U.S. patent number RE32,311 [Application Number 06/780,130] was granted by the patent office on 1986-12-16 for derivatives of a-21978c cyclic peptides.
This patent grant is currently assigned to Eli Lilly and Company. Invention is credited to Manuel Debono.
United States Patent |
RE32,311 |
Debono |
December 16, 1986 |
Derivatives of A-21978C cyclic peptides
Abstract
A-21978C cyclic peptide derivatives of the formula ##STR1##
wherein R is hydrogen, a specified aminoacyl or N-alkanoylaminoacyl
group, 8-methyldecanoyl, 10-methyldodecanoyl, 10-methylundecanoyl,
the specific C.sub.10 -alkanoyl group of A-21978C.sub.0 or the
specific C.sub.12 -alkanoyl groups of A-21978C factors C.sub.4 and
C.sub.5 or an amino-protecting group; and R.sup.1 is hydrogen, an
amino-protecting group, or a specified aminoacyl or
N-alkanoylaminoacyl group; provided that, when R is other than
aminoacyl or N-alkanoylaminoacyl, R.sup.1 must be aminoacyl or
N-alkanoylaminoacyl; and, when R.sup.1 is an amino-protecting
group, R must be aminoacyl or N-alkanoylaminoacyl; and the salts
thereof, are useful as antibacterial agents or as intermediates to
antibacterial agents.
Inventors: |
Debono; Manuel (Indianapolis,
IN) |
Assignee: |
Eli Lilly and Company
(Indianapolis, IN)
|
Family
ID: |
27009014 |
Appl.
No.: |
06/780,130 |
Filed: |
September 25, 1985 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
Reissue of: |
380499 |
May 21, 1982 |
04396543 |
Aug 2, 1983 |
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Current U.S.
Class: |
530/317; 930/190;
930/20; 930/200; 930/21; 930/270 |
Current CPC
Class: |
C07K
7/08 (20130101); A61K 38/00 (20130101) |
Current International
Class: |
C07K
7/08 (20060101); C07K 7/00 (20060101); A61K
38/00 (20060101); C07K 005/12 () |
Field of
Search: |
;260/112.5R |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
J Shoji et al., ibid. 29 (12) 1268-1274, 1275-1280 (1976). .
D. Storm et al., "Polymyxin and Related Antibiotics," Ann. Rev.
Biochem. 46: 731-732 (1977). .
J. M. Weber et al., J. Antibiotics 31 (4) 373-374 (1978). .
J. Shoji et al., ibid. 28, 764-769 (1975). .
J. Shoji et al., ibid. 29 (4) 380-389 (1976). .
S. Chihara et al., ibid. 38 (3), 521-529 (1974). .
S. Chihara et al., ibid. 38 (10) 1767-1777 (1974). .
T. Suzuki et al., J. Biochem. 56 (4) 335-343 (1964). .
T. Kato et al., J. Antibiotics 29 (12) 1339-1340 (1976). .
S. Chihara et al., Agr. Biol. Chem. 37 (11) 2455-2463 (1973). .
S. Chihara et al., ibid. 37 (12) 2709-2717 (1973)..
|
Primary Examiner: Phillips; Delbert R.
Attorney, Agent or Firm: Harrison; Nancy J.
Claims
I claim:
1. An A-21978C cyclic peptide derivative of the formula: ##STR75##
wherein R is hydrogen 8-methyldecanoyl, 10-methyldodecanoyl,
10-methylundecanoyl, the specific C.sub.10 -alkanoyl group of
A-21978C.sub.0 or the specific C.sub.12 -alkanoyl groups of
A-21978C factors C.sub.4 and C.sub.5, an amino-protecting group, an
aminoacyl group of the formula ##STR76## wherein Q is C.sub.1
-C.sub.16 alkylene, or an N-alkanoylamino acyl group of the formula
##STR77## wherein:
W is a divalent aminoacyl radical of the formula: ##STR78## wherein
A is C.sub.1 -C.sub.10 alkylene or C.sub.5 -C.sub.6 cycloalkylene;
##STR79## 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 -alkoxy, C.sub.1 -C.sub.3 alkylthio, carbamyl, or
C.sub.1 -C.sub.3 alkylcarbamyl; ##STR80## wherein X is hydrogen
chloro, bromo, iodo, amino, 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; ##STR81## wherein
X.sup.1 is chloro, bromo, iodo, amino, hydroxy, C.sub.1 -C.sub.3
alkyl or C.sub.1 -C.sub.3 alkoxy; ##STR82## wherein B is a divalent
radical of the formula: --(CH.sub.2).sub.n -- and n is an integer
from 1 to 3; --CH.dbd.CH--; --CH.dbd.CH--CH.sub.2 --; or ##STR83##
R.sup.2 is C.sub.1 -C.sub.17 alkyl or C.sub.2 -C.sub.17 alkenyl;
and R.sup.1 is hydrogen, an amino-protecting group, an aminoacyl
group of the formula ##STR84## as herein defined, or an
N-alkanoylaminoacyl group of the formula ##STR85## as herein
defined; provided that, when R is other than aminoacyl or
N-alkanoylaminoacyl, R.sup.1 must be aminoacyl or
N-alkanoylaminoacyl; and, when R.sup.1 is an amino-protecting
group, R must be aminoacyl or N-alkanoylaminoacyl; and the salts
thereof.
2. A compound of claim 1 wherein R is ##STR86## A is C.sub.1
-C.sub.10 alkylene and R.sup.2 is straight chain C.sub.1 -C.sub.17
alkyl.
3. A compound of claim 2 wherein R.sup.1 is hydrogen.
4. The compound of claim 3 wherein R is
5-[N-(n-dodecanoyl)amino]-n-pentanoyl.
5. The compound of claim 3 wherein R is
11-[N-(n-dodecanoyl)amino]-n-undecanoyl.
6. The compound of claim 3 wherein R is
11-[N-(n-heptanoyl)amino]-n-undecanoyl.
7. A compound of claim 1 wherein R is ##STR87## X is hydrogen and
R.sub.2 is straight chain C.sub.1 -C.sub.17 alkyl.
8. A compound of claim 7 wherein R.sup.1 is hydrogen.
9. The compound of claim 8 wherein R is
p-[N-(n-dodecanoyl)amino]benzoyl.
10. The compound of claim 8 wherein R is
m-[N-(n-dodecanoyl)amino]benzoyl.
11. The compound of claim 8 wherein R is
p-[N-(n-tetradecanoyl)amino]benzoyl.
12. A compound of claim 1 wherein R is ##STR88## X.sup.1 is chloro,
bromo, iodo, amino, C.sub.1 -C.sub.3 alkyl, hydroxy, or C.sub.1
-C.sub.3 alkoxy, and R.sup.2 is straight chain C.sub.1 -C.sub.17
alkyl.
13. A compound of claim 12 wherein R.sup.1 is hydrogen.
14. The compound of claim 13 wherein R is
5-amino-4-[N-(n-dodecanoyl)amino]-2-hydroxybenzoyl.
15. The compound as defined in claim 13 wherein R is
3-[N-(n-dodecanoyl)amino]-2,5-dichlorobenzoyl.
16. The compound of claim 1 wherein R is
p-[N-(n-dodecanoyl)amino]phenylacetyl and R.sup.1 is hydrogen.
17. The compound of claim 1 wherein R is
p-[N-(n-dodecanoyl)amino]cinnamoyl and R.sup.1 is hydrogen.
18. The compound of claim 1 wherein R is
p-[N-(n-dodecanoyl)amino]hippuryl and R.sup.1 is hydrogen.
19. The compound of claim wherein R is
2-[N-(n-dodecanoyl)amino]nicotinoyl and R.sup.1 is hydrogen.
20. A compound of claim 1 wherein R is ##STR89## R.sup.2 is C.sub.1
-C.sub.17 alkyl and R.sup.3 is phenyl, benzyl or
3-indolemethyl.
21. A compound of claim 20 wherein R.sup.1 is hydrogen.
22. The compound of claim 21 wherein R is
N-(n-dodecanoyl)phenylalanyl.
23. The compound of claim 21 wherein R is
N-(n-octanoyl)phenylalanyl.
24. The compound of claim 21 wherein R is
N-(n-nonanoyl)phenylalanyl.
25. The compound of claim 21 wherein R is
N-(n-decanoyl)phenylalanyl.
26. The compound of claim 21 wherein R is
N-(n-undecanoyl)phenylalanyl.
27. The compound of claim 21 wherein R is
N-(n-tridecanoyl)phenylalanyl.
28. The compound of claim 21 wherein R is
N-(n-tetradecanoyl)phenylalanyl.
29. The compound of claim 21 wherein R is
N-(n-hexanoyl)tryptophanyl.
30. The compound of claim 21 wherein R is
N-(n-dodecanoyl)tryptophanyl.
31. The compound of claim 20 wherein R is
N-(n-hexanoyl)tryptophanyl.
32. The compound of claim 32 wherein R.sup.1 is
8-methyldecanoyl.
33. The compound of claim 31 wherein R.sup.1 is
10-methyldodecanoyl.
34. The compound of claim 31 wherein R.sup.1 is
10-methylundecanoyl.
Description
SUMMARY OF THE INVENTION
This invention relates to derivatives of A-21978C cyclic peptides
which have formula 1: ##STR2## wherein R is hydrogen, a specified
aminoacyl or N-alkanoylaminoacyl group, 8-methyldecanoyl,
10-methylundecanoyl, 10-methyldodecanoyl, the specific C.sub.10
-alkanoyl group of A-21978C factor C.sub.0 or the specific C.sub.12
-alkanoyl groups of A-21978C factors C.sub.4 and C.sub.5 or an
amino-protecting group; and R.sup.1 is hydrogen, a specified
aminoacyl or N-alkanoylaminoacyl group or an amino-protecting
group; provided that, when R is other than aminoacyl or
N-alkanoylaminoacyl, R.sup.1 must be aminoacyl or
N-alkanoylaminoacyl; and, when R.sup.1 is an amino-protecting
group, R must be aminoacyl or N-alkanoylaminoacyl; and the salts of
these peptides. The A-21978C cyclic peptide derivatives and salts
of this invention are useful semi-synthetic antibacterial agents or
are useful as intermediates to such agents.
DETAILED DESCRIPTION OF THE INVENTION
In this specification the following abbreviations, most of which
are commonly known in the art, are used:
Ala: alanine
Asp: aspartic acid
Gly: glycine
Kyn: kynurenine
Orn: ornithine
Ser: serine
Thr: threonine
Trp: tryptophan
t-BOC: tert-butoxycarbonyl
Cbz: benzyloxycarbonyl
DMF: dimethylformamide
THF: tetrahydrofuran
HPLC: high performance liquid chromatography
NMR: .sup.1 H nuclear magnetic resonance
TLC: thin-layer chromatography
UV: ultraviolet
FIELD OF THE INVENTION
Although there are many known antibacterial agents, the need for
improved antibiotics continues. Antibiotics differ in their
effectiveness against pathogenic organisms. Organism strains which
are resistant to known antibiotics continually develop. In
addition, individual patients often suffer serious reactions to
specific antibiotics, due to hypersensitivity and/or to toxic
effects. There is, therefore, a continuing need for new and
improved antibiotics.
THE PRIOR ART
The A-21978C antibiotics are closely related, acidic peptide
antibiotics. Members of this class of antibiotics which were
previously known include crystallomycin, amphomycin, zaomycin,
aspartocin, and glumamycin [see T. Korzybski, Z. Kowszyk-Gindifer
and W. Kurylowicz, "Antibiotics-Origin, Nature and Properties,"
Vol. I, Pergamon Press, New York, N.Y., 1967, pp. 397-401 and
404-408]; tsushimycin [J. Shoji, et al., J. Antibiotics 21, 439-443
(1968)]; laspartomycin [H. Naganawa, et al., J. Antibiotics 21,
55-62 (1968); brevistin [J. Shoji and T. Kato, J. Antibiotics 29,
380-389 (1976)]; cerexin A [J. Shoji, et al., J. Antibiotics 29,
1268-1274 (1976)] and cerexin B [J. Shoji and T. Kato, J.
Antibiotics 29, 1275-1280 (1976)]. Of these antibiotics, brevistin,
cerexin A and cerexin B appear to be most closely related to the
A-21978C antibiotics.
The A-21978C antibiotics are described by Robert L. Hamill and
Marvin M. Hoehn in U.S. Pat. No. 4,208,403, issued June 17, 1980,
which is incorporated herein by reference. As described in U.S.
Pat. No. 4,208,403, the A-21978 antibiotic complex contains a major
component, factor C, which is itself a complex of closely related
factors. A-21978 factor C, which is called the A-21978C complex,
contains individual factors C.sub.0, C.sub.1, C.sub.2, C.sub.3,
C.sub.4 and C.sub.5. Factors C.sub.1, C.sub.2 and C.sub.3 are major
factors; and factors C.sub.0, C.sub.4 and C.sub.5 are minor
factors. The structure of the A-21978C factors is shown in formula
2: ##STR3## wherein 3MG represents L-threo-3-methylglutamic acid,
and R.sup.N represents a specific fatty acid moiety. The specific
R.sup.N groups of the factors are as follows:
______________________________________ A-21978C Factor R.sup.N
Moiety ______________________________________ C.sub.1
8-methyldecanoyl C.sub.2 10-methylundecanoyl C.sub.3
10-methyldodecanoyl C.sub.0 C.sub.10 --alkanoyl* C.sub.4 C.sub.12
--alkanoyl* C.sub.5 C.sub.12 --alkanoyl*
______________________________________ *Identity not yet
determined
Kleinschmidt et al. in U.S. Pat. No. 3,150,059, issued in 1964,
described an enzyme elaborated by the Actinoplanaceae which was
capable of deacylating penicillin antibiotics. Abbott and Fukuda in
U.S. Pat. No. 4,293,482, issued in 1981, reported that an
Actinoplanaceae enzyme was capable of deacylating the A-30912 type
of cyclic peptide antibiotic.
In 1967 Kimura and Tatsuki, in Japanese Patent No. 4058/67 (Derwent
Abstr. 26695), described the enzymatic deacylation of the peptide
antibiotic glumamycin. The microorganism catalyzing the deacylation
was identified as closely related to Pseudomonas dacunhae. They
stated that "deacylated derivatives of the compounds are useful as
the material for synthesis of the related compounds, as in the case
of 6-aminopenicillanic acid for penicillin", but gave no examples
of re-acylation.
In 1965, Kimura and coworkers reported that a bacterium isolated
from soil catalyzed the deacylation of the peptide antibiotic
colistin (polymyxin E) (see Kimura, et al., Abstracts of Papers,
21st Meeting of the Pharmaceutical Society of Japan, Tokushima,
October, 1965, p. 422). They reported that new derivatives of
colistin were prepared by acylation of the deacylated nucleus, but
did not discuss whether these derivatives had any activity.
Kato and Shoji [J. Antibiotics 29 (12), 1339-1340 (1976)] attempted
to use the enzyme described by Kimura et al. to deacylate the
cyclic peptide antibiotic octapeptin C.sub.1. The enzyme did not
catalyze the desired reaction. It was subsequently found that
deacylation could be accomplished chemically by oxidation of the
.beta.-hydroxyl group of the fatty acid followed by treatment with
hydroxylamine.
In 1973 Chihara and coworkers reported their work with colistin. In
this work two plant proteases, ficin and papain, were used to
hydrolyze colistin to a nonapeptide and a fatty acyl
.alpha.,.gamma.-diaminobutyric acid residue. The plant enzymes,
however, in addition to removing the fatty acid acyl substituent
also removed the terminal amino acid of the colistin molecule [See
S. Chihara et al., Agr. Biol. Chem. 37 (11), 2455-2463 (1973);
ibid. 37 (12), 2709-2717 (1973); ibid. 38 (3), 521-529 (1974); and
ibid. 38 (10), 1767-1777 (1974)]. The colistin nonapeptide was
isolated and then reacylated with a variety of fatty acid
chlorides. Subsequently, Chihara's group produced N-fatty acyl
monoacyl derivatives of colistin nonapeptide. These derivatives
restored a tenth amino acid to the colistin nonapeptide and were
used to study structure-activity relationships.
The polymyxin antibiotics have been hydrolyzed with the enzyme
subtilopeptidase A [See T. Suzuki et al., J. Biochem. 56 (4),
335-343 (1964)]. This enzyme deacylated the peptides, but in
addition hydrolyzed some of the peptide bonds so that a variety of
peptide products resulted.
In 1978 Weber and Perlman reported that a Corynebacterium isolated
from soil inactivated the peptide antibiotic amphomycin by
deacylation of the isotridecanoic acid side chain [see J.
Antibiotics 31 (4), 373-374 (1978)].
Kuwana et al. in U.S. Pat. No. 4,050,989, issued in 1977, described
the deacylation of pepsin-inhibiting peptides (pepsidines) by an
enzyme from Bacillus pumilus and the use of these products to
prepare N-acyl-pentapeptide homologs.
Shoji and coworkers deacylated the cyclic peptide antibiotics
cerexin A, cerexin B, and brevistin in order to determine the
structures of these antibiotics [see J. Shoji and T. Kato, J.
Antibiotics 28, 764-769 (1975) and ibid. 29 (4), 380-389 (1976);
and J. Shoji et al., ibid. 29 (12), 1268-1274 (1976); and ibid. 29
(12), 1275-1280 (1976)]. Deacylation was accomplished with an
enzyme preparation prepared from Pseudomonas sp. M-6-3 (polymyxin
acylase) and by chemical means. Chemical deacylation, however,
resulted in extensive side reactions.
Despite the contributions of these groups, it is extremely
difficult, when confronted with the problem of deacylating a
peptide antibiotic having a different structure, to know whether an
enzyme exists which can be used for this purpose. Finding such an
enzyme is even more difficult when the substrate antibiotic
contains a cyclic peptide nucleus. Enzymes have a high degree of
specificity. Differences in the peptide moiety and in the side
chain of the substrate antibiotic will affect the outcome of the
deacylation attempt. In addition, many microorganisms make a large
number of peptidases which attack different portions of the peptide
moiety. This frequently leads to intractable mixtures of
products.
Thus, it was most surprising that what may be the same enzyme which
was used to deacylate penicillins and the A-30912 antibiotics could
also be used successfully to deacylate the A-21978C antibiotics. In
each of the A-21978C antibiotics (formula 2), the fatty acid side
chain (R.sup.N) is attached at the .alpha.-amino group of the
tryptophan residue. In the co-pending application, of Bernard J.
Abbott, Manuel Debono and David S. Fukuda entitled "A-21978C CYCLIC
PEPTIDES", Ser. No. 380,497 filed May 21, 1982, the full disclosure
of which is incorporated herein by reference, is described the
discovery that the fatty acid side chain can be cleaved by an
enzyme without affecting the chemical integrity of the remainder of
the A-21978C peptide.
The enzyme used 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, an antibiotic selected from
A-21978C complex, A-21978C factors C.sub.0, C.sub.1, C.sub.2,
C.sub.3, C.sub.4, and C.sub.5, blocked A-21978C complex, and
blocked A-21978C factors C.sub.0, C.sub.1, C.sub.2, C.sub.3,
C.sub.4, and C.sub.5 is added to a culture of the microorganism.
The culture is allowed to incubate with the substrate until the
deacylation is substantially complete. The corresponding A-21978C
cyclic peptide thereby obtained is separated from the fermentation
broth by methods known in the art.
The cyclic peptides obtained by these enzymatic deacylations are
shown in formula 3. ##STR4## wherein R.degree. and R' are,
independently, hydrogen or an amino-protecting group; and the salts
thereof.
Removal of the acyl moiety from the A-21978C complex or A-21978C
individual factors C.sub.0, C.sub.1, C.sub.2, C.sub.3, C.sub.4, and
C.sub.5 gives the compound of formula 3 wherein R.degree. and R'
each represent hydrogen, which is the common cyclic peptide present
in antibiotic A-21978C factors. For convenience herein, this
compound will be called A-21978C nucleus. This compound can also be
represented by formula 4: ##STR5## wherein 3MG represents
L-threo-3-methylglutamic acid.
The compounds of formula 3 wherein R.degree. or R' are other than
hydrogen are prepared by deacylation of appropriately blocked
antibiotic A-21978C factors C.sub.0, C.sub.1, C.sub.2, C.sub.3,
C.sub.4 and C.sub.5. For convenience herein, these compounds will
be called blocked A-21978C nuclei. These blocked compounds are
useful intermediates to certain peptides of formula 1, e.g. those
compounds wherein R.sup.1 is an amino-protecting group.
As will be apparent to those skilled in the art, A-21978C nucleus
and blocked A-21978C nuclei can be obtained either in the form of
free amines or of acid addition salts. Although any suitable acid
addition salt may be used, those which are non-toxic and
pharmaceutically acceptable are preferred.
The method of preparing the A-21978C nuclei of formula 3 from the
corresponding A-21978C antibiotic by means of fermentation using
Actinoplanes utahensis NRRL 12052 is described in the co-pending
application of Abbott et al., Ser. No. 380,497. A. utahensis NRRL
12052 is available to the public from the Agricultural Research
Culture Collection (NRRL) Northern Regional Research Center, U.S.
Department of Agriculture, 1815 N. University St., Peoria, Ill.
61604, U.S.A., under the accession number NRRL 12052. Preparation 1
herein illustrates the preparation of A-21978C nucleus by
fermentation using the A-21978C complex as the substrate and
Actinoplanes utahensis NRRL 12052 as the microorganism.
Other Actinoplanaceae cultures which can be used to prepare the
A-21978C nuclei of formula 3 are available to the public from the
Northern Regional Research Laboratory under the following accession
numbers:
______________________________________ Actinoplanes missouriensis
NRRL 12053 Actinoplanes sp. NRRL 8122 Actinoplanes sp. NRRL 12065
Streptosporangium roseum NRRL 12064 var. hollandensis
______________________________________
The effectiveness of any given strain of microorganism within the
family Actinoplanaceae for carrying out the deacylation 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 Micrococcus luteus 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) re-acylation
with an appropriate side chain (e.g. lauroyl, n-decanoyl or
n-dodecanoyl) to restore activity.
The present invention relates to novel compounds derived by
acylating an A-21978C nucleus (compound of formula 3). The
compounds of the present invention have the chemical structure
depicted in formula 1: ##STR6## wherein R is hydrogen,
8-methyldecanoyl, 10-methyldodecanoyl, 10-methylundecanoyl, the
specific C.sub.10 -alkanoyl group of A-21978C.sub.0 or the specific
C.sub.12 -alkanoyl groups of A-21978C factors C.sub.4 and C.sub.5,
an amino-protecting group, an aminoacyl group of the formula
##STR7## wherein Q is C.sub.1 -C.sub.16 alkylene, or an
N-alkanoylaminoacyl group of the formula ##STR8## wherein:
W is a divalent aminoacyl radical of the formula: ##STR9## wherein
A is C.sub.1 -C.sub.1 alkylene or C.sub.5 -C.sub.6 cycloalkylene;
##STR10## 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 alkoxy, C.sub.1 -C.sub.3 alkylthio, carbamyl, or
C.sub.1 -C.sub.3 alkylcarbamyl; ##STR11## wherein X is hydrogen
chloro, bromo, iodo, amino, 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; ##STR12## wherein
X.sup.1 is chloro, bromo, iodo, amino, hydroxy, C.sub.1 -C.sub.3
-alkyl or C.sub.1 -C.sub.3 -alkoxy; ##STR13## wherein B is a
divalent radical of the formula: --(CH.sub.2).sub.n -- and n is an
integer from 1 to 3; --CH.dbd.CH--; --CH.dbd.CH--CH.sub.2 --; or
##STR14##
R.sup.2 is C.sub.1 -C.sub.17 alkyl or C.sub.2 -C.sub.17 alkenyl;
and
R.sup.1 is hydrogen, amino-protecting group, an aminoacyl group of
the formula ##STR15## as herein defined, or an N-alkanoylaminoacyl
group of the formula ##STR16## as herein defined; provided that,
when R is other than aminoacyl or N-alkanoylaminoacyl, R.sup.1 must
be aminoacyl or N-alkanoylaminoacyl; and, when R.sup.1 is an
amino-protecting group, R must be aminoacyl or N-alkanoylaminoacyl;
and the salts thereof.
As used 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 or C.sub.1 -C.sub.16 alkylene
radicals which are preferred for purposes of this invention are:
--CH.sub.2 --; ##STR17## 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 ##STR18## in which p is an integer from 1
to 8 and q is an integer from 0 to 7, provided that p+q 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 ##STR19## 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 ##STR20## it will be
recognized by those skilled in the art that the ##STR21## 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 ##STR22## and --NH-- functions are
oriented in the para configuration.
The terms "substituted phenyl" and "substituted benzyl", as defined
by R.sup.3 in formula 1, 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.sup.3 or X in formula
1 includes the methyl, ethyl, n-propyl, or i-propyl groups.
Illustrative R and/or R.sup.1 aminoacyl and N-alkanoylaminoacyl
groups are provided in the Examples, infra. Other such illustrative
R and/or R.sup.1 groups are:
4-[N-(n-octanoyl)amino]cyclohexan-1-carbonyl,
7-[N-(n-heptanoyl)amino]-n-octanoyl,
.alpha.-hydroxymethyl-.alpha.-[N-(n-pentadecanoyl)amino]acetyl;
.alpha.-(m-methoxyphenyl)-.alpha.-[N-(n-heptanoyl)amino]acetyl,
m-chloro-p-[N-(n-nonanoyl)amino]benzoyl,
2,4-dihydroxy-5-[N-(n-decanoyl)amino]benzoyl,
4-[N-(3-methylbutanoyl)amino]nicotinoyl,
4-[N-(n-heptadecanoyl)amino]phenylpropionyl and
p-[N-(n-hexadecanoyl)amino]hippuryl.
The compounds of formula 1 are capable of forming salts. These
salts are also part of this invention. Such salts are useful, for
example, for separating and purifying the compounds.
Pharmaceutically-acceptable alkali-metal, alkaline-earth-metal,
amine and acid-addition salts are particularly useful.
For example, the compounds of formula 1 have five free carboxyl
groups which can form salts. Partial, mixed and complete salts of
these carboxyl groups are, therefore, contemplated as part of this
invention. In preparing these salts, pH levels greater than 10
should be avoided due to the instability of the compounds at such
levels.
Representative and suitable alkali-metal and alkaline-earth metal
salts of the compounds of formula 1 include the sodium, potassium,
lithium, cesium, rubidium, barium, calcium and magnesium salts.
Suitable amine salts of the formula 1 compounds include the
ammonium and the primary, secondary, and tertiary C.sub.1 -C.sub.4
-alkylammonium and hydroxy-C.sub.2 -C.sub.4 -alkylammonium salts.
Illustrative amine salts include those formed by reaction of a
formula 1 compound with ammonium hydroxide, methylamine,
sec-butylamine, isopropylamine, diethylamine, di-isopropylamine,
cyclohexylamine, ethanolamine, triethylamine, 3-amino-1-propanol
and the like.
The alkali-metal and alkaline-earth-metal cationic salts of the
compounds of formula 1 are prepared according to procedures
commonly used for the preparation of cationic salts. For example,
the free acid form of a formula 1 compound is dissolved in a
suitable solvent such as warm methanol or ethanol; a solution
containing the stoichiometric quantity of the desired inorganic
base is aqueous methanol is added to this solution. The salt thus
formed can be isolated by routine methods, such as filtration or
evaporation of the solvent.
The salts formed with organic amines can be prepared in a similar
manner. For example, the gaseous or liquid amine can be added to a
solution of a formula 1 compound in a suitable solvent such as
ethanol; the solvent and excess amine can be removed by
evaporation.
The compounds of this invention also have free amino groups and
can, therefore, form acid addition salts. Such salts are also part
of this invention. Representative and suitable acid-addition salts
of the compounds of formula 1 include those salts formed by
standard reaction with both organic and inorganic acids such as,
for example, hydrochloric, sulfuric, phosphoric, acetic, succinic,
citric, lactic, maleic, fumaric, palmitic, cholic, pamoic, mucic,
D-glutamic, d-camphoric, glutaric, glycolic, phthalic, tartaric,
lauric, stearic, salicylic, methanesulfonic, benzenesulfonic,
sorbic, picric, benzoic, cinnamic and like acids.
The compounds of formula 1 are prepared by acylating a compound of
formula 3 at the .alpha.-amino group of tryptophan with the
appropriate N-alkanoylaminoacyl or N-alkanoylaminoacyl side chain
using methods conventional in the art for forming an amide bond.
The acylation is accomplished, in general, by reacting the formula
3 compound with an activated derivative of the acid (formula 5)
corresponding to the desired acyl side chain group. ##STR23## (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, an 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.
It will be recognized by those skilled in the art that the
compounds of formula 1 are prepared using selective acylation
procedures and with the assistance of amino-protecting groups. For
example, when a compound of formula 3 wherein R.degree. and R' are
hydrogen is the starting material, acylation can occur at both the
.alpha.-amino group of tryptophan and the .delta.-amino group of
ornithine to give N.sub.Trp, N.sub.Orn -diacyl derivatives. To
obtain derivatives monoacylated at the .alpha.-amino group of
tryptophan, therefore, it is preferable to acylate a compound of
formula 3 wherein the .delta.-amino group of ornithine (the
R.degree. position) is blocked by an amino-protecting group. Such
starting materials are preferably obtained by blocking the A-21978C
factor at this position before it is deacylated. The aromatic
antibiotic group of kynurenine (the R' position) is the least
reactive of the three free amino groups in the A-21978C nucleus.
Thus, acylation at the R or R.sup.1 position does not usually
involve blocking this amino group.
Scheme I outlines general procedures for the preparation of the
compounds of formula 1. In this Scheme the following symbols are
used:
[*]=remainder of A-21978C
N.sub.T =.alpha.-amino group of tryptophan
N.sub.O =.delta.-amino group of ornithine
N.sub.K =aromatic amino group of kynurenine
R, R.sup.1 =substituents as defined
R.sub.N =acyl group of natural factor
B=amino-protecting group
Acyl=an acylation step
Deacyl=a deacylation step
Block=acylation with an amino-protecting group
Deblock=removal of an amino-protecting group
In Scheme I the N.sub.Trp -monoacyl derivatives of A-21978C are
represented by general formula 3 and the N.sub.Trp, N.sub.Orn
-diacyl derivatives of A-21978C are represented by general formula
4. Those N.sub.Trp, N.sub.Orn -diacyl derivatives wherein the
N.sub.Trp -acyl group is that of a natural A-21978C factor are
represented by general formula 8. ##STR24##
A preferred method for preparing the compounds of formula 1 is by
the active ester method, using the compound of formula 3 wherein
R'=H and R.degree.=t-BOC, i.e. the A-21978C N.sub.Orn t-BOC nucleus
or "tBOC nucleus". The use of the 2,4,5-trichlorophenyl ester of
the desired N-alkanoylamino acid or N-alkenoylamino acid (formula
5) as the acylating agent is most preferred. In this method, an
excess amount of the active ester is reacted with the t-BOC nucleus
at room temperature in a non-reactive organic solvent such as DMF,
THF, diethyl ether or dichloromethane. 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. A particularly useful
purification method is column chromatography, using silica gel as
the stationary phase and ethyl acetate:methanol (3:2, v:v) as the
solvent system. The t-BOC group is removed by treatment with
trifluoroacetic acid/anisole/triethylsilane or, preferably,
trifluoroacetic acid/1,2-ethanedithiol for from about three to
about five minutes at room temperature. After the solvent is
removed, the residue is purified by reversed-phase HPLC.
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 5) 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 desired 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 used 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 used 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 used 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 used as
starting materials and will give products which are within the
contemplation of this invention.
When an A-21978C cyclic peptide of this invention is used as an
antibacterial agent, it may be administered either orally or
parenterally. As will be appreciated by those skilled in the art,
the A-21978C compound is commonly administered together with a
pharmaceutically acceptable carrier or diluent. The dosage of
A-21978C compound will depend upon a variety of considerations,
such as, for example, the nature and severity of the particular
infection to be treated. Those skilled in the art will recognize
that appropriate dosage ranges and/or dosage units for
administration may be determined by considering the MIC and
ED.sub.50 values and toxicity data herein provided together with
factors such as pharmacokinetics, the patient or host and the
infecting microorganism.
The methods of making and using the compounds of the present
invention are illustrated in the following examples:
PREPARATION 1
Preparation of A-21978C Nucleus
A. Fermentation of Actinoplanes utahensis
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
______________________________________ Pre-cooked 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 g 2 ml conc HCl) 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 3.0 g casein* Beef extract
0.5 g Glucose 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--ZAmine
A, Humko Sheffield Chemical, Lyndhurst, NJ.
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
______________________________________ Pre-cooked 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, NY.
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 abovedescribed.
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-mount 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 (80 ml), prepared as
above-described, is used to inoculate 10 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 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
14-liter fermentation vessel at a temperature of about 30.degree.
C. for about 66 hours. The fermentation medium is stirred with
conventional agitators at about 600 RPM and aerated with sterile
air to maintain the dissolved oxygen level above 30% of air
saturation at atmospheric pressure.
B. Deacylation of A-21978C
A fermentation of A. utahensis is carried out as described in
Section A, using slant medium A and production medium I and
incubating the production medium for about 67 hours. Crude A-21978C
complex (100 g), prepared as described in U.S. Pat. No. 4,208,403,
is added to the fermentation medium.
Deacylation of the A-21978C complex is monitored by assay against
Micrococcus luteus. The fermentation is allowed to continue until
deacylation is complete as indicated by disappearance of activity
vs. M. luteus, a period of about 24 hours.
C. Isolation of A-21978C Nucleus
Whole fermentation broth (20 liters), obtained as described in
Section B, was filtered with a filter aid (Hyflo Super-Cel, Johns
Manville Corp.). The mycelial cake was discarded. The filtrate thus
obtained was passed through a column containing 1.5 liters of HP-20
resin (DIAION High Porous Polymer, HP-Series, Mitsubishi Chemical
Industries Limited, Tokyo, Japan). The effluent thus obtained was
discarded. The column was then washed with deionized water (10 L.)
to remove residual filtered broth. This wash water was discarded.
The column was then eluted with water:acetonitrile mixtures (10 L.
each of 95:5, 9:1, and 4:1), collecting 1-liter fractions.
Elution was monitored by analytical HPLC, using silica gel/C.sub.18
and a solvent system of water:methanol (3:1) containing 0.1%
ammonium acetate, detecting the nucleus with a UV monitor at 254
nm. Fractions containing the nucleus were combined, concentrated
under vacuum to remove the acetonitrile and freeze-dried to give
40.6 g of semi-purified A-21978C nucleus.
D. Purification of A-21978C Nucleus
Semi-purified A-21978C nucleus (15 g), obtained as described in
Section C, was dissolved in 75 ml of water:methanol:acetonitrile
(82:10:8) containing 0.2% acetic acid and 0.8% pyridine. This
solution was pumped onto a 4.7-.times.192-cm column containing 3.33
L. of silica gel (Quantum LP-1)/C.sub.18. The column was developed
with the same solvent system. Fractions having a volume of 350 ml
were collected. Separation was monitored at 280 nm with a UV
monitor. Fractions containing the nucleus were combined,
concentrated under vacuum to remove solvents and freeze-dried to
give 5.2 g of purified A-21978C nucleus.
E. Characteristics of A-21978C Nucleus
A-21978C nucleus has the following characteristics:
(a) Form: white amorphous solid which fluoresces under short-wave
UV
(b) Empirical formula: .[.C.sub.62 H.sub.82 N.sub.16 O.sub.26 .].
.Iadd.C.sub.62 H.sub.83 N.sub.17 O.sub.25 .Iaddend.
(c) Molecular weight: .[.1466.]. .Iadd.1465 .Iaddend.
(d) Solubility: soluble in water
(e) Infrared absorption spectrum (KBr disc) shows absorption maxima
at the following frequencies (cm.sup.-1): 3300 (broad), 3042
(weak), 2909 (weak), 1655 (strong), 1530 (strong), 1451 (weak),
1399 (medium), 1222 (medium), 1165 (weak), 1063 (weak) and 758
(medium to weak)
(f) UV absorption spectrum in methanol shows maxima at 223 nm
(.epsilon. 41,482) and 260 nm (.epsilon. 8,687)
(g) Electrometric titration in 66% aqueous dimethylformamide
indicates the presence of four titratable groups with pK.sub.a
values of about 5.2, 6.7, 8.5 and 11.1 (initial pH 6.12).
PREPARATION 2
Alternate Preparation of A-21978C Nucleus
A-21978C nucleus was prepared according to the method of
Preparation 1 except for certain changes in Section B. The A.
utahensis culture was incubated initially for about 48 hours; the
substrate was semipurified A21978C complex (50 g); and incubation
after addition of the substrate was about 16 hours. The broth
filtrate was passed over a column containing 3.1 liters of HP-20
resin. The column was washed with 10 volumes of water and then was
eluted with water:acetonitrile (95:5). Elution was monitored as in
Preparation 1. After collecting 24 liters, the eluting solvent was
changed to weater:acetonitrile (9:1). Fractions containing the
nucleus were eluted with this solvent. These fractions were
combined, concentrated under vacuum to remove acetonitrile, and
freeze-dried to give 24.3 g of semi-purified A-21978C nucleus.
This semi-purified A-21978C nucleus (24.3 g) was dissolved in water
(400 ml). The solution was pumped onto a 4.7-.times.192-cm steel
column containing 3.33 liters of silica gel (Quantum LP-1)/C.sub.18
prepared in water:methanol:acetonitrile (8:1:1) containing 0.2%
acetic acid and 0.8% pyridine. The column was developed with the
same solvent at a pressure of about 2000 psi, collecting 350 ml
fractions. Elution was monitored by UV at 280 nm. Fractions
containing the nucleus were combined, concentrated under vacuum to
remove solvents, and freeze-dried to give 14 g of highly purified
A-21978C nucleus.
PREPARATION 3
Preparation of N.sub.Orn -t-BOC-A-21978C Factors C.sub.2 and
C.sub.3
A mixture of A-21978C factors C.sub.2 and C.sub.3 (10 g), prepared
as described in U.S. Pat. No. 4,208,403, was dissolved in water (50
ml) with sonication (200 mg/ml). The pH of the solution was
adjusted from 4.05 to 9.5 with 5N NaOH (3.6 ml). Di-tert-butyl
dicarbonate (3.0 ml) was added, and the reaction mixture was
stirred at room temperature for 2 hours. The pH of the reaction was
maintained at 9.5 by manual addition of 5N NaOH (6.5 ml added in 2
hours).
The reaction was monitored periodically by TLC on silica gel, using
CH.sub.3 CN:H.sub.2 O (7:3 and 8:2) solvent systems and detecting
by UV.
After about 10 minutes and reaction solution became rapidly turbid,
and base consumption increased. After 30 minutes, the rate of
increase in turbidity and the rate of base consumption decreased,
indicating that the reaction was complete. Nevertheless, the
reaction was continued for an additional 90 minutes to insure
completion. At the end of the two-hour reaction, the reaction
material was lyophilized immediately to give 12.7 g of N.sub.Orn
-t-BOC-A-21978C factors C.sub.2 and C.sub.3.
Using similar procedures, two 10-g reactions and a 30-g reaction
were run. In each of these the reaction time was only 40 minutes.
The two 10-g experiments gave 11.9 and 12.1 g of product,
respectively. The 30-g reaction gave 35.4 g of product.
PREPARATION 4
Preparation of A-21978C N.sub.Orn -t-BOC Nucleus
A. Fermentation of A. utahensis
A fermentation of A. utahensis was carried out as described in
Preparation 1, Section A, using slant medium A and production
medium I and incubating the production medium for about 66
hours.
B. Deacylation of N.sub.Orn -t-BOC Complex
The A-21978C N.sub.Orn-t-BOC complex (1185 g of crude substrate
which contained about 176 g of A-21978C complex) was added to the
fermentation medium. Deacylation was carried out as described in
Preparation 1, Section B. Deacylation was complete, as indicated by
HPLC, after about 24 hours.
C. Isolation of A-21978C N.sub.Orn -t-BOC Nucleus
Fermentation broth (100 L.), obtained as described in Section B,
was filtered with a filter aid (Hyflo Super-cel). The filtrate was
passed over a column containing 7.5 L. of HP-20 resin (DIAION); the
column was washed with water (38 L.). Elution was monitored by
silica gel/C.sub.18 HPLC with UV detection at 254 nm. Some nucleus
was eluted in the wash. Subsequent elution of nucleus was carried
out with water:acetonitrile mixtures as follows: (95:5)-40 L;
(9:1)--40 L.; and (85:15)--100 L. Fractions containing the nucleus
were combined, concentrated under vacuum to remove solvent, and
freeze-dried to give 298.5 g of semi-purified A-21978C N.sub.Orn
-t-BOC Nucleus
D. Purification of A-21978C N.sub.Orn -t-BOC Nucleus
Semi-purified A-21978C N.sub.Orn -t-BOC nucleus (30 g), obtained as
described in section C, was dissolved in water:acetonitrile (9:1)
containing 0.2% acetic acid and 0.8% pyridine (75 ml). This
solution was applied to a 4.7.times.192-cm steel column containing
3.33 L. of silica gel (Quantum LP-1)/C.sub.18 equilibrated in the
same solvent system. The column was developed under pressure with
water:acetonitrile:methanol (80:15:5) containing 0.2% acetic acid
and 0.8% pyridine, collecting 350-ml fractions and detecting
product by UV at 280 nm. Fractions containing the product were
combined, concentrated under vacuum to remove solvent and
freeze-dried to give 18.4 g of purified A-21978C N.sub.Orn -t-BOC
nucleus.
A-21978C t-BOC nucleus has the following characteristics:
(a) Form: white amorphous solid which fluoresces under short-wave
UV
(b) Empirical formula: .[.C.sub.67 H.sub.90 N.sub.16 O.sub.28 .].
.Iadd.C.sub.67 H.sub.91 N.sub.17 O.sub.27 .Iaddend.
(c) Molecular weight: .Badd..[.1566.]..Baddend. .Iadd.1565
.Iaddend.
(d) Solubility: soluble in water
(e) Infrared absorption spectrum (KBr disc) shows absorption maxima
at the following frequencies (cm.sup.-1): 3345 (broad), 3065
(weak), 2975 (weak) 2936 (weak), .about.1710 (shoulder), 1660
(strong), 1530 (strong), 1452 (weak), 1395 (medium, 1368 (weak),
1341 (weak), 1250 (medium), 1228 (medium), 1166 (medium to weak)
and 1063 (weak)
(f) UV absorption spectrum in 90% ethanol shows maxima at: 220 nm
(.epsilon.42,000) and 260 nm (.epsilon.10,600).
(g) HPLC retention time=6 min on 4.6-.times.300-mm silica-gel
C.sub.18 column, using H.sub.2 O/CH.sub.3 CN/CH.sub.3 OH (80:15:5)
solvent containing 0.2% NH.sub.4 OAc at a flow rate of 2 ml/min
with UV detection.
PREPARATION 5
Alternative Purification of A-21978C N.sub.Orn -t-BOC Nucleus
Semi-purified A-21978C N.sub.Orn -t-BOC nucleus (10.8 g), obtained
as described in Preparation 4, Section C, was dissolved in water
and applied to a column containing 80 ml of Amberlite IRA-68
(acetate cycle). The column was washed with water and, at a flow
rate of 5 ml/min, was eluted sequentially with 0.05N acetic acid
(1080 ml), 0.1N acetic acid (840 ml), and 0.2N acetic acid (3120
ml), collecting 120-ml fractions. The column was monitored with
analytical HPLC over silica gel/C.sub.18, using a system of
water:acetonitrile:methanol (80:15:5) containing 0.2% ammonium
acetate and detecting product with UV at 254 nm. Fractions
containing the product were combined; the pH of the solution was
adjusted to 5.8 with pyridine; the resulting solution was
concentrated under vacuum to a volume of about 200 ml. Water was
added to the concentrate, and the resulting solution was
reconcentrated to remove pyridine. This concentrate was
freeze-dried to give 3.46 g of purified A-21978C N.sub.Orn -t-BOC
nucleus.
PREPARATIONS 6-14
The preparation of a number of useful N-alkanoylamino acids is
described in U.S. Pat. No. 4,293,483 (see Table 1, columns 9-16).
Such compounds 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 used should be sufficient to make the
concentration of reactants between 0.1 to 0.2M. 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.
Other N-alkanoylamino acids prepared by this procedure are
summarized in Table 1.
TABLE I
__________________________________________________________________________
Preparation of NAlkanoyl Amino Acids Prep. Alkanoic acid chloride
Amino Acid NAlkanoyl Amino Acid No. Formula wt. (g) Formula wt. (g)
Formula wt. (g)
__________________________________________________________________________
6 CH.sub.3 (CH.sub.2).sub.6 COCl 3.25 L-phenylalanine 3.30 CH.sub.3
(CH.sub.2).sub.6 CONHCH(CH.sub.2 C.sub.6 H.sub.5)COOH 4.85 7
CH.sub.3 (CH.sub.2).sub.7 COCl 2.0 " 1.82 CH.sub.3 (CH.sub.2).sub.7
CONHCH(CH.sub.2 C.sub.6 H.sub.5)COOH 2.8 8 CH.sub.3
(CH.sub.2).sub.8 COCl 3.9 " 3.30 CH.sub.3 (CH.sub.2).sub.8
CONHCH(CH.sub.2 C.sub.6 H.sub.5)COOH 5.35 9 CH.sub.3
(CH.sub.2).sub.9 COCl 4.0 " 3.23 CH.sub.3 (CH.sub.2).sub.9
CONHCH(CH.sub.2 C.sub.6 H.sub.5)COOH 4.5 10 CH.sub.3
(CH.sub.2).sub.10 COCl 6.54 " 4.95 CH.sub.3 (CH.sub.2).sub.10
CONHCH(CH.sub.2 C.sub.6 H.sub.5)COOH 5.2 11 CH.sub.3
(CH.sub.2).sub.11 COCl 2.0 " 1.42 CH.sub.3 (CH.sub.2).sub.11
CONHCH(CH.sub.2 C.sub.6 H.sub.5)COOH 1.7 12 CH.sub.3
(CH.sub.2).sub.12 COCl 4.8 " 3.30 CH.sub.3 (CH.sub.2).sub.12
CONHCH(CH.sub.2 C.sub.6 H.sub.5)COOH 6.6 13 CH.sub.3
(CH.sub.2).sub.4 COCl 6.2 L-tryptophan 10.2 ##STR25## 6.82 14
CH.sub.3 (CH.sub.2).sub.10 COCl 10.9 " 10.2 ##STR26## 13.8
__________________________________________________________________________
PREPARATIONS 15-24
The 2,4,5-trichlorophenyl esters of the N-alkylamino acids
described in U.S. Pat. No. 4,293,483 are also described in that
patent (see Table 2, columns 17-20). Such compounds are prepared
according to the following general procedure:
The N-alkanoylamino acid (1 mole), 2,4,5-trichlorophenol (1.1
mole), and DCC (1 mole) are dissolved in dichloromethane, diethyl
ether or THF. 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
acetonitrile-water or diethyl ether-petroleum ether.
The preparation of other 2,4,5-trichlorophenyl esters of
N-alkanoylamino acids prepared by this method is summarized in
Table II.
TABLE II
__________________________________________________________________________
Preparation of 2,4,5-Trichlorophenyl Esters NAlkanoyl Amino Acid
2,4,5-Trichlorophenyl Preparation No. Formula wt (g) Ester Product
wt
__________________________________________________________________________
(g) 15 CH.sub.3 (CH.sub.2).sub.6 CONHCH(CH.sub.2 C.sub.6
H.sub.5)COOH 2.9 4.1 16 CH.sub.3 (CH.sub.2).sub.7 CONHCH(CH.sub.2
C.sub.6 H.sub.5)COOH 2.8 3.86 17 CH.sub.3 (CH.sub.2).sub.8
CONHCH(CH.sub.2 C.sub.6 H.sub.5)COOH 3.19 1.5 18 CH.sub.3
(CH.sub.2).sub.9 CONHCH(CH.sub.2 C.sub.6 H.sub.5)COOH 3.29 5.01 19
CH.sub.3 (CH.sub.2).sub.11 CONHCH(CH.sub.2 C.sub.6 H.sub.5)COOH 1.7
2.01 20 CH.sub.3 (CH.sub.2).sub.12 CONHCH(CH.sub.2 C.sub.6
H.sub.5)COOH 3.75 4.18 21 ##STR27## 2.1 1.6 22 CH.sub.3
(CH.sub.2).sub.10 CONHCH(CH.sub.2 C.sub.6 H.sub.5)COOH 3.47 3.98 23
##STR28## 6.04 1.87 24 ##STR29## 7.76 4.03
__________________________________________________________________________
EXAMPLES 1-33
The N-alkanoylamino acid derivatives of A-21978C of formula 1 are
prepared according to the following general procedure which
involves acylating the nucleus using the activated-ester
method:
A solution of N.sub.Orn -t-BOC-blocked-A-21978C nucleus (t-BOC
nucleus) in DMF was treated with the 2,4,5-trichlorophenyl ester
("active ester") of the corresponding N-alkanoylamino acid. The
reaction mixture was stirred at room temperature for about 18 to
about 24 hours under an atmosphere of nitrogen and then was
evaporated to dryness under reduced pressure to give a residue. A
small amount of methanol was added to the residue; a solid
(N,N'-dicyclohexylurea) which did not dissolve in the methanol was
removed by filtration and discarded. The filtrate was evaporated
under vacuum to give a solid, the crude N.sub.Orn -t-BOC-N.sub.Trp
-acyl-A-21978C analog. This analog was purified using a "Prep
LC/System 500" unit (Waters Associates, Inc., Milford, Mass.)
equipped with a Prep Pak-500/C.sub.18 column (Waters Associates
Inc.) as a stationary phase. The column was eluted isocratically,
using a water:methanol:acetonitrile (2:1:2) solvent system
containing 0.1% pyridinium acetate, and eluting one 250-ml fraction
per minute for approximately 40 fractions. Amounts of sample
applied varied from about 1 g to about 5 g. Early fractions
containing unreacted starting materials were discarded. The desired
product was always the major peak (using a UV detector) following
the early fractions. Individual fractions were pooled on the basis
of TLC [reversed-phase silica gel (C.sub.18), a
water:methanol:acetonitrile (3:3:4) solvent system, and Von Urk
spray for detection] and bioautographic analysis [silica-gel TLC
plates (Merck), an acetonitrile:acetone:water (2:2:1) solvent
system and Micrococcus luteus as the assay organism].
The N.sub.Orn -t-BOC-N.sub.Trp -acyl-A-21978C analog, obtained as a
single component by this method, was lyophilized and treated with
anhydrous trifluoroacetic-acid (10 ml per 0.3-0.5 g of analog)
containing 2% anisole at 0.degree. C. After about five minutes the
reaction mixture was evaporated to dryness under reduced pressure.
The residue obtained was triturated with a small amount of ether.
The solid precipitate was collected and air-dried. This material
was dissolved in water; the pH of the solution was raised to about
6 to 7 by the addition of pyridine; and the solution was then
lyophilized. The resulting product was obtained as a single
component and was characterized by its chromatographic properties
and its amino-acid analysis.
Table III summarizes a group of N.sub.Trp -alkanoylaminoacyl
A-21978C derivatives prepared by this procedure.
TABLE III
__________________________________________________________________________
N.sub.TrpAlkanoylamino Acid Derivatives of A-21978C Cyclic Peptides
##STR30## 1 .]. ##STR31## 1 t-BOC A-21978C A-21978C Example
Product.sup.a Ester Nucleus Product Product No. R in formula 1 (mg)
(mg) (mg).sup.b (mg) R.sub.f.sup.c
__________________________________________________________________________
1 (D)CH.sub.3 (CH.sub.2).sub.10 CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO
900 900.sup.d 556 436 0.82 2 CH.sub.3 (CH.sub.2).sub.10
CONH(CH.sub.2).sub.4CO 900 900.sup.d 489 485 0.65 3 CH.sub.3
(CH.sub.2).sub.10 CONH(CH.sub.2).sub.10CO 600 900.sup.d 326 242
0.83 ##STR32## 416 1000.sup.c -- 195 0.34 5 ##STR33## 900 900.sup.d
352 263 0.57 6 ##STR34## 800 800.sup.d 76 24 0.23 7 ##STR35## 800
800.sup.d 389 312 0.17 8 (L)CH.sub.3 (CH.sub.2).sub.10
CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO 800 800.sup.d 421 324 0.82 9
##STR36## 1000 1000 -- 230 0.68 10 ##STR37## 900 900.sup.d 633 485
0.74 11 ##STR38## 800 800.sup.d 245 186 0.58 12 ##STR39## 350
700.sup.d 376 304 0.38 13 ##STR40## 900 900.sup.d 320 218 0.65 14
CH.sub.3 (CH.sub.2).sub.5 CONH(CH.sub.2).sub.10CO 900 900.sup.d 503
428 0.60 15 ##STR41## 900 900.sup.d 273 212 0.83 16 ##STR42## 1000
1000.sup.d 426 286 0.73 17 (L)CH.sub.3 (CH.sub.2).sub.6
CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO 800 800.sup.d 444 343 0.48 18
(L)CH.sub.3 (CH.sub.2).sub.7 CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO 800
800.sup.d 412 312 0.50 19 (L)CH.sub.3 (CH.sub.2).sub.8
CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO 800 800.sup.d 519 453 0.52 20
(L)CH.sub.3 (CH.sub.2).sub.9 CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO 800
800.sup.d 371 287 0.53 21 (L)CH.sub.3 (CH.sub.2).sub.11
CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO 800 800.sup.d 412 335 22
(L)CH.sub.3 (CH.sub.2).sub.12 CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO
800 800.sup.d 468 328
__________________________________________________________________________
.sup.a R.sup.1 = H, .sup.b N.sub.Ornt-BOCN.sub.TrpAcyl-Nucleus,
.sup.c Thin-layer chromatography on silica gel (Merck), using a
water:CH.sub. 3 CN:acetone (1:2:2) solvent system .sup.d
N.sub.Ornt-BOCNucleus, .sup.e A21978C Nucleus.
Other N-alkanoylamino acid derivatives of formula 1 prepared in a
similar manner are summarized in Table IV.
TABLE IV
__________________________________________________________________________
A-21978C Cyclic Peptides of Formula 1 Example No. R R.sup.1
__________________________________________________________________________
23 ##STR43## H 24 ##STR44## H 25 H ##STR45## 26 H CH.sub.3
(CH.sub.2).sub.9 CONHCH(CH.sub. 2 C.sub.6 H.sub.5)CO
__________________________________________________________________________
Table V summarizes a group of A-21978C derivatives prepared
according to the general procedure, but using A-21978C factors as
starting materials.
TABLE V
__________________________________________________________________________
Diacyl Derivatives of A-21978C Example Starting A-21978C Product
No. R in formula 1 R.sup.1 in formula 1 Factor Ester (mg) Factor
(mg) (mg) R.sub.f.sup.a
__________________________________________________________________________
27 CH.sub.3 CH.sub.2 CH(CH.sub.3)(CH.sub.2).sub.6 CO ##STR46##
C.sub.1 100 48 43 0.81 28 CH.sub.3 CH(CH.sub.3)(CH.sub.2).sub.8 CO
" C.sub.2 100 48 25 0.73 29 CH.sub.3 CH.sub.2
CH(CH.sub.3)(CH.sub.2).sub.8 CO " C.sub.3 400 1000 413 0.87 30
CH.sub.3 CH.sub.2 CH(CH.sub.3)(CH.sub.2).sub.6 CO H.sub.2
N(CH.sub.2).sub.10CO C.sub.1 1000 1000 732 0.65
__________________________________________________________________________
.sup.a TLC on silica gel (Merck) using a water:CH.sub.3 CN:acetone
(1:2:2 solvent system
Other N.sub.Trp -N.sub.Orn -diacyl derivatives of A-21978C prepared
according to the general procedure are listed in Table VI.
TABLE VI ______________________________________ Diacyl Derivatives
of A-21978C Ex- am- ple No. R in Formula 1 R.sup.1 in Formula 1
______________________________________ 31 ##STR47## ##STR48## 32
##STR49## t-BOC 33 CH.sub.3 (CH.sub.2).sub.10 CONHCH(CH.sub.2
C.sub.6 H.sub.5)CO t-BOC ______________________________________
EXAMPLE 34
Preparation of N.sub.Trp -[N-(n-Decanoyl)-L-phenylalanyl]-A-21978C
Nucleus (Compound of Example 19)
This example illustrates the large-scale preparation of compounds
by the active-ester method.
A. Preparation of
N-(n-Decanoyl)-L-Phenylalanyl-2,4,5-Trichlorophenolate
A solution of N-(n-decanoyl)-L-phenylalanine (31.9 g, 0.1 mole) and
2,4,5-trichlorophenol (19.7 g, 0.1 mole) in 1 liter of anhydrous
ether was treated with N,N'-dicyclohexylcarbodiimide (20.6 g, 0.1
mole). The reaction was stirred overnight at room temperature. The
precipitated N,N'-dicyclohexylurea was removed by filtration and
discarded. The filtrate was concentrated under vacuum to dryness.
The residue obtained was triturated with ether, and the solids
(residual cyclohexylurea) were removed by filtration. The filtrate
was evaporated to dryness under reduced pressure. The residue was
crystallized from acetonitrile to give 36.9 g of crystalline
N-(n-decanoyl)-L-phenylalanyl-2,4,5-trichlorophenolate, m.p.
122.degree.-124.degree. C.
B. Preparation of N.sub.Trp
-[N-(N-Decanoyl)-L-phenylalanyl]-N.sub.Orn -t-BOC-A-21978C
Nucleus
A solution of
N-(n-decanoyl)-L-phenylalanyl-2,4,5-trichlorophenolate (10 g, 0.02
mole), N.sub.Orn -t-BOC-A-21978C nucleus (10 g, 0.006 mole) in
anhydrous DMF (1 L) was stirred at room temperature for 96 hours
under an atmosphere of nitrogen. The solvent was removed by
evaporation under reduced pressure. The residual material was
stirred with a mixture of diethyl ether (800 ml) and chloroform
(200 ml) for 2 hours. The product was separated by filtration to
give a light brown powder (10.3 g). This material (9.9 g) was
dissolved in methanol (200 ml) and purified by preparative HPLC,
using a "Prep LC/System 500" unit and a PrePak-500/C.sub.18 Column
as the stationary phase. The column was eluted isocratically, using
a water:methanol:acetonitrile (2:1:2) solvent system and collecting
250-ml fractions at a rate of one fraction per minute. The desired
compound was eluted in the 9th through the 22nd fractions.
Fractions were combined on the basis of TLC [reversed phase silica
gel/C.sub.18 ; developed with water:methanol:acetonitrile (3:3:4);
detected with Van Urk spray]. Combined fractions were examined by
bioautography [silica gel TLC acetonitrile:acetone:water (2:2:1)
solvent system and Micrococcus luteus as the detecting organism]
and were shown to consist of a single bioactive component. This
procedure gave 6.02 g of N.sub.Trp
-[N-(n-decanoyl)-L-phenylalanyl]-N.sub.Orn -t-BOC-A-21978C nucleus
[compound of formula 1: R=N-(n-decanoyl-L-phenylalanyl); R.sup.1
=t-BOC].
C. Preparation of N.sub.Trp
-[N-(n-Decanoyl)-L-phenylalanyl]-A-21978C Nucleus
A flask (100 ml) was cooled to 5.degree. C. in an icebath.
N.sub.Trp -[N-(n-decanoyl)-L-phenylalanyl]-N.sub.Orn
-t-BOC-A-21978C nucleus (6.02 g. 0.008 mole), prepared as described
in Section B, and then anhydrous trifluoroacetic acid containing 2%
anisole (50 ml) were added to the flask. The mixture, which went
into solution in approximately two miniutes, was stirred under an
atmosphere of nitrogen for ten minutes. The solution was evaporated
to dryness under reduced pressure at below 40.degree. C. to give a
gummy solid which was triturated twice with a diethyl
ether:dichloromethane (4:1) solution (two 100-ml volumes). The
solids were collected by filtration and washed with diethyl ether
to give the TFA salt. This was dissoled in water (50 ml), and the
pH of the solution was adjusted to 5.4 with pyridine. The solution
was then lyophilized to give 6.1 g of off-white lyophilizate.
The lyophilizate, dissolved in methanol (35 ml), was purified using
a reverse-phase C.sub.18 silica-gel column (Waters Associates, Prep
500), eluting in stepwise gradient with H.sub.2 O:CH.sub.3
OH:CH.sub.3 CN containing 0.1% pyridinium acetate at ratios of
3:1:2, 2:1:2 and 1:2:2 and collecting fractions having a volume of
250 ml. The desired product was eluted during the 2:1:2 elution.
The fractions containing the product were lyophilized to give 2.23
g of cream-colored N.sub.Trp
-[N-(n-decanoyl)-L-phenylalanyl]-A-21978C nucleus (compound of
formula 1: R=N-(n-decanoyl)-L-phenylalanyl; R.sup.1 =H).
The product was evaluated by analytical HPLC [reversed-phase
C.sub.18 silica-gel column, MeOH:CH.sub.3 CN:H.sub.2 O:PyOAc
(15:35:49:1) solvent and UV detection at 230 nm], by TLC
[reversed-phase C.sub.18 silica-gel plates (Whatman), H.sub.2
O:CH.sub.3 OH:CH.sub.3 CN (3:3:4) solvent and Van Urk spray and
short-wave UV for detection] and by bioautography [silica-gel TLC
(Merck), an H.sub.2 O:CH.sub.3 CN:acetone (1:2:2) solvent, and
Micrococcus luteus as the detecting organism]. Each of these
methods demonstrated that the product was homogenous. Substitution
at the tryptophan N-terminus position was confirmed by 360 MHz PMR.
Amino-acid analysis confirmed the incorporation of one equivalent
of L-phenylalanine into the product.
EXAMPLE 34
The antibacterial activity of the compounds of formula 1 can be
demonstrated in vitro, using standard agar-dilution tests. The
results of the antibacterial testing of representative compounds of
formula 1 are set forth in Table VII. In Table VII activity is
measured by the minimal inhibitory concentration (MIC), i.e. the
lowest concentration of compound at which growth of the
microorganism is inhibited by the test compound.
TABLE VII
__________________________________________________________________________
Antibiotic Activity of A-21978C Cyclic Peptides Test Organism
MIC.sup.a of Test Compound.sup.b
__________________________________________________________________________
1 2.sup.e 3 4.sup.f 5 6 7 8 9.sup.f 10.sup.f 11
__________________________________________________________________________
Staphylococcus aureus X1.1 0.5 4 2 4,8 0.5 1 0.5 0.25 32,64
0.5,0.25 0.5 Staphylococcus aureus V41.sup.c 0.5 4 2 4,16 0.5 2 1
0.25 64,128 0.5,0.5 0.25 Staphylococcus aureus X400.sup.d 1 4 8
8,16 1 4 2 0.5 64,128 1,0.5 2 Staphylococcus aureus S13E 0.5 4 2
4,8 0.5 2 0.5 0.25 32,64 0.5,0.5 0.5 Staphylococcus epidermidis
EP11 2 4 8 16,64 2 2 2 1 128, 2.1 0.5 >128 Staphylococcus
epidermidis EP12 1 2 8 16,64 1 2 2 1 128, 1,0.5 0.5 >128
Streptococcus pyogenes C203 0.125 1 2 4,8 0.25 0.5 1 0.25 16,16
0.125, 0.125 0.06 Streptococcus pneumoniae Park I 0.125 4 0.5 4,16
0.25 2 0.125 0.125 32,64 0.25, 0.06 0.25 Streptococcus Group D X66
1 32 8 32,>64 4 16 2 1 >128, 4,4 2 >128 Streptococcus
Group D 9960 0.5 8 8 8,32 1 2 2 0.25 128, 2.1 4 >128
__________________________________________________________________________
12 13 14 15 16 17 18 19.sup.g 20 21 22
__________________________________________________________________________
Staphylococcus aureus X1.1 1 0.25 4 1 0.5 4 2 0.5 0.5 0.5 0.5
Staphylococcus aureus V41.sup.c 2 0.25 4 2 0.5 8 2 0.5 0.5 0.5 1
Staphylococcus aureus X400.sup.d 4 0.5 8 8 1 8 4 1 2 2 2
Staphylococcus aureus S13E 2 0.25 4 2 0.5 8 2 0.5 0.5 0.5 1
Staphylococcus epidermidis EP11 4 0.5 8 4 1 8 4 1 2 2 4
Staphylococcus epidermidis EP12 4 0.25 8 4 1 8 2 0.5 2 2 4
Streptococcus pyogenes C203 2 0.03 2 1 0.5 2 0.5 0.25 0.125 0.125
0.25 Streptococcus pneumoniae Park I 1 0.03 8 0.25 0.125 -- -- 0.75
-- -- -- Streptococcus Group D X66 16 1 >128 4 1 128 32 8 4 1 1
Streptococcus Group D 9960 4 0.25 64 4 0.5 32 8 2 2 0.25 0.5
__________________________________________________________________________
23 24 25 26 27 28 29.sup.f 30 31.sup.f 32 33
__________________________________________________________________________
Staphylococcus aureus X1.1 0.5 1 8 8 0.5 0.5 0.5,1 1 16,2 1 0.5
Staphylococcus aureus V41.sup.c 1 2 16 >128 2 2 4,4 1 32,4 1 2
Staphylococcus aureus X400.sup.d 4 4 16 >128 1 1 1,1 2 32,8 2 2
Staphylococcus aureus S13E 1 2 8 16 0.5 0.5 0.5,1 1 16,4 1 1
Staphylococcus epidermidis EP11 2 4 16 >128 4 4 8,16 2 128,16 4
8 Staphylococcus epidermidis EP12 2 4 16 >128 4 4 8,16 1 64,8 4
8 Streptococcus pyogenes C203 0.25 2 4 1 0.5 0.5 1,0.5 0.25 8,4 1 1
Streptococcus pneumoniae Park I 0.25 1 8 -- 0.5 0.25 0.25,0.5 0.25
8,4 0.5 0.5 Streptococcus Group D X66 4 16 128 >128 8 4 8,16 64
>128, 8 16 32 Streptococcus Group D 9960 2 4 32 32 4 4 32,32 32
64,8 4 4
__________________________________________________________________________
.sup.a MIC in mcg/ml .sup.b Compound numbers = example numbers in
Tables III-VI .sup.c Penicillin-resistant strain .sup.d
Methicillin-resistant-strain .sup.e Median of five experiments
.sup.f Two experiments .sup.g Median of three experiments
The A-21978C cyclic peptides of formula 1 have shown in vivo
antimicrobial activity against experimental bacterial infections.
When two doses of test compound were administered subcutaneously or
orally to mice in illustrative infections, the activity observed
was measured as an ED.sub.50 value [effective dose in mg/kg to
protect fifty percent of the test animals: See Warren Wick, et al.,
J. Bacteriol. 81, 233-235 (1961)]. The ED.sub.50 values observed
for A-21978C compounds are given in Tables VIII and IX.
TABLE VIII
__________________________________________________________________________
In Vivo Activity of A-21978C Cyclic Peptides ED.sub.50 Values.sup.b
Staphylococcus Streptococcus Compound Formula 1 Compound.sup.a
aureus pyogenes No. R Subcutaneous Subcutaneous Oral
__________________________________________________________________________
1 (D)CH.sub.3 (CH.sub.2).sub.10 CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO
1.4,2.05.sup.c <0.25,0.21 >200 2 CH.sub.3 (CH.sub.2).sub.10
CONH(CH.sub.2).sub.4CO 0.65,0.93 <0.25,0.107 117 3 CH.sub.3
(CH.sub.2).sub.10 CONH(CH.sub.2).sub.10CO >18 6.2 >200
##STR50## <5 18.8 >200 5 ##STR51## 1.67 >0.25,0.46 >200
7 ##STR52## 19 0.23 >200 8 (L)CH.sub.3 (CH.sub.2).sub.10
CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO 2.35 0.32 150 10 ##STR53##
3.56,4.12.sup.c 0.69 >200 11 ##STR54## 0.65 0.04 59 12 ##STR55##
2.3 <0.25,0.12.sup.c >200 13 ##STR56## 0.54 0.05 69 14
CH.sub.3 (CH.sub.2).sub.5 CONH(CH.sub. 2).sub.10CO >18 >9
>200 15 ##STR57## >18 1.02 >200 16 ##STR58## 5.19 3.14 200
17 (L)CH.sub.3 (CH.sub.2).sub.6 CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO
2.58 1.48 >200 18 (L)CH.sub.3 (CH.sub.2).sub.7 CONHCH(CH.sub.2
C.sub.6 H.sub.5)CO 1.38 0.59 184 19 (L)CH.sub.3 (CH.sub.2).sub.8
CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO 0.7,0.98.sup.c 0.39 >200 20
(L)CH.sub.3 (CH.sub.2).sub.9 CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO
1.25 0.35 >200 21 (L)CH.sub.3 (CH.sub.2).sub.11 CONHCH(CH.sub.2
C.sub.6 H.sub.5)CO 0.76 0.14 >200 22 (L)CH.sub.3 (CH.sub.2).sub.
12 CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO 4.8 <0.27,0.36.sup.c
>200 24 ##STR59## 2.3 <0.25,0.12.sup.c >200
__________________________________________________________________________
.sup.a R.sup.1 = H .sup.b mg/kg .times. 2 .sup.c Two
experiments
TABLE IX
__________________________________________________________________________
In Vivo Activity of A-21978C Cyclic Peptides ED.sub.50 Values.sup.a
Staphylococcus Streptococcus Compound Formula 1 Compound aureus
pyogenes No. R R.sup.1 Subcutaneous Subcutaneous Oral
__________________________________________________________________________
25 H ##STR60## >70 >22 >200 26 H CH.sub.3 (CH.sub.2).sub.9
CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO 9.2 >18 >200 29 CH.sub.3
CH.sub.2 CH(CH.sub.3)(CH.sub.2).sub.8 CO ##STR61##
>2.2>70.sup.b 10.6,6.0 >200
__________________________________________________________________________
.sup.a mg/kg .times. 2 .sup. b Two experiments
The results of toxicity tests on some of the A-21978C cyclic
peptides are summarized in Table X.
TABLE X
__________________________________________________________________________
Toxicity of A-21978C Cyclic Peptides Compound Formula 1 Compound
LD.sub.50 (mg/kg) No. R R.sup.1 in
__________________________________________________________________________
Mice.sup.a 1 (D)CH.sub.3 (CH.sub.2).sub.10 CONHCH(CH.sub.2 C.sub.6
H.sub.5)CO H 250 2 CH.sub.3 (CH.sub.2).sub.10
CONH(CH.sub.2).sub.4CO H >600 3 CH.sub.3 (CH.sub.2).sub.10
CONH(CH.sub.2).sub.10CO H 600 ##STR62## H >300 5 ##STR63## H 277
7 ##STR64## H 200 8 (L)CH.sub.3 (CH.sub.2).sub.10 CONHCH(CH.sub.2
C.sub.6 H.sub.5)CO H 250 9 ##STR65## H 450.sup.b 10 ##STR66## H 450
11 ##STR67## H 450 12 ##STR68## H 450 13 ##STR69## H 300 14
CH.sub.3 (CH.sub.2).sub.5 CONH(CH.sub.2).sub.10CO H >600 15
##STR70## H 250 16 ##STR71## H 225 17 (L)CH.sub.3 (CH.sub.2).sub.6
CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO H 450 18 (L)CH.sub.3
(CH.sub.2).sub.7 CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO H >600 19
(L)CH.sub.3 (CH.sub.2).sub.8 CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO H
600 20 (L)CH.sub.3 (CH.sub.2).sub.9 CONHCH(CH.sub.2 C.sub.6
H.sub.5)CO H 400 21 (L)CH.sub.3 (CH.sub.2).sub.11 CONHCH(CH.sub.2
C.sub.6 H.sub.5)CO H 225 22 (L)CH.sub.3 (CH.sub.2).sub.12
CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO H 225 24 ##STR72## H 450 25 H
##STR73## 300 26 H CH.sub.3 (CH.sub.2).sub.9 CONHCH(CH.sub.2
C.sub.6 H.sub.5)CO 225 29 CH.sub.3 CH.sub.2
CH(CH.sub.3)(CH.sub.2).sub.8 CO ##STR74## 37.5
__________________________________________________________________________
.sup.a Administered intravenously .sup.b Material was in
suspension
* * * * *