of bacteria6 which play an important role in digestive
processes. Elimination or reduction of the populations of
bacteria in the gastrointestinal tract can influence digestion
and facilitate opportunistic infections by pathogenic bacteria.7
Oral ingestion of antimicrobial agents that are stable in the
gastrointestinal tract can cause significant perturbations in
bacterial populations.8
reduced.4 To ensure that unmodified cholic acid would be
released upon decomposition of the new compounds, the
C-24 position of the steroid was not reduced. To observe
the properties of compounds with hydrophobic and polar
functionality at C-24, octyl esters and choline esters of cholic
acid derivatives were prepared, giving 1-6 (Figure 1).
We have developed and now report a series of compounds,
based on a cholic acid scaffolding, that are effective
antibiotics yet decompose to endogenous and nontoxic
compounds under mild conditions. An attractive potential
use for compounds of this type is in controlling bacterial
growth in food. Because these compounds decompose under
mild conditions, they are expected to become inactive upon
ingestion and therefore presumably not adversely affect the
natural flora of the digestive system.
Nisin, a peptide antibiotic, is currently the only antimi-
crobial agent approved in many countries for use in food.9
Nisin is active against Gram-positive strains but is nearly
inactive against Gram-negative organisms such as Escheri-
chia coli and strains of Salmonella.10 Selected compounds
in the series of new antimicrobials that we have developed
are very active against these Gram-negative bacteria as well
as against Gram-positive organisms.
To provide compounds capable of decomposition under
mild conditions, we esterified the hydroxyl groups at C-3,
C-7, and C-12 of cholic acid with amino acids including
glycine, â-alanine, and γ-aminobutyric acid. The resulting
compounds contained both ester and amine functionality, and
this combination was expected to cause decomposition of
the compounds under mildly basic conditions. Because of
the possibility of pyrrolidone formation, we expected the
cholic acid derivatives containing γ-aminobutyric acid to
decompose especially rapidly.
Figure 1. Structures of cholic acid derivatives 1-7.
Compound 7 was also prepared to determine the effects of
a carboxylate group at C-24 on antimicrobial activity.
Compounds 1-3 were prepared as described in Scheme
1. The carboxylic acid at C-24 of cholic acid was esterified
Scheme 1.a Preparation of 1-3
In addition to consideration of functionalization of the
hydroxyl groups at C-3, C-7, and C-12 of cholic acid, we
took into account the influence of groups attached at C-24.
Previous work4a established that the nature of the groups
included at C-24 greatly influences the biological activity
of the compounds: hydrophobic groups made the compounds
active against Gram-negative strains, while shorter chains
resulted in compounds capable of permeabilizing the outer
membranes of Gram-negative bacteria and killing Gram-
positive organisms. In previously reported cholic acid-derived
antimicrobial agents, the C-24 position of the steroid was
a (a) Octanol, TsOH (73%); (b) Bocglycine, Boc-â-alanine or
Boc-γ-aminobutyric acid, DCC, DMAP, CH2Cl2 (91-95%); (c)
HCl, dioxane (84-99%).
with octanol followed by incorporation of the Boc-protected
amino acids, giving 9-11. Cleavage of the Boc groups with
HCl in dioxane yielded 1-3. Preparation of 4-6 (Scheme
2) required protection of the carboxylic acid of cholic acid
as the benzyl ester. Incorporation of the Boc-protected amino
acids was followed by deprotection of the carboxylic acid,
yielding 16-18. Attempts to esterify the acid with choline
failed using a variety of standard acid-activating reagents.
The choline group was successfully incorporated via a two-
step procedure: esterification with N,N-dimethylethanola-
mine using DCC and DMAP followed by treatment with
methyl iodide. Removal of the Boc groups yielded 4-6.
Preparation of 7 only required removal of the Boc protecting
groups of 16. Because 7 proved to be inactive (vide infra),
a complete series of acids, derived from deprotection of 17
and 18, was not prepared.
(6) Bacterial populations have been estimated to reach 1014 cells at all
sites of the human body (the human body contains ca. 1013 cells). Tannock,
G. W. In Normal Microfluora. An Introduction to Microbes Inhabiting the
Human Body; Tannock, G. W., Ed.; Chapman and Hall: London, 1994; pp
1-36.
(7) For example see: (a) Vollaard, E. J.; Clasener, H. A. L. Antimicrob.
Agents Chemother. 1994, 38, 409. (b) Hentges, D. J. In Human Intestinal
Microflora in Health and Disease; Hentges, D. J., Ed.; Academic Press:
New York, 1983; pp 311-328.
(8) For example, see: Finegold, S. M.; Mathisen, G. E.; George, W. L.
In Human Intestinal Microflora in Health and Disease; Hentges, D. J., Ed.;
Academic Press: New York, 1983; pp 356-438.
(9) Delves-Broughton, J.; Blackburn, P.; Evans, R. J.; Hugenholtz, J.
Antonie Van Leeuwenhoek 1996, 69, 193.
(10) De Vuyst, L.; Vandamme, E. J. In Bacteriocins of Lactic Acid
Bacteria; De Vuyst, L.; Vandamme, E. J., Ed.; Blackie Academic and
Professional: Glasgow, 1994; pp 151-221.
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Org. Lett., Vol. 2, No. 18, 2000