Derivatives of A-30912A nucleus

BACKGROUND OF THE INVENTION 
This invention relates to novel semisynthetic antifungal compounds which 
are prepared by the acylation of the cyclic peptide nucleus produced by 
the enzymatic deacylation of antibiotic A30912 factor A. 
Antibiotic A-30912 factor A is an antifungal cyclic peptide having the 
formula: 
##STR9## 
wherein R is the linoleoyl group 
##STR10## 
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 B, C, D, E, and G. The A-30912 complex and 
the individual factors A through G are described by M. Hoehn and K. Michel 
in U.S. Pat. No. 4,024,245. 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 A has also been found to be identical to antibiotic echinocandin B 
[see F. Benz et al., Helv. Chim. Acta, 57, 2459 (1974) and Swiss Pat. No. 
568,386] and to antibiotic SL 7810/F [see C. Keller-Juslen et al. 
Tetrahedron Letters, 4147 (1976) and Belgium Pat. No. 834,289]. 
Antibiotic A-30912 factor A 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 NRRL 8112 (see 
U.S. Pat. No. 4,024,246); (c) Aspergillus nidulans var. echinulatus 
A-32204 as described in Swiss Pat. No. 568,386; (d) Aspergillus rugulosus 
NRRL 8039 (see Belgian Pat. No. 834,289); or (e) 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 Mar. 
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 A, 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 96, 97, and 98, herein, illustrate the preparation 
of the A-42355 complex and the isolation and purification of A-30912 
factor A 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 Ser. No. 46,875, filed June 8, 1979 (now abandoned). 
In the A-30912 factor A molecule (Formula I), the linoleoyl side chain (R) 
is attached at the cyclic peptide nucleus at the .alpha.-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 A is added to a culture of the microorganism and 
the culture is allowed to incubate with the substrate until the 
deacylation is substantially complete. The cyclic nucleus thereby obtained 
is separated from the fermentation broth by methods known in the art. 
Unlike antibiotic A-30912 factor A, 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 A, is depicted in Formula II. 
##STR11## 
Removal of the side chain group affords a free primary .alpha.-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 "A-30912A nucleus." As will be apparent to those skilled in the 
art, A-30912A 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-30912A nucleus from antibiotic A-30912 factor A 
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 "A-30912A NUCLEUS", Ser. No. 103,017, which was filed 
Dec. 13, 1979. A continuation-in-part application of this application, 
with the corresponding Ser. No. 103,149, is being filed herewith this even 
date, the full disclosure of which is incorporated herein by reference. 
Example 93, herein, illustrates the preparation of A-30912A nucleus by 
fermentation using antibiotic A-30912 factor A 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.degree. 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 other antibiotic substances possess the same nucleus as 
that of antibiotic A-30912 factor A. These antibiotics differ from 
antibiotic A-30912 factor A in that different acyl groups are present in 
place of the linoleoyl group (R) in Formula I. Such antibiotics are: (a) 
tetrahydro-A-30912 factor A (tetrahydro-SL 7810/F; tetrahydro echinocandin 
B) described in Belgium Pat. No. 834,289 and by F. Benz et al., Helv. 
Chim. Acta, 57 2459 (1974), which compound is depicted in Formula I when R 
is stearoyl; and (b) aculaecin A, which is a component of the aculaecin 
complex (prepared by fermentation using Aspergillus aculeatus NRRL 8075) 
and is described by K. Mizuno et al., in U.S. Pat. No. 3,978,210. As is 
discussed in Belgium Pat. No. 859,067, in aculaecin A the palmitoyl side 
chain is present in place of linoleoyl. Tetrahydro-A-30912 factor A can be 
prepared from antibiotic A-30912 factor A by catalytic hydrogenation using 
PtO.sub.2 in ethanol under positive pressure. Both tetrahydro-A-30912 
factor A and aculaecin A can be employed as substrates 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 the A-30912A nucleus (Formula II). The compounds of the present 
invention have the chemical structure depicted in Formula III: 
##STR12## 
wherein R.sup.1 is an N-alkanoyl amino acyl group of the formula 
##STR13## 
wherein: W is a divalent aminoacyl radical of the formula: 
##STR14## 
wherein A is C.sub.1 -C.sub.10 alkylene or C.sub.5 -C.sub.6 cycloalkylene; 
##STR15## 
wherein R.sup.3 is hydroxymethyl, hydroxyethyl, mercaptomethyl, 
mercaptoethyl, methylthioethyl, 2-thienyl, 3-indole-methyl, phenyl, 
benzyl, or substituted phenyl or substituted benzyl in which the benzene 
ring thereof is substituted with chloro, bromo, iodo, nitro, C.sub.1 
-C.sub.3 alkyl, hydroxy, C.sub.1 -C.sub.3 alkylthio, carbamyl, or C.sub.1 
-C.sub.3 alkylcarbamyl; 
##STR16## 
wherein X is hydrogen chloro, bromo, iodo, nitro, C.sub.1 -C.sub.3 alkyl, 
hydroxy, C.sub.1 -C.sub.3 alkoxy, mercapto, C.sub.1 -C.sub.3 alkylthio, 
carbamyl, or C.sub.1 -C.sub.3 alkylcarbamyl; 
##STR17## 
wherein X.sup.1 is chloro, bromo, or iodo; 
##STR18## 
wherein B is a divalent radical of the formula: --(CH.sub.2).sub.n --, 
wherein n is an integer from 1 to 3; --CH.dbd.CH--; --CH.dbd.CH--CH.sub.2 
--; or 
##STR19## 
and R.sup.2 is C.sub.1 -C.sub.17 alkyl or C.sub.2 -C.sub.17 alkenyl. 
As employed herein the terms "alkylene", "alkyl", "alkoxy", "alkylthio", 
and "alkenyl" comprehend both straight and branched hydrocarbon chains. 
"Alkyl" means a univalent saturated hydrocarbon radical. "Alkenyl" means a 
univalent unsaturated hydrocarbon radical containing one, two, or three 
double bonds, which may be oriented in the cis or trans configuration. 
"Alkylene" means a divalent saturated hydrocarbon radical. "Cycloalkylene" 
means a divalent cyclic saturated hydrocarbon radical. 
Illustrative C.sub.1 -C.sub.10 alkylene radicals, which are preferred for 
purposes of this invention are: 
--CH.sub.2 --; 
##STR20## 
in which R.sup.5 is C.sub.1 -C.sub.4 alkyl (i.e., methyl, ethyl, n-propyl, 
i-propyl, n-butyl, t-butyl, i-butyl, or 1-methylpropyl); 
--(CH.sub.2).sub.m in which m is an integer from 2 to 10; and 
##STR21## 
in which p is an integer from 1 to 8 and q is an integer from 0 to 7, 
provided that n+m must be no greater than 8. 
Illustrative C.sub.1 -C.sub.17 alkyl groups which are preferred for the 
purposes of this invention are: 
(a) CH.sub.3 --; 
(b) --(CH.sub.2).sub.n CH.sub.3 wherein n is an integer from 1 to 16; and 
##STR22## 
wherein r and s are independently, an integer from 0 to 14 provided that 
r+s can be no greater than 14. Illustrative C.sub.2 -C.sub.17 alkenyl 
radicals, which are preferred for the purpose of this invention, are 
(a) --(CH.sub.2).sub.t --CH.dbd.CH--(CH.sub.2).sub.u --CH.sub.3 wherein t 
and u are independently, an integer from 0 to 14 provided that t+u can be 
no greater than 14. 
(b) --(CH.sub.2).sub.v --CH.dbd.CH--(CH.sub.2).sub.y 
--CH.dbd.CH--(CH.sub.2).sub.z --CH.sub.3 wherein v and z are 
independently, an integer from 0 to 11 and y is an integer from 1 to 12 
provided that v+y+z can be no greater than 11. 
In particular, the following embodiments of the C.sub.1 -C.sub.17 alkyl 
groups are preferred: 
CH.sub.3 -- 
CH.sub.3 (CH.sub.2).sub.5 -- 
CH.sub.3 (CH.sub.2).sub.6 -- 
CH.sub.3 (CH.sub.2).sub.8 -- 
CH.sub.3 (CH.sub.2).sub.10 -- 
CH.sub.3 (CH.sub.2).sub.12 -- 
CH.sub.3 (CH.sub.2).sub.14 -- 
CH.sub.3 (CH.sub.2).sub.16 -- 
In particular, the following embodiments of the C.sub.2 -C.sub.17 alkenyl 
groups are preferred: 
cis-CH.sub.3 (CH.sub.2).sub.5 CH.dbd.CH(CH.sub.2).sub.7 -- 
trans-CH.sub.3 (CH.sub.2).sub.5 CH.dbd.CH(CH.sub.2).sub.7 -- 
cis-CH.sub.3 (CH.sub.2).sub.10 CH.dbd.CH(CH.sub.2).sub.4 -- 
trans-CH.sub.3 (CH.sub.2).sub.10 CH.dbd.CH(CH.sub.2).sub.4 -- 
cis-CH.sub.3 (CH.sub.2).sub.7 CH.dbd.CH(CH.sub.2).sub.7 -- 
trans-CH.sub.3 (CH.sub.2).sub.7 CH.dbd.CH(CH.sub.2).sub.7 -- 
cis-CH.sub.3 (CH.sub.2).sub.5 CH.dbd.CH(CH.sub.2).sub.9 -- 
trans-CH.sub.3 (CH.sub.2).sub.5 CH.dbd.CH(CH.sub.2).sub.9 -- 
cis, cis-CH.sub.3 (CH.sub.2).sub.4 CH.dbd.CHCH.sub.2 
CH.dbd.CH(CH.sub.2).sub.7 -- 
trans, trans-CH.sub.3 (CH.sub.2).sub.4 CH.dbd.CHCH.sub.2 
CH.dbd.CH(CH.sub.2).sub.7 -- 
cis,cis,cis-CH.sub.3 CH.sub.2 CH.dbd.CHCH.sub.2 CH.dbd.CHCH.sub.2 
CH.dbd.CH--(CH.sub.2).sub.7 --. 
When "W" is a divalent radical of the formula 
##STR23## 
it will be recognized by those skilled in the art that the 
##STR24## 
function and the --NH-- function may be oriented on the benzene ring in 
the ortho, meta, or para configuration relative to each other. The 
substituent represented by X may be substituted at any available position 
of the benzene ring. Preferred embodiments are those in which X is 
hydrogen and the 
##STR25## 
and --NH-- functions are oriented in the para configuration. 
The terms "substituted phenyl" and "substituted benzyl", as defined by 
R.sub.3 in Formula III, contemplate substitution of a group at any of the 
available positions in the benzene ring--i.e. the substituent may be in 
the ortho, meta, or para configuration. The term "C.sub.1 -C.sub.3 alkyl" 
as defined by R.sub.3 or X in Formula III includes the methyl, ethyl, 
n-propyl, or i-propyl groups. 
DETAILED DESCRIPTION OF THE INVENTION 
The compounds of Formula III inhibit the growth of pathogenic fungi as 
evidenced by standard biological test procedures. The compounds are 
useful, therefore, for controlling the growth of fungi on environmental 
surfaces (as an antiseptic) or in treating infections caused by fungi. The 
antifungal activity of the compounds has been demonstrated against Candida 
albicans in vitro in agar plate disc diffusion tests and in agar dilution 
tests, or in vivo in tests in mice infected with C. albicans. Thus, the 
compounds are particularly useful in treating infections caused by strains 
of C. albicans (candidosis). The compounds of Formula III have also shown 
activity in vitro in agar-plate disc diffusion tests against Trichophyton 
mentagrophytes, a dermatophytic organism. Activity has also been found in 
in vitro agar plate disc diffusion tests against Saccharomyces 
pastorianus, and Neurospora crassa. Certain compounds (as shown in Example 
92, Table 8) give significant blood levels upon oral administration in 
mice. 
When given to a dog by intravenous administration, 100 mg/kg per day for 
five days, the compound of Formula III wherein R is 
n-dodecanoyl-p-aminobenzoyl showed no outward signs of toxicity, although 
increased SGPT levels were observed. 
The compounds of Formula III are prepared by acylating A-30912A nucleus at 
the .alpha.-amino group of dihydroxyornithine with the appropriate 
N-alkanoyl aminoacyl or N-alkenoyl amino acyl 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 acid (Formula IV) corresponding to the desired acyl side 
chain group. 
##STR26## 
(W and R.sup.2 have the meaning described herein supra). By the term 
"activated derivative" 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. 
an alkoxyformic acid anhydride or aryloxyformic acid anhydride) or (c) an 
activated ester (e.g. a 2,4,5-trichlorophenyl ester, a 
N-hydroxybenztriazole ester, or an N-hydroxysuccinimide 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'-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 N-alkanoylamino acid or N-alkenoylamino acid (Formula IV) 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 
nonreactive organic solvent such as dimethyl formamide (DMF). The reaction 
time is not critical, although a time of about 15 to about 18 hours is 
preferred. At the conclusion of the reaction, the solvent is removed, and 
the residue is purified such as by column chromatography using silica gel 
as the stationary phase and a mixture of ethyl acetate/methanol (3:2, v/v) 
as the solvent system. 
The 2,4,5-trichlorophenyl esters of the N-alkanoylamino acids or 
N-alkenoylamino acids can be prepared conveniently by treating the desired 
amino acid (Formula IV) with 2,4,5-trichlorophenol in the presence of a 
coupling agent, such as N,N'-dicyclohexylcarbodiimide. Other methods 
suitable for preparing amino acid esters will be apparent to those skilled 
in the art. 
The N-alkanoylamino acids or N-alkenoylamino acids are either known 
compounds or they can be made by acylating the appropriate amino acid with 
the appropriate alkanoyl or alkenoyl group using conventional methods, 
such as those described herein supra. A preferred way of preparing the 
N-alkanoylamino acids is by treating the appropriate amino acid with an 
alkanoic acid chloride in pyridine. The alkanoic acids or alkenoic acids, 
the activated derivatives thereof, and the amino acids employed in the 
preparation of the products of this invention are either known compounds 
or they can be made by known methods or by modification of known methods 
which will be apparent to those skilled in the art. 
If a particular amino acid contains an acylable functional group other than 
the amino group, it will be understood by those skilled in the art that 
such a group must be protected prior to reaction of the amino acid with 
the reagent employed to attach the alkanoyl or alkenoyl group. Suitable 
protecting groups can be any group known in the art to be useful for the 
protection of a side chain functional group in peptide synthesis. Such 
groups are well known, and the selection of a particular protecting group 
and its method of use will be readily known to one skilled in the art 
[see, for example, "Protective Groups In Organic Chemistry", M. McOmie, 
Editor, Plenum Press, N.Y., 1973]. 
It will be recognized that certain amino acids employed in the synthesis of 
the products of this invention may exist in optically active forms, and 
both the natural configuration (L-configuration) and unnatural 
configuration (D-configuration) may be employed as starting materials and 
will give products which are within the contemplation of this invention. 
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. 
Certain compounds of Formula III give significant blood levels after oral 
administration (see Example 92, Table 8) and can be administered 
systemically by the oral route. For oral use, such compounds can be 
administered in combination with pharmaceutically acceptable carriers or 
excipients in the form of capsules, tablets or powders. The nature and 
proportion of such carriers or excipients of which will be recognized by 
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 
EXAMPLES 1-30 
Table 1, below, gives the preparation of various N-alkanoyl amino acids. 
The compounds shown in Table 1 are prepared according to the following 
general procedure: 
The appropriate alkanoic acid chloride is added dropwise to the appropriate 
amino acid (1:1 mole ratio) dissolved in pyridine. The amount of pyridine 
employed should be such as to make the concentration of reactants between 
0.1 to 0.2 M. The solution is stirred at room temperature for about 3 to 6 
hours, after which it is poured into a large volume of water. The product 
precipitates from solution and is collected by filtration and crystallized 
from methanol. 
TABLE 1 
__________________________________________________________________________ 
Preparation of N-alkanoyl Amino Acids 
__________________________________________________________________________ 
Example 
Alkanoic acid chloride 
Amino Acid 
No. Formula wt. Formula wt. 
__________________________________________________________________________ 
1 CH.sub.3 (CH.sub.2).sub.10 COCl 
3.00 
g NH.sub.2 CH(CH.sub.2 C.sub.6 H.sub.5)CO.sub.2 
2.0 
g. 
2 CH.sub.3 (CH.sub.2).sub.10 COCl 
234 
mg NH.sub.2 (CH).sub.4 CO.sub.2 H 
482 
mg. 
3 CH.sub.3 (CH.sub.2).sub.10 COCl 
21.85 
g. NH.sub.2 (CH.sub.2).sub.10 CO.sub.2 H 
20.1 
mg. 
4 CH.sub.3 (CH.sub.2).sub.10 COCl 
11.09 
g. 
##STR27## 6.2 
g. 
5 CH.sub.3 (CH.sub.2).sub.10 COCl 
2.19 
g. 
##STR28## 1.37 
g. 
6 CH.sub.3 (CH.sub.2).sub.10 COCl 
437 
mg. 
##STR29## 306 
mg. 
7 CH.sub.3 (CH.sub.2).sub.10 COCl 
2.17 
g. 
##STR30## 2.06 
g. 
8 CH.sub.3 (CH.sub.2).sub.10 COCl 
2.17 
g. 
##STR31## 3.89 
g. 
9 CH.sub.3 (CH.sub.2).sub.10 COCl 
2.17 
g. 
##STR32## 1.51 
g. 
10 CH.sub.3 (CH.sub.2).sub.10 COCl 
2.17 
g. 
##STR33## 1.51 
g. 
11 CH.sub.3 (CH.sub.2).sub.10 COCl 
2.17 
g. 
##STR34## 1.67 
g. 
12 CH.sub.3 (CH.sub.2).sub.10 COCl 
21.85 
g. 
##STR35## 15.1 
g. 
13 CH.sub.3 (CH.sub.2).sub.10 COCl 
2.19 
g. 
##STR36## 1.6 
g. 
14 CH.sub.3 (CH.sub.2).sub.10 COCl 
21.85 
g. 
##STR37## 19.4 
g. 
15 CH.sub.3 (CH.sub.2).sub.10 COCl 
8.52 
g. 
##STR38## 5.4 
g. 
16 CH.sub.3 (CH.sub.2).sub.5 COCl 
14.85 
g. NH.sub.2 (CH.sub.2).sub.10 CO.sub.2 H 
20.1 
g. 
17 CH.sub.3 COCl 
785 
mg. 
##STR39## 1.37 
g. 
18 CH.sub.3 (CH.sub.2).sub.5 COCl 
1.49 
g. 
##STR40## 1.37 
g. 
19 CH.sub.3 (CH.sub.2).sub.8 COCl 
1.91 
g. 
##STR41## 1.37 
g. 
20 CH.sub.3 (CH.sub.2).sub.12 COCl 
2.46 
g. 
##STR42## 1.37 
g. 
21 CH.sub.3 (CH.sub.2).sub.14 COCl 
2.74 
g. 
##STR43## 1.37 
g. 
22 CH.sub.3 (CH.sub.2).sub.6 COCl 
3.42 
g. 
##STR44## 3.43 
g. 
23 CH.sub.3 (CH.sub.2).sub.10 COCl 
4.60 
g. 
##STR45## 3.43 
g. 
24 CH.sub.3 (CH.sub.2).sub.6 COCl 
3.42 
g. 
##STR46## 3.43 
g. 
25 CH.sub.3 (CH.sub.2).sub.10 COCl 
4.60 
g. 
##STR47## 3.43 
g. 
26 CH.sub.3 (CH.sub.2).sub.6 COCl 
3.42 
g. 
##STR48## 3.43 
g. 
27 CH.sub.3 (CH.sub.2).sub.10 COCl 
4.60 
g. 
##STR49## 3.43 
g. 
28 CH.sub.3 (CH.sub.2).sub.6 COCl 
3.42 
g. 
##STR50## 6.18 
g. 
29 CH.sub.3 (CH.sub.2).sub.8 COCl 
4.01 
g. 
##STR51## 4.12 
g. 
30 CH.sub.3 (CH.sub.2).sub.12 COCl 
5.18 
g. 
##STR52## 4.12 
g. 
__________________________________________________________________________ 
Example 
N-Alkanoyl Amino Acid 
No. Formula wt. 
__________________________________________________________________________ 
1 CH.sub.3 (CH.sub.2).sub.10 CONHCH(C.sub.5 H.sub.5)CO.sub.2 
H 2.5 
g. 
2 CH.sub.3 (CH.sub.2).sub.10 CONH(CH.sub.2).sub.4 CO.sub.2 
598 
mg. 
3 CH.sub. 3 (CH.sub.2).sub.10 CONH(CH.sub.2).sub.10 CO.sub.2 
H 19.97 
g. 
4 
##STR53## 6.0 
g. 
5 
##STR54## 3.19 
g. 
6 
##STR55## 670 
mg. 
7 
##STR56## 3.58 
g. 
8 
##STR57## 5.23 
g. 
9 
##STR58## 3.04 
g. 
10 
##STR59## 2.87 
11 
##STR60## 3.34 
g. 
12 
##STR61## 33.3 
g. 
13 
##STR62## 2.76 
g. 
14 
##STR63## 37.6 
g. 
15 
##STR64## 7.6 
g. 
16 CH.sub.3 (CH.sub.2).sub.5 CONH(CH.sub.2).sub.10 CO.sub.2 
31.3 
g. 
17 
##STR65## 1.56 
g. 
18 
##STR66## 2.25 
g. 
19 
##STR67## 2.52 
g. 
20 
##STR68## 2.84 
g. 
21 
##STR69## 3.16 
g. 
22 
##STR70## 3.20 
g. 
23 
##STR71## 2.89 
g. 
24 
##STR72## 3.19 
g. 
25 
##STR73## 4.26 
g. 
26 
##STR74## 4.76 
g. 
27 
##STR75## 6.23 
g. 
28 
##STR76## 4.26 
g. 
29 
##STR77## 3.638 
g. 
30 
##STR78## 4.187 
g. 
__________________________________________________________________________ 
EXAMPLES 31-60 
Table 2, below, gives the preparation of the 2,4,5-trichlorophenyl esters 
of the N-alkanoyl amino acids shown in Table 1. The compounds set forth in 
Table 2 are prepared according to the following general procedure: 
The N-alkanoylamino acid (1 mole), 2,4,5-trichlorophenol (1.1 mole), and 
dicyclohexylcarbodiimide (1 mole) are dissolved in methylene chloride, 
ether or tetrahydrofuran. The solution is stirred at room temperature for 
about 16 to about 20 hours after which it is filtered. The filtrate is 
taken to dryness, and the product is crystallized from either 
acetonitrilewater or diethyl ether-petroleum ether. 
TABLE 2 
__________________________________________________________________________ 
Preparation of 2,4,5-trichlorophenyl esters 
N-Alkanoyl Amino Acid Wt. of 2,4,5-trichlorophenol 
Example No. 
Formula wt ester product 
__________________________________________________________________________ 
31 CH.sub.3 (CH.sub.2).sub.10 CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO.sub.2 
H 333 mg. 
500 mg. 
32 CH.sub.3 (CH.sub.2).sub.10 CONH(CH.sub.2).sub.4 CO.sub.2 H 
598 mg. 
955 mg 
33 CH.sub.3 (CH.sub.2).sub.10 CONH(CH.sub.2).sub.10 CO.sub.2 H 
3.83 g. 
1.02 g. 
34 
##STR79## 638 mg. 
410 mg. 
35 
##STR80## 3.19 g. 
2.43 g. 
36 
##STR81## 670 mg. 
1.03 g. 
37 
##STR82## 3.58 g. 
2.20 g. 
38 
##STR83## 5.23 g. 
1.41 g. 
39 
##STR84## 3.04 g. 
4.7 g. 
40 
##STR85## 2.87 g. 
4.45 g. 
41 
##STR86## 3.34 g. 
3.86 g. 
42 
##STR87## 3.33 g. 
2.6 g. 
43 
##STR88## 2.76 g. 
2.14 g. 
44 
##STR89## 3.76 g. 
1.0 g. 
45 
##STR90## 3.28 g. 
4.4 g. 
46 CH.sub.3 (CH.sub.2).sub.5 CONH(CH.sub.2).sub.10 CO.sub.2 H 
4.8 g. 
1.68 g. 
47 
##STR91## 895 mg. 
1.48 g. 
48 
##STR92## 1.245 g. 
1.59 g. 
49 
##STR93## 2.52 g. 
2.97 g. 
50 
##STR94## 2.84 g. 
2.44 g. 
51 
##STR95## 3.16 g. 
1.33 g. 
52 
##STR96## 2.08 g. 
2.436 g. (700 mg. after recryst.) 
53 
##STR97## 2.65 g. 
2.373 g. 
54 
##STR98## 2.68 g. 
1.619 g. 
55 
##STR99## 3.19 g. 
1.605 g. 
56 
##STR100## 2.38 g. 
1.716 g. 
57 
##STR101## 2.83 g. 
1.575 g. 
58 
##STR102## 4.19 g. 
2.02 g. 
59 
##STR103## 2.88 g. 
3.507 g. 
60 
##STR104## 3.33 g. 
1.897 g. 
__________________________________________________________________________ 
EXAMPLES 61-90 
Table 3, below, gives the preparation of the derivatives of A-30912A 
nucleus prepared from the N-alkanoyl amino acid 2,4,5-trichlorophenyl 
esters set forth in Table 2. The compounds set forth in Table 3 are 
prepared in general according to the following procedure: 
To A-30912A nucleus, dissolved in dimethylformamide (DMF) is added the 
2,4,5-trichlorophenyl ester of the N-alkanoyl amino acid. The reaction 
mixture is stirred for 15-18 hours after which it is taken to dryness to 
give a residue. The residue is washed (two times each) with ethyl ether 
and by methylene chloride. The washings are discarded. The remaining 
residue is dissolved in ethyl acetate-methanol (3:2, v/v) and is 
chromatographed on a silica gel (Woelm 70-150 mesh) column using the 
aforesaid solvent system as the eluent. The fractions from the 
chromatograph are monitored by TLC on silica gel (Merck) using ethyl 
acetate-methanol (3:2, v/v) as the solvent system. Fractions containing 
the desired product are combined, and solvent is removed to give the 
product as a residue. The product may be analyzed by reversed phase HPLC 
as follows: The sample dissolved in H.sub.2 O/CH.sub.3 OH/CH.sub.3 CN 
(1:2:2 v/v)(1 mg./ml.) is injected into a 1/4 inch by 12 inch stainless 
steel column packed with C.sub.18 Micro Bondapak resin (Waters Associates, 
Inc., Milford, Mass) and the column is eluted with a solvent system 
comprising H.sub.2 O/CH.sub.3 OH/CH.sub.3 CN (1:2:2 v/v). The elution is 
ml./minute using a Waters 600A pump (Waters Associates, Inc.) and chart 
speed of 0.2 in./minute. Eluent is monitored with a Varian Vari-Chrom UV 
detector at 230 nm. 
The products may also be analyzed by field desorption mass spectrometry 
(FDMS). 
TABLE 3 
__________________________________________________________________________ 
N-Alkanoylamino Acid Derivatives of A-30912A Nucleus 
##STR105## 
Exam- HPLC 
ple Product Ester A30912A Retention 
No. R.sup.1 in Formula III 
Example 
Wt. (mg) 
nucleus (mg) 
Product (mg) 
M.sup.+ (cm) 
__________________________________________________________________________ 
61 CH.sub.3 (CH.sub.2).sub.10 CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO 
31 141 250 158 1148(M.sup.+ + 
--) 
62 CH.sub.3 (CH.sub.2).sub.10 CONH(CH.sub.2).sub.4CO 
32 795 250 132 1101(M.sup.+ + 22) 
-- 
63 CH.sub.3 (CH.sub.2).sub.10 CONH(CH.sub.2).sub.10CO 
33 462 400 327 1185(M.sup.+ + 
1.23 
64 
##STR106## 34 515 400 247 1121(M.sup.+ + 23) 
-- 
65 
##STR107## 35 515 400 302 1120(M.sup.+ + 22) 
-- 
66 
##STR108## 36 515 400 354 1137(M.sup.+ + 23) 
1.33 
67 
##STR109## 37 570 400 196 1197(M.sup.+ + 30) 
1.65 
68 
##STR110## 38 750 400 291 -- 1.20 
69 
##STR111## 39 512 400 182 1135(M.sup.+ + 23) 
-- 
70 
##STR112## 40 512 400 166 1143(M.sup.+ + 31) 
1.43 
71 
##STR113## 41 530 400 120 1151(M.sup.+ + 23) 
-- 
72 
##STR114## 42 497 400 452 1135(M.sup.+ + 23) 
1.32 
73 
##STR115## 43 535 400 286 1148(M.sup.+ + 24) 
1.40 
74 
##STR116## 44 540 400 453 1170(M.sup.+ + 25) 
1.40 
75 
##STR117## 45 492 400 277 -- -- 
76 CH.sub.3 (CH.sub.2).sub.5 CONH(CH.sub.2).sub.10CO 
46 493 400 273 1115(M.sup.+ + 23) 
0.95 
77 
##STR118## 47 360 400 213 980(M.sup.+ + 
1.75 
78 
##STR119## 48 430 400 218 -- -- 
79 
##STR120## 49 500 400 162 1093(M.sup.+ + 23) 
0.90 
80 
##STR121## 50 527 400 234 1149(M.sup.+ + 23) 
2.35 
81 
##STR122## 51 555 400 350 1177(M.sup.+ + 23) 
3.23 
82 
##STR123## 52 477 400 176 1099(M.sup.+ + 
0.7 
83 
##STR124## 53 533 400 127 1155(M.sup.+ + 23) 
2.3 
84 
##STR125## 54 477 400 319 1099(M.sup.+ + 23) 
1.0 
85 
##STR126## 55 533 400 214 1155(M.sup.+ + 23) 
3.1 
86 
##STR127## 56 477 400 290 1.0 
87 
##STR128## 57 533 400 325 3.4 
88 
##STR129## 58 512 400 324 1.3 
89 
##STR130## 59 540 400 281 1162(M.sup.+ + 23) 
1.8 
90 
##STR131## 60 596 400 269 1217(M.sup.+ + 23) 
7.7 
__________________________________________________________________________ 
EXAMPLE 91 
The following procedure illustrates the larger-scale preparation of the 
compounds of Formula III. The specific compound prepared by the procedure 
given below is the compound of Formula III wherein R.sup.1 is 
N-(n-dodecanoyl)-p-aminobenzoyl. 
A. Preparation of N-(n-Dodecanoyl)-p-aminobenzoic acid 
n-dodecanoyl chloride (8.74 g.; 40 mmoles) is added dropwise to a solution 
of dissolved p-aminobenzoic acid (5.5 g.; 40 mmoles) dissolved in pyridine 
(100 ml.). The mixture is stirred for 3 hours and poured into water (3 
l.). The precipitate which forms is filtered and dried in vacuo to give 
N-(n-dodecanoyl)-p-aminobenzoic acid (11.01 g.). 
B. Preparation of the 2,4,5-trichlorophenyl ester of 
N-(n-dodecanoyl)-p-aminobenzoic acid 
N-(n-Dodecanoyl)-p-aminobenzoic acid (11.01 g.; 34.5 mmole), 
2,4,5-trichlorophenol (7.5 g.; 38 mmole), and dicyclohexylcarbodiimide 
(6.94 g.; 34.5 mmole) are dissolved in methylene chloride (250 ml). The 
mixture is stirred at room temperature for 3.5 hours and then filtered. 
The filtrate is evaporated in vacuo to give a residue which is 
crystallized from acetonitrile/water to afford the 2,4,5-trichlorophenyl 
ester of N-(n-dodecanoyl)-p-aminobenzoic acid (12.84 g.). 
C. Acylation of A-30912A nucleus 
A-30912A nucleus (8.16 g.; 10.2 mmole) and the 2,4,5-trichlorophenyl ester 
of N-(n-dodecanoyl)-p-aminobenzoic acid (4.72 g.; 10.2 mmole) are 
dissolved in dimethylformamide (100 ml.). The solution is stirred at room 
temperature for 15 hours. Solvent is removed in vacuo to give a residue 
which is washed twice with diethylether. The washes are discarded. The 
washed residue is dissolved in methanol (50 ml.) and is purified by 
reversed phase HPLC by means of a "Prep LC/System 500" unit (Waters 
Associates, Inc., Milford, Mass.) using a Prep Pak-500/C.sub.18 Column 
(Waters Associates, Inc.) as the stationary phase. The column is eluted 
isocratically with H.sub.2 O/CH.sub.3 OH/CH.sub.3 CN (25:65:10 v/v) at 500 
psi. The fractions are analyzed by TLC using silica gel plates and H.sub.2 
O/CH.sub.3 OH/CH.sub.3 CN (25:65:10 v/v) as the solvent system. Fractions 
containing the desired product are combined and lyophilized to give the 
N-(n-dodecanoyl)-p-aminobenzoyl derivative of A-30912A nucleus (3.5 g). 
EXAMPLE 92 
The antifungal activity of the compounds of Formula III can be demonstrated 
and elicited in vitro in standard disc-diffusion tests and agar-dilution 
tests, and in vivo in standard tests in mice which assess effectiveness 
against a systemic fungal infection. The results of the antifungal testing 
of representative compounds of Formula III (Example 61-90) are set forth 
in Tables 4, 5, 6 and 7. 
Tables 4 and 5 give the results of the testing in vitro of the compounds of 
Examples 61-81 by agar-plate disc-diffusion methods. In Table 4 activity 
is measured by the size (diameter in mm.) of the observed zone of 
inhibition of the microorganism produced by the test compound. In Table 5, 
activity is measured by the minimal inhibitory concentration (MIC) of the 
substance (.mu.g/disc) required to inhibit growth of the test organism. 
Table 6 gives the results of the testing in vitro of the 
N-(n-dodecanoyl)-p-aminobenzoyl derivative of A30912A nucleus (Formula 
III, R.sup.1 is N-(dodecanoyl)-p-aminobenzoyl) against five strains of 
Candida albicans by the agar dilution method. In Table 6 activity is 
measured by the minimal inhibitory concentration (MIC) of the substance 
(.mu.g/ml) required to inhibit the test organism. 
The results of in vivo tests to evaluate the effectiveness of the compound 
of Examples 61-81, 86 and 88 against an infection caused by Candida 
albicans A-26 in mice are given in Table 7, where activity is measured by 
the ED.sub.50 value (the dose in mg/kg. required to cure 50% of the test 
animals). Where an ED.sub.50 value was not obtained, activity is indicated 
by the lowest dose at which a significant anti-fungal effect is observed. 
In this test, groups of male albino mice (specific pathogen free), 
weighing 18 to 20 grams, are infected intravenously with Candida albicans 
A-26. The animals are X-irradiated 24 hours prior to infection at about 50 
roentgens per minute for 8 minutes (400 total dose) to reduce immune 
responses to the infecting organism. At 0, 4, and 24 hours post infection 
each group of mice is given graded doses subcutaneously of the test 
compound as a suspension in 33% polyethylene glycol-water. The day of 
death for each animal is recorded. Student's t test statistical comparison 
of the average day of death is made between each group of infected-treated 
animals at a particular dosage level and 10 infected-untreated animals to 
determine if treatment significantly extends survival time. 
Table 8 gives the results of the testing of compounds for absorption after 
oral administration. In this test, mice are gavaged with a dose of 416 
mg/kg of the test compound suspended in 33% PEG 400-water. At time 
intervals, blood samples are taken from the orbital sinus and are assayed 
for antibiotic activity as follows: A 7 mm. disc containing 20 .mu.l of 
whole blood is placed on agar seeded with Aspergillus montevidensis 
A35137. After 40 hours incubation at 30.degree. C. zones of inhibition 
from the blood samples are compared to a standard obtained from the test 
compound, and the amount of compound in the blood sample is calculated. 
TABLE 4 
__________________________________________________________________________ 
Antifungal Activity By the Agar Plate Disc Diffusion Test 
Compound Size of Zone of Inhibition (mm).sup.(a) 
Example Saccharomyces 
Neurospora 
Trichophyton 
Candida 
No. R.sup.1 of Formula III pastorianus X-52 
Crassa 846 
mentagrophytes 
albicans 
__________________________________________________________________________ 
A-26 
61 CH.sub.3 (CH.sub.2).sub.10 CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO 
18 42* 55* 25 
62 CH.sub.3 (CH.sub.2).sub.10 CONH(CH.sub.2).sub.4CO 
15 27* 60* 25 
63 CH.sub.3 (CH.sub.2).sub.10 CONH(CH.sub.2).sub.10CO 
15 28* 55* 24 
18 35 63* 25 
15 28 56 24 
64 
##STR132## 21 17 33* 35* 
55* 56* 23 19 
65 
##STR133## 17 -- -- 20 
66 
##STR134## 18 -- -- 23 
67 
##STR135## 19 35* 44* 27 
68 
##STR136## 17 30* 60 25 
69 
##STR137## 17 30* -- 16 
70 
##STR138## 19 25* 45* 26 
71 
##STR139## 10 32* 50* 20 
72 
##STR140## 20 30* 55* 19 
73 
##STR141## 17 29* 24* 60* 
74 
##STR142## 13 28* -- 15 
75 
##STR143## 14 30* 54* 24 
76 CH.sub.3 (CH.sub.2).sub. 5 CONH(CH.sub.2).sub.10CO 
-- 20* 35* 10 
77 
##STR144## 17 24* 51* 24 
78 
##STR145## 20 25 14 45* 
79 
##STR146## 17 30* -- 24 
80 
##STR147## 17 -- -- 22 
81 
##STR148## 24 -- -- 25 
__________________________________________________________________________ 
.sup.(a) Compounds were tested as suspension in methanol. The compounds 
were tested at a concentration of 1 mg/ml by a dipping 7mm disc into the 
suspension and placing it on the agar surface. Incubation: 24-48 hours at 
25-37.degree. C. 
*Measurable zone of inhibition with regrowth of organism around disc. 
TABLE 5 
__________________________________________________________________________ 
Antifungal Activity By the Agar Plate Disc Diffusion Test 
Compound MIC (.mu.g/disc)* 
Example No. 
R.sup.1 or Formula III Candida albicans A-26 
Trychophyton mentagrophytes 
#6 
__________________________________________________________________________ 
61 CH.sub.3 (CH.sub.2).sub.10 CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO 
0.625 &lt;0.039 
62 CH.sub.3 (CH.sub.2).sub.10 CONH(CH.sub.2).sub.4CO 
1.25 0.078 
63 CH.sub.3 (CH.sub.2).sub.10 CONH(CH.sub.2).sub.10CO 
2.5 0.156 
1.25 0.156 
64 
##STR149## 0.625 1.25 &lt;0.039 0.678 
65 
##STR150## 5.0 &lt;0.039 
66 
##STR151## 1.25 &lt;0.039 
67 
##STR152## 0.625 &lt;0.039 
68 
##STR153## 1.25 0.078 
69 
##STR154## &gt;20 &lt;0.039 
70 
##STR155## 20 &lt;0.039 
71 
##STR156## -- -- 
72 
##STR157## 2.5 0.078 
73 
##STR158## 1.25 &lt;0.039 
74 
##STR159## 10 0.078 
75 
##STR160## 2.5 0.078 
76 CH.sub.3 (CH.sub.2).sub.5 CONH(CH.sub.2).sub.10CO 
&gt;20;80 0.625 
77 
##STR161## &gt;20;40 0.312 
78 
##STR162## &gt;20;160 1.25 
79 
##STR163## 0.625 -- 
80 
##STR164## 1.25 &lt;0.039 
81 
##STR165## 0.312 &lt;0.039 
__________________________________________________________________________ 
*Compounds were suspended in 0.01M sodium borate solution, pH 7.5. The 
compounds were tested at 20 .mu.g/disc at top level and at twofold 
dilutions until end points were reached. Incubation: 24 hours at 
30.degree. C. 
**Measurable zones of inhibition with regrowth of organism around disc. 
TABLE 6 
In vitro activity of the N-(n-dodecanoyl)-p-aminobenzoyl (Example 64) and 
the N-(n-dodecanoyl)-5-amino-n-pentanoyl (Example 62) derivatives of 
A-30912A nucleus against 5 strains of Candida albicans by the agar 
dilution assay. 
______________________________________ 
MIC (.mu.g/ml) 
Compound A26 SBH 16 SBH 31 SBH 28 SBH 29 
______________________________________ 
Ex. 64 0.312 0.625 0.625 0.625 0.625 
Ex. 62 1.25 2.5 2.5 2.5 2.5 
______________________________________ 
TABLE 7 
__________________________________________________________________________ 
Therapeutic Activity Against Candida Albicans A-26 in Mice* 
Lowest 
Compound Active 
Example ED.sub.50 
Dose 
No. R.sup.1 of Formula III (mg/kg)** 
(mg/kg) 
__________________________________________________________________________ 
61 CH.sub.3 (CH.sub.2).sub.10 CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO 
&gt;40 &gt;40 
62 CH.sub.3 (CH.sub.2).sub.10 CONH(CH.sub.2).sub.4CO 
22 20 
63 CH.sub.3 (CH.sub.2).sub.10 CONH(CH.sub.2).sub.10CO 
&gt;40 &gt;40 
64 
##STR166## 15 15 10 .ltoreq.5 
65 
##STR167## &gt;40 &gt;40 
66 
##STR168## &gt;40 40 
67 
##STR169## 14 10 
68 
##STR170## &gt;40 40 
69 
##STR171## &gt;40 &gt;40 
70 
##STR172## &gt;40 &gt;40 
71 
##STR173## &gt;40 &gt;40 
72 
##STR174## &gt;40 40 
73 
##STR175## 24 20 
74 
##STR176## &gt;40 &gt;40 
75 
##STR177## &gt;40 &gt;40 
76 CH.sub.3 (CH.sub.2).sub.5 CONH(CH.sub.2).sub.10CO 
&gt;40 &gt;40 
77 
##STR178## &gt;40 &gt;40 
78 
##STR179## &gt;40 &gt;40 
79 
##STR180## 26 5 
80 
##STR181## 11 2.5 
81 
##STR182## 7 5 
86 
##STR183## 29 10 
88 
##STR184## &gt;40 20 
__________________________________________________________________________ 
*Dosage Schedule: 40, 20, 15, and 10 mg/kg. Dosages given 0, 4, and 24 
hours post injection as suspension of test compound in 30% PEGH.sub.2 O. 
Number of mice receiving test compounds at each dosage level: 6 mice per 
group. Number of mice in control (untreated) group: 10 mice per group. 
**As measured by increase in survival time of treated animals versus 
control, calculated by method of Reed V. Mueuch, American J. Hygiene, 493 
(1938). 
TABLE 8 
__________________________________________________________________________ 
Blood Levels after Administration in Mice 
Compound 
Example 
No. R.sup.1 in Formula III Blood Levels* (.mu.g/ml) 
__________________________________________________________________________ 
61 CH.sub.3 (CH.sub.2).sub.10 CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO 
0 
62 CH.sub.3 (CH.sub.2).sub.10 CONH(CH.sub.2).sub.4CO 
0 
63 CH.sub.3 (CH.sub.2).sub.10 CONH(CH.sub.2).sub.10 
0.40(0) 
64 
##STR185## 0.83 
65 
##STR186## 0 
66 
##STR187## 0 
67 
##STR188## 0.53 
68 
##STR189## 0.34 
69 
##STR190## 0 
70 
##STR191## 0 
71 
##STR192## -- 
72 
##STR193## 0.34 
73 
##STR194## 1.31 
74 
##STR195## 0.64 
75 
##STR196## 0 
76 CH.sub.3 (CH.sub.2).sub.5 CONH(CH.sub.2).sub.10CO 
0 
77 
##STR197## 0 
78 
##STR198## -- 
79 
##STR199## -- 
80 
##STR200## 6.5 
81 
##STR201## 36 
__________________________________________________________________________ 
*Four hours after administration of test compound at dose of 416 mg/kg by 
gavage as suspension of compound in 33% PEG 400H.sub.2 O). Compound 
determined by bioassay vs. Aspergillus montevidensis A35137. 
EXAMPLE 93 
Preparation of A-30912A 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'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's mineral stock has the following composition: 
Ingredient Amount 
______________________________________ 
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'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.degree. 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's Dried Grain* 
5.0 g 
K.sub.2 HPO.sub.4 1.0 g 
Czapek'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.degree. 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, "Preservation and Storage of Microorganisms in 
the Gas Phase of Liquid Nitrogen, 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.degree. 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.degree. 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 II 
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.degree. 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 A 
A fermentation of A. utahensis is carried out as described in Sect. A, 
using slant medium A and production medium I and incubating the production 
medium for about 42 hours. A-30912 factor A (340 g. of crude substrate 
which contained about 19.7 g. of A-30912 factor A, dissolved in 1.5 L 
ethanol) is added to the fermentation medium. 
Deacylation of A-30912 factor A is monitored by assay against Candida 
albicans. The fermentation is allowed to continue until deacylation is 
complete as indicated by disappearance of activity vs. C. albicans. 
c. Isolation of A-30912A Nucleus 
Whole fermentation broth (100 liters), obtained as described in Sect. B and 
containing nucleus from about 20 g of A-30912 factor A, is filtered. The 
mycelial cake is discarded. The clear filtrate thus obtained (about 93 
liters) is passed through a column containing 4.5 liters of HP-20 resin 
(DIAION High Porous Polymer, HP-Series, Mitsubishi Chemical Industries 
Limited, Tokyo, Japan) at a rate of 200 ml/minute. 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 (85 liters) at a rate of 200-300 ml/minute. 
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-30912A nucleus. The eluate containing the A-30912A nucleus is 
concentrated under vacuum to a small volume and lyophilized to give 
approximately 97 grams of crude nucleus. 
D. Purification of A-30912A Nucleus by Reversed-Phase Liquid Chromatography 
Crude A-30912A nucleus (25 grams), obtained as described in Section C, is 
dissolved in 300 ml of water:acetonitrile:acetic acid:pyridine (96:2:1:1). 
This solution is chromatographed on a 4-liter stainless-steel column (8 
cm.times.80 cm) filled with Lichroprep RP-18, particle size 25-40 microns 
(MC/B Manufacturing Chemists, Inc. E/M, Cincinnati, OH). The column is 
part of a Chromatospac Prep 100 unit (Jobin Yvon, 16-18 Rue du Canal 91160 
Longjumeau, France). The column is operated at a pressure of 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, Instrument Specialist Co., 4700 Superior Ave., 
Lincoln, Nebr. 68504) with an optical unit (ISCO Type 6). Fractions having 
a volume of about 500 ml are collected each minute. 
On the basis of absorption at 280 nm, fractions containing A-30912A nucleus 
are combined, evaporated under vacuum and lyophilized to give 2.6 grams of 
nucleus. The amount of solvent required to complete this chromatographic 
separation process varies from 7-8 liters. 
E. Characteristics of A30912A nucleus 
(a) Empirical formula: C.sub.34 H.sub.51 N.sub.7 O.sub.15. 
(b) Molecular weight: 797.83 
(c) Soluble in water, dimethylformamide, dimethylsulfoxide, and methanol; 
insoluble in chloroform, toluene, and diethylether. 
(d) Infrared absorption spectrum (KBr disc.) 
Shows absorption maxima at: 
3340 broad (OH, H-bonded); 2970, 2930, and 2890 (CH stretch, aliphatic 
CH.sub.3, CH.sub.2, CH groups) 1660 and 1625 (several carbonyls C.dbd.O); 
1510-1550; 1430-1450 (CH wag); 1310-1340; 1230-1260; 1080; 835, 650 broad, 
and 550 broad cm.sup.-1. 
(e) Electrometric titration in 66% aqueous dimethylformamide indicates the 
presence of a titratable group with a pK.sub.a value of about 7.35 
(initial pH 7.32). 
(f) HPLC retention time (K'):11.52 min. under following conditions. 
Column: 4.times.300 mm 
Packing: silica gel/C.sub.18 
Solvent: ammonium acetate:acetonitrile: 
water (1:2:97) 
Flow Rate: 3 ml/min 
Pressure: 2500 psi 
Detector: variable wavelength UV at 230 nm 
Sensitivity: 0-0.4 A.U.F.S. 
EXAMPLE 94 
A-30912A nucleus is prepared and purified by the method of Example 93 
except that tetrahydro-A-30912A is used as the substrate. 
EXAMPLE 95 
A-30912A nucleus is prepared and purified by the method of Example 93 
except that aculeacin A is used as the substrate. 
EXAMPLE 96 
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.degree. 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.degree. 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.degree. 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.degree. 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 "A", Dow Corning 
The inoculated production medium is allowed to ferment in a 165-liter 
fermentation tank at a temperature of 25.degree. 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 97 
Separation of the A-42355 Antibiotic Complex 
Whole fermentation broth (4127 liters), obtained by the method described in 
Example 96 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 l 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 98 
Isolation of A-30912 Factor A 
The co-pending application of Karl H. Michel entitled RECOVERY PROCESS FOR 
A-30912 ANTIBIOTICS, Ser. No. 103,014, filed Dec. 13, 1979, describes the 
reversed-phase high performance, low pressure liquid chromatography 
(HPLPLC) using silica gel/C.sub.18 adsorbent as a preferred method for the 
final purification of A-30912 factor A. 
A-42355 antibiotic complex (1 g), prepared as described in Example 97, is 
dissolved in 7 ml of methanol:water:acetonitrile (7:2:1). This solution is 
filtered and introduced onto a 3.7-cm I.D..times.35-cm glass column 
[Michel-Miller High Performance Low Pressure (HPLPLC) Chromatography 
Column, Ace Glass Incorporated, Vineland, NJ 08360] packed with 
LP-1/C.sub.18 silica gel reversed-phase resin (10-20 microns), prepared as 
described in Example 99, through a loop with the aid of a valve system. 
The column is packed in methanol:water:acetonitrile (7:2:1) by the 
slurry-packing procedure described in Example 100. An F.M.I. pump with 
valveless piston design (maximum flow 19.5 ml/minute) is used to move the 
solvent through the column at a flow rate of 9 ml/minute at ca. 100 psi, 
collecting fractions every minute. Elution of the antibiotic is monitored 
at 280 nm by using a UV monitor (ISCO Model UA-5, Instrument Specialist 
Co., 4700 Superior Ave., Lincoln, Nebr. 68504) with an optical unit (ISCO 
Type 6). 
The individual A-30912 factors can be identified by the use of thin-layer 
chromatography (TLC). The R.sub.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 9. 
TABLE 9 
______________________________________ 
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.sub.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.times.20 cm) and Candida albicans bioautography, are 
given in Table 10. 
TABLE 10 
______________________________________ 
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 .times. 15.0 cm 
Packing: Nucleosil.RTM. 10-C.sub.18 (Machery- 
Nagel and Company); packed 
using slurry-packing pro- 
cedure of Example 73 
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 11. 
TABLE 11 
______________________________________ 
Retention Time 
A-30912 Factor (seconds) 
______________________________________ 
A 792 
B 870 
H 990 
D 1,140 
______________________________________ 
EXAMPLE 99 
Preparation of Silica Gel/C.sub.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.degree. 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.degree. 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.degree. 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.degree. C. overnight (16-20 hours). 
EXAMPLE 100 
Slurry Packing Procedure for Michel-Miller Columns 
General Information 
This procedure is employed for packing reversed phase silica gel C.sub.18 
resins, such as that described in Example 99. 
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 flow-rate; 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 precolumn 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 101 
Preparation of Tetrahydro-A-30912A 
A-30912 factor A is dissolved in ethanol. PtO.sub.3 in absolute ethanol is 
reduced to form Pt, which in turn is used to reduce the A-30912 factor A 
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-30912A.