Synthesis and antibacterial properties of novel hydrolyzable cationic amphiphiles. Incorporation of multiple head groups leads to impressive antibacterial activity

Article abstract of DOI:10.1021/jm049106l

Two sets of novel multiheaded cationic amphiphiles bearing one, two, and three trimethyl-ammonium headgroups (T1, T2, and T3) and pyridinium headgroups (P1, P2, and P3), have been synthesized and tested for antimicrobial activities against both Gram-positive and Gram-negative bacteria. The multicationic headgroups in these amphiphiles were attached covalently via scissile ester-type linkages. The results were compared with those for known surface-active, nonhydrolyzable compounds cetyltrimethylammonium bromide (CTAB) and cetylpyridinium bromide (CPB). The killing effects of the new single-headed amphiphiles (T1 and P1) were lower than those of CTAB and CPB, but with an increase in the number of headgroups in the amphiphiles, the killing effects increased for both sets of compounds. It was found that amphiphiles with triple headgroups (T3 and P3) were most active among all the amphiphiles, whereas amphiphile P1 had a very poor killing effect on both types of bacteria. The multiheaded pyridinium amphiphiles were more active compared to their trimethylammonium counterparts. The time needed to kill bacteria with multiheaded amphiphiles was significantly less than that of single-headed amphiphiles. Owing to the presence of a cleavable ester moiety, these new amphiphiles are hydrolyzed spontaneously at physiological conditions. This property enables them to be readily metabolized and therefore have the potential to be superior disinfectants and antiseptics for food and body surfaces.

Full text of DOI:10.1021/jm049106l

J. Med. Chem. 2005, 48, 3823-3831
3823
Synthesis and Antibacterial Properties of Novel Hydrolyzable Cationic
Amphiphiles. Incorporation of Multiple Head Groups Leads to Impressive
Antibacterial Activity
Jayanta Haldar,† Paturu Kondaiah,‡ and Santanu Bhattacharya*,†
Departments of Organic Chemistry and of Molecular Reproduction, Development and Genetics,
Indian Institute of Science, Bangalore 560012, India
Received November 5, 2004
Two sets of novel multiheaded cationic amphiphiles bearing one, two, and three trimethyl-
ammonium headgroups (T1, T2, and T3) and pyridinium headgroups (P1, P2, and P3), have
been synthesized and tested for antimicrobial activities against both Gram-positive and Gram-
negative bacteria. The multicationic headgroups in these amphiphiles were attached covalently
via scissile ester-type linkages. The results were compared with those for known surface-active,
nonhydrolyzable compounds cetyltrimethylammonium bromide (CTAB) and cetylpyridinium
bromide (CPB). The killing effects of the new single-headed amphiphiles (T1 and P1) were
lower than those of CTAB and CPB, but with an increase in the number of headgroups in the
amphiphiles, the killing effects increased for both sets of compounds. It was found that
amphiphiles with triple headgroups (T3 and P3) were most active among all the amphiphiles,
whereas amphiphile P1 had a very poor killing effect on both types of bacteria. The multiheaded
pyridinium amphiphiles were more active compared to their trimethylammonium counterparts.
The time needed to kill bacteria with multiheaded amphiphiles was significantly less than
that of single-headed amphiphiles. Owing to the presence of a cleavable ester moiety, these
new amphiphiles are hydrolyzed spontaneously at physiological conditions. This property
enables them to be readily metabolized and therefore have the potential to be superior
disinfectants and antiseptics for food and body surfaces.
Introduction
Amphiphilic cationic compounds have been known to
exhibit strong antimicrobial activity.1,2 They exhibit
rapid activity against a broad range of microorganisms
such as bacteria (both Gram-positive and Gram-nega-
tive),3 fungi,4 and certain viruses.5,6 But because of the
high affinity of the amphiphilic compounds toward
biological membranes, it has been shown that these
compounds are not quite suitable for certain applica-
tions, as they lead to skin irritation and hypersensitiv-
ity. To overcome these side effects, the concept of “soft”
antimicrobial agents,7 which possess a cleavable moiety
in the molecule, such as an ester of betaine or choline,
have been introduced. These esters when subjected to
base- or enzyme-catalyzed hydrolysis, produce signifi-
cantly less toxic components.8,9 The antimicrobial activ-
ity of quaternary ammonium compounds bearing be-
taine esters is quite similar to that of the corresponding
nonhydrolyzable quaternary ammonium compound cetyl-
trimethylammonium bromide (CTAB), but they hydro-
lyze spontaneously into betaine and fatty alcohol, which
are readily metabolized. The hydrolysis of betaine esters
is enhanced at higher pH and in cationic micellar
aggregates8,10 and reduced at higher ionic strength and
in aggregates with fatty acids and hydrophobic alcohol.11
The ester bond in such cationic compounds is hydro-
lyzable at higher pH, and the pH of the sex organ vagina
Figure 1. Molecular structures of various cationic surfactants
bearing trimethylammonium headgroups.
is low, within the range of 2-4. Therefore, these ester
bonds are quite stable within this pH range and hence
are able to kill bacteria and viruses in the vaginal
lumen. At the vaginal wall, however, the pH value
increases significantly, and when the cationic com-
pounds come into contact with the tissues of the wall,
the ester bond is likely to be hydrolyzed into less toxic
substances which can be easily metabolized. Therefore,
these types of compounds do not cause irritation if used
as prophylactic agents, and they are the better choice
than stable quaternary compounds.12 Covalent attach-
ment of multiple headgroups into an amphiphilic mol-
ecule13 allows fine control of the molecular architecture
and hence aggregate morphology. When such linkages
are based on scissile ester bonds, then the resulting
systems become attractive choices as potential anti-
* To whom correspondence should be addressed. Phone: 91 80 2293
2664. Fax: 91 80 2360 0529. E-mail: sb@orgchem.iisc.ernet.in. Also a
recipient of the National Bioscience Career Development Award, DBT.
† Department of Organic Chemistry.
‡ Department of Molecular Reproduction, Development and Genet-
ics.
10.1021/jm049106l CCC: $30.25 © 2005 American Chemical Society
Published on Web 05/06/2005

3824 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Haldar et al.
Scheme 2a
Figure 2. Molecular structures of various cationic surfactants
bearing pyridinium headgroups.
Scheme 1a
a Reagents, conditions, and yields: (a) diethyl malonate, NaH,
THF, 0 °C (100 min), rt (2 h) and 70 °C (15 h), 71%; (b) LiAlH4,
AlCl3, Et2O, 0 °C (20 min) and rt (1 h), 96%; (c) BrCH2COBr,
DMAP, Et3N, CH2Cl2, 0 °C (10 min) and rt (3 h), 64%; (d) NMe3,
acetone, pressure tube, rt (24 h), 87%; (e) pyridine, acetone,
pressure tube, 50 °C (24 h), 78%.
a Reagents, conditions, and yields: (a) BrCH2COBr, DMAP,
Et3N, CH2Cl2, 0 °C (10 min) and room temperature (rt) (1 h), 91%;
(b) NMe3, acetone, pressure tube, rt (12 h), 98%; (c) pyridine,
acetone, pressure tube, 50 °C (12 h), 90%.
with pyridine in dry acetone at 50 °C in a screw-top
pressure tube with heating for 12 h (Scheme 1). A white
solid was formed which was filtered and washed repeat-
edly with dry acetone, and finally pure P1 (90%) was
obtained upon repeated recrystallizations from CHCl3/
n-hexane.
microbial agents. Therefore, it is worthwhile to develop
novel soft antimicrobial agents bearing esters, which can
be easily hydrolyzed to produce less toxic compounds,
and also have multiple charges, enabling them to
interact with the bacterial cell surface more strongly
than their monomeric counterparts. Hence, they are
expected to have better antimicrobial properties. Herein
we present the synthesis of multiheaded cationic am-
phiphiles. Since the main objective of the present study
is the elucidation of how structural modification of
cationic amphiphilic systems influences their antibacte-
rial properties, we also present the results of anti-
microbial studies of two novel sets of multiheaded
amphiphiles possessing cleavable ester moieties, against
both Gram-positive and Gram-negative bacteria.
For the synthesis of the double-headed amphiphiles
T2 (METAB) and P2 (MEPB), diethyl malonate was
monalkylated with 1-bromotetradecane in dry THF in
the presence of NaH. This afforded diethyl n-tetra-
decylmalonate (5) in 71% yield upon purification by
gravity-driven column chromatography over silica gel.
Compound 5 was then reduced using LiAlH4 and AlCl3
in dry Et2O to obtain the corresponding diol 6 in 96%
yield. The diol was then reacted with >2 equiv of
bromoacetyl bromide in the presence of dry Et3N and a
catalytic amount of DMAP in CH2Cl2 first at 0 °C for
10 min and then at room temperature for 3 h. Workup
followed by purification over a silica gel column afforded
the diester 7 in 64% yield. Pure ester 7 was finally
quaternized with trimethylamine in dry acetone in a
screw-top pressure tube with stirring at room temper-
ature for 24 h (Scheme 2). A white solid formed which
was filtered and washed repeatedly with dry acetone.
Pure T2 (87%) was obtained upon several recrystalli-
zations from a mixture of CHCl3/EtOAc.
For the synthesis of P2, pure ester 7 was reacted with
pyridine in dry acetone in a screw-top pressure tube
with heating at 50 °C for 24 h (Scheme 2). A gummy
solid was formed on the walls of the pressure tube. The
required compound P2 (78%) was purified by repetitive
precipitations upon solubilization of the gummy mate-
rial using a 1:1 mixture of CHCl3/acetone and then upon
dropwise addition of n-hexane. Both T2 and P2 were
found to be highly hygroscopic in nature.
Results
Synthesis. The synthesis of single-headed am-
phiphiles T1 (HETAB) and P1 (HEPB) began with the
esterification of 1-hexadecanol with bromoacetyl bro-
mide in the presence of Et3N and a catalytic amount of
4-(N,N-dimethylamino)pyridine (DMAP) in CH2Cl2 to
afford hexadecyl bromoacetate (4) in 91% isolated yield.
After purification by column chromatography over silica
gel, 4 was quaternized using trimethylamine in dry
acetone in a screw-top pressure tube with stirring at
room temperature (rt) for 12 h (Scheme 1). A white solid
was formed which was filtered and washed repeatedly
with dry acetone. The pure amphiphile T1 (98%) was
obtained upon several recrystallizations from a mixture
of CHCl3/n-hexane. To synthesize P1, 4 was reacted

Synthesis of Hydrolyzable Cationic Amphiphiles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3825
Scheme 3a
Table 1. MBCs (µM) of Cleavable Multiheaded Cationic
Amphiphilesa
amphiphile
E. coli
S. sonnei
S. aureus
E. faecalis
CTAB
T1
16.9
20.6
9.7
18.3
33.8
14.9
9.6
18.9
24.5
15.1
11.3
15.6
18.5
22.5
12.0
9.0
T2
T3
CPB
P1
P2
P3
7.3
10.4
12.4
16.3
8.2
6.5
8.9
3.5
13.9
5.2
10.4
4.9
a MBC (µM) determined by the spread plate method. The
average values are reported on the basis of three separate
experiments where the variance was <10%. Amphiphile P1 was
not able to kill the bacteria completely even at concentrations as
high as 300 µM.
dissolving the sticky material in a 1:1 mixture of CHCl3/
EtOAc and then upon dropwise addition of n-hexane.
To synthesize P3, 10 was reacted with pyridine in dry
acetone in a screw-top pressure tube with heating at
50 °C for 48 h (Scheme 3). A brown sticky solid formed
on the walls of the pressure tube and was dissolved in
CHCl3 and precipitated several times upon addition of
n-hexane. During the process of precipitation the com-
pound initially came out of the solution as a dense
yellow liquid. The two phases could be separated from
this by careful decantation to afford the dense material,
which eventually solidified into a sticky mass, P3 (62%).
Both the surfactants were found to be highly hygro-
scopic in nature. All the final products and key inter-
mediates were appropriately characterized as given in
the Experimental Section.
Antibacterial Property. Antibacterial activity of the
multiheaded amphiphiles was investigated, and the
results were compared with those of the nonhydrolyz-
able surfactants CTAB and cetylpyridinium bromide
(CPB). The solubility of both single-headed surfactants
(T1 and P1) was less in both water and Luria-Burtani
(LB) medium compared to those of other amphiphiles.
Between the two single-headed amphiphiles, P1 was
even less soluble than T1. The stock solution of all the
amphiphiles was made in sterile water. The facile
solubility of amphiphiles with multiple headgroups in
both water and LB medium could be a consequence of
the increased hydrophilicity of the resulting am-
phiphiles with a larger number of headgroups.
To investigate the influence of systematically enhanc-
ing the number of headgroup units incorporated at one
end of a single hydrocarbon chain of the amphiphilic
molecule and also the nature of the headgroups on the
bactericidal effect, we have tested these two sets of
amphiphiles against both Gram-positive and Gram-
negative bacteria. The minimum bactericidal concentra-
tions (MBCs) were determined by the spread plate
method.14 The MBC data for the amphiphiles against
both Gram-negative and Gram-positive bacteria are
shown in Table 1. The MBC value was considered as
the lowest amphiphile concentration at which there was
no viable cell present. The relevant data for CTAB and
CPB are also included in Table 1 for comparison. The
killing effect was similar for both Gram-positive and
Gram-negative bacteria and increased with an increase
in the number of headgroups on the amphiphile for both
sets of compounds (Figure 3). In general, the am-
phiphiles bearing multiheaded pyridinium headgroups
a Reagents, conditions, and yields: (a) PCC, CH2Cl2, rt (5 h),
95%; (b) formaldehyde solution, KOH, 50% aqueous EtOH, rt (4
h) and 50 °C (2 h), 41%; (c) BrCH2COBr, DMAP, Et3N, CH2Cl2, 0
°C (10 min) and rt (5 h), 53%; (d) NMe3, acetone, pressure tube, rt
(48 h), 65%; (e) pyridine, acetone, pressure tube, 50 °C (48 h), 62%.
The synthesis of the triple-headed amphiphiles T3
(BETAB) and P3 (BEPB) began with the oxidation of
1-hexadecanol to the corresponding aldehyde 8 (95%)
by pyridinium chlorochromate (PCC) in CH2Cl2. 1-Hexa-
decanal (8) was then converted to 2,2-bis(hydroxymeth-
yl)hexadecanol (9) by reaction with formaldehyde solu-
tion in the presence of KOH and 50% aqueous EtOH
initially at room temperature for 4 h and then at 50 °C
for 2 h. After removal of EtOH under vacuum, the
resulting mixture was taken up in Et2O and allowed to
settle. This resulted in the separation of the organic
layer from this mixture, which was dried over anhy-
drous Na2SO4 and concentrated to produce a white
residue. After column chromatography over silica gel,
the alcohol 9 (41%) was isolated as a pure white solid.
The required compound was obtained first by two aldol
reactions at room temperature followed by a cross-
Cannizzaro reaction at 50 °C with formaldehyde. Then
the triol 9 was fully esterified to the corresponding
triester 10 (53%) upon reaction with 3.6 equiv of
bromoacetyl bromide in the presence of dry Et3N and a
catalytic amount of DMAP in CH2Cl2 first at 0 °C for
10 min and then at room temperature for 5 h. After
workup and purification by column chromatography
over silica gel, pure triester 10 was isolated. Finally 10
was quaternized with trimethylamine in dry acetone in
a screw-top pressure tube with stirring at room tem-
perature for 48 h (Scheme 3). A sticky solid formed on
the walls of the pressure tube. The required compound
T3 (65%) was purified by repetitive precipitations by

3826 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Haldar et al.
medium. The concentration of T1 and P1 was kept at
50 µM, whereas the concentration of the other am-
phiphiles was maintained at 20 µM against all organ-
isms.
Among the amphiphiles with trimethylammonium
headgroups, T3 was found to be the most active one
against both types of bacteria (Figure 4). When exposed
to the triple-headed amphiphile T3, all bacterial cells
were killed within 20-30 min for both types of bacteria.
In the case of T2 and CTAB, the killing of bacteria
required a longer time compared to that of T3. There-
fore, the time taken for killing of the bacterial cells
became progressively less with an increase in the
number of headgroups. From parts C and D of Figure
4, which show the killing effect on Gram-positive
bacteria S. aureus and E. faecalis, respectively, it is
evident that with these compounds the time required
to kill the Gram-positive bacteria is a little less than
that for Gram-negative bacteria.
Figure 5 shows the log(survivors) versus exposure
time plots for the multiheaded amphiphiles with pyri-
dinium headgroups against both types of bacteria. In
this set of compounds, it was observed that the am-
phiphile with triple headgroups, P3, was also the most
active compound. The activity of P1 was very poor,
which was also found during the estimation of the MBC
value (Table 1). When 20 µM P3 was treated against
both types of bacteria, all bacterial cells were killed
within ∼10 min. Thus, with an increase in the number
of headgroups of pyridinium amphiphiles, the killing
effect became even faster. In this case, it was also found
that the compounds killed the Gram-positive bacteria
faster than the Gram-negative bacteria (Figure 5).
Figure 3. Comparison of bactericidal activity of various
cationic amphiphiles with (A) trimethylammonium headgroups
and (B) pyridinium headgroups against both Gram-positive
and Gram-negative bacteria.
were found to be more active than the amphiphiles
bearing trimethylammonium headgroups against either
type of bacteria.
Discussion
The MBC values for T1 against Escherichia coli and
Shigella sonnei were 20.6 and 33.8 µM, respectively, and
7.3 and 9.6 µM for T3. Against Staphylococcus aureus
and Enterococcus faecalis, the MBC values were 24.5
and 22.5 µM, respectively, for T1 and 11.3 and 9.0 µM,
respectively, for T3. The MBC values for double-headed
amphiphile T2 were between those of the single- and
triple-headed amphiphiles against all the bacteria. In
the case of single-headed amphiphiles, however, CTAB
was more effective than T1 (Figure 3A).
Among the series of amphiphiles with pyridinium
headgroups, P1 had a very poor killing effect on both
types of bacteria. Even at concentrations as high as 300
µM, it merely reduced the cell count for E. coli from 5.7
× 109 to 1.6 × 104 cells/mL, for S. sonnei from 3.2 × 107
to 2.4 × 102 cells/mL, for S. aureus from 4.9 × 109 to
8.3 × 102 cells/mL, and for E. faecalis from 1.2 × 108 to
4.5 × 103 cells/mL. The MBC values of amphiphile P3
against E. coli and S. sonnei were 6.5 and 3.5 µM,
respectively, and against S. aureus and E. faecalis, they
were 5.2 and 4.9 µM, respectively. The MBC values for
double-headed pyridinium amphiphile P2 ranged be-
tween those of of CPB and P3 against both Gram-
negative and Gram-positive bacteria (Figure 3B).
Figures 4 and 5 show the log(survivors) versus
exposure time plots for the multiheaded amphiphiles
bearing trimethylammonium headgroups and pyri-
dinium headgroups, respectively, against both Gram-
negative and Gram-positive bacteria. Exposure of the
amphiphiles to bacterial cells was carried out in LB
The sequence of elementary events in the lethal action
of the cationic disinfectants15 may be summarized as
follows: (i) adsorption onto the bacterial cell surface,
(ii) diffusion through the cell wall, (iii) binding to the
cytoplasmic membrane, (iv) disruption of the cytoplas-
mic membrane, (v) release of K+ ions and other cyto-
plasmic constituents, and (vi) precipitation of the cell
contents and the death of the cell. Electrophoretic
measurements have clearly demonstrated that the
bacterial cell surface is usually negatively charged, and
hence, the adsorption of cationic amphiphiles onto the
negatively charged cell surface is facilitated by electro-
static interaction, along with the hydrophobic inter-
action.16
The MBC values decrease with an increase in the
number of headgroups in the amphiphile, implying that
the antibacterial activity of amphiphiles with multiple
headgroups is higher than that of single-headed am-
phiphiles. This might be explained by taking into
consideration each of the elementary processes involved
in the lethal action.15 The adsorption of amphiphiles
with multicationic headgroups onto the negatively
charged bacterial cell surface is expected to take place
at a greater extent than that of a monocationic am-
phiphile through electrostatic interaction because of the
much higher charge density carried by multiheaded
cationic amphiphiles.16 Furthermore, binding to the
cytoplasmic membrane is also expected to be facilitated
by the multiheaded cationic amphiphiles, compared to

Synthesis of Hydrolyzable Cationic Amphiphiles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3827
Figure 4. Log(survivors) versus exposure time plots for different amphiphiles with trimethylammonium headgroups against (A)
E. coli, (B) S. sonnei, (C) S. aureus, and (D) E. faecalis: 1, control (without any amphiphile); 2, T1 (50 µM); 3, CTAB (20 µM); 4,
T2 (20 µM); 5, T3 (20 µM).
single-headed amphiphiles, because of the presence of
a large number of negatively charged species (such as
acidic phospholipids and some membrane proteins) in
the membrane.17,18 Thus, the disruption of the mem-
brane and the subsequent leakage of K+ ions and other
cytoplasmic constituents would be enhanced by the
multiheaded cationic amphiphiles.
have lower solubility in water compared to CTAB and
CPB. From our previous studies it was found that they
have a greater tendency to form open lamellar or other
larger aggregates.13,20 Therefore, they have a greater
binding affinity in excessive amounts to a lesser number
of cells. This in turn enhances their tendency to associ-
ate with compounds released from the cells. In contrast,
the multiheaded amphiphiles that are highly soluble in
water and form much smaller and more dynamic
aggregates13 can bind to a greater number of cells. They
can also easily diffuse through the cell wall and interact
with the cytoplasmic membrane to kill the bacteria more
efficiently.
All the amphiphiles are almost equally active for both
Gram-positive and Gram-negative bacteria. It is known
that the Gram-positive strains have a rather simple cell
wall composed of a rigid peptidoglycan layer, which
allows foreign molecules to come inside without much
difficulty.19 Thus, it is expected that any foreign sub-
stances diffuse easily through the cell walls of Gram-
positive bacteria. On the other hand, in the case of
Gram-negative bacteria, there is another bilayer mem-
brane outside the peptidoglycan layer (outer mem-
brane). Because of this outer membrane, foreign mol-
ecules cannot easily diffuse through the cell walls of
Gram-negative bacteria.19 But these types of low mo-
lecular mass amphiphiles because of their lipidlike
properties can easily diffuse through both the outer
membrane and the cell wall to reach the cytoplasmic
membrane of the Gram-negative species and hence are
able to kill the bacteria with equal efficiency.
Conclusion
Two sets of novel multiheaded cationic amphiphiles
bearing one, two, and three trimethylammonium head-
groups and pyridinium headgroups were synthesized
and characterized. The antibacterial activity of multi-
headed amphiphiles against both Gram-negative and
Gram-positive bacteria is significantly enhanced com-
pared to that of single-headed amphiphiles. This could
be due to their higher solubility in water and greater
positive charge density per molecule, which enable them
to interact better with the bacterial cell surface, leading
to more efficient killing of the bacteria. These com-
pounds have their cationic headgroups connected to a
hydrocarbon chain via cleavable ester linkages and
The activity of single-headed surfactants T1 and P1
is lower than that of CTAB and CPB. This could be due
to the fact that single-headed amphiphiles T1 and P1

3828 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Haldar et al.
Figure 5. Log(survivors) versus exposure time plots for different amphiphiles with pyridinium headgroups against (A) E. coli,
(B) S. sonnei, (C) S. aureus, and (D) E. faecalis: 1, control (without any amphiphile); 2, P1 (50 µM); 3, CPB (20 µM); 4, P2 (20
µM); 5, P3 (20 µM).
washed with NaHCO3 (0.5 M, 10 mL) and saturated brine
solution (20 mL). The organic layer was separated, dried over
anhydrous Na2SO4, and concentrated to afford a residue which
was purified by column chromatography over silica gel using
an EtOAc/hexane (1:99) solvent mixture. A low-melting-point,
white compound (Rf ≈ 0.8 in an EtOAc/hexane (1:99) solvent
mixture) was isolated in 91% yield. FT-IR (neat): 1740 cm-1
(CdO str). 1H NMR (CDCl3, 300 MHz): δ 0.88 (t, terminal
-CH3, 3H), 1.25 (br m, (-CH2-)13, 26H), 1.59-1.7 (m, -CH2-
CH2O-, 2H), 3.85 (s, -OC(O)CH2-, 2H), 4.17 (t, -CH2CH2O-,
2H). MALDI-TOF: m/z 386.4 [M + Na]+.
Hexadecyl N-Ethanoate N,N,N-Trimethylammonium
Bromide (HETAB, T1). Dry NMe3 gas was passed into dry
acetone (5.0 mL) in a screw-top pressure tube at 0 °C till the
volume of the resulting solution was 15 mL. Then 4 (1.06 g,
2.92 mmol) was dissolved in dry acetone (3.0 mL) and added
to the pressure tube at 0 °C. Immediately after the addition
of the bromide, a white precipitate was formed. The reaction
mixture was stirred at room temperature for 3-4 h for
completion of the reaction. The precipitate was filtered and
washed several times with dry acetone to give a white solid.
Pure surfactant T1 (98%) was obtained upon repeated recrys-
tallizations from CHCl3/hexane. FTIR (KBr): 1751 cm-1
(CdO str). 1H NMR (300 MHz, CDCl3): δ 0.88 (t, terminal
-CH3, 3H), 1.25 (br s, (CH2)13, 26H), 1.63-1.70 (m, -CH2-
CH2O-, 2H), 3.67 (s, -+N(CH3)3, 9H), 4.18 (t, -CH2O-, 2H),
4.99 (s, -CH2N+-, 2H). 13C NMR (133.3 MHz, CDCl3): δ 14.05,
22.66, 25.66, 28.31, 29.16, 29.32, 29.45, 29.68, 31.91, 54.34,
63.22, 66.915, 164.71. ESI-MS: m/z M+ [C21H44NO2]+, calcd
342.3, found 342.2. Anal. Calcd for C21H44NO2Br: C, 59.7; H,
10.5; N, 3.31. Found: C, 59.54; H, 10.63; N, 2.95.
can therefore be hydrolyzed to form readily metabolized
substances.8,9 This property renders this class of mul-
tiheaded amphiphiles potent candidates to replace the
stable cationic compounds.
Experimental Section
All the starting materials were obtained from Aldrich or
Fluka and used as received. PCC was synthesized as described
by Corey et al.21 CTAB and CPB used in this study were
purchased from Aldrich. All the solvents were reagent grade
and dried prior to use. Column chromatography was performed
using 60-120 mesh silica gel. NMR spectra were recorded
1
using a JEOL JNM λ-300 (300 MHz for H) or Bruker AMX-
400 (400 MHz for 1H and 133.3 MHz for 13C) spectrometer.
The chemical shifts (δ) are reported in parts per million
downfield from the peak for the internal standard TMS for
1H NMR and 13C NMR. Mass spectra were recorded on a
Kratos PCKompact SEQ V1.2.2 MALDI-TOF spectrometer, a
MicroMass ESI-TOF spectrometer, or a Shimadzu tabletop
GC-MS or ESI-MS (HP1100LC-MSD) instrument. Infrared
(IR) spectra were recorded on a Jasco FT-IR 410 spectrometer
using KBr pellets or neat.
Hexadecyl Bromoethanoate (4). 1-Hexadecanol (1.0 g,
4.13 mmol) was dissolved in dry CH2Cl2 (8 mL). DMAP (0.068
g, 0.56 mmol) and dry Et3N (0.74 mL) were added to the
reaction mixture, and the resulting mixture was cooled in an
ice-water bath. Bromoacetyl bromide (0.43 mL, 4.95 mmol)
was added dropwise to the reaction mixture, after 10 min the
ice bath was removed, and the mixture was stirred for 1 h at
room temperature. The reaction mixture was diluted with
CHCl3 (50 mL) and successively washed with dilute HCl
solution (2 M, 10 mL) and with water (10 mL). Finally, it was
Hexadecyl N-Ethanoate Pyridinium Bromide (HEPB,
P1). 4 (1.45 g, 3.99 mmol) was dissolved in dry acetone (10

Synthesis of Hydrolyzable Cationic Amphiphiles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3829
mL) and added to a screw-top pressure tube. Dry pyridine (0.96
mL, 11.97 mmol) was added to the pressure tube, and the
mixture was heated at 50 °C for 12 h. A white solid was formed
which was filtered and washed repeatedly with dry acetone.
Pure surfactant P1 (1.59 g, 90%) was obtained upon several
recrystallizations from CHCl3/n-hexane. FT-IR (KBr): 1748
cm-1 (CdO str). 1H NMR (300 MHz, CDCl3): δ 0.88 (t, terminal
-CH3, 3H), 1.25 (br m, (-CH2-)13, 26H), 1.64-1.69 (m, -CH2-
CH2O-, 2H), 4.21 (t, -CH2O-, 2H), 6.3 (s, -COCH2-, 2H),
8.08 (t, m-H, 2H), 8.53 (t, p-H, 1H), 9.42 (d, J ) 6.6 Hz, o-H,
2H). 13C NMR (133.3 MHz, CDCl3): δ 14.07, 22.65, 25.68,
28.40, 29.34, 29.50, 29.69, 31.91, 61.28, 127.75, 146.11, 146.78,
165.81. ESI-MS: m/z M+ [C23H40NO2]+, calcd 362.5, found
362.5. Anal. Calcd for C23H40NO2Br: C, 62.4; H, 9.1; N, 3.16.
Found: C, 62.2; H, 9.2; N, 3.05.
Ethyl 2-(Ethoxycarbonyl)hexadecanoate (5).22 NaH
(1.25 g, 26 mmol, 50% dispersion in oil) was added to a two-
necked round-bottom flask closed with a septum to which dry
n-hexane (5 mL) was added, and the mixture was stirred for
5 min to dissolve the mineral oil. The supernatant in n-hexane
was removed by a syringe, dry THF (10 mL) was added, and
the resulting mixture was stirred at 0 °C for 15 min. Diethyl
malonate (5.66 g, 35.3 mmol) in dry THF (10 mL) was then
slowly added over 30 min at 0 °C to the resulting suspension.
The solution became clear after 10 min of stirring. The stirring
was continued for another 30 min. 1-Bromotetradecane (4 g,
14.4 mmol) in dry THF (15 mL) was added dropwise over 30
min to the stirred solution, resulting in the formation of a
white viscous residue which was diluted with dry THF (10
mL), and this mixture was stirred at room temperature for 2
h, followed by refluxing for 15 h at 70 °C to ensure the
completion of the reaction. The mixture was then taken up in
CHCl3 (200 mL) and washed first with water (100 mL × 3)
and then with 10% (w/v) brine solution (150 mL). The CHCl3
layer was separated and dried using anhydrous Na2SO4. The
solvent was evaporated to leave a gumlike residue which was
adsorbed on silica gel, and the required compound (Rf ≈ 0.6
in an EtOAc/hexane (4:96) solvent mixture) was purified by
column chromatography. Pure compound was isolated in 71%
(3.6 g) yield. FT-IR (neat): 1735 cm-1 (CdO str). 1H NMR (300
MHz, CDCl3): δ 0.88 (t, terminal -CH3, 3H), 1.2-1.3 (br m,
(-CH2-)12, -OCH2CH3 × 2, 30H), 1.8 (m, -CH2CH-, 2H), 3.3
(t, -CH2CH-, 1H), 4.15 (q, -OCH2CH3 × 2, 4H). LR-MS: m/z
357 [M + H]+.
2-(Hydroxymethyl)hexadecanol (6). Anhydrous AlCl3
(0.634 g, 4.76 mmol) was added slowly to a solution of LiAlH4
(0.639 g, 16.8 mmol) in dry diethyl ether (20 mL) at 0 °C. The
color of the mixture became white, and 5 (1 g, 2.8 mmol) in
the form of a solution in dry diethyl ether (10 mL) was added
dropwise. The reaction mixture was stirred at 0 °C for 20 min
and then at room temperature for 1 h. At the end of this period,
moist Et2O (10 mL) was added slowly, and the mixture was
stirred for 10 min. The reaction mixture was then washed with
HCl solution (2 N, 10 mL), and the aqueous layer was
extracted with EtOAc (50 mL × 2). All the organic layers were
obtained and dried over anhydrous Na2SO4, and the solvent
was evaporated to leave a white solid, which was separated
by column chromatography using silica gel. A white solid was
obtained (0.73 g, 96%) toward the end. FT-IR (Nujol): 3300
cm-1 (O-H str). 1H NMR (300 MHz, CDCl3): δ 0.85 (t,
terminal -CH3, 3H), 1.23 (br m, (CH2)13, 26 H), 1.7-1.8 (m,
-CHCH2-, 1H), 3.6-3.8 (m, -CHCH2OH × 2, 4H). LR-MS:
m/z 273 [M + H]+.
2-(Methyl bromoethanoate)hexadecyl Bromoetha-
noate (7). DMAP (0.05 g, 0.41 mmol) and Et3N (0.53 mL, 3.82
mmol) were added to 6 (0.4 g, 1.47 mmol) dissolved in CH2Cl2
(6.0 mL), which was cooled at 0 °C. Bromoacetyl bromide (0.3
mL, 3.52 mmol) was then added dropwise at this temperature.
After ∼10 min the ice bath was removed, and stirring was
continued at room temperature for 3 h. The solvent from the
reaction mixture was evaporated, and the required compound
was purified by column chromatography over silica gel using
an EtOAc/hexane (4:96) solvent mixture (Rf ≈ 0.5 in an EtOAc/
hexane (6:94) solvent mixture). A low-melting-point white solid
was obtained (0.48 g, 64%). FT-IR (neat): 1743 and 1730 cm-1
(CdO str). 1H NMR (300 MHz, CDCl3): δ 0.88 (t, terminal
-CH3, 3H), 1.25 (br m, (CH2)13, 26 H), 2.06-2.17 (m,
-CHCH2O-, 1H), 3.85 (s, -OC(O)CH2- × 2, 4H), 4.15 (dd,
J1 ) 11.25 Hz, J2 ) 6.3 Hz, -CH(CHaHbO-)2, 2H), 4.22 (dd,
J1 ) 11.1 Hz, J2 ) 5.4 Hz, -CH(CHaHbO-)2, 2H). MALDI-
TOF: m/z 537.8 [M + Na]+.
2-(Methyl N-ethanoate N,N,N-trimethylammonium
bromide)hexadecyl N-ethanoate N,N,N-Trimethyl-
ammonium Bromide (METAB, T2). Dry NMe3 gas was
passed into dry acetone (2.0 mL) at 0 °C in a screw-top
pressure tube till the volume of the resulting solution was 8.0
mL. Then 7 (0.26 g, 0.506 mmol) was dissolved in dry acetone
(2.0 mL) and added to the pressure tube at 0 °C. Immediately
after addition of the bromide, a white precipitate was formed,
and the reaction mixture was stirred at rt for 12 h for
completion. The precipitate was filtered and washed several
times with dry acetone to give a white solid. NMe3 was
removed from the filtrate by heating with hot water, and then
the solvent was removed to give a white solid also. Pure
surfactant T2 (87%) was obtained upon repeated recrystalli-
zations from CHCl3/EtOAc. FTIR (KBr): 1749 cm-1 (CdO str).
1H NMR (300 MHz, CDCl3): δ 0.88 (t, terminal -CH3, 3H),
1.26 (s, (CH2)13, 26H), 2.12-2.18 (m, -CHCH2O-, 1H), 3.62
(s, -+N(CH3)3 × 2, 18H), 4.03 (dd, J1 ) 7.2 Hz, J2 ) 10.8 Hz,
-CH(CHaHbO-)2, 2H), 4.44 (dd, J1 ) 3.4 Hz, J2 ) 10.8 Hz, -
CH(CHaHbO-)2, 2H), 5.51 (ABq, J ) 17.7 Hz, -+NCHaHb ×
2, 4H). 13C NMR (133.3 MHz, CDCl3): δ 14.05, 22.65, 26.87,
28.05, 29.33, 29.50, 29.67, 31.91, 36.94, 54.27, 62.98, 68.12,
165.06. ESI-MS: m/z M2+ [C27H56N2O4]2+ Br- (78.91), calcd
551.3, found 551.3; M2+ Br- (80.91), calcd 553.3, found 553.4;
M
2+/2, calcd 236.2, found 236.1. Anal. Calcd for C27H56N2O4-
Br2,H2O: C, 49.85; H, 8.99; N, 4.3. Found: C, 49.53; H, 8.97;
N, 3.9.
2-(Methyl N-ethanoate pyridinium bromide)hexa-
decyl N-Ethanoate Pyridinium Bromide (MEPB, P2).
Inside a screw-top pressure tube, 7 (0.77 g, 1.49 mmol) was
dissolved in dry acetone (8 mL). Dry pyridine (0.72 mL, 8.94
mmol) was added to the pressure tube, and the mixture was
heated at 50 °C for 24 h. A gummy solid was formed on the
wall of the pressure tube. CHCl3 was added to the reaction
mixture, and the resulting mixture was transferred into a
round-bottom flask. The solvent and extra pyridine were
removed under vacuum. The required compound P2 (78%) was
purified by repetitive precipitations by dissolving the solid in
a 1:1 mixture of CHCl3/acetone and then upon dropwise
addition of n-hexane. The compound was found to be a
hygroscopic solid. FT-IR (KBr): 1750 cm-1 (CdO str). 1H NMR
(300 MHz, CDCl3): δ 0.88 (t, terminal -CH3, 3H), 1.26 (br m,
(-CH2-)13, 26H), 2.02-2.17 (m, -CHCH2O-, 1H), 4.15 (dd,
J1 ) 7.2 Hz, J2 ) 10.8 Hz, -CH(CHaHbO-)2, 2H), 4.37 (dd, J1
) 3.3 Hz, J2 ) 10.8 Hz, -CH(CHaHbO-)2, 2H), 6.43 (ABq, J
) 17.4 Hz, -+NCHaHb × 2, 4H), 8.12 (t, m-H, 4H), 8.59 (t,
p-H, 2H), 9.55 (d, J ) 6 Hz, o-H, 4H). 13C NMR (133.3 MHz,
CDCl3): δ: 14.07, 22.68, 27.145, 27.305, 27.90, 28.08, 29.34,
29.71, 29.94, 31.93, 39.82, 42.24, 61.56, 61.78, 65.86, 67.45,
128.035,146.36,146.765,165.97.ESI-MS: m/zM2+[C31H48N2O4]2+
Br- (78.91), calcd 591.3, found 591.5; M2+ Br- (80.91), calcd
593.3, found 593.5; M2+/2, calcd 256.2, found 256.2. Anal. Calcd
for C31H48N2O4Br2,H2O: C, 53.9; H, 7.2; N, 4.1. Found: C, 53.8;
H, 7.0; N, 3.9.
1-Hexadecanal (8).21 PCC (6.68 g, 30.99 mmol) was taken
up in dry CH2Cl2 (100 mL) and the mixture stirred at room
temperature for 5 min. After addition of hexadecanol (5 g,
20.66 mmol) to the reaction mixture, it was again stirred for
3 h at room temperature. The reaction mixture was diluted
with dry ether, and the upper portion of the solvent was
decanted. The insoluble residue was washed 3-4 times with
dry ether, and the solvent from all the washings along with
the main Et2O fraction was evaporated to give a solid residue.
The compound from this residue was purified by column
chromatography over silica gel using an EtOAc/hexane (2:98)
solvent mixture (Rf ≈ 0.7 in an EtOAc/hexane (2:98) solvent
mixture). A white solid compound (4.68 g, 95%) was obtained.

3830 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Haldar et al.
FT-IR (Nujol): 1720 cm-1 (CdO str), 3370 cm-1 (C-H ald str).
1H NMR (300 MHz, CDCl3): δ 0.85 (t, terminal -CH3, 3H),
1.23-1.26 (br m, (-CH2-)13, 26H), 2.36-2.42 (m, -CH2CHO-,
2H), 9.74 (br s, -CH2CHO-, 1H). LR-MS: m/z 241 [M + H]+.
(0.07 g, 0.105 mmol) was dissolved in dry acetone (4 mL), and
the resulting mixture added to a screw-top pressure tube. Dry
pyridine (1 mL) was added, and the mixture was heated at 50
°C for 48 h. A brown solid was formed on the wall of the
pressure tube. The reaction mixture was cooled at room
temperature, and solvent was decanted carefully from the
pressure tube. The solid residue was dissolved in CHCl3 and
precipitated upon addition of n-hexane several times. During
the process of precipitation the compound initially came out
of the solution as a dense yellow liquid. The solvent could be
separated from this by careful decantation to afford a sticky
solid material, P3 (62%). The compound was found to be an
extremely hygroscopic solid. FT-IR (KBr): 1750 cm-1 (CdO
str). 1H NMR (300 MHz, CDCl3): δ 0.86 (t, terminal -CH3,
3H), 1.26 (br m, (-CH2-)13, 26H), 4.26 (s, -CH2O- × 3, 6H),
6.51 (s, -COCH2- × 3, 6H), 8.09 (t, m-H, 6H), 8.56 (t, p-H,
3H), 9.75 (d, J ) 5.4 Hz, o-H, 6H). 13C NMR (133.3 MHz,
CDCl3): δ 14.09, 22.71, 27.16, 27.32, 27.91, 28.09, 29.36, 29.72,
29.96, 31.94, 39.84, 42.26, 61.57, 61.79, 65.87, 67.46, 128.04,
146.37, 146.79, 165.99. ESI-MS: m/z M3+ [C39H56N3O6]3+ 2Br-
(78.91), calcd 820.7, found 820.6; M3+ Br- (80.91) Br- (78.91),
calcd 822.6, found 822.3; M3+ 2Br- (80.91), calcd 824.7, found
824.2; (M3+ Br- (78.91))/2, calcd 370.8; found 370.8; (M3+ Br-
(80.91))/2, calcd 371.8, found 371.6; M3+/3, calcd 220.9, found
220.9. Anal. Calcd for C39H56N3O6Br3‚3H2O: C, 48.9; H, 6.5;
N, 4.4. Found: C, 48.7; H, 6.4; N, 4.5.
Microorganisms and Culture Conditions. The micro-
bicidal activity of these new multiheaded amphiphiles was
tested against both Gram-negative and Gram-positive bacteria.
The Gram-negative bacteria examined in the present study
were E. coli (DH5R) and S. sonnei (AK1). The Gram-positive
bacteria were S. aureus (clinical isolate) and E. faecalis (clinical
isolate). The LB medium (10 g of tryptone, 5 g of yeast extract,
and 10 g of NaCl in 1000 mL of sterile distilled water (pH ≈
7)) was used as a liquid medium. The LB agar (10 g of tryptone,
5 g of yeast extract, 10 g of NaCl, and 15 g of agar in 1000 mL
of sterile distilled water) was used as a solid medium for all
the experiments. The stock solution of the amphiphiles was
made in autoclaved sterile water.
Freeze-dried ampules of E. coli were opened, and a loopful
of culture was spread to give single colonies on LB agar and
incubated at 37 °C for 24 h. A single colony was picked up
with a sterile micropipet tip, placed in 10 mL of liquid LB
medium, and inoculated with constant shaking at 37 °C for
10 h to give a working concentration of 108-109 cells/mL before
every experiment.
A single colony of S. sonnei was picked up with a small tip,
placed in 10 mL of liquid LB medium, and inoculated with
constant shaking at 37 °C for 12 h to give a working
concentration of 107-108 cells/mL before every experiment.
S. aureus and E. faecalis grown on one blood agar plate were
used to isolate a single colony with a small tip, placed in 10
mL of liquid LB, and inoculated with constant shaking at 37
°C for 10 h for S. aureus to give a working concentration of
109-1010 cells/mL and 12 h for E. faecalis to give a working
concentration of 107-108 cells/mL before every experiment.
2,2-Bis(hydroxymethyl)hexadecanol (9).23 To a solution
of 1-hexadecanal (4.09 g, 17.04 mmol) were added formalde-
hyde solution (3.54 mL, 1.0 mL contains 1.075 g) and 50%
aqueous ethanol (50 mL), and the resulting mixture was cooled
at 0 °C. KOH (0.954 g, 17.04 mmol) dissolved in 50% aqueous
ethanol (50 mL) was added to the reaction mixture dropwise.
The reaction mixture was stirred at room temperature for 4
h, after which it was heated at 50 °C for 2 h. Ethanol was
evaporated, and the reaction mixture was extracted with Et2O
4-5 times. The organic layers were collected together and
dried over anhydrous Na2SO4. A colorless gumlike material
was obtained after evaporation of the solvent. The required
compound (Rf ≈ 0.3 in an EtOAc/hexane mixture (3:2)) was
purified by column chromatography using silica gel. A white
solid compound (2.1 g, 41%) was isolated. FT-IR (Nujol): 3340
1
cm-1. H NMR (300 MHz, CDCl3): δ 0.85 (t, terminal -CH3,
3H), 1.15-1.22 (br m, (-CH2-)13, 26H), 2.43 (br s, -C(CH2OH)3,
3H), 3.72 (s, -C(CH2OH)3, 6H). LR-MS: m/z 303 [M + H]+.
2,2-Bis(methyl bromoethanoate)hexadecyl Bromo-
ethanoate (10). 9 (0.302 g, 1.22 mmol) was dissolved in CH2-
Cl2 (7.0 mL). DMAP (0.089 g, 0.73 mmol) and Et3N (0.66 mL,
4.76 mmol) were added, and the reaction mixture was cooled
at 0 °C. Bromoacetyl bromide (0.38 mL, 4.4 mmol) was then
added dropwise at this temperature. The ice bath was removed
after 10 min, and the reaction mixture was allowed to stir at
room temperature for 5 h. The reaction mixture was diluted
with CHCl3 (30 mL) and successively washed with dilute HCl
solution (2 M, 10 mL) and with water (10 mL). Finally it was
washed carefully with NaHCO3 (0.5 M, 10 mL) and saturated
brine solution. The organic layer was separated and dried over
anhydrous Na2SO4. The desired product was isolated by
column chromatography over silica gel using an EtOAc-
hexane solvent mixture. A colorless liquid (Rf ≈ 0.4 in an
EtOAc/hexane (8:92) solvent mixture) was obtained (0.43 g,
53%). FT-IR (neat): 1741 cm-1 (br CdO str). 1H NMR (300
MHz, CDCl3): δ 0.88 (t, terminal -CH3, 3H), 1.26 (br m,
(-CH2-)12, 24H), 1.47 (br m, -CH2C-, 2H), 3.85 (s, (-COCH2-
)3, 6H), 4.2 (s, (-CH2O-)3, 6H). MALDI-TOF: m/z 689.7 [M +
H]+.
2,2-Bis(methyl N-ethanoate N,N,N-trimethylammoni-
um bromide)hexadecyl N-Ethanoate N,N,N-Trimeth-
ylammonium bromide (BETAB, T3). Dry NMe3 gas was
passed into dry acetone (2.0 mL) in a screw-top pressure tube
at 0 °C till the volume of the resulting solution was 10 mL.
Then the corresponding bromide 10 (0.28 g, 0.42 mmol) was
dissolved in dry acetone (2.0 mL) and added to the pressure
tube at 0 °C. The reaction mixture was allowed to stir at room
temperature for 12 h. During the reaction, a gummy solid was
formed which stacks to the walls of the pressure tube. CHCl3
was added to the reaction mixture, and the resulting mixture
was transferred into a round-bottom flask. First, NMe3 was
removed by heating with hot water, and then the solvent was
evaporated. Final compound T3 (65%) was purified by repeti-
tive precipitations from a CHCl3/EtOAc/n-hexane mixture. The
compound was found to be an extremely hygroscopic solid.
FTIR (KBr): 1751 cm-1 (CdO str). 1H NMR (300 MHZ,
CDCl3): δ 0.88 (t, terminal -CH3, -3H), 1.26 (br s, (-CH2-
)13, 26H), 3.36 (s, -+N(CH3)3 × 3, 27H), 4.25 (s, -CH2O- × 3,
6H), 5.57 (s, -CH2N+- × 3, 6H). 13C NMR (133.3 MHz,
CDCl3): δ 14.085, 22.67, 22.92, 29.35, 29.53, 29.72, 30.35,
31.92, 32.29, 39.93, 44.82, 54.335, 62.55, 69.12, 165.26. ESI-
MS: m/z M3+ [C33H68N3O6]3+ 2Br- (78.91), calcd 760.3, found
760.3; M3+ Br- (78.91) Br- (80.91), calcd 762.3, found 762.4;
MBC. A 100 µL sample of the overnight culture was added
to 12-15 glass test tubes (autoclaved) with different amounts
of liquid LB medium, and then different amounts of stock
amphiphile solution were added such that the overall volume
of the samples was 2 mL and the same amount of bacteria
was present in every test tube. For control experiments, sterile
water was added to one of the test tubes instead of amphiphile
solution. Then all the tubes were inoculated at 37 °C for 5 h
with constant shaking. After that each tube was diluted
depending upon the requirement, and 100 µL was spread on
an LB agar plate inside the laminar flow. The plates were
incubated at 37 °C for 24 h before the colonies were counted.
The counting was done three times every time. The MBC was
taken as the lowest amphiphile concentration at which there
was no colony present in the plate after 24 h of incubation at
37 °C.
M
3+ 2Br- (80.91), calcd 764.3, found 764.3, (M3+ Br- (78.91))/
2, calcd 340.7; found 340.7; (M3+ Br- (80.91))/2, calcd 341.7,
found 341.6; M3+/3, calcd 200.8, found 200.9. Anal. Calcd for
C33H68N3O6Br3,H2O: C, 46.05; H, 8.2; N, 4.88. Found: C, 45.67;
H, 8.05; N, 4.43.
2,2-Bis(methyl N-ethanoate pyridium bromide)hexa-
decyl N-Ethanoate Pyridinium Bromide (BEPB, P3). 10
Bacterial Killing Assays. Overnight cultures were diluted
with liquid LB medium to give a working concentration of

Synthesis of Hydrolyzable Cationic Amphiphiles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3831
108-1010 cells/mL. The amphiphile stock solution was added
at higher than MBC values, and this suspension was incubated
at room temperature inside the laminar flow. The concentra-
tion of T1 and P1 used was 50 µM, whereas 20 µM was used
for all other amphiphiles. At regular intervals after amphiphile
addition, 10 µL samples were removed every time, and kept
at 4 °C. After the experiments, all the samples were diluted
accordingly, and 100 µL was spread onto LB agar plates. Then
the plates were incubated at 37 °C for 24 h, and the viable
cells were counted. The control experiment was also performed
where amphiphile was not added.
(10) Thompson, R. A.; Allenmark, S. Effects of molecular association
on the rates of hydrolysis of long-chain alkyl betaines (alkoxy-
carbonyl-N,N,N-trialkylmethanaminium halides. Acta Chem.
Scand. 1989, 43, 690-693.
(11) Thompson, R. A.; Allenmark, S. Factors influencing the micellar
catalyzed hydrolysis of long-chain betainates. J. Colloid Interface
Sci. 1992, 148, 241-246.
(12) Allenmark, S.; Lindstedt, M.; Edebo, L. Prophylactic agent for
controlling venereal diseases. Eur. Pat. 0298065A2, 1989.
(13) Haldar, J., V. K. Aswal, P. S. Goyal; S. Bhattacharya. Molecular
modulation of surfactant aggregation in water: effect of the
incorporation of multiple head groups on micellar properties.
Angew. Chem., Int. Ed. 2001, 40, 1228-1232.
(14) Ikeda, T.; Yamaguchi, H.; Tazuke, S. New polymer biocides:
synthesis and antibacterial activities of polycations with pendant
biguanide groups. Antimicrob. Agents Chemother. 1984, 26, 139-
144.
(15) Franklin, T. J., Snow, G. A., Eds. Antiseptics, antibiotics and
the cell membrane. Biochemistry of Antimicrobial Action; Chap-
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Acknowledgment. This work was supported by
grants from the Department of Biotechnology and
Chemical Biology Unit, JNCASR.
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