Bicephalic amphiphile architecture affects antibacterial activity

Article abstract of DOI:10.1016/j.ejmech.2011.06.026

A series of cationic amphiphiles, each with an aromatic core, was prepared and investigated for antimicrobial properties. The synthesized amphiphiles in this study are bicephalic (double-headed) in that they each possess two trimethylammonium head groups

Full text of DOI:10.1016/j.ejmech.2011.06.026

European Journal of Medicinal Chemistry 46 (2011) 4219e4226
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Bicephalic amphiphile architecture affects antibacterial activity
Jade E. LaDowa, David C. Warnock b, Kristina M. Hamill b, Kaitlin L. Simmons b, Robert W. Davis b,
Christian R. Schwantes b, Devon C. Flaherty b, Jon A.L. Willcox b, Kelsey Wilson-Henjum a,
,
b
a
Kevin L. Caran b , Kevin P.C. Minbiole , Kyle Seifert
*
a James Madison University, Department of Biology, 820 Madison Drive, MSC 7801, Harrisonburg, VA 22807, USA
b James Madison University, Department of Chemistry and Biochemistry, 901 Carrier Drive, MSC 4501, Harrisonburg, VA 22807, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of cationic amphiphiles, each with an aromatic core, was prepared and investigated for anti-
microbial properties. The synthesized amphiphiles in this study are bicephalic (double-headed) in that
they each possess two trimethylammonium head groups and a single linear alkoxy tail. Minimum
inhibitory and minimum bactericidal concentrations of these amphiphiles were in the low micromolar
range. Antimicrobial activities are highly sensitive to the chain length of the hydrophobic region, and
modestly reliant on the relative positioning of the head groups on the aromatic core. These trends were
more pronounced in time kill assays, wherein longer chain compounds required significantly shorter
times to completely kill bacteria. Microscopy suggested that the mode of cell death was lysis. Strong
inhibition was observed with both biscationic compounds and monocationic comparisons against Gram-
positive bacteria; only biscationic amphiphiles maintained good activity versus the Gram-negative
bacteria tested. These observations provide direction for future antimicrobial structural investigations.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Received 26 February 2011
Received in revised form
10 June 2011
Accepted 16 June 2011
Available online 25 June 2011
Keywords:
Double-headed amphiphiles
Bicephalic amphiphiles
Antibacterial
Antiseptic
Antimicrobial
1. Introduction
It has been suggested that the use of certain classes of anti-
septics, with effective hygiene, to prevent or treat infections will
Antibiotic resistance has increased dramatically in the last 20
years, particularly in human pathogens such as methicillin-
resistant Stapyhlococcus aureus (MRSA), Clostridium difficile,
vancomycin-resistant enterococci, and Mycobacterium tuberculosis.
This increase has threatened the ability to treat hospital- and
community-acquired infections [1e4]. During this time, pharma-
ceutical research has moved away from discovering new antimi-
crobials in favor of developing more lucrative drugs [5]. Recently,
widespread resistance to antibacterial compounds such as triclosan
has been observed, necessitating new antimicrobial compounds,
particularly novel antibiotics and antiseptics [6]. In addition to
developing new antimicrobials by modifying existing drugs, novel
structures must also be developed, particularly compounds that
will be difficult for organisms to inactivate or to resist via mutation.
Compounds that present a unique strategy of activity are of
premium importance.
delay the increase in antibiotic resistance [2]. Specifically, cationic
antimicrobial compounds have been used to combat microorgan-
isms for over 50 years with little evidence of widespread resistance
[2]. For example, benzylalkylammonium chlorides have long served
as effective antiseptic agents in contact lens solutions and many
Lysol formulations; chlorhexidine is a standard in mouthwashes [7]
and surgical preparation [8]. The strong antimicrobial properties of
many cationic antiseptics are likely due to their Coulombic attrac-
tion to anionic biological surfaces and their ability to incorporate
into (and consequently disrupt the packing of) the cell membrane
[2,9e18]. Additionally, cationic antiseptics can displace cell surface
cations such as Mg2þ and Ca2þ, which are essential for proper cell
function [2]. Notably, it was recently reported that the antibacterial
activity of an antiseptic increases with the number of cationic
groups due to the multi-dentate interactions with the bacterial
surface; activity was shown against both Gram-positive and Gram-
negative bacteria [9].
Amphiphilic molecules, those with both hydrophilic and lipo-
philic segments, have long been utilized in the service of human-
kind for tasks ranging from the routine (washing one’s hands) to
more technologically challenging endeavors (transfection of genes
into cells) [19]; cationic variants possess key structural elements for
Abbreviations: MBC, Minimum Bactericidal Concentration; MIC, Minimum
Inhibitory Concentration; MRSA, methicillin-resistant Staphylococcus aureus; NBS,
N-bromosuccinimide; SDS, Sodium dodecyl sulfate; TSA, trypticase soy agar.
* Corresponding author. Tel.: þ1 540 568 6632; fax: þ1 540 568 7938.
E-mail address: carankl@jmu.edu (K.L. Caran).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.06.026

4220
J.E. LaDow et al. / European Journal of Medicinal Chemistry 46 (2011) 4219e4226
antimicrobial activity. The antimicrobial activity of a particular
cationic amphiphile is strongly dependent on its structure, and
structural variation can take a significant number of forms. The
hydrophobic section is commonly composed of one or more linear
saturated hydrocarbon chains, but it can also contain branches,
points of unsaturation, or rings [20]. The hydrophilic head group of
most classes of cationic amphiphiles is either the salt of an amine
(with a charge that is pH-dependent) or a quaternary ammonium
(with a pH-independent positive charge).
A number of studies have demonstrated the relationship
between an amphiphile’s hydrophobic chain length and its activity
against microbes, with optimal effectiveness typically being found
at some intermediate chain length, above and below which activity
decreases [2,10,14]. For example, Roy and Das studied a class of
cationic amphiphiles capable of forming hydrogels with tail lengths
from 10e18 carbons and found the greatest bactericidal activity
against Gram-positive bacteria for compounds between 12e14
carbons [14]. Others report that multiple lipophilic chains, chain
branching, and cis-unsaturation generally lead to diminished
antimicrobial activity [21].
While a diversity of headgroup arrangements in multi-headed
amphiphile architecture are known (Fig. 1), including gemini
amphiphiles (two tails and two head groups connected via
a spacer) [22,23], bolaamphiphiles (two heads separated by a long
hydrophobe) [24,25] and polycephalic amphiphiles (multi-headed,
single-tailed) [26e28], the last class has received relatively little
consideration by the scientific community.
Fig. 1. Architectures of multi-headed amphiphiles with two head groups: Gemini
amphiphiles (top), bolaamphiphiles (middle), and polycephalic amphiphiles (bottom).
Blue circles represent hydrophilic headgroups, red lines represent lipophilic tails, and
black lines represent linkers.
core) of our polycephalic amphiphiles will more profoundly alter
the optimal curvature of the resulting aggregate when incorporated
into the membrane (Fig. 2) [21,30]. Furthermore, presentation of
a series of related compounds wherein the cationic groups are
methodically adjusted in relative position, specifically on an arene
core, will allow for optimization of the architecture for potent
antimicrobial activity.
2. Chemistry
Headgroup architecture also profoundly affects antimicrobial
activity, as the number, positioning, and nature of cationic groups
will dictate the interaction with the cell surface [9e11,15]. This fact,
in addition to another recent study demonstrating that amphi-
philes with large head groups and a single hydrophobic tail have
significant membrane lysing potential [21], suggest that further
study on multi-headed single-tailed amphiphiles is needed. Such
compounds display a low critical packing parameter1 [29], indi-
cating they are approximately cone-shaped, and thus have the
ability to disrupt biological or synthetic membranes by incorpo-
rating into and eventually lysing the lipid bilayer, potentially in
lower concentrations (Fig. 2). Incorporation of polycephalic, single-
tailed amphiphiles (shown as two large filled heads with single
tails) may allow more efficient membrane disruption than those
with only a single headgroup.
Here, we report the synthesis and antimicrobial properties of
a series of biscationic bicephalic (double-headed) amphiphiles
designed and produced in our laboratory, each with two trime-
thylammonium head groups and a single linear alkoxy tail,
attached to an arene core (Fig. 3, 1e10) [28]. Structural variety in
this series includes compounds with chain lengths ranging from
10e18 carbons, as well as those with various positioning of the
head groups on the arene relative to the tail. For comparison, we
have also included four cationic single-headed derivatives (11e14)
and one anionic single-headed derivative (15) in our study. We
hypothesized that the amphiphiles in this study would serve as
particularly efficient antimicrobial compounds, likely through
membrane disruption. The rationale behind this was two-fold: (1)
The multiple cationic groups would form strong, multi-dentate
attractive interactions with anionic biological surfaces [9] and (2)
the geometry (with an effective headgroup size significantly larger
than a conventional single-headed amphiphile) and rigidity (arene
2.1. Preparation of amphiphiles
Syntheses of amphiphilic species 1e8, 11, and 12 were per-
formed according to our previously published method [28].
Accordingly, a series of commercially available dimethylphenols as
well as p-cresol were exposed to Williamson ether synthesis
conditions, resulting in phenol alkylation in high yields (compound
A, 72e99%, Scheme 1). Light-promoted benzylic bromination (NBS,
benzoyl peroxide) subsequently generated the corresponding
benzylic bromides B in low yields. Exposure to trimethylamine in
ethanol at reflux completed the syntheses of these amphiphiles (C),
which were purified by recrystallization (EtOH/Et2O). Complete
experimental details for all new compounds are presented in the
Supporting Information.
Amphiphiles 9 and 10, the 3,5-disubstituted analogs, were
prepared via an alternate method, as benzylic bromination of the
3,5-dimethylalkoxyphenol derivatives yielded a complex mixture
of products.2 We thus prepared
9
and 10 from 5-
hydroxyisophthalate dimethylester (14, Scheme 2). Accordingly,
Williamson ether synthesis (tetradecyl or hexadecyl bromide,
K2CO3, CH3CN, reflux) followed by lithium aluminum hydride
reduction yielded the diol (E). Subsequent reaction with phos-
phorus tribromide provided bis-benzylic bromides B, which were
transformed to the corresponding trimethylammonium cations in
good yield as described above.
3. Antibacterial properties
3.1. Minimum inhibitory concentrations (MIC), minimum
bactericidal concentrations (MBC), and time killing assays
Encouraged by promising preliminary results wherein the
synthesized amphiphiles inhibited growth of Gram-positive and
Gram-negative pathogenic bacteria via disk diffusion assays, the
1
The critical packing parameter is defined as V/aolc where V is the volume of the
nonpolar chain, ao is the area of the headgroup and lc is the length of the chain.
Polycephalic surfactants with a large headgroup area (ao) relative to the chain
volume (V) have a low critical packing parameter.
2
This is perhaps due to competing electrophilic aromatic substitution for this
compound, wherein all three substituents activate the unsubstituted positions.

J.E. LaDow et al. / European Journal of Medicinal Chemistry 46 (2011) 4219e4226
4221
E. faecalis, CTAB (14) was the most effective compound (4
mM), and
compounds 1, 2, and 5e12 had MIC and MBC values ranging from 16
to 50 M. 2,5-C10 (3) and SDS (15) were again ineffective at 500 M.
m
m
The concentration of compounds necessary to kill E. coli were
between those necessary to kill E. faecalis and P. aeruginosa,
generally between 31 and 63 mM with 2,5-C10 (3), MC14 (12) and
SDS (15) ineffective. P. aeruginosa was only killed by relatively high
concentrations of some compounds; others were ineffective.
The amphiphiles 2,4-C14 (2) and 3,5-C14 (9) had the lowest MIC
and MBC values against all 4 strains, with 3,5-C16 (10) and MC10
(11) being nearly as effective across the strains. CTAB (14) was the
most effective amphiphile against both gram-positive organisms;
however, it was much less effective versus gram-negative organ-
isms. Only 3 and 15 were relatively ineffective against the gram-
positive strains, while 3, 4, 8, 12, 13, and 15 were ineffective
against both Gram-negative strains. The monocationic compounds
(11e14) were much more effective at inhibiting Gram-positive
organisms than Gram-negatives. There were no compounds that
preferentially killed Gram-negative organisms over Gram-positive
organisms.
Fig. 2. Top: Cross section of a bilayer composed of lipids (open heads with two tails)
with conventional amphiphiles (each shown as a filled headgroup with one tail)
incorporating into (a), and subsequently disrupting (b) the membrane. Bottom:
Incorporation (a’) of bicephalic amphiphiles (shown as double filled heads and single
tails) may allow more efficient membrane disruption (b’).
MIC values were determined by the broth microdilution method
as described in the Experimental Section (Table 1; see Supporting
Information for same data reported as mg/mL). Using the Gram-
positive pathogens S. aureus and Enterococcus faecalis, and the
Gram-negative organisms Pseudomonas aeruginosa and Escher-
ichia coli, MICs were determined as the lowest concentration
which prevented visible growth at 72 h. After microtiter plates
were incubated and MICs determined, aliquots from each of the
wells were placed on TSA plates and incubated to determine the
MBC values for each of the compounds. The MBC was determined
as the lowest concentration of compound at which there were no
colonies growing on the plate. The MBC and MIC values were
identical in every case, indicating that the compounds are
effective at killing the bacteria, not just inhibiting growth.
Dodecyltrimethyl ammonium bromide (DTAB, 13), cetyltrimethyl
ammonium bromide (CTAB, 14), and sodium dodecyl sulfate
(SDS, 15) were used for comparison in these studies. For the MICs
and MBCs, there was variability both with various compounds
against individual strains, and with a single compound across
strains.
In addition to determining MIC and MBC values, compounds
1e12 were assayed for the time necessary to kill 2.5 ꢀ106 cfu/mL at
100 mM (Table 2). The time kill results were similar for S. aureus
and E. faecalis. 2,5-C18 (7), 3,5-C14 (9), 3,5-C16 (10), and MC14 (12)
each killed within 15 min, while MC10 (11) killed within 30 min.
Compound 3,5-C16 (10) killed E. coli within 15 min, while 2,3-C14
(1), 2,5-C18 (7), and MC10 (11) killed within 30 min. Although
a number of compounds were effective at killing within 72 h,
clearly 3,5-C16 (10) was the most efficient at killing the 3 strains
tested, while 2,5-C18 (7) was nearly as efficient. MC14 (12) was
very effective against the Gram-positive strains, but ineffective
against E. coli.
Additionally, aliquots of cells killed by the compounds were
placed on slides and visualized microscopically to determine if any
intact cells were present after treatment. When treated with inef-
fective compounds such as 3 and 4, intact cells were observed on
the slides. No intact cells were observed when bacteria were
treated with effective compounds, suggesting that the effective
compounds kill via cell lysis.
All compounds except for 2,5-C10 (3), DTAB (13), and SDS (15)
were effective at killing S. aureus at low micromolar concentrations,
with compounds 2,3-C14 (1), 2,4-C14 (2), 3,5-C14 (9), 3,5-C16 (10),
MC10 (11), MC14 (12), and CTAB (14) effective at < 10 mM. Against
N
N
N
N
N
N
N
N
O
O
O
O
2 Br
2 Br
2 Br
2 Br
13
13
n-1
13
1
2,3-C14
2
3
4
5
6
7
2,5-C10 n=10
2,5-C12 n=12
2,5-C14 n=14
2,5-C16 n=16
2,5-C18 n=18
8
2,6-C14
2,4-C14
N
N
N
N
O3SO
15
Br
Na
n-1
11
Br
13
14 CTAB n=16
DTAB n=12
SDS
O
2 Br
O
n-1
n-1
9
3,5-C14 n=14
11
12 MC14 n=14
MC10 n=10
10 3,5-C16 n=16
Fig. 3. Biscationic and monocationic amphiphile series with anionic comparison.

4222
J.E. LaDow et al. / European Journal of Medicinal Chemistry 46 (2011) 4219e4226
4. Discussion
Me3N
RO
NMe3
4.1. General trends of antimicrobial activity
2,3
2,4
4C spacer
This series of biscationic amphiphiles displayed antibacterial
activity against two pathogenic Gram-negative bacteria and two
Gram-positive bacteria with few exceptions. MIC and MBC values
were equivalent in all cases, indicating that inhibition was due to
death of bacterial cells, and were in the micromolar range,
Me3N
RO
NMe3
NMe3
extending down to 4 mM. Of the Gram-positive organisms tested,
compounds were more effective at killing S. aureus than E. faecalis,
with the exception of CTAB (14). Higher MIC and MBC values were
observed with E. coli and P. aeruginosa, both Gram-negative
organisms, with one exception. Compound 9 was equally effective
Me3N
3,5
2,6
5C spacer
against E. faecalis and E. coli (31 mM). This difference in effectiveness
OR
OR
between the Gram-positive and Gram-negative organisms could be
due to the inherent differences of the two cell types. Gram-positive
cells have a cytoplasmic membrane surrounded by a thick cell wall
comprised of peptidoglycan. Gram-negative cells have a cyto-
plasmic membrane, surrounded by a thin layer of peptidoglycan,
and an additional outer membrane composed of glycer-
ophospholipids and lipopolysaccharides (LPS) [31]. The LPS
component forms a tight layer with decreased permeability, espe-
cially to hydrophobic molecules, which may make it more difficult
for the amphiphiles to diffuse across and disrupt the cytoplasmic
membrane [32].
In the MIC and MBC assays, P. aeruginosa was only killed with
relatively high concentrations of compounds, and in some cases
was not killed at all. This was not surprising, as P. aeruginosa is
reported to be relatively insensitive to quaternary ammonium
compounds in general, likely due to the decreased permeability of
the outer membrane [2]. This, along with multi-drug efflux
pumps, also confers resistance to P. aeruginosa against multiple
antibiotics [33].
Me3N
Me3N
NMe3
NMe3
2,5
6C spacer
RO
Fig. 4. Headgroup isomers with spacing between cationic groups in bold.
NBS
Br
N
m
m
m
m
Me3N
benzoyl peroxide
RBr, K2CO3
EtOH
reflux
CCl4, hv
CH3CN, reflux
m Br
OR
B
OR
C
OR
A
OH
1 8 11 12
( - ,
,
)
Chain
length (R)
% yield % yield of
of A bromide B amphiphile C
% yield of
Amphiphile m Substitution
1
2
2
2
2
2
2
2
2
2
1
1
2,3
2,4
C14H29
C14H29
C10H21
C12H25
C14H29
C16H33
C18H37
C14H29
C10H21
C14H29
78%
39%
22%
14%
25%
23%
28%
25%
19%
30%
15%
68%
11%
76%
81%
87%
78%
83%
56%
76%
74%
91%
90%
88%
86%
72%
78%*
99%
92%
80%
3
2,5
4
2,5
5
2,5
6
2,5
7
2,5
8
2,6
11
12
para (4)
para (4)
Scheme 1. Preparation of cationic amphiphiles 1e8 and 11e12. * ¼ acetone used as solvent.

J.E. LaDow et al. / European Journal of Medicinal Chemistry 46 (2011) 4219e4226
4223
O
O
OH
OH
O
O
LiAlH4
RBr, K2CO3
MeO
OMe
MeO
OMe
Et2O, reflux
CH3CN, reflux
OH
14
OR
E
OR
D
R = C14H29, 53%
R = C16H33, 38%
R = C14H29, 60%
R = C16H33, 85%
Br
Br
N
N
Me3N
PBr3
2 Br
EtOH, reflux
THF, RT
OR
B
OR
C
9, R = C14H29, 88%
R = C14H29, 46%
R = C16H33, 44%
10
, R = C16H33, 77%
Scheme 2. Preparation of amphiphiles 9 and 10.
In the time kill assays, again there was considerable variability.
Three biscationic compounds, 7, 9, and 10, and two monocationic
compounds, 11 and 12, were the most efficient at killing the two
Gram-positive organisms. Against Gram-negative bacteria,
however, the most rapid killing came from compounds 1, 7, 10, and
11. Thus one compound, 2,3-C14 (1), was more time-efficient at
killing E. coli than either of the Gram-positive organisms; no
compound had been observed with a lower MIC or MBC for E. coli
than either of the Gram-positive organisms. It is unknown why the
discrepancy exists for the two assays, but it is likely due to what the
various methods are measuring. The MIC and MBC assays are
determining the concentration of compound necessary to inhibit
and kill a given number of cells, while the time kill assays measure
how quickly a defined concentration of compound kills a pop-
ulation of cells. The difference in the results of the two assays also
suggests that multiple methods may be necessary when deter-
mining antimicrobial effectiveness of compounds.
Additionally, in the time kill assays, there were 3 instances
where a compound was unable to kill 2.5 ꢀ 106 cfu/mL of S. aureus
and/or E. faecalis after 72 h. These same compounds were able to
kill 5 ꢀ 105 cells in the MIC and MBC assays. This discrepancy in the
effectiveness of these compounds used in the two methods for
assessing antimicrobial activity was likely due to the difference in
the number of cells used for each of the assays.
4.2. Effects of biscationic substitution patterns on activity
MIC values, MBC values, and the time killing results with
S. aureus, E. faecalis, and E. coli were evaluated based on positioning
of the head groups of the C14 compounds (Fig. 5). The series of
biscationic amphiphiles was developed with a variety of place-
ments of the head groups on the arene core, in the positions of 2,3-,
2,4-, 2,5-, 2,6-, and 3,5-disubstitution relative to the alkoxy group.
The geometry and rigidity of our polycephalic amphiphiles was
expected to profoundly alter the optimal curvature of the resulting
aggregate when incorporated into the cytoplasmic membrane
(Fig. 2), and thus influence activity. Modest structure-activity
relationship for the MIC and MBC values was observed in regards
to the relative positioning (e.g., compounds 1 vs 2); in each series
there was ꢁ4-fold difference in MIC and MBC values amongst
regioisomeric biscationic amphiphiles of equal chain length.
Although all amphiphiles were effective at killing, 2,4-C14 (2) and
3,5-C14 (9) were the two most effective C14 compounds against all
3 strains, with 2,5-C14 (5) and 2,6-C14 (8) marginally less effective.
The time killing data with the varying C14 head groups suggest
that the 3,5 (9) arrangement is the fastest at killing both Gram-
positive organisms, taking 15 min to kill (Fig. 5, open symbols).
The 2,3 (1) arrangement was the fastest at killing E. coli (0.5 h), but
the 2,4 (2) and 3,5 arrangements were also effective, killing all cells
Table 1
MICs and MBCs (
Table 2
m
M) of Amphiphilesa.
Time Killing of Bacterial Strains by Amphiphiles. Data are reported as time (h) when
all cells were killed by 100 m
M of amphiphilea.
Amphiphile S. aureus (Gþ) E. faecalis (Gþ
)
E. coli (Gꢂ) P. aeruginosa (Gꢂ
)
Amphiphile
S. aureus (Gþ
)
E. faecalis (Gþ
)
E. coli (Gꢂ
)
2,3-C14 (1)
2,4-C14 (2)
2,5-C10 (3)
2,5-C12 (4)
2,5-C14 (5)
2,5-C16 (6)
2,5-C18 (7)
2,6-C14 (8)
3,5-C14 (9)
3,5-C16 (10)
MC10 (11)
MC14 (12)
DTAB (13)
CTAB (14)
SDS (15)
6
6
31
16
63
31
125
125
2,3-C14 (1)
2,4-C14 (2)
2,5-C10 (3)
2,5-C12 (4)
2,5-C14 (5)
2,5-C16 (6)
2,5-C18 (7)
2,6-C14 (8)
3,5-C14 (9)
3,5-C16 (10)
MC10 (11)
MC14 (12)
1
1
NK
NK
48
24
0.25
48
0.25
0.25
0.5
0.25
24
2
0.5
1
NK
NK
72
2
0.5
NK
1
0.25
0.5
NK
250
31
16
16
16
16
4
500
125
31
50
31
>500
500
63
>500
>500
250
NK
NK
48
NK
0.25
NK
0.25
0.25
0.5
0.25
63
125
63
250
50
31
125
31
500
63
8
31
63
125
8
16
63
250
6
16
>500
250
63
>500
>500
>500
>500
63
4
250
125
4
500
a
Times were determined as described in the Experimental section. All experi-
ments were performed a minimum of three times. NK ¼ 100% of cells not killed after
treatment. P. aeruginosa was not tested because the MIC values for all compounds
>500
a
Gþ ¼ Gram-positive, Gꢂ ¼ Gram-negative. All experiments were performed
a minimum of three times. See Supporting Information (Table 1S) for same data
except for 3,5-C14 (9) were greater than 100
not effective at 100 M.
mM. SDS was not used because it was
reported as
m
g/mL.
m

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J.E. LaDow et al. / European Journal of Medicinal Chemistry 46 (2011) 4219e4226
Fig. 5. MIC, MBC, and Time Killing Comparisons Based on Relative Position of Head Groups with C14 tails. Panel A. S. aureus, Panel B, E. faecalis, Panel C, E. coli. Filled symbols
(A, -, C) indicate the MIC and MBC for each organism, while open symbols of the same shape (>, ,, B) indicate the time necessary to kill 100% of the organism. * ¼ time
necessary to kill was >72 h.
within 1 h. The 2,5 (5) and 2,6 (8) arrangements were clearly
inferior at killing all organisms.
where C18 (7) was still the fastest killer, with C16 (6) unable to kill
at 72 h, and C14 (5) able to kill at 48 h. Generally, the longer chain
length provided superior activity against Gram-positive and Gram-
negative cells. Chain lengths of 12 carbons or less were ineffective
in the time kill assays.
The longer optimal chain length in our series of compounds
relative to compounds with a single cationic charge (which show
maximum activity around 12e14 C for Gram-negative and 14e16 C
for Gram-positive bacteria) [2] can be explained by the increased
water solubility of our derivatives owing to the two cationic head
groups. In other words, the decreased water solubility at higher
chain lengths exhibited by the single-headed derivatives (which
may lead to lower activity) [34] is less significant for the double-
headed compounds of a similar chain length.
At this point, we are uncertain as to the origin of the differences
between the activities of these head group isomers but it may be in
part related to the spacing between the head groups and the
approximate distance between anionic groups at the cell surface,
thus suggesting an optimal relative geometry between the biological
and synthetic counterparts. Fig. 4 highlights the number of carbons
separating the two cationic charges in each of the isomers. Our more
efficient headgroup isomers (with 2,4 and 3,5 arrangements) have
a 5C spacer, while those with a 4C (2,3 arrangement) or 6C (2,5-
arrangement) spacer were less efficient. The inferiority of the 2,6
arrangement (which, like our best two isomers, also has a 5C spacer)
may be due to unfavorable steric interactions of the arene as it
approaches the cytoplasmic membrane. This spacer-length depen-
dence is similar to what has been suggested for bisguanidinium
antimicrobials, where a 6C spacer between headgroups is more
active than longer or shorter spacers [2].
4.4. Effect of number of cations on activity
Finally, comparison of our biscationic series to four mono-
cationic amphiphiles (11e14) did not completely support the
hypothesis and literature precedent [9] suggesting that multiple
cationic moieties would increase efficacy in bacterial growth inhi-
bition; however, only biscationic amphiphiles maintained good
activity versus both of the Gram-negative bacteria tested. As eval-
uated in the MIC and MBC assays, three monocationic species (11,
12, 14) were amongst the most potent compounds prepared against
S. aureus and E. faecalis, but generally less effective than other
biscationic compounds against the Gram-negative organisms.
Biscationic and monocationic amphiphiles showed variable
effectiveness in time kill assays, with each group providing one of
the fastest killers. The biscationic compound 3,5-C16 (10) was
better than MC10 (11) by 15 min against all 3 strains, and was
equally good as MC14 (12) at killing S. aureus and E. facalis, but was
much better at killing E. coli. When assaying the effectiveness of the
two monocationic compounds to kill E. coli, MC10 (11) was able to
4.3. Effect of alkoxy chain length on activity
MIC values, MBC values, and the time killing results against
S. aureus, E. faecalis, and E. coli were also evaluated based on the
alkoxy chain length of the 2,5 headgroup series (3e7). Clear trends
emerged that correlated chain length to antimicrobial activity in
the MIC and MBC assays, specifically with the 2,5-disubstituted
compounds (Fig. 6, filled symbols). The shorter C10 (3) and C12
(4) derivatives were not as effective as 2,5 derivatives with longer
chain lengths; C18 (7) was the most effective. This trend held true
for all organisms tested.
Comparing the time effectiveness of killing on chain length of
the 2,5 derivatives, the C18 (7) variant was easily the most effective.
Compounds were generally less effective as the chain length was
shortened. The only discrepancy with this trend was with E. faecalis,
Fig. 6. MIC, MBC and Time Killing Comparisons Based on Tail Length of the 2,5 Derivatives. Panel A. S. aureus, Panel B, E. faecalis, Panel C, E. coli. Filled symbols (A, -, C) indicate
the MIC and MBC for each organism, while open symbols of the same shape (>, ,, B) indicate the time necessary to kill 100% of the organism. * ¼ time necessary to kill was >72 h
y ¼ MIC values > 500
mM.

J.E. LaDow et al. / European Journal of Medicinal Chemistry 46 (2011) 4219e4226
4225
kill within 30 min, while MC14 (12) was unable to kill the pop-
ulation of cells at 72 h. The drastic difference in efficiency at killing
E. coli by the two monocations (11, 12) suggests that tail length is
critical for killing this Gram-negative organism, with the shorter
chain length more effective. Tail length of the monocations may not
be as important when killing Gram-positive bacteria since the
difference was minimal.
48, and 72 h, one-hundred microliter aliquots were plated on TSA
plates, and the plates were incubated overnight at 37 ꢃC. Data
reported are times when no colonies were observed growing on
plates after overnight incubation. Additionally, 100
ml of cells
treated with compounds for 72 h were placed on slides, Gram-
stained, and visualized microscopically to determine if intact cells
were still present after treatment.
5. Conclusions
Acknowledgements
Our results indicate a moderate set of structure-activity relation-
ships for this synthesized series of bicephalic amphiphiles, specifi-
cally favoring the 3,5- and 2,4-substitution series, as well as extended
chain length for speed in bacterial killing assays. Only biscationic
amphiphiles maintained good activity versus the Gram-negative
bacteria tested. Armed with this knowledge, we aim to extend our
investigations into optimized and novel architectures in this series.
Longer chain derivatives of the 3,5-substitution pattern are high
priorities for future investigations, as well as compounds with alter-
nate head group structures. Finally, we aim to broaden our under-
standing of the interdependence of activity against specific bacteria,
thus a larger battery of test organisms will also be investigated.
This work was primarily supported by a Multi-Investigator
Cottrell College Science Award from the Research Corporation for
Science Advancement (to KLC, KPCM, and KS; MICCSA 10709).
Additional support came from the American Chemical Society
Petroleum Research Fund (to KLC; 43543-GB4), Research Corpora-
tion (to KLC, CC5948), the US Department of Defense ASSURE
program (to KLC; DMR-0851367), and the National Science Foun-
dation e REU program (CHE-0754521).
Appendix. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2011.06.026. These data
include synthetic details, MOL files and InChIKeys of the most
important compounds described in this article.
6. Experimental section
6.1. Bacterial strains and culture conditions
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