Antibacterial activities of aminoglycoside antibiotics-derived cationic amphiphiles. Polyol-modified neomycin B-, Kanamycin A-, Amikacin-, and Neamine-based amphiphiles with potent broad spectrum antibacterial activity

Article abstract of DOI:10.1021/jm1000437

Cationic amphiphiles containing multiple positively charged amino functions define a structurally diverse class of antibacterials with broad-spectrum activity and different modes of action. Oligocationic amphiphiles have been used as antibiotics to treat infections and as antiseptics and disinfectants for decades with little or no occurrence of resistance. We have prepared a novel class of cationic amphiphiles termed aminoglycoside antibiotics-derived amphiphiles in which the polyol scaffold of the aminoglycosides neomycin B, kanamycin A, amikacand neamine has been uniformly decorated with hydrophobic residues in the form of polycarbamates and polyethers. Our results show that the nature of the polyol modification as well as the nature of the aminoglycoside antibiotics has a strong effect on the antibacterial potency. The most potent antibacterials are polyol-modified neomycin B-based amphiphiles containing unsubstituted aromatic rings. These analogues exhibit up to 256-fold enhanced antibacterial activity against resistant strains when compared to neomycin B while retaining most of their activity against neomycin B-susceptible strains.

Full text of DOI:10.1021/jm1000437

3626 J. Med. Chem. 2010, 53, 3626–3631
DOI: 10.1021/jm1000437
Antibacterial Activities of Aminoglycoside Antibiotics-Derived Cationic Amphiphiles. Polyol-Modified
Neomycin B-, Kanamycin A-, Amikacin-, and Neamine-Based Amphiphiles with Potent Broad
Spectrum Antibacterial Activity
Smritilekha Bera,† George G. Zhanel,‡ and Frank Schweizer*,†,‡
†Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, R3T 2N2, Canada, and ‡Department of Medical Microbiology,
University of Manitoba, Winnipeg, Manitoba, R3E 3P4, Canada
Received January 13, 2010
Cationic amphiphiles containing multiple positively charged amino functions define a structurally diverse
class of antibacterials with broad-spectrum activity and different modes of action. Oligocationic amphiphiles
have been used as antibiotics to treat infections and as antiseptics and disinfectants for decades with little or
no occurrence of resistance. We have prepared a novel class of cationic amphiphiles termed aminoglycoside
antibiotics-derived amphiphiles in which the polyol scaffold of the aminoglycosides neomycin B, kanamycin
A, amikacin, and neamine has been uniformly decorated with hydrophobic residues in the form of
polycarbamates and polyethers. Our results show that the nature of the polyol modification as well as the
nature of the aminoglycoside antibiotics has a strong effect on the antibacterial potency. The most potent
antibacterials are polyol-modified neomycin B-based amphiphiles containing unsubstituted aromatic rings.
These analogues exhibit up to 256-fold enhanced antibacterial activity against resistant strains when
compared to neomycin B while retaining most of their activity against neomycin B-susceptible strains.
Introduction
defensins, gramicidin S variants, and others) transit the outer
membrane by interacting at sites at which divalent cations
cross-bridge adjacent polyanionic polymers. This causes a
destabilization of the outer membrane that is proposed to lead
to self-promoted uptake of the OCAs and/or other extracellular
molecules.4a,9 After transit through the outer membrane OCAs
contact the anionic surface of the cytoplasmic membrane. Here
depending on the structure of the OCA, several scenarios can be
envisaged. Amphiphilic OCAs can insert themselves into the
cytoplasmic membrane, thereby either disrupting the physical
integrity of the bilayer, via membrane thinning, transient
poration, and/or disruption of the barrier function, or translo-
cating across the membrane and acting on internal targets.4a
This mode of action has been shown to limit the risk of cross-
resistance,4,10 and several amphiphilic OCAs including chlor-
hexidine and polymyxins are in use as antiseptics, disinfectants,
and antibiotics for several decades with little or no occurrence
of resistance.11,30 Nonamphiphilic OCAs such as aminoglyco-
side antibiotics must cross the bacterial membrane in order to
bind to intracellular targets such as RNA, DNA, and proteins.
In this case coadministration with membrane permeabilizing
agents such as ionic lipids can result in synergistic enhance-
ments of the antibacterial action.12,13 It is generally believed
that the selective bacterial cytotoxicity of OCAs is caused by the
affinity of the net negative charge found on bacterial cell
membranes in contrast to eukaryotic lipid bilayers which are
typically made up of zwitterionic phospholipids.4a
The explosive growth of multidrug-resistant bacteria in
hospitals and the community have led to an emerging crisis
where an increasing number of antibiotics cease to be of
microbiological and clinical usefulness.1 As a result, there is a
pressing need for novel classes of antibacterial agents with new
or combined mechanisms of action that are active against
multidrug-resistant bacteria and possess reduced likelihood
for the development of resistance. Oligocationic antibacterials
(OCAsa) containing multiple positively charged amino func-
tions or other cationic groups define a structurally diverse class
of antibacterials with broad-spectrum activity and different
modes of action.2,31 This class of antibacterial agents can be
further subdivided into nonamphiphilic OCAs such as amino-
glycosides3 but also amphiphilic OCAs comprising the natu-
rally occurring cationic antimicrobial peptides,4 synthetic
mimics of antimicrobial peptides (SMAMPs),5 synthetic oligo-
cationic lipopeptides,6 oligocationic lipids,7 and polymers.8
The cationic charges of the OCA ensure accumulation
at polyanionic microbial cell surfaces that contain acidic
polymers, such as lipopolysaccharides, and wall-associated
teichoic acids in Gram-negative and Gram-positive bacteria,
respectively.4a Several OCAs including aminoglycoside anti-
biotics (gentamicin) and antimicrobial peptides (polymyxin B,
*To whom correspondence should be addressed. Phone: þ 204-474-
7012. Fax: þ 204-474-7608. E-mail: schweize@cc.umanitoba.ca.
a Abbreviations: ATCC, American Type Culture Collection; AMPs,
antimicrobial peptides; Boc, tert-butyl carbamate; CAN-ICU, Canadian
Intensive Care Unit; CLSI, Clinical Laboratory Standards Institute;
MIC, minimal inhibitory concentration; MRSA, methicillin resistant
Staphylococcus aureus; MRSE, methicillin-resistant Staphylococcus epi-
dermidis; OCAs, oligocationic antibacterials; SMAMPs, synthetic
mimics of antimicrobial peptides; TFA, trifluoroacetic acid.
Aminoglycoside antibiotics constitute a large family of
clinically important nonamphiphilic OCAs used in the treat-
ment of bacterial infections.3 Aminoglycosides effect their
antibacterial activity by interfering with ribosomal func-
tion (via binding to the A-site region on the 16S subunit of
rRNA), which ultimately results in the disruption of protein
r
pubs.acs.org/jmc
Published on Web 04/07/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3627
Figure 1. Amphiphilic aminoglycoside antibiotics-derived polycarbamates 1-10 and polyethers 11-20.
biosynthesis.14,15 Although aminoglycoside antibiotics exhibit
potent bactericidal activity, their widespread use has been
compromised by the worldwide emergence and spread of
aminoglycoside-resistant strains1 and toxicity.16,17 Several
mechanisms cause resistance including decreased uptake into
cells, as a result of activation of drug efflux pumps, modified
membrane potential, changes in membrane composition,
covalent modification of the drug, and others.18-20 Recently,
we and others have shown that chemical modifications at the
single primary C500-hydroxyl group in neomycin B via attach-
ment of hydrophobic substituents including lipid chains, aro-
matic residues, hydrophobic amino acids, and steroids resulted
in the formation of potent antibacterials.21-23 Analogues of
this type display an amphiphilic surface comprising an oligo-
cationic neomycin B-derived headgroup and a segregated lipid
tail. Inspired by this unexpected result, we became interested in
exploring whether hydrophobic modifications could be ex-
tended to other hydroxyl groups in neomycin B. Because of the
synthetic difficulties associated with the regioselective differ-
entiation of all remaining six secondary hydroxyl groups in
neomycin B, we focused on neomycin B analogues in which all
hydroxyl groups of the polyol are derivatized with hydropho-
bic polycarbamates or polyethers. Compounds of this type are
expected to display polyamphiphilic surfaces when compared
to the monoamphiphilic surface of cationic head-and-tail
amphiphiles such as C500-modified neomycin B-lipid conju-
gates. Subsequently, we were interested in extending our study
to other members of aminoglycoside antibiotics including
kanamycin A, amikacin, and neamine that differ in size,
cationic charge, and structure of the polyol scaffold.
Figure 2. Structures of unprotected and Boc-protected aminogly-
coside antibiotics used for conversion into amphiphilic polycarba-
mates and polyethers.
low antibacterial activity of neomycin B and kanamycin
A toward several multidrug resistant bacteria including
methicillin-resistant Staphylococcus aureus (MRSA), Pseudo-
monas aeruginosa, and Stenotrophomonas maltophilia was an
additional incentive to develop novel analogues with reduced
resistance and increased activity. Amikacin was selected
because of its potent Gram-positive and Gram-negative
activity, while neamine was chosen as a minimal neomycin
B mimetic to explore size effects (Figure 2). The amphiphilic
neomycin B-derived polycarbamates 1-4, the kanamycin
A-derived polycarbamates 5-7, the amikacin-derived poly-
carbamates 8 and 9, and the neamine-derived polycarbamate
10 were prepared from the corresponding amino protected
tert-butylcarbamates(Boc) of the corresponding aminoglyco-
sides 21-2435-38 (Figure 1) via carbamoylation of the hydro-
xy groups with various commercially available hydrophobic
isocyanates including phenyl isocyanate, 4-chlorophenyl
isocyanate, hexyl isocyanate, and 4-N,N-dimethylphenyl
isocyanate in pyridine (Scheme 1).33 Deblocking of the Boc
Results
In this paper we report on the synthesis and antibacterial
activities of neomycin B-, kanamycin A-, amikacin-, and
neamine-derived amphiphilic cationic polycarbamates and
polyethers (Figure 1). Neomycin B and kanamycin A
(Figure 2) were selected because of their commercial avail-
ability in multigram quantities and low price. Moreover, the

3628 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Bera et al.
group using trifluoroacetic acid afforded the cationic and
amphiphilic polycarbamates 1-10 in the form of their TFA
salts. Analogously, the cationic and amphiphilic polyethers
11-20 were prepared form the corresponding Boc-protected
aminoglycosides 21-23 via O-alkylation with reactive alkyl
bromides such as benzyl bromide, 4-chlorobenzyl bromide,
4-tert-butylbenzyl bromide, and methyl iodide using barium
hydroxide in DMF34 (Scheme 1) followed by deblocking of
the amino protecting groups to produce neomycin B-derived
polyethers 11-14, kanamycin A-derived polyethers 15-17,
and amikacin-derived polyethers 18-20 (Figure 1).
All compounds were tested against American Type Culture
Collection (ATCC) reference strains as well as clinical iso-
lates from the Canadian Intensive Care Unit (CAN-ICU)
study.25 Isolates tested included S. aureus ATCC 29213,
methicillin-resistant Staphylococcus aureus (MRSA) ATCC
33592, S. epidermidis ATCC 14990, methicillin-resistant
Staphylococcus epidermidis (MRSE) (cefazolin-CZ MIC >
32 μg/mL) CAN-ICU 61589, Enterococcus faecalis ATCC
29212, E. faecium ATCC 27270, Streptococcus pneumoniae
ATCC 49619, E. coli ATCC 25922, E. coli (gentamicin-
resistant MIC > 32 μg/mL) CAN-ICU 61714, E. coli
(amikacin MIC 32 μg/mL) CAN-ICU 63074, Pseudomonas
aeruginosa ATCC 27853 and P. aeruginosa (Gent-R MIC >
32 μg/mL) CAN-ICU 62308, Stenotrophomonas maltophilia
(CAN-ICU 62585), Acinetobacter baumanni (CAN-ICU
63169), and S. pneumoniae ATCC 13883. Antibacterial acti-
vity against Gram-positive and Gram-negative microorgan-
isms was performed via broth macrodilution tests using
CLSI methodology.24 The minimum inhibitory concentra-
tions (MICs) in μg/mL of the aminoglycoside-derived amphi-
philic polycarbamates and polyethers were determined using
established methods24 and are shown in Tables 1 and 2,
respectively. Our results show that the nature of the cationic
scaffold (neomycin B, kanamycin A, amikacin, and neamine)
and the nature of the polyol substituent (hydrophobic carba-
mate or ether) influence the antibacterial activity and strain
specific susceptibility. The most potent neomycin B-derived
heptaphenyl carbamate 1 exhibits excellent Gram-positive
activity against S. aureus, MRSA, S. epidermidis, and MRSE
(MIC < 1 μg/mL). A remarkable 256-fold enhancement of
1 when compared to unmodified neomycin B against MRSA
is observed, while at the same time the potent activity against
neomycin B-susceptible strains such as S. aureus, S. epidermi-
dis, MRSE, and S. pneumoniae is maintained and also the
molecular weight of 1 is more than doubled. Introduction of
p-chloro and p-dimethylamino substituents into the phenyl
ring of the polycarbamates results in significant loss of Gram-
positive activity as observed for compounds 2-4. Slightly
reduced activities are observed for 1 against Gram-negative
E. coli, while very little improvements are seen against
P. aeruginosa. A very intriguing result is the potent activity
of 1 against multidrug-resistant S. maltophilia (MIC =
4 μg/mL) demonstrating an over 128-fold increased suscepti-
bility over neomycin B and a slightly increased activity against
A. baumannii (MIC = 32 μg/mL).
Replacement of the neomycin B-based hexacationic scaf-
fold by a tetracationic kanamycin A-based scaffold results in
reduced antibacterial activity. Kanamycin A-based amphi-
philic heptacarbamates 5-7 exhibit significantly reduced
Gram-positive activity (up to 32-fold) and little Gram-
negative activity (MIC > 64 μg/mL) for most strains (except
MRSA for compound 8) when compared to neomycin
B-based carbamates 1-4. Moreover, modifications on the
kanamycin A-based tetracationic scaffold influence the anti-
bacterial activity. For instance, amikacin-based tetracationic
octaphenyl carbamate 8 and octahexyl carbamate 9 exhibit
significantly improved Gram-positive activity (2- to 16-fold)
when compared to their respective kanamycin A-based hepta-
carbamates 5 and 6. Amikacin-derived tetracationic octacar-
bamates 8 and 9 are structurally related to kanamycin
A-derived tetracationic heptacarbamates 5 and 6 via amida-
tion of N-3 of kanamycin A with a L-hydroxyaminobuteroyl
substituent. In addition, tetracationic octahexylcarbamate 9
shows 8- to 32-fold improvements against the Gram-negatives
E. coli and S. maltophilia when compared to 6, indicating
that subtle modifications on the kanamycin A-based tetracat-
ionic scaffold can lead to significant improvements in the
antibacterial activity. Moreover, reduction in the size of the
Scheme 1a
a Reagents and conditions: (a) R2NCO, pyridine, DMF; (b) TFA,
0 °C, 3 min; (c) Ba(OH)2, DMF, benzyl bromide or MeI.
Table 1. Minimal Inhibitory Concentrations (MIC) in μg/mL for Various Bacterial Strains against Neomycin B-, Kanamycin A-, Amikacin-, and
Neamine-Derived Oligocationic Polycarbamates 1-11
control
organism
genta- neomy-
cin B
kanamy-
cin A
amika-
cin
micin
1
2
3
4
5
6
7
8
9
10
16
S. aureusa
MRSAb
1
1
1
1
64
64
16
32
2
16
16
8
4
32
32
4
32
64
16
32
16
32
16
32
4
8
4
2
256
0.25
0.5
32
256
1
>512
2
8
16
2
4
16
8
S. epidermidisc
MRSEd
0.25
32
4
0.5
0.5
16
32
16
16
1
4
2
8
32
128
8
8
64
2
2
32
4
8
S. pneumoniaee
E. colif
128
8
128
>256
256
256
8
32
4
>256
32
32
1
4
>256
>256
256
8
128
128
64
8
16
>256 128
>256 64
>256 64
>256 128
>256 32
4
256
256
256
E. colig
E. colih
128
8
8
8
32
2
8
ndn
512
512
>512
64
32
256
>256
256
32
4
4
32
P. aeruginosai
P. aeruginosaj
S. maltophiliak
A. baumanniil
8
256 >256 >256 128
128 256 256 128
>256 256
>256 >256 >256
>512
>512
>512
32
>256 64
128
128
64
128
>512
128
128
>512
128
0.5
256
128
256
256
16
16
4
128
>256 >256 >256
>256 >256 >256
>256 >256 >256
32
>128 256
>128 64
e
S. pneumoniaem 0.25
1
256 256
4
>256
1
aATCC 29213. b Methicillin-resistant S. aureus ATCC 33592. ATCC 1490. d Methicillin-resistant S. epidermidis (ATCC 14990). ATCC 49619.
c
fATCC 25922. gATCC 6174 (gentamicin resistant). hCAN-ICU 63074. ATCC 27853. j CAN-ICU 62308. kCAN-ICU 62584. CAN-ICU 63169.
i
l
mATCC 13883. nnd = not determined.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3629
Table 2. Minimal Inhibitory Concentrations (MIC) in μg/mL for Various Bacterial Strains against Neomycin B-, Kanamycin A-, and Amikacin-
Derived Oligocationic Polyethers 12-21
control
organism
genta-
micin
neomy-
cin B
kanamy-
cin A
amika-
cin
11
12
64
13
14
15
16
32
17
18
19
128
20
S. aureusa
MRSAb
1
1
4
128
128
>256
>256
256
4
8
8
4
8
>256
>256
256
4
4
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
2
256
0.25
0.5
32
4
64
32
>512
2
32
32
8
4
128
128
S. epidermidisc
MRSEd
0.25
32
2
64
64
1
1
2
64
128
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
c
128
8
16
64
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
2
1
64
128
S. pneumoniaee
E. colif
4
16
8
256
128
32
32
8
32
16
16
16
128
64
32
32
16
1
4
128
>128
64
8
16
128
128
4
>256
>256
256
E. colig
E. colih
128
8
8
16
4
128
128
32
16
2
ndn
512
512
>512
64
32
64
32
4
P. aeruginosai
P. aeruginosaj
S. maltophiliak
A. baumanniil
S. pneumoniaem
8
32
64
8
>128
>128
>128
>128
128
256
128
>512
>512
>512
32
>64
>64
>64
>64
>64
>128
>128
>128
>128
>128
>256
>256
>256
>256
>256
e
128
>512
128
0.25
128
>512
128
0.5
>128
>128
>128
16
8
1
1
aATCC 29213. b Methicillin-resistant S. aureus ATCC 33592. ATCC 1490. d Methicillin-resistant S. epidermidis (ATCC 14990). ATCC 49619.
fATCC 25922. gATCC 6174 (gentamicin resistant). h CAN-ICU 63074. ATCC 27853. j CAN-ICU 62308. k CAN-ICU 62584. CAN-ICU 63169.
i
l
mATCC 13883. n nd = not determined.
aminoglycoside antibiotic does not abolish antibacterial acti-
vity. For instance, the neamine-based tetracationic tetraphenyl
carbamate 10 displays improved antibacterial activity against
most Gram-positive strains and all Gram-negative strains
when compared to kanamycin A-based tetracationic hexaphe-
nyl carbamate analogue 5. However, 10 shows significantly
reduced Gram-positive activity when compared to related
hexacationic heptaphenyl carbamate 1 (16-fold reduction in
MIC). This indicates that the neamine portion of neomycin
and the lower portion of neomycin are required for optimal
antibacterial activity. However, the relative potent antibacter-
ial activity of 10 may indicate that the upper portion of
neomycin is more important for antibacterial activity.
A similar trend in the antibacterial activities is observed for
the neomycin B-, kanamycin A-, and amikacin-based oligocat-
ionic polyethers 11-20 (Table 2). A variety of substituents
including methyl, benzyl, p-chlorobenzyl, and p-tertbutyl ethers
were evaluated. The most potent cationic polyether analogues
in the order of activity are the neomycin B-based hexacationic
heptabenzyl ether 11, the amikacin-based tetracationic octa-
benzyl ether 18, and the kanamycin A-based tetracationic
heptaphenyl ether 15. Similar to our previous observation with
cationic polycarbamates, the introduction of a chloro or tert-
butyl substituent into the para-position of the phenyl ring
greatly diminishes (16- to 32-fold increase in MIC) antibacterial
activity. The most potent cationic polyether analogue 11
exhibits broad-spectrum activity against Gram-positive and
most Gram-negative strains tested. Of particular interest are
the potent activities of 11 against the emerging multidrug
resistant superbugs such as MRSE (MIC=2 μg/mL), MRSA
(MIC=4 μg/mL) S. maltophilia (MIC=8 μg/mL), A. bau-
mannii (MIC = 16 μg/mL), and P. aeruginosa (two strains;
MIC = 32-64 μg/mL) strains while retaining good activity
against nonresistant strains such as S. epidermidis (MIC =
2 μg/mL), S. aureus (MIC=4 μg/mL), E. coli ATCC25922
(MIC = 8 μg/mL), and S. pneumoniae (MIC = 16 μg/mL).
Moreover, the requirement for a hydrophobic ether substituent
on the oligocationic aminoglycoside scaffold is due to the fact
that cationic polymethyl ethers 14, 17, and 20 do not exhibit
antibacterial activities below an MIC of 256 μg/mL.
Four different oligocationic aminoglycoside-based polyol
scaffolds (neomycin B, kanamycin A, amikacin, and neamine)
were selected in order to study how chemical modifications on
aminoglycoside-based polyol scaffolds affect the antibacterial
activity against Gram-positive, Gram-negative, and multi-
drug-resistant strains of bacteria. We focused on chemically
easily accessible polyol modifications such as single-step con-
version into hydrophobic polycarbamates and polyethers.
Inspired by the antibacterial activity of other cationic amphi-
philes (short cationic antimicrobial peptides, synthetic mimics
of antimicrobial peptides, cationic lipids and polymers), we
hypothesized that conversion of polar aminoglycoside anti-
biotics into oligocationic amphiphiles provides a general tool
to restore antibacterial activity in this old class of antibiotics.
Previous results obtained by us21 and others22,23 have shown
that C500-modified neomycin B-based polycationic lipids bear-
ing C16- or C20-lipid chains exhibit strong Gram-positive but
reduced Gram-negative activity. The most potent neomycin
B-C16-lipid conjugate in the form of the hexacationic TFA salt
exhibited antibacterial activities against S. aureus (MIC = 8
μg/mL), MRSE(MIC=2μg/mL), E. coli ATCC 25922 (MIC=
32 μg/mL), and P. aeruginosa (MIC= 128 μg/mL).21 However,
exposure of this polycationic lipid to human red blood cells
resulted in significant hemolytic activity (50% hemolysis at
200 μg/mL).23 The high hemolytic activity of this cationic lipid
very likely does not permit systemic use, and potential anti-
bacterial applications are limited to use as disinfectants and
antiseptics and as antibacterial agent in topical applications.
Interestingly, combinational studies using neomycin B-C16-
lipid conjugate in combination with amikacin and vancomycin
demonstrate synergistic effects against certain Gram-positive
and Gram-negative strains indicating a different mode of
action.23 Because of the structural similarity of the neomycin
B-C16-lipid conjugate and the here described cationic poly-
carbamates and polyethers with other oligocationic amphi-
philes, a membranolytic mode of action appears to be likely.
Previous studies on (oligo)cationic surfactants, peptides, pepti-
domimetics, lipopeptides, and lipids have shown that cationic
amphiphiles target the lipid bilayer of bacteria, resulting in
enhanced permeability of the bacterial cell wall,4,6g,8,11 al-
though other mechanisms such as enzyme inactivation,
denaturation of cell proteins, and RNA-binding have been
proposed.26 Evidence for a different antibacterial mode of
action of polyol substituted aminoglycoside antibiotics-derived
Discussion
In this paper we explored the antibacterial activities of 20
aminoglycoside antibiotics-derived cationic amphiphiles.

3630 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Bera et al.
amphiphiles when compared to their parent aminoglycosides is
provided by the inactivity of the cationic polymethyl ethers 14,
17, and 20. The necessity for a hydrophobic polyol substituent
is consistent with the amphiphilic nature of other cationic
antibacterials with proven membranolytic mode of actions
such as cationic antibacterial peptides, lipopeptides, and catio-
nic lipids and surfactants. However, because of the polycationic
nature of aminoglycoside antibiotics-derived amphiphiles,
RNA-mediated interactions are likely especially after enhanced
permeability of the bacterial cell membrane. Synergistic effects
of aminoglycoside antibiotics with membrane permeabilizing
agents are well documented.12,13
Without any doubt a very intriguing finding of our study
is the potent antibacterial activity of neomycin B-based
hexacationic heptaphenyl carbamate 1 against multidrug-
resistant Gram-positive MRSE (MIC = 0.5 μg/mL) and MRSA
(MIC = 1 μg/mL). Incontrast, the tetracationic kanamycin A
(5)-, amikacin (8)-, and neamine (10)-based polyphenyl car-
bamates exhibit up to 64-fold reduced Gram-positive activity,
indicating that the nature of the polyol scaffold, the number of
positive charges, and number of hydrophobic groups influ-
ence the antibacterial Gram-positive activity. Compound 1
(MW = 2132.6) also exhibits good Gram-negative activity
against three strains of E. coli (MIC 16-32 μg/mL) especially
when considering the high molecular weight. Although the
MIC is 2- to 8-fold reduced when compared to neomycin B
sulfate (MW = 908.9) susceptible E. coli, the activity is
comparable to neomycin B when the increase in molecular
weight is taken into account. A rather surprising result is the
observed potent activity of 1 against neomycin B-resistant
Gram-negative S. maltophilia (MIC = 4 μg/mL) which is 32-
to 64-fold lower when compared to amphiphilic di- and
tetracationic lipopeptides.27 In addition, good activity is also
seen against A. baumannii (MIC = 32 μg/mL). A. baumannii
and S. maltophilia are opportunistic nosocomial pathogens
found mostly in intensive care units.28 These microorganisms
are known to be frequently resistant to many commonly used
antimicrobial classes including the β-lactams and fluoroqui-
nolones, and as a result, new chemotherapeutic options are in
high demand.29
Replacement of the polyphenyl carbamate substituent by
polybenzyl ethers also produces highly potent antibacterials.
The most potent hexacationic neomycin B-based heptabenzyl
ether 11 displays slightly decreased Gram-positive activity
(MIC 2-4 μg/mL) against S. aureus, MRSA, S. epidermidis,
and MRSE when compared to its neomycin B-based poly-
carbamate analogue 1. However, enhanced Gram-negative
activity is observed against E. coli, P. aeruginosa and
A. baumannii while a slight 2-fold decrease is observed against
S. maltophilia. A rather unexpected result is the observed
aromatic substitution effect on the antibacterial activity in
both the cationic polycarbamates and polyethers. For in-
stance, we consistently observed that incorporation of a
para-substituent into the aromatic ring of polyol-substituted
phenyl carbamates and benzyl ethers in the neomycin B,
kanamycin A, and amikacin scaffolds results in a 4- to 64-
fold decrease in antibacterial activity. The cause for this
surprising substituent effect is not understood and requires
additional work including time-dependent cell death studies,
studies on bacterial cell wall permeability, combinational
studies with other classes of antibiotics, and extended struc-
ture activity relationships in order to provide a deeper under-
standing of this effect. Nevertheless, the observed substituent
effect is rather unexpected and indicates that other factors
besides the cationic charges and amphiphilicity contribute to
the observed antibacterial activity in this class of compounds.
On the basis of the potent antibacterial activity of neomycin
B-based amphiphiles 1 and 11 against neomycin B-susceptible
and resistant strains relative to other aminoglycoside-based
and polyol-modified amphiphiles such as kanamycin A,
amikacin, and neamine, it appears that the neomycin B-based
scaffold exhibits superior antibacterial properties.
Conclusion
In the present study, we have established that polyol-
substituted aminoglycoside antibiotics-derived cationic am-
phiphiles form a novel class of antibacterial agents with broad
spectrum Gram-positive and Gram-negative activity. The
amphiphilic nature of the oligocationic aminoglycoside anti-
biotics-derived amphiphile is crucial for their antibacterial
activity, while O-methylation of the polyol scaffold abolishes
antibacterial activity. Neomycin B and amikacin-based
polyol-modified cationic amphiphiles display consistently
higher antibacterial activities when compared to kanamycin
A, indicating that the nature of the aminoglycoside antibiotics-
derived polyol influences the antibacterial potency. The
most potent compounds 1, 9, and 11 exhibit significantly
improved antibacterial activity against resistant strains when
compared to their parent compounds neomycin B and
amikacin while retaining their antibacterial activity against
most neomycin B- and amikacin-susceptible bacterial strains.
Unsubstituted phenyl rings in the form of polyphenyl carba-
mates or polyphenyl ether consistently exhibit higher anti-
bacterial activities when compared to their para-substituted
analogues. The observed aromatic substitution effects suggest
that the antibacterial mode of action of polyol-substituted
aminoglycoside antibiotics-derived amphiphiles involves
other factors besides polycationic charges and amphiphilicity.
Other potential modes of action may involve selective binding
to lipid A and other lipopolysaccharides,32 as well as wall-
associated teichoic acids, and RNA-mediated interactions.3
Acknowledgment. The authors thank the Natural Sciences
and Engineering Research Council of Canada (NSERC), the
Canadian Foundation for Innovation (CFI), the Canadian
Institutes for Health Research (CIHR), the Manitoba Health
Research Council (MHRC), and the University of Manitoba,
Canada, for financial support.
Supporting Information Available: Synthetic procedures,
spectral and analytical data for new compounds, and antibac-
terial testing protocols. This material is available free of charge
via the Internet at http://pubs.acs.org.
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