Preparation and characterization of cholic acid-derived antimicrobial agents with controlled stabilities

Article abstract of DOI:10.1021/ol0062704

Novel cholic acid-derived antimicrobial agents that decompose under mildly basic conditions have been prepared. These compounds range in biological properties from potent antibacterial activity to effective permeabilization of the outer membranes of Gram-negative bacteria.

Full text of DOI:10.1021/ol0062704

ORGANIC
LETTERS
2000
Vol. 2, No. 18
2837-2840
Preparation and Characterization of
Cholic Acid-Derived Antimicrobial
Agents with Controlled Stabilities
Qunying Guan,† Chunhong Li,† Erica J. Schmidt,‡ J. Scott Boswell,‡
,†
Joshua P. Walsh,‡ Glenn W. Allman,‡ and Paul B. Savage*
Departments of Chemistry and Biochemistry and Microbiology, Brigham Young
UniVersity, ProVo, Utah 84602
paul_saVage@byu.edu
Received June 28, 2000
ABSTRACT
Novel cholic acid-derived antimicrobial agents that decompose under mildly basic conditions have been prepared. These compounds range
in biological properties from potent antibacterial activity to effective permeabilization of the outer membranes of Gram-negative bacteria.
With the emergence of numerous strains of pathogenic
multidrug-resistant bacteria comes the need for new antimi-
crobials to which bacteria are unlikely to develop resistance.
A group of antimicrobial agents that has received consider-
able attention recently is membrane active cationic peptide
antibiotics.1 These compounds disrupt or permeabilize prokary-
otic membranes.2 Endogenous examples have been identified
in many diverse organisms including animals and plants.
Bacteria rarely develop resistance to membrane-active com-
pounds because resistance requires alterations to the bulk
membrane structure of the organisms. For example, few
strains have been isolated that are resistant to polymyxin B,
a commonly used cationic peptide antibiotic. Those that have
been isolated exhibit membrane structural changes that
influence the permeability of their membranes.3
We recently reported several series of cholic acid-derived
compounds that, although not composed of amino acids,
display a facial amphiphilicity similar to that of many
cationic peptide antibiotics and mimic aspects of their
behavior including membrane activity.4 The antimicrobial
activities of these compounds range from potent antibacterial
activity to permeabilization of the outer membranes of Gram-
negative bacteria and sensitization of these organisms to
hydrophobic antibiotics.4,5
As an extension of our earlier work, we have become
interested in antimicrobial agents that decompose into
endogenous and nontoxic compounds under mild conditions
including those encountered in the human gastrointestinal
tract. Found within the gastrointestinal tract are large numbers
(4) (a) Li, C.; Budge, L. P.; Driscoll, C. D.; Willardson, B. M.; Allman,
G. W.; Savage, P. B.J. Am. Chem. Soc. 1999, 121, 931. (b) Rehman, A.;
Li, C.; Budge, L. P.; Street, S. E.; Savage, P. B. Tetrahedron Lett. 1999,
40, 1865. (c) Li, C.; Peters, A. S.; Meredith, E. L.; Allman, G. H.; Savage,
P. B. J. Am. Chem. Soc. 1998, 120, 2961.
(5) Li, C.; Lewis, M. R.; Gilbert, A. B.; Noel, M. D.; Scoville, D. H.;
Allman, G. W.; Savage, P. B. Antimicrob. Agents Chemother. 1999, 43,
1347.
† Department of Chemistry and Biochemistry.
‡ Department of Microbiology.
(1) For a recent review, see: Epand, R. M.; Vogel, H. J. Biochim.
Biophys. Acta 1999, 1462, 11.
(2) Shai, Y. Biochim. Biophys. Acta 1999, 1462, 55.
(3) Groisman, E. A.; Kayser, J.; Soncini, F. C. J. Bacteriol. 1997, 179,
7040.
10.1021/ol0062704 CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/17/2000

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.
2838
Org. Lett., Vol. 2, No. 18, 2000

charge on their membranes.11 Consequently, the negative
charge of 7 likely inhibits interaction with the membranes
of the bacteria.
Scheme 2.a Preparation of 4-6
The outer membranes of Gram-negative bacteria provide
a permeability barrier to many hydrophobic molecules.
Consequently, many hydrophobic antibiotics, such as eryth-
romycin, are only weakly active against Gram-negative
strains. Because cholic acid derivatives were expected to
permeabilize the outer membranes of Gram-negative bacteria,
measurements were made of concentrations of selected
compounds that were necessary to lower the MIC of
erythromycin from 30 µg/mL to a clinically useful level of
1 µg/mL (Table 1). Because 1-3 were active alone against
E. coli, the concentrations at which they caused permeabi-
lization were very near their MICs. Compounds 4-6 were
effective permeabilizers of the outer membrane, while 7 was
inactive.
A primary issue that may determine the utility of membrane-
active antimicrobials in systemic applications is their selec-
tivity for prokaryotic over eukaryotic membranes. Because
prokaryotic membranes typically carry a net negative charge
and their eukaryotic counterparts are generally composed of
zwitterionic phospholipids,11 it would be expected that
compounds bearing positive charges would display selectivity
for prokaryotic membranes. Minimum hemolytic concentra-
tions (MHCs) can be compared to concentrations of com-
pounds required for antibacterial activity as a measure of
membrane selectivity. The MHC values of compounds 1-7
were measured as previously reported5 and are included in
Table 1. Compounds 1-3 exhibit very low MHCs; however,
compounds 4-6 appear to be nonhemolytic presumably due
to the additional positive charge at C-24. Nevertheless, if
compounds 1-6 find use in preventing microbial growth in
food, it is unlikely that their hemolytic behavior would be
an issue because the compounds would likely be degraded
to inactive components before absorption occurs.
a (a) Benzyl alcohol, TsOH (81%); (b) Bocglycine, Boc-â-alanine
or Boc-γ-aminobutyric acid, DCC, DMAP, CH2Cl2 (68-78%); (c)
H2, Pd/C (97-99%); (d) (CH3)2N(CH2)2OH, DCC, DMAP, CH2Cl2
or THF (62-82%); (e) MeI, CH2Cl2. f) HCl, dioxane (83-90%
for two steps).
The antimicrobial activities of the compound 1-7 were
tested with standard strains of Gram-negative and Gram-
postive bacteria, E. coli (ATCC 25922) and Staphylococcus
aureus (ATCC 25923), respectively (Table 1). Minimum
Table 1. Antimicrobial Activities of Compounds 1-7
MICa
MICb
c
MHCd
compd
(µg/mL)
(µg/mL)
(µg/mL)
(mg/mL)
1
2
3
4
5
6
7
1.8
4.0
1.2
15
11
1.0
7.0
3.5
60
30
0.7
3.0
3.5
10
2.0
4.0
2.0
<10
>200
>200
>200
>100
To test the stabilities of the ester groups at C-3, C-7, and
C-12, compounds 22-244b (Figure 2) were used because they
14
23
2.0
>100
>100
>100
a Mimum inhibition concentration (MIC) against S. aureus (ATCC
25923). b MIC against E. coli (ATCC 25922). c Concentration of the
compounds required to lower the MIC of erythromycin from 30 to 1 µg/
mL with E. coli (ATCC 25922). d Minimum hemolytic concentration.
inhibition concentrations (MICs) were measured as described
previously.5 As anticipated, the compounds containing the
hydrophobic chain at C-24 (1-3) were the most active
against the Gram-negative strain. The compounds with
choline at C-24 (4-6) were much less active alone against
the Gram-negative strain yet retained the ability to inhibit
the growth of the Gram-positive strain. This difference was
likely due to the inability of compounds 4-6 to traverse the
outer membrane of the Gram-negative strain. With a
negatively charged group at C-24, 7 was inactive against
either strain. Prokaryotes generally exhibit a net negative
Figure 2. Structures of cholic acid derivatives 22-24.
contained a chromophore that facilitated monitoring of HPLC
experiments. Stabilities of the compounds were tested at pH
2.0, 7.0, and 12.0 in a phosphate buffer at room temperature.
Decomposition through ester hydrolysis yielded compounds
(11) Matsuzaki, K.; Sugishita, K.; Fugii, N.; Miyajima, K. Biochemistry
1995, 34, 3423.
Org. Lett., Vol. 2, No. 18, 2000
2839

that were less polar and easily separable from the starting
compounds. Initially, only one benzene-containing decom-
position product was observed; at longer reaction times two
other decomposition products were observed which presum-
ably corresponded to sequential ester hydrolysis. The half-
lives of compounds 22-24 at the three pH values are shown
in Table 2. At low pH the amine groups were protonated
values. Likewise, loss of amino acids resulted in a significant
loss of antibacterial activity in compounds 1-6; these gave
relatively high MIC values after storage in solution for
extended periods (>2 weeks).
Because compounds 1-6 are active antimicrobial agents
and because their stability can be manipulated by controlling
pH, these compounds may find use in situations in which
antimicrobial compounds are required but long-term exposure
is undesirable. As mentioned, these compounds may be ideal
for use in controlling bacterial growth in food. The com-
pounds could be applied to food, and after ingestion, the
compounds would break down to endogenous and/or non-
toxic components. Investigation of the ability of the com-
pounds to inhibit microbial growth in food is ongoing and
will be reported in due course.
Table 2. Stabilities (half-lives) of Compounds 22-24 in
Phosphate Buffer
compd
pH 2.0
pH 7.0
pH 12.0
22
23
24
>37 days
>37 days
33 days
28 days
37 days
12 days
<30 min
<30 min
<30 min
Acknowledgment. This work was generously supported
by the National Institutes of Health (GM 54619) and the
National Science Foundation (CAREER).
and the compounds were relatively stable. At higher pH the
amines were less strongly protonated and became involved
in ester hydrolysis. As expected the γ-aminobutyric acid
derived compound was especially susceptible to hydrolysis,
presumably yielding pyrrolidone.
As we have shown,4c partial loss of facial amphiphilicity
through inversion at C-3 in other cholic acid-derived
antimicrobial agents results in dramatic increases in MIC
Supporting Information Available: Experimental details
for the preparation of 1-7 and stability experiments. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL0062704
2840
Org. Lett., Vol. 2, No. 18, 2000