Patent Publication Number: US-4322338-A

Title: Derivatives of A-30912B nucleus

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application is a continuation-in-part of application Ser. No. 103,314, filed Dec. 13, 1979, now abandoned. 
    
    
     BACKGROUND OF THE INVENTION 
     This invention relates to novel semi-synthetic antifungal compounds which are prepared by the acylation of the cyclic peptide nucleus produced by the enzymatic deacylation of antibiotic A30912 factor B. 
     Antibiotic A-30912 factor B is an antifungal cyclic peptide having the formula: ##STR2## wherein R is the linoleoyl group ##STR3## Throughout this application, the cyclic peptide formulas, such as formula I, assume that the amino acids represented are in the L-configuration. The factor is isolated from the A30912 complex which contains other factors arbitrarily designated factors A, C, D, E, F, and G. The A-30912 complex and the individual factors A through G are disclosed by M. Hoehn and K. Michel in U.S. Pat. No. 4,024,245. Antibiotic A-30912 factor A is identical to antibiotic A-22802 which is described by C. Higgins and K. Michel in U.S. Pat. No. 4,024,246. Factor B has also been found to be identical to antibiotic echinocandin C [see R. Traber et al., Helv. Chim. Acta, 62, 1252 (1979)] and to antibiotic SL 7810/F-II [See Belgium Pat. No. 834,289, Derwent Abstract 30159X]. 
     Antibiotic A-30912 factor B is prepared by fermentation using one of several different organisms, namely: (a) Aspergillus rugulosus NRRL 8113 (see U.S. Pat. No. 4,024,245); (b) Aspergillus nidulans var. echinulatus A-32204, NRRL 3860, as described in Swiss Pat. No. 568,386; (c) Aspergillus rugulosus NRRL 8039 (see Belgian Pat. No. 834,289); or (d) Aspergillus nidulans var. roseus NRRL 11440 (see co-pending application of L. Boeck and R. Kastner, METHOD OF PRODUCING THE A-30912 ANTIBIOTICS, Ser. No. 126,078, filed March 3, 1980, which is a continuation-in-part of application Ser. No. 46,744, filed June 8, 1979, (now abandoned), the entire disclosure of which is incorporated herein by reference. 
     A subculture of A. nidulans var. roseus has been deposited and made a part of the permanent culture collection of the Northern Regional Research Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Peoria, Ill. 61604, from which it is available to the public under the number NRRL 11440. 
     When a strain of A nidulans var. roseus NRRL 11440 is used to produce A-30912 factor B, a complex of factors is obtained which for convenience is called the A-42355 antibiotic complex. A-30912 factor A is the major factor of the A-42355 antibiotic complex, while factors B, D and H are minor factors. Examples 5, 6, and 7 herein, illustrate the preparation of the A-42355 complex and the isolation and purification of A-30912 factor B therefrom. A-30912 factor H is further described in a co-pending application of Karl H. Michel entitled ANTIBIOTIC A-30912 FACTOR H, Ser. No. 117,739, filed Feb. 1, 1980, which is a continuation-in-part of application Serial No. 46,875, filed June 8, 1979 (now abandoned). 
     In the antibiotic A-30912 factor B molecule (Formula I), the linoleoyl side chain (R) is attached at the cyclic peptide nucleus at the α-amino group of the dihydroxyornithine residue. Surprisingly, it has been found that the linoleoyl side chain can be cleaved from the nucleus by an enzyme without affecting the chemical intregity of the nucleus. The enzyme employed to effect the deacylation reaction is produced by a microorganism of the family Actinoplanaceae, preferably the microorganism Actinoplanes utahensis NRRL 12052, or a variant thereof. To accomplish deacylation, antibiotic A30912 factor B is added to a culture of the microorganism and the culture is allowed to incubate with the substrate until the deacylation is subtantially complete. The cyclic nucleus thereby obtained is separated from the fermentation broth by methods known in the art. Unlike antibiotic A-30912 factor B, the cyclic nucleus (lacking the linoleoyl side chain) is substantially devoid of antifungal activity. 
     The cyclic nucleus afforded by the aforedescribed enzymatic deacylation of antibiotic A-30912 factor B, is depicted in Formula II. ##STR4## 
     A-30912B nucleus has an empirical formula of C 34  H 51  N 7  O 14  and a molecular weight of 781.81. 
     Removal of the side chain group affords a free primary α-amino group in the dihydroxyornithine residue of the cyclic peptide. For convenience, the compound having the structure given in Formula II will be referred to herein as &#34;A-30912B nucleus.&#34; As will be apparent to those skilled in the art, A-30912B nucleus can be obtained either in the form of the free amine or of the acid addition salt. Although any suitable acid addition salt may be employed, those which are non-toxic and pharmaceutically acceptable are preferred. 
     The method of preparing A-30912B nucleus from antibiotic A-30912 factor B by means of fermentation using Actinoplanes utahensis NRRL 12052 is described in the co-pending application of Bernard J. Abbott and David S. Fukuda, entitled &#34;A-30912B NUCLEUS&#34;, Ser. No. 103,121, was filed Dec. 13, 1979. A continuation-in-part application of this application, with the corresponding Ser. No. 181,437 is being filed herewith this even date, the full disclosure of which is incorporated herein by reference. Example 3 herein, illustrates the preparation of A-30912B nucleus by fermentation using antibiotic A-30912 factor B as the substrate and Actinoplanes utahensis NRRL 12052 as the microorganism. 
     The enzyme produced by Actinoplanes utahensis NRRL 12052 may be the same enzyme which has been used to deacylate penicillins (see Walter J. Kleinschmidt, Walter E. Wright, Frederick W. Kavanagh, and William M. Stark, U.S. Pat. No. 3,150,059, issued Sept. 22, 1964). 
     Cultures of representative species of Actinoplanaceae are available to the public from the Northern Regional Research Laboratory under the following accession numbers: 
     
         ______________________________________                                    
Actinoplanes utahensis                                                    
                      NRRL 12052                                          
Actinoplanes missouriensis                                                
                      NRRL 12053                                          
Actinoplanes sp.      NRRL 8122                                           
Actinoplanes sp.      NRRL 12065                                          
Streptosporangium roseum                                                  
var. hollandensis     NRRL 12064                                          
______________________________________                                    
 
    
     The effectiveness of any given strain of microorganism within the family Actinoplanaceae for carrying out the deacylation of this invention is determined by the following procedure. A suitable growth medium is inoculated with the microorganism. The culture is incubated at about 28° C. for two or three days on a rotary shaker. One of the substrate antibiotics is then added to the culture. The pH of the fermentation medium is maintained at about pH 6.5. The culture is monitored for activity using a Candida albicans assay. Loss of antibiotic activity is an indication that the microorganism produces the requisite enzyme for deacylation. This must be verified, however, using one of the following methods: (1) analysis by HPLC for presence of the intact nucleus; or (2) reacylation with an appropriate side chain (e.g. linoleoyl, stearoyl, or palmitoyl) to restore activity. 
     It is known that another antibiotic substance possesses the same nucleus as that of antibiotic A-30912 factor B. This substance, which differs from antibiotic A-30912 factor B in that a different acyl group is present in place of the linoleoyl group (R) in Formula I, is tetrahydro-A-30912 factor B (tetrahydro-SL 7810/F-II; tetrahydro echinocandin C) described by R. Traber et al., Helv. Chim. Acta, 62 1252 (1979). Tetrahydro-A-30912 factor B is depicted in Formula I when R is stearoyl. Tetrahydro-A-30912 factor B can be prepared from antibiotic A-30912 factor B by catalytic hydrogenation using PtO 2  in ethanol under positive pressure. Tetrahydro-A-30912 factor B can be employed as a substrate in place of antibiotic A-30912 factor B for the enzymatic deacylation using the procedures herein described. 
     SUMMARY OF THE INVENTION 
     The invention sought to be patented comprehends novel compounds derived by acylating A-30912B nucleus (Formula II). The compounds of the present invention have the chemical structure depicted in Formula III: ##STR5## wherein R 1  is C 6  -C 24  alkyl or C 6  -C 24  alkenyl; provided that when R 1  is alkyl, R 1  cannot be n-heptadecyl; and, when R 1  is alkenyl, R 1  cannot be cis, cis--CH 3  (CH 2 ) 4  CH═CHCH 2  --CH═CH(CH 2 ) 7 )--. 
     By the term &#34;alkyl&#34; is meant a univalent saturated, straight-chain or branched-chain hydrocarbon radical. By the term &#34;alkenyl&#34; is meant a univalent, unsaturated, straight-chain or branched-chain hydrocarbon radical containing not more than three double bonds. The double bonds of the unsaturated hydrocarbon chain may be either in the cis or trans configuration. By &#34;C 6  -C 24  &#34; is meant a hydrocarbon (including straight and branched chains) containing from six to 24 carbon atoms. 
     In subgeneric aspects, the invention contemplates the following preferred embodiments of the compounds of Formula III: 
     (a) The compounds wherein R 1  is alkyl of the formula CH 3  (CH 2 ) n  --, wherein n is an integer from 5 to 23, provided that n cannot be 16. 
     (b) The compounds wherein R 1  is alkyl of the formula CH 3  (CH 2 ) n  --, wherein n is 10, 11, 12, 13, 14, 15, 17, 18, 19, or 20. 
     (c) The compounds wherein R 1  is alkyl of the formula ##STR6##  wherein n and m are each, independently, an integer from 0 to 21 provided that n+m must be no less than 3 and no greater than 21. 
     (d) The compounds wherein R 1  is alkenyl containing one cis or trans double bond. 
     (e) The compounds wherein R 1  is cis or trans alkenyl of the formula 
     
         CH.sub.3 (CH.sub.2).sub.n CH═CH(CH.sub.2).sub.m -- 
    
      wherein n and m are each, independently, an integer from 0 to 21, provided that n+m must be no less than 3 and no greater than 21. 
     (f) The compounds wherein R 1  is alkenyl containing two cis or trans double bonds. 
     (g) The compounds wherein R 1  is cis or trans alkenyl of the formula 
     
         CH.sub.3 (CH.sub.2).sub.n CH═CH(CH.sub.2).sub.m CH═CH(CH.sub.2).sub.p -- 
    
      wherein n and p are each, independently, an integer of from 0 to 18 and m is an integer from 1 to 19, provided that m+n+p must be no less than 1 and no greater than 19 and that R 1  cannot be linoleoyl. 
     (h) The compounds wherein R 1  is: 
     cis--CH 3  (CH 2 ) 5  CH═CH(CH 2 ) 7  -- 
     trans--CH 3  (CH 2 ) 5  CH═CH(CH 2 ) 7  -- 
     cis-CH 3  (CH 2 ) 10  CH═CH(CH 2 ) 4  -- 
     trans--CH 3  (CH 2 ) 10  CH═CH(CH 2 ) 4  -- 
     cis--CH 3  (CH 2 ) 7  CH═CH(CH 2 ) 7  -- 
     trans--CH 3  (CH 2 ) 7  CH═CH(CH 2 ) 7  -- 
     cis--CH 3  (CH 2 ) 5  CH═CH(CH 2 ) 9  -- 
     trans--CH 3  (CH 2 ) 5  CH═CH(CH 2 ) 9  -- 
     cis--CH 3  (CH 2 ) 7  CH═CH(CH 2 ) 9  -- 
     trans--CH 3  (CH 2 ) 7  CH═CH(CH 2 ) 9  -- 
     cis--CH 3  (CH 2 ) 7  CH═CH(CH 2 ) 11  -- 
     trans--CH 3  (CH 2 ) 7  CH═CH(CH 2 ) 11  -- 
     trans,trans--CH 3  (CH 2 ) 4  CH═CHCH 2  CH═CH(CH 2 ) 7  -- 
     cis,cis,cis--CH 3  CH 2  CH═CHCH 2  CH═CHCH 2  CH═CH--(CH 2 ) 7  --. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The compounds of Formula III inhibit the growth of pathogenic fungi, and are useful, therefore, for controlling the growth of fungi on environmental surfaces (as an antiseptic) or in treating infections caused by fungi. In particular, the compounds are active against Candida albicans and are, thus, especially useful for treating candidosis. The activity of the compounds can be assessed in standard microbiological test procedures, such as in vitro in agar plate disc diffusion tests or in agar dilution tests, or in vivo in tests in mice infected with C. albicans. The compounds are also active against Trichophyton mentagrophytes (a dermatophytic organism), Saccharomyces pastorianus, and Neurospora crassa. 
     The compounds of Formula III are prepared by acylating A-30912B nucleus at the α-amino group of dihydroxyornithine with the appropriate alkanoyl or alkenoyl side chain using methods conventional in the art for forming an amide bond. The acylation is accomplished, in general, by reacting the nucleus with an activated derivative of the alkanoic acid or alkenoic acid (R 1  CO 2  H) corresponding to the desired acyl side chain group (R 1  CO--). By the term &#34;activated derivative&#34; is meant a derivative which renders the carboxyl function of the acylating agent reactive to coupling with the primary amino group to form the amide bond which links the acyl side chain to the nucleus. Suitable activated derivatives, their methods of preparation, and their methods of use as acylating agents for a primary amine will be recognized by those skilled in the art. Preferred activated derivatives are: (a) an acid halide (e.g. acid chloride), (b) an acid anhydride (e.g. a alkoxyformic acid anhydride or aryloxyformic acid anhydride) or (c) an activated ester (e.g. a 2,4,5-trichlorophenyl ester). Other methods for activating the carboxyl function include reaction of the carboxylic acid with a carbonyldiimide (e.g. N,N-dicyclohexylcarbodiimide or N,N&#39;-diisopropylcarbodiimide) to give a reactive intermediate which, because of instability, is not isolated, the reaction with the primary amine being carried out in situ. 
     A preferred method for preparing the compounds of Formula III is by the active ester method. The use of the 2,4,5-trichlorophenyl ester of the desired alkanoic or alkenoic acid as the acylating agent is most preferred. In this method, an excess amount of the active ester is reacted with the nucleus at room temperature in a non-reactive organic solvent such as dimethyl formamide (DMF). The reaction time is not critical, although a time of about 6 to about 20 hours is preferred. At the conclusion of the reaction, the solvent is removed, and the residue is purified such as by reversed phase HPLC using LP-1/C18 as the stationary/phase and a mixture of H 2  O/CH 3  OH/CH 3  CN as the solvent system. 
     An alternative acylation method is a modified Schotten-Baumann procedure in which the nucleus is treated with the acid chloride of the desired alkanoic acid or alkenoic acid at an alkaline pH. In this method, an excess of the acid chloride in a non-reactive organic solvent (such as acetone) is added slowly to a solution of the nucleus in [KHPO 4  ]-buffer (pH 7.5 to 8.0) and acetone. The crude reaction product is separated from the reaction product by extraction into an immiscible organic solvent (chloroform and ethyl acetate). Final purification is by reversed-phase HPLC, as described above. 
     The alkanoic and alkenoic acids employed as starting materials for the acylation reaction, and the activated derivatives thereof (in particular, the acid chlorides and the 2,4,6-trichlorophenyl esters), are known compounds or can be prepared from known compounds by known methods. The 2,4,5-trichlorophenyl esters are conveniently made by treating the acid chloride of the alkanoic or alkenoic acid with 2,4,5-trichlorophenol in the presence of pyridine or by treating the free alkanoic or alkenoic acid with 2,4,5-trichlorophenol in the presence of N,N&#39;dicyclohexylcarbodiimide employed as a coupling agent. The 2,4,5-trichlorophenyl ester derivative can be purified by column chromatography over silica gel in toluene. 
     When employed systemically, the dosage of the compounds of Formula III will vary according to the particular compound being employed, the severity and nature of the infection, and the physical condition of the subject being treated. Therapy should be initiated at low dosages, the dosage 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 may be added, if desired, various conventional pharmaceutically acceptable preserving, buffering, solubilizing, or suspending agents. Other additives, such as saline or glucose may be added to make the solutions isotonic. The proportions and nature of such additives will be apparent to those skilled in the art. 
     When employed to treat vaginal candida infections, the compounds of Formula III can be administered in combination with pharmaceutically acceptable conventional excipients suitable for intravaginal use. Formulations adapted for intravaginal administration will be known to those skilled in the art. 
    
    
     The methods of making and using the compounds of the present invention are illustrated in the following examples: 
     EXAMPLE 1 
     n-Tridecanoyl Derivative of A-30912B Nucleus 
     The following procedure, which gives the preparation of the compound of Formula III wherein R 1  is CH 3  (CH 2 ) 11  --, illustrates the method of preparation of the compounds of Formula III by the &#34;active ester&#34; method. 
     A. Preparation of 2,4,5-trichlorophenyl n-tridecanoate. 
     A solution of n-tridecanoic acid (Sigma Chemical Co.) (12.5 g.), 2,4,5-trichlorophenol (11.5 g.), and N,N-dicyclohexylcarbodiimide (12.0 g.) in methylene chloride (650 ml.) is stirred at room temperature for 16 hours. The reaction mixture is then filtered and dried in vacuo to give 2,4,5-trichlorophenyl n-tridecanoate (22 g.). The material is purified by column chromatography over silica gel (Woelm) using toluene as the eluent. Fractions are monitored by TLC using a shortwave UV light for detection. Fractions containing the purified product are pooled and concentrated in vacuo to dryness. 
     B. Acylation of A-30912B Nucleus with 2,4,5-trichlorophenyl n-tridecanoate. 
     A solution of 2,4,5-trichlorophenyl n-tridecanoate (3.3 mmoles) and A-30912B nucleus (1 mmole) in dimethylformamide (DMF) (200 ml.) is stirred at room temperature for 16 hours. Removal of solvent in vacuo affords a residue. The residue is slurried with methylene chloride (300 ml.) for 45 minutes, and the mixture is filtered. The filtrate is discarded. The remaining solids are extracted with methanol (300 ml.) and the methanol extract is filtered and concentrated in vacuo to give a crude product. 
     The crude product is purified by reversed-phase HPLC as follows: 
     A sample of the crude product (1 g.), dissolved in methanol (5 ml.), is injected into a 1×32 in. stainless steel column packed with LP-1/C 18  resin (see Example 8). The column is eluted with a solvent system comprising 3:3:4 H 2  O/CH 3  OH/CH 3  CN. The elution is performed at a pressure of 1000-1500 psi with a flow rate of 11-12 ml./min using a LDC duplex pump (Milton-Roy). The effuent is monitered by an ultraviolet detector (ISCO-UA-5) at 280 nm. Fractions are collected every two minutes (21-24 ml.). The fractions containing the desired product are pooled and dried in vacuo to afford the title product. The purified product is analyzed by thin layer chromatography (TLC) using reversed-phase C 18  plates (Whatman KC 18 ) and a solvent system comprising 1:2:2 (v/v) H 2  O/CH 3  OH/CH 3  CN. After development, the plates are observed under U.V. light to detect the product. 
     EXAMPLE 2 
     Employing the method of Example 1, but substituting the appropriate alkanoic acid or alkenoic acid in Step A and the appropriate alkanoic or alkenoic acid 2,4,5-trichlorophenyl ester in Step B, there are obtained the derivatives of A-30912B nucleus shown below: 
     
         ______________________________________                                    
Alkanoyl and Alkenoyl Derivatives of A-30912B Nucleus                     
 ##STR7##                                                                 
R.sup.1                                                                   
______________________________________                                    
CH.sub.3 (CH.sub.2).sub.10                                                
CH.sub.3 (CH.sub.2).sub.12                                                
CH.sub.3 (CH.sub.2).sub.13                                                
CH.sub.3 (CH.sub.2).sub.14                                                
CH.sub.3 (CH.sub.2).sub.15                                                
CH.sub.3 (CH.sub.2).sub.17                                                
CH.sub.3 (CH.sub.2).sub.18                                                
CH.sub.3 (CH.sub.2).sub.19                                                
CH.sub.3 (CH.sub.2).sub.20                                                
cis-CH.sub.3 (CH.sub.2).sub.5 CHCH(CH.sub.2).sub.7                        
trans-CH.sub.3 (CH.sub.2).sub.5CHCH(CH.sub.2).sub.7                       
cis-CH.sub.3 (CH.sub.2).sub.10 CHCH(CH.sub.2).sub.4                       
trans-CH.sub.3 (CH.sub.2).sub.10 CHCH(CH.sub.2).sub.4                     
cis-CH.sub.3 (CH.sub.2).sub.7 CHCH(CH.sub.2).sub.7                        
trans-CH.sub.3 (CH.sub.2).sub.7 CHCH(CH.sub.2).sub.7                      
cis-CH.sub.3 (CH.sub.2).sub.5 CHCH(CH.sub.2).sub.9                        
trans-CH.sub.3 (CH.sub.2).sub.5 CHCH(CH.sub.2).sub.9                      
cis-CH.sub.3 (CH.sub.2).sub.7 CHCH(CH.sub.2).sub.9                        
trans-CH.sub.3 (CH.sub.2).sub.7 CHCH(CH.sub.2).sub.9                      
cis-CH.sub.3 (CH.sub.2).sub.7 CHCH(CH.sub.2).sub.11                       
trans-CH.sub.3 (CH.sub.2).sub.7 CHCH(CH.sub.2).sub.11                     
trans,trans-CH.sub.3 (CH.sub.2).sub.4 CHCHCH.sub.2 CHCH(CH.sub.2).sub.7   
cis,cis,cis-CH.sub.3 CH.sub.2 CHCHCH.sub.2 CHCHCH.sub.2 CHCH(CH.sub.2).sub
.7                                                                        
______________________________________                                    
 
    
     EXAMPLE 3 
     Preparation of A30912B Nucleus 
     A. Fermentation of Actinoplanes utahensis NRRL 12052 
     A stock culture of Actinoplanes utahensis NRRL 12052 is prepared and maintained on an agar slant. The medium used to prepare the slant is selected from one of the following: 
     
         ______________________________________                                    
MEDIUM A                                                                  
Ingredient             Amount                                             
______________________________________                                    
Baby oatmeal           60.0 g                                             
Yeast                   2.5 g                                             
K.sub.2 HPO.sub.4       1.0 g                                             
Czapek&#39;s mineral stock*                                                   
                        5.0 ml                                            
Agar                   25.0 g                                             
Deionized water        q.s. to 1 liter                                    
pH before autoclaving is about 5.9; adjust to pH 7.2                      
by addition of NaOH; after autoclaving, pH is about                       
6.7.                                                                      
______________________________________                                    
 *Czapek&#39;s mineral stock has the following composition:                   
 
    
     
         FeSO.sub.4 . 7H.sub.2 O (dissolved in                                     
2 ml conc HCl)          2 g                                               
KCl                    100 g                                              
MgSO.sub.4 . 7H.sub.2 O                                                   
                       100 g                                              
Deionized water        q.s. to 1 liter                                    
______________________________________                                    
 
    
     
         ______________________________________                                    
MEDIUM B                                                                  
Ingredient             Amount                                             
______________________________________                                    
Potato dextrin         5.0 g                                              
Yeast extract          0.5 g                                              
Enzymatic hydrolysate of casein*                                          
                       3.0 g                                              
Beef extract           0.5 g                                              
Dextrose               12.5 g                                             
Corn starch            5.0 g                                              
Meat peptone           5.0 g                                              
Blackstrap molasses    2.5 g                                              
MgSO.sub.4 . 7H.sub.2 O                                                   
                       0.25 g                                             
CaCO.sub.3             1.0 g                                              
Czapek&#39;s mineral stock 2.0 ml                                             
Agar                   20.0 g                                             
Deionized water        q.s. to 1 liter                                    
______________________________________                                    
 *N-Z-Amine A, Humko Sheffield Chemical, Lyndhurst, N.J.                  
 
    
     The slant is inoculated with Actinoplanes utahensis NRRL 12052, and the inoculated slant is incubated at 30° C. for about 8 to 10 days. About 1/2 of the slant growth is used to inoculate 50 ml of a vegetative medium having the following composition: 
     
         ______________________________________                                    
Ingredient            Amount                                              
______________________________________                                    
Baby oatmeal          20.0 g                                              
Sucrose               20.0 g                                              
Yeast                 2.5 g                                               
Distiller&#39;s Dried Grain*                                                  
                      5.0 g                                               
K.sub.2 HPO.sub.4     1.0 g                                               
Czapek&#39;s mineral stock                                                    
                      5.0 ml                                              
Deionized water       q.s. to 1 liter                                     
adjust to pH 7.4 with NaOH; after autoclaving, pH is                      
about 6.8.                                                                
______________________________________                                    
 *National Distillers Products Co., 99 Park Ave., New York, N.Y.          
 
    
     The inoculated vegetative medium is incubated in a 250-ml wide-mouth Erlenmeyer flask at 30° C. for about 72 hours on a shaker rotating through an arc two inches in diameter at 250 RPM. 
     This incubated vegetative medium may be used directly to inoculate a second-stage vegetative medium. Alternatively and preferably, it can be stored for later use by maintaining the culture in the vapor phase of liquid nitrogen. The culture is prepared for such storage in multiple small vials as follows: In each vial is placed 2 ml of incubated vegetative medium and 2 ml of a glycerol-lactose solution [see W. A. Dailey and C. E. Higgens, &#34;Preservation and Storage of Microorganisms in the Gas Phase of Liquid Nitrogen,&#34; Cryobiol 10, 364-367 (1973) for details]. The prepared suspensions are stored in the vapor phase of liquid nitrogen. 
     A stored suspension (1 ml) thus prepared is used to inoculate 50 ml of a first-stage vegetative medium (having the composition earlier described). The inoculated first-stage vegetative medium is incubated as above-described. 
     In order to provide a larger volume of inoculum, 10 ml of the incubated first-stage vegetative medium is 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 two-liter wide-mouth Erlenmeyer flask at 30° C. for about 48 hours on a shaker rotating through an arc two inches in diameter at 250 RPM. 
     Incubated second-stage vegetative medium (800 ml), prepared as above-described, is used to inoculate 100 liters of sterile production medium selected from one of the following: 
     
         ______________________________________                                    
MEDIUM I                                                                  
Ingredient           Amount (g/L)                                         
______________________________________                                    
Peanut meal          10.0                                                 
Soluble meat peptone 5.0                                                  
Sucrose              20.0                                                 
KH.sub.2 PO.sub.4    0.5                                                  
K.sub.2 HPO.sub.4    1.2                                                  
MgSO.sub.4 . 7H.sub.2 O                                                   
                     0.25                                                 
Tap water            q.s. to 1 liter                                      
______________________________________                                    
 
    
     The pH of the medium is about 6.9 after sterilization by autoclaving at 121° C. for 45 minutes at about 16-18 psi. 
     
         ______________________________________                                    
MEDIUM II                                                                 
Ingredient          Amount (g/L)                                          
______________________________________                                    
Sucrose             30.0                                                  
Peptone             5.0                                                   
K.sub.2 HPO.sub.4   1.0                                                   
KCl                 0.5                                                   
MgSO.sub.4 . 7H.sub.2 O                                                   
                    0.5                                                   
FeSO.sub.4 . 7H.sub.2 O                                                   
                    0.002                                                 
Deionized water     q.s. to 1 liter                                       
______________________________________                                    
 
    
     Adjust to pH 7.0 with HCl; after autoclaving, pH is about 7.0. 
     
         ______________________________________                                    
MEDIUM III                                                                
Ingredient          Amount (g/L)                                          
______________________________________                                    
Glucose             20.0                                                  
NH.sub.4 Cl         3.0                                                   
Na.sub.2 SO.sub.4   2.0                                                   
ZnCl.sub.2          0.019                                                 
MgCl.sub.2 . 6H.sub.2 O                                                   
                    0.304                                                 
FeCl.sub.3 . 6H.sub.2 O                                                   
                    0.062                                                 
MnCl.sub.2 . 4H.sub.2 O                                                   
                    0.035                                                 
CuCl.sub.2 . 2H.sub.2 O                                                   
                    0.005                                                 
CaCO.sub.3          6.0                                                   
KH.sub.2 PO.sub.4 * 0.67                                                  
Tap water           q.s. to 1 liter                                       
______________________________________                                    
 *Sterilized separately and added aseptically                             
 Final pH about 6.6.                                                      
 
    
     The inoculated production medium is allowed to ferment in a 165-liter fermentation tank at a temperature of about 30° C. for about 42 hours. The fermentation medium is stirred with conventional agitators at about 200 RPM and aerated with sterile air to maintain the dissolved oxygen level above 30% of air saturation at atmospheric pressure. 
     B. Deacylation of Antibiotic A-30912 Factor B 
     A fermentation of A. utahensis is carried out as described in Sect. A, using production medium I. After the culture is incubated for about 48 hours, A-30912 factor B, dissolved in a small amount of methanol, is added to the fermentation medium. 
     Deacylation of A-30912 factor B is monitored by paper-disc assay against Candida albicans or Neurospora crassa. The fermentation is allowed to continue until deacylation is complete as indicated by disappearance of activity. 
     C. Isolation of A-30912B Nucleus 
     Whole fermentation broth, obtained as described in Sect. B is filtered. The mycelial cake is discarded. The clear filtrate thus obtained is passed through a column containing HP-20 resin (DIAION High Porous Polymer, HP-Series, Mitsubishi Chemical Industries Limited, Tokyo, Japan). The effluent thus obtained is discarded. The column is then washed with up to eight column volumes of deionized water at pH 6.5-7.5 to remove residual filtered broth. This wash water is discarded. The column is then eluted with a water:methanol (7:3) solution. Elution is monitored using the following procedure: Two aliquots are taken from each eluted fraction. One of the aliquots is concentrated to a small volume and is treated with an acid chloride such as myristoyl chloride. This product and the other (untreated) aliquot are assayed for activity against Candida albicans. If the untreated aliquot does not have activity and the acylated aliquot does have activity, the fraction contains A-30912B nucleus. The eluate containing A-30912B nucleus is concentrated under vacuum to a small volume and lyophilized to give crude nucleus. 
     D. Purification of A-30912B Nucleus by Reversed-Phase Liquid Chromatography 
     Crude A-30912B nucleus, obtained as described in Section C, is dissolved in water:acetonitrile:acetic acid:pyridine (96:2:1:1). This solution is chromatographed on a column filled with Lichroprep RP-18, particle size 25-40 microns (MC/B Manufacturing Chemists, Inc. E/M, Cincinnati, Ohio). 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 90-100 psi, giving a flow rate of about 60 ml/minute, using the same solvent. Separation is monitored at 280 nm using a UV monitor (ISCO Absorption Monitor Model UA-5, Instrumentation Specialties Co., 4700 Superior Ave., Lincoln, Nebr. 68504) with an optical unit (ISCO Type 6). 
     On the basis of absorption at 280 nm, fractions containing A-30912B nucleus are combined, evaporated under vacuum and lyophilized to give purified A-30912B nucleus. 
     EXAMPLE 4 
     A-30912B nucleus is prepared and purified by the method of Example 3 except that tetrahydro-A-30912B is used as the substrate. 
     EXAMPLE 5 
     Preparation of the A-42355 Antibiotic Complex 
     A. Shake-Flask Fermentation 
     A culture of Aspergillus nidulans var. roseus NRRL 11440 is prepared and maintained on an agar slant prepared with medium having the following composition: 
     
         ______________________________________                                    
Ingredient           Amount                                               
______________________________________                                    
Glucose              5 g                                                  
Yeast extract        2 g                                                  
CaCO.sub.3           3 g                                                  
Vegetable juice*     200 ml                                               
Agar**               20 g                                                 
Deionized water      q.s. to 1 liter                                      
(initial pH 6.1)                                                          
______________________________________                                    
 *V-8 Juice, Campbell Soup Co., Camden, N.J.                              
 **Meer Corp.                                                             
 
    
     The slant is inoculated with Aspergillus nidulans var. roseus NRRL 11440, and the inoculated slant is incubated at 25° C. for about seven days. The mature slant culture is covered with water and scraped with a sterile loop to loosen the spores. The resulting suspension is further suspended in 10 ml of sterile deionized water. 
     One ml of the suspended slant growth is used to inoculate 55 ml of vegetative medium in a 250-ml flask. The vegetative medium has the following composition: 
     
         ______________________________________                                    
Ingredient             Amount                                             
______________________________________                                    
Sucrose                25     g                                           
Blackstrap molasses    36     g                                           
Corn-steep liquor      6      g                                           
Malt extract           10     g                                           
K.sub.2 HPO.sub.4      2      g                                           
Enzymatic hydrolysate                                                     
of casein*             10     g                                           
Tap water              1100   ml                                          
(initial pH 6.5-6.7)                                                      
______________________________________                                    
 *N-Z-Case, Humko Sheffield Chemical, Lyndhurst, N.J.                     
 
    
     The inoculated vegetative medium is incubated at 25° C. for 48 hours at 250 rpm on a rotary-type shaker. After 24 hours, the medium is homogenized for one minute at low speed in a blender (Waring type) and then returned to incubation 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 may be used to inoculate shake-flask fermentation culture medium or to inoculate a second-stage vegetative medium. Alternatively, it can be stored for later use by maintaining the culture in the vapor phase of liquid nitrogen. The culture is prepared for such storage in multiple small vials as follows: The vegetative cultures are mixed volume/volume with a suspending solution having the following composition: 
     
         ______________________________________                                    
Ingredient           Amount                                               
______________________________________                                    
Glycerol             20 ml                                                
Lactose              10 g                                                 
Deionized water      q.s. to 100 ml                                       
______________________________________                                    
 
    
     The prepared suspensions are distributed in small sterile screw-cap tubes (4 ml per tube). These tubes are stored in the vapor phase of liquid nitrogen. 
     A stored suspension thus prepared can be used to inoculate either agar slants or liquid seed media. Slants are incubated at 25° C. in the light for 7 days. 
     B. Tank Fermentation 
     In order to provide a larger volume of inoculum, 10 ml of incubated first-stage vegetative culture is 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° C. for 24 hours on a shaker rotating through an arc two inches in diameter at 250 rpm. 
     Incubated second-stage medium (800 ml), prepared as above described, is used to inoculate 100 liters of sterile production medium selected from one of the following: 
     
         ______________________________________                                    
MEDIUM IV                                                                 
Ingredient         Amount                                                 
______________________________________                                    
ZnSO.sub.4 . 7H.sub.2 O                                                   
                   0.00455 g/L                                            
Soluble meat peptone*                                                     
                   30.5 g/L                                               
Soybean meal       15.5 g/L                                               
Tapioca dextrin**  2.0 g/L                                                
Blackstrap molasses                                                       
                   10.5 g/L                                               
Enzymatic hydrolysate                                                     
of casein***       8.5 g/L                                                
Na.sub.2 HPO.sub.4 4.5 g/L                                                
MgSO.sub.4 . 7H.sub.2 O                                                   
                   5.5 g/L                                                
FeSO.sub.4 . 7H.sub.2 O                                                   
                   0.1 g/L                                                
Cottonseed oil     40.0 ml                                                
(Antifoam)****     1.0 ml                                                 
Tap water          1000.0 ml                                              
(initial pH 6.8-7.0)                                                      
______________________________________                                    
 *O.M. Peptone, Amber Laboratories, Juneau, Wisc.                         
 **Stadex 11, A.E. Staley Co., Decatur, Ill.                              
 ***N-Z-Amine A, Humko Sheffield Chemical, Lyndhurst, N.J.                
 ****P2000, Dow Corning, Midland, Michigan                                
 
    
     
         ______________________________________                                    
MEDIUM V                                                                  
Ingredient            Amount                                              
______________________________________                                    
Glucose               2.5%                                                
Starch                1.0%                                                
Soluble meat peptone* 1.0%                                                
Blackstrap molasses   1.0%                                                
CaCO.sub.3            0.2%                                                
MgSO.sub.4 . 7H.sub.2 O                                                   
                      0.05%                                               
Enzymatic hydrolysate of                                                  
casein**              0.4%                                                
(Antifoam)***         0.02%                                               
Tap water             q.s. to volume                                      
______________________________________                                    
 *O.M. Peptone                                                            
 **N-Z-Amine A                                                            
 ***Antifoam &#34;A&#34;, Dow Corning                                             
 
    
     The inoculated production medium is allowed to ferment in a 165-liter fermentation tank at a temperature of 25° C. for about 7 days. The fermentation medium is aerated with sterile air, maintaining the dissolved oxygen level above approximately 50 percent of air saturation. 
     C. Third-Stage Vegetative Medium 
     Whenever the fermentation is carried out in tanks larger than those used for 100-liter fermentation, it is recommended that a third-stage vegetative culture be used to seed the larger tank. A preferred third-stage vegetative medium has the following composition: 
     
         ______________________________________                                    
Ingredient             Amount                                             
______________________________________                                    
Sucrose                25     g                                           
Blackstrap molasses    25     g                                           
Corn-steep liquor      6      g                                           
Enzymatic hydrolysate                                                     
of casein*             10     g                                           
Malt extract           10     g                                           
K.sub.2 HPO.sub.4      2      g                                           
Tap water              1000   ml                                          
(initial pH 6.1)                                                          
______________________________________                                    
 *N-Z-Case                                                                
 
    
     EXAMPLE 6 
     Separation of the A-42355 Antibiotic Complex 
     Whole fermentation broth (4127 liters), obtained by the method described in Example 5 using production medium V, is stirred thoroughly with methanol (4280 liters) for one hour and then is filtered, using a filter aid (Hyflo Super-cel, a diatomaceous earth, Johns-Manville Products Corp.). The pH of the filtrate is adjusted to pH 4.0 by the addition of 5 N HCl. The acidified filtrate is extracted twice with equal volumes of chloroform. The chloroform extracts are combined and concentrated under vacuum to a volume of about 20 liters. This concentrate is added to about 200 liters of diethyl ether to precipitate the A-42355 complex. The precipitate is separated by filtration to give 2775 g of the A-42355 complex as a gray-white powder. 
     EXAMPLE 7 
     Isolation of A-30912 Factor B 
     A-42355 complex is separated as described in Example 6 except that the concentrated chloroform extracts (285 L) are chromatographed over a silica-gel column (150 L of Grace silica-gel, grade 62) at a flow rate of 2 L/min. The column is washed with chloroform (200 L), eluted with acetonitrile (500 L), and then continuously eluted with acetonitrile:water (98:2) at a flow rate of 1 L/min. Fractions having a volume of approximately 200 L are collected and analyzed individually for biological activity. The bioassay is performed by a paper-disc assay on agar plates seeded with Candida albicans. Fractions 77 through 103 (1365 L) are combined and concentrated under vacuum. The concentrated solution (4.5 L) contains a precipitate which is removed by filtration to give 119 g of factor B-enriched A-42355 complex. The filtrate is concentrated to dryness; the residue obtained is redissolved in an appropriate volume of methanol. The methanol solution is added to diethyl ether (10 volumes) to precipitate the factor-B-containing antibiotic complex. This precipitate is also separated by filtration and dried to give an additional 24 g of factor-B-enriched A-42355 complex as a gray powder. 
     Factor-B-enriched A-42355 complex thus obtained (1.0 g) is dissolved in 8 ml of methanol:water:acetonitrile (7:2:1). This solution is filtered and introduced onto a silica-gel column (3.7-cm I.D.×33-cm Michel-Miller Column) through a loop with the aid of a valve system. The column is packed with LP-1/C 18  silica-gel reversed-phase resin (10-20 microns), prepared as described in Example 8, in methanol:water:acetonitrile (7:2:1) through a loop with the aid of a valve system. The slurry packing procedure described in Example 9 is used. The solvent is moved through the column at a flow rate of 10 ml/min at ca. 100 psi, using an F.M.I. pump with valveless piston design. One fraction is collected every minute. Elution of the antibiotic is monitored using a UV monitor at 280 nm as in Example 3. Fractions 102-110 are combined and concentrated under vacuum to give an oil. The oil is dissolved in a small volume of tert-butanol and lyophilized to give 22 mg of A-30912 factor B. 
     The individual A-30912 factors can be identified by the use of thin-layer chromatography (TLC). The R f  values of A-30912 factors A-G, using silica gel (Merck, Darmstadt) TLC, a benzene:methanol (7:3) solvent system, and Candida albicans bioautography are given in Table I. 
     
                       TABLE I                                                     
______________________________________                                    
A-30912 Factor     R.sub.f Value                                          
______________________________________                                    
A                  0.35                                                   
B                  0.45                                                   
C                  0.54                                                   
D                  0.59                                                   
E                  0.27                                                   
F                  0.18                                                   
G                  0.13                                                   
______________________________________                                    
 
    
     The approximate R f  values of A-30912 factors A, B, C, D, and H in different solvent systems, using silica gel TLC (Merck-Darmstadt silica gel #60 plates, 20×20 cm) and Candida albicans bioautography, are given in Table II. 
     
                       TABLE II                                                    
______________________________________                                    
           R.sub.f Values - Solvent Systems                               
A-30912 Factor                                                            
             a        b        c      d                                   
______________________________________                                    
Factor A     0.28     0.14     0.28   0.43                                
Factor B     0.39     0.21     0.42   0.47                                
Factor C     0.46     0.31     0.51   0.58                                
Factor D     0.50     0.38     0.57   0.61                                
Factor H     0.42     0.27     0.36   0.53                                
______________________________________                                    
 Solvent Systems                                                          
 a: ethyl acetate:methanol (3:2)                                          
 b: ethyl acetate:methanol (7:3)                                          
 c: acetonitrile:water (95:5)                                             
 d: ethyl acetate:ethanol:acetic acid (40:60:0.25)                        
 
    
     A-30912 factors A, B, D and H can also be indentified by analytical HPLPLC using the following conditions: 
     
         ______________________________________                                    
Column:         glass, 0.8 × 15.0 cm                                
Packing:        Nucleosil® 10-C.sub.18 (Machery-                      
                Nagel and Company); packed                                
                using slurry-packing pro-                                 
                cedure of Example 8                                       
Solvent:        methanol:water:aceto-                                     
                nitrile (7:2:1)                                           
Sample Volume:  8 mcl                                                     
Sample Size:    8 mcg                                                     
Column Temperature:                                                       
                ambient                                                   
Flow Rate:      1.8 ml/min                                                
Pressure:       ca. 200 psi                                               
Detector:       UV at 222 nm (ISCO Model                                  
                1800 Variable Wavelength                                  
                UV-Visible Absorbance                                     
                Monitor)                                                  
Pump:           LDC Duplex Minipump                                       
Injection:      loop injection                                            
______________________________________                                    
 
    
     The approximate retention times for A-30912 factors A, B, D, and H under these conditions are summarized in Table III. 
     
                       TABLE III                                                   
______________________________________                                    
                  Retention Time                                          
A-30912 Factor    (seconds)                                               
______________________________________                                    
A                 792                                                     
B                 870                                                     
H                 990                                                     
D                 1,140                                                   
______________________________________                                    
 
    
     EXAMPLE 8 
     Preparation of Silica Gel/C 18  Reversed Phase Resin 
     Step 1: Hydrolysis 
     LP-1 silica gel (1000 g from Quantum Corp., now Whatman) is added to a mixture of concentrated sulfuric acid (1650 ml) and concentrated nitric acid (1650 ml) in a 5-L round-bottom flask and shaken for proper suspension. The mixture is heated on a steam bath overnight (16 hours) with a water-jacketed condenser attached to the flask. 
     The mixture is cooled in an ice bath and carefully filtered using a sintered-glass funnel. The silica gel is washed with deionized water until the pH is neutral. The silica gel is then washed with acetone (4 L) and dried under vacuum at 100° C. for 2 days. 
     Step 2: First Silylation 
     The dry silica gel from Step 1 is transferred to a round-bottom flask and suspended in toluene (3.5 L). The flask is heated on a steam bath for 2 hours to azeotrope off some residual water. Octadecyltrichlorosilane (321 ml, Aldrich Chemical Company) is added, and the reaction mixture is refluxed overnight (16 hours) with slow mechanical stirring at about 60° C. Care is taken so that the stirrer does not reach near the bottom of the flask. This is to prevent grinding the silica gel particles. 
     The mixture is allowed to cool. The silanized silica gel is collected, washed with toluene (3 L) and acetone (3 L), and then air-dried overnight (16-20 hours). The dried silica gel is suspended in 3.5 L of acetonitrile:water (1:1) in a 5-L flask, stirred carefully at room temperature for 2 hours, filtered, washed with acetone (3 L) and air-dried overnight. 
     Step 3: Second Silylation 
     The procedure from the first silylation is repeated using 200 ml of octadecyltrichlorosilane. The suspension is refluxed at 60° C. for 2 hours while stirring carefully. The final product is recovered by filtration, washed with toluene (3 L) and methanol (6 L), and then dried under vacuum at 50° C. overnight (16-20 hours). 
     EXAMPLE 9 
     Slurry Packing Procedure for Michel-Miller Columns General Information 
     This procedure is employed for packing a silica gel C 18  reversed phase resin such as that prepared by the method of Example 8. 
     Generally, a pressure of less than 200 psi and flow rates between 5-40 ml/minute are required for this slurry packing technique; this is dependent on column volume and size. Packing pressure should exceed the pressure used during actual separation by 30-50 psi; this will assure no further compression of the adsorbent during separation runs. 
     A sudden decrease in pressure may cause cracks or channels to form in the packing material, which would greatly reduce column efficiency. Therefore, it is important to let the pressure drop slowly to zero whenever the pump is turned off. 
     The approximate volume of columns (Ace Glass Cat. No., unpacked) are No. 5795-04, 12 ml; No. 5795-10, 110 ml; No. 5795-16, 300 ml; No. 5795-24, 635 ml; and No. 5796-34, 34 ml. 
     The time required to pack a glass column will vary from minutes to several hours depending on column size and the experience of the scientist. 
     EXAMPLE 
     1. Connect glass column to a reservoir column via coupling (volume of reservoir column should be twice that of the column). Place both columns in vertical positions (reservoir column above). 
     2. Weigh out packing material (ca. 100 g for 200 ml column). 
     3. Add ca. five volumes of solvent to packing material; use a mixture of 70-80% methanol and 20-30% water. 
     4. Shake well until all particles are wetted, let stand overnight or longer to assure complete soaking of particles by solvent. Decant supernatant liquid. 
     5. Slurry the resin with sufficient solvent to fill reservoir column. Pour swiftly into reservoir. The column must be pre-filled with the same solvent and the reservoir column should be partly filled with solvent before slurry is poured. The use of larger slurry volumes may also provide good results; however, this will require (a) larger reservoir or (b) multiple reservoir fillings during the packing procedure. 
     6. Close reservoir with the Teflon plug beneath the column (see FIG. 1 of U.S. Pat. No. 4,131,547, plug No. 3); connect to pump; and immediately start pumping solvent through system at maximum flow rate if Ace Cat. No. 13265-25 Pump or similar solvent-delivery system is used (ca. 20 ml/minute). 
     7. Continue until column is completely filled with adsorbent. Pressure should not exceed maximum tolerance of column during this operation (ca. 200 psi for large columns and 300 psi for analytical columns). In most cases, pressures less than 200 psi will be sufficient. 
     8. Should pressure exceed maximum values, reduce flowrate; pressure will drop. 
     9. After column has been filled with adsorbent, turn off pump; let pressure drop to zero; disconnect reservoir; replace reservoir with a pre-column; fill pre-column with solvent and small amount of adsorbent; and pump at maximum pressure until column is completely packed. For additional information, see general procedure. 
     Always allow pressure to decrease slowly after turning off pump--this will prevent formation of any cracks or channels in the packing material. 
     10. Relieve pressure and disconnect pre-column carefully. With small spatula remove a few mm (2-4) of packing from top of column; place 1 or 2 filter(s) in top of column; gently depress to top of packing material, and place Teflon plug on top of column until seal is confirmed. Connect column to pump, put pressure on (usually less than 200 psi) and observe through glass wall on top of column if resin is packing any further. If packing material should continue to settle (this may be the case with larger columns), some dead space or channelling will appear and step 9 should be repeated. 
     EXAMPLE 10 
     Preparation of Tetrahydro-A-30912B 
     A-30912 factor B is dissolved in ethanol. PtO 2  in absolute ethanol is reduced to form Pt, which in turn is used to reduce the A-30912 factor B catalytically, using hydrogenation under positive pressure until the reaction is complete (about 2-3 hours). The reaction mixture is filtered and concentrated under vacuum. The residue is dissolved in a small amount of tert-butanol and lyophilized to give tetrahydro-A-30912B.