, 4.0 g of the core of A-30912A are reacted with 2,4,5-trichlorophenyl n-tridecanoate to obtain 2.6 g of the purified title product. The materials of the two preparations are combined (5.4 g). Mass ion by field desorption mass spectrometry: (M + Na +):

  
  <EMI ID = 232.1>

  
(This Micro Bondapak, Waters Co.) with a 2: 1: 2 mixture of water, methanol and acetonitrile as the eluent system, has only a peak.

  
EXAMPLE 18

  
N-tridecanoylated derivative of the core of A-30912B

  
A solution of 2,4,5-trichlorophenyl n-tridecanoate (prepared as in Example 17, Step A) (3.3 mmol) and one mole of the nucleus of A is stirred at ambient temperature for 16 hours. -30912B (see Preparations 15 and 16) in 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. The filtrate is discarded, the resulting 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 follows.

  
A sample of the crude product (1 g), dissolved in 5 ml of methanol, is injected into a 2.5 x 81 cm stainless steel column packed with LP-1 / C resin (see Preparation 11). The column is eluted with a solvent system comprising a 3: 3: 4 mixture of water, methanol and

  
  <EMI ID = 233.1>

  
103.5 bar with a flow rate of 11-12 ml / minute using a LDC duplex pump (Milton-Roy). The taper is checked by an ultra-violet detector (ISCO-UA 5) at 280 nm. We

  
  <EMI ID = 234.1>

  
The fractions containing the desired product are combined and dried under vacuum to obtain the title product. The purified product is analyzed by thin layer chromatography using reverse phase C18 plates.

  
  <EMI ID = 235.1>

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

  
EXAMPLE 19

  
Using the method of Example 18 but

  
  <EMI ID = 236.1>

  
Step A and the appropriate 2,4,5-trichlorophenyl ester of alkanol or alkenoic acid in Step B, the derivatives of the core of A-30912B are indicated below:
Alkanoylated and alkenoylated derivatives of the core of A-30912B

  

  <EMI ID = 237.1>
 

  

  <EMI ID = 238.1>
 

  
  <EMI ID = 239.1>

  
  <EMI ID = 240.1>

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

  
  <EMI ID = 241.1>

  
Step A) and Immole of the nucleus of A-30912D (see Preparations 17 and 18) in 200 ml of DMF. Removal of the solvent in vacuo gives a residue. We dilute the residue with

  
  <EMI ID = 242.1>

  
filters the mixture. We reject the filtrate. The resulting 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 described in Example 18.

  
EXAMPLE 21

  
Using the method of Example 20 but using the appropriate alkanoic acid or alkenol acid

  
  <EMI ID = 243.1>

  
obtains the derivatives of the core of A-30912D indicated below:
Alkanoylated and alkenoylated derivatives of the core of A-30912D

  

  <EMI ID = 244.1>
 

  

  <EMI ID = 245.1>

EXEMPT 22

  
N-tridecanoylated derivative of the core of A-30912H

  
A solution of 3.3 mmol of 2,4,5-trichlorophenyl n-tridecanoate (prepared as in Example 17, Step A) and of Immole of the core of A-30912H are stirred at ambient temperature for 16 hours. see the

  
  <EMI ID = 246.1>

  
Removal of the solvent in vacuo gives a residue. The residue is diluted with 300 ml of methylene chloride for 45 minutes and then the mixture is filtered. We reject the filtrate.

  
The resulting 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 described in Example 18.

  
EXAMPLE 23

  
Using the method of Example 22 but using the appropriate alkanol or alkenoic acid in Step A and the appropriate 2,4,5-trichlorophenyl ester of alkanoic or alkenol acid in Step B, the derivatives of the core of A-30912H indicated below:
Alkanoylated and alkenoylated derivatives of the core of A-30912H
  <EMI ID = 247.1>
 
  <EMI ID = 248.1>
 EXAMPLE 2 4

  
The following procedure illustrates the preparation of the N-n-tridecanoylated derivative of the core of A-30912H from the core of A-30912A.

  
The core of A-30912A is treated with 2,4,5-trichlorophenyl n-tridecanoate according to the procedure of Example 17. The derivative thus obtained is methylated by treating a sample (20 mg) with 0.06 ml 3% HCl in methanol in dimethvlformamide. The solution is allowed to 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 reverse phase CLPE in

  
  <EMI ID = 249.1>

  
EXAMPLE 25

  
N-tridecanoylated derivative of the S31794 / F-1 nucleus

  
A solution of 3.3 mmol of 2,4,5-trichlorophenyl n-tridecanoate (prepared as in Example 17, Step A) and 1 mmol of S31794 / F-1 core is stirred at room temperature for 16 hours. (see Preparation 21) in 200 ml of DMF. Removal of the solvent in vacuo leaves 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

  
  <EMI ID = 250.1>

  
described in Example 18.

  
EXAMPLE 26

  
Using the method of Example 25 but substituting the appropriate alkanoic or alkenol acid in Step A and the 2,4,5-trichlorophenyl ester of the acid

  
  <EMI ID = 251.1>

  
the derivatives of the S31794 / F-1 nucleus indicated below:

  
  <EMI ID = 252.1>
  <EMI ID = 253.1>
 
  <EMI ID = 254.1>
 EXAMPLE 27

  
The antifungal activity of the compounds of formula III can be demonstrated and studied in vitro in disk diffusion and dilution tests on conventional agar or in conventional tests in vivo in mice which. show the efficacy against a systemic fungal infection. The results of the antifungal tests for representative compounds of formula IV (Examples 1 to 16) are given in Tables II, III, IV and V.

  
  <EMI ID = 255.1>

  
the in vitro test of the compounds of Examples 1 to 16 by methods of diffusion on disc on agar plate. In Table II, the activity is measured by the dimension
(diameter in mm) of the zone of inhibition of the microorganism observed produced by the test compound. In Table III, the activity is measured by the inhibitory concentration

  
  <EMI ID = 256.1>

  
inhibit the growth of the test organism. Table IV gives the results of the in vitro test of the ntridecanoylated derivative of the nucleus of A-3Q912A (formula III, R5 is

  
  <EMI ID = 257.1>

  
the agar dilution method. In Table III, activity is measured by the minimum inhibitory concentration (MIC) of the substance (pg / ml) required to inhibit the test organism.

  
The results of in vivo tests intended to evaluate the effectiveness of the compound of Examples 1 to 16 with regard to

  
  <EMI ID = 258.1>

  
mice are given in Table V, where the activity is

  
  <EMI ID = 259.1>

  
to cure 50% of the test animals). When DE 5 is not obtained, the activity is indicated by the lowest

  
dose at which a significant antifungal effect is observed. 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 4 minutes (total dose of
400) to reduce the body's immunological reactions to infection. At 0, 4 and 24 hours after infection, one

  
gives each group of mice progressive doses subcutaneously of the test compound as a

  
  <EMI ID = 260.1>

  
the water. The day of death is noted for each animal. A statistical comparison by Student's "t" test of the average day of death is carried out between each group of infected animals treated at a particular dose and a group of 10 infected animals not treated, to determine whether the treatment prolongs significantly the duration of.

  
Table VI gives the results of the test of the compounds of Examples 1 to 16 to determine their absorption after oral administration. In this essay, we

  
gave the mice a dose of 416 mg / kg of the test compound in suspension in a 33% mixture of PEG 400 in water.

  
At time intervals, blood samples are taken from the orbital sinus and the antibiotic activity is determined as follows: a 7 mm disc containing 20 μl of whole blood is placed on agar seeded with Aspergillus

  
  <EMI ID = 261.1>

  
compares the zones of inhibition from the blood samples with a reference obtained from the test compound, and the amount of compound in the blood sample is calculated.

  

  <EMI ID = 262.1>


  

  <EMI ID = 263.1>
 

  

  <EMI ID = 264.1>


  

  <EMI ID = 265.1>
 

  

  <EMI ID = 266.1>


  

  <EMI ID = 267.1>


  

  <EMI ID = 268.1>
 

TABLE IV

  
In vitro activity of the n-tridecanoylated derivative of the nucleus of A-30912A with respect to five strains of Candida albicans.

  

  <EMI ID = 269.1>
 

  

  <EMI ID = 270.1>


  

  <EMI ID = 271.1>
 

  

  <EMI ID = 272.1>
 

  

  <EMI ID = 273.1>


  

  <EMI ID = 274.1>


  

  <EMI ID = 275.1>



    

Claims (25)

1. Compound of formula:  <EMI ID = 276.1> where R is H or OH and <EMI ID = 277.1> H or both OH, and  <EMI ID = 278.1> and R5 is a C6-C24 alkyl or alkenyl group  <EMI ID = 279.1>  <EMI ID = 280.1> cannot be an n-tridecyl, n-tetradecyl, n-pentadecyl or n-heptadecyl group; and, when R is an alkenyl group, R cannot  <EMI ID = 281.1> and when R and R are both H, R and R are all  <EMI ID = 282.1> <EMI ID = 283.1>  <EMI ID = 284.1>  <EMI ID = 285.1>  <EMI ID = 286.1> and when R <1>, R <3> and R4 are all OH, R <2> is -CO-NH, and R 5 is an alkyl group, R 5 cannot be an n-tridecyl group. 2. Compound according to claim 1, characterized in that R is a C6-C24 'alkyl group 3. Compound according to claim 2, characterized in that R is an alkyl group of formula CH3 (CH2) n where n is an integer from 5 to 23, provided that n cannot be  <EMI ID = 287.1> cannot be 12, 13 or 14. 4. Compound according to claim 3, characterized.  <EMI ID = 288.1> 5. Compound according to claim 2, characterized in that R is an alkyl group of formula  <EMI ID = 289.1> in which n and m are independently an integer from 0 to 21,  <EMI ID = 290.1>  <EMI ID = 291.1> 6. Compound according to claim 1, characterized in that R is a C6-C24 alkenyl group containing a cis or trans double bond. 7. Compound according to claim 6, characterized in that R is a cis- or trans-alkenyl group of formula  <EMI ID = 292.1>   <EMI ID = 293.1> as long as the sum n + m is not less than 3 and is not greater &#65533; 21. 8. Compound according to claim 7, characterized  <EMI ID = 294.1> 9. Compound according to claim 1, characterized in that R is a C6-C24 alkenyl group containing two cis or trans double bonds. 10. Compound according to claim 9, characterized  <EMI ID = 295.1> CH (CH2) 7- 11. Compound according to claim 1, characterized in that R is a C6-C24 alkenyl group containing three cis or trans double bonds. 12. Compound according to claim 11, characterized in that R5 is cis, cis, cis-CH3CH2CH = CHCH2CH = CHCH2-  <EMI ID = 296.1> 13. Process for the preparation of a compound of formula III, characterized in that it consists in acylating with a  <EMI ID = 297.1> of formula  <EMI ID = 298.1>  in which R is H or OH and  <EMI ID = 299.1> H or both OH, and  <EMI ID = 300.1> C -Cg alkyloxy and R 4 is OH, or R <2> is 0 <EMI ID = 301.1> 14. Method according to claim 13, characterized  <EMI ID = 302.1> 15. Method according to claim 14, characterized  <EMI ID = 303.1> where n is an integer from 5 to 23, provided that n is not  <EMI ID = 304.1> cannot be 12, 13 or 14. 16. Method according to claim 14, characterized  <EMI ID = 305.1> 17. Method according to claim 14, characterized  <EMI ID = 306.1>  <EMI ID = 307.1>  <EMI ID = 308.1> provided that the sum n + m is not less than 3 and not more than 21. 18. The method of claim 13, characterized in that R is a C6-C24 alkenyl group containing a cis or trans double bond. 19. The method of claim 18, characterized in that R is a cis- or trans-alkenyl group of formula  <EMI ID = 309.1> where n and m are independently an integer from 0 to 21, provided that the sum n + m is not less than 3 and is not more than 21. 20. Method according to claim 19, characterized  <EMI ID = 310.1> 21. The method of claim 13, characterized in that R is a C6-C24 alkenyl group containing two cis or trans double bonds. 22. Method according to claim 21, characterized  <EMI ID = 311.1> 23. The method of claim 13, characterized in that R is a C6-C24 alkenyl group containing three cis or trans double bonds. 24. Method according to claim 23, characterized  <EMI ID = 312.1> 25. Antifungal composition containing as active ingredient a compound according to any one of claims 1 to 12.