Broad-spectrum antibacterial amphiphilic aminoglycosides: A new focus on the structure of the lipophilic groups extends the series of active dialkyl neamines

Article abstract of DOI:10.1016/j.ejmech.2018.08.022

Amphiphilic aminoglycosides (AAGs) constitute a new class of antibacterial compounds targeting the bacterial membranes. We have identified the 3′,6-dinonyl neamine 9 as a broad spectrum antibacterial AAG. Here, we report on the synthesis, antibacterial activity and eukaryotic cytotoxicity of new 3′,6-dialkyl neamines designed in order to finely delineate the structure-activity relationships relating their activity to a lipophilicity window. New broad-spectrum antibacterial derivatives were obtained carrying two identical linear or branched alkyl groups or two different linear alkyl groups. Two fluorescent antibacterial 3′,6-heterodialkyl neamines carrying a pyrenylbutyl fluorophore were also identified as potential tools for mechanistic study. Homodialkyl and heterodialkyl neamines appeared to be more active on Gram-negative bacteria than dinaphthylalkyl neamines. However, branched dialkyl neamines or heterodialkyl derivatives were found to be more cytotoxic on mammalian cells than 9. The exposure of P. aeruginosa over one month to half-MIC of one of the most active derivatives 9 demonstrated the high difficulty of resistance emergence to AAGs.

Full text of DOI:10.1016/j.ejmech.2018.08.022

Accepted Manuscript
Broad-spectrum antibacterial amphiphilic aminoglycosides: A new focus on the
structure of the lipophilic groups extends the series of active dialkyl neamines
Louis Zimmermann, Julie Kempf, Florian Briée, Jitendriya Swain, Marie-Paule
Mingeot-Leclercq, Jean-Luc Décout
PII:
S0223-5234(18)30680-9
10.1016/j.ejmech.2018.08.022
EJMECH 10633
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 May 2018
Revised Date: 4 August 2018
Accepted Date: 7 August 2018
Please cite this article as: L. Zimmermann, J. Kempf, F. Briée, J. Swain, M.-P. Mingeot-Leclercq, J.-L.
Décout, Broad-spectrum antibacterial amphiphilic aminoglycosides: A new focus on the structure of the
lipophilic groups extends the series of active dialkyl neamines, European Journal of Medicinal Chemistry
(2018), doi: 10.1016/j.ejmech.2018.08.022.
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ACCEPTED MANUSCRIPT
Broad-Spectrum Antibacterial Amphiphilic Aminoglycosides: A New Focus on
the Structure of the Lipophilic Groups Extends the Series of Active Dialkyl
Neamines
Louis Zimmermann,† Julie Kempf,† Florian Briée,† Jitendriya Swain,§ Marie-Paule Mingeot-
Leclercq,*,§ and Jean-Luc Décout*,†
†Univ. Grenoble Alpes, CNRS, Département de Pharmacochimie Moléculaire, 38058
Grenoble, France.
§Unité de Pharmacologie Cellulaire et Moléculaire, Louvain Drug Research Institute,
Université catholique de Louvain, Avenue E. Mounier 73, B1.73.05, B-1200 Brussels,
Belgium
Highlights: Antibacterial amphiphilic aminoglycosides targeting membranes less susceptible
to resistance
Keywords: amphiphilic aminoglycosides, neamine, antibacterial, structure-activity
relationships, resistance, P. aeruginosa
ABSTRACT
Amphiphilic aminoglycosides (AAGs) constitute a new class of antibacterial compounds
targeting the bacterial membranes. We have identified the 3´,6-dinonyl neamine 9 as a broad
spectrum antibacterial AAG. Here, we report on the synthesis, antibacterial activity and
eukaryotic cytotoxicity of new 3´,6-dialkyl neamines designed in order to finely delineate the
structure-activity relationships relating their activity to a lipophilicity window. New broad-
spectrum antibacterial derivatives were obtained carrying two identical linear or branched
alkyl groups or two different linear alkyl groups. Two fluorescent antibacterial 3´,6-
heteroalkyl neamines carrying a pyrenylbutyl fluorophore were also identified as potential
tools for mechanistic study. Homodialkyl and heterodialkyl neamines appeared to be more
active on Gram-negative bacteria than dinaphthylalkyl neamines. However, branched dialkyl
neamines or heterodialkyl derivatives were found to be more cytotoxic on mammalian cells
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than 9. The exposure of P. aeruginosa over one month to half-MIC of one of the most active
derivatives 9 demonstrated the high difficulty of resistance emergence to AAGs.
Graphical Abstract
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INTRODUCTION
Antibacterial amphiphilic aminoglycosides (antibacterial AAGs) constitute a new class of
antibiotic drugs candidates obtained by chemical modification of the polycationic antibiotic
drugs aminoglycosides (AGs) such as neomycin B 1 (Figure 1), kanamycins,
tobramycin…that target the A site of bacterial 16S rRNA to cause protein synthesis
alteration.1,6 AGs are highly potent broad-spectrum antibiotics and decades of widespread
clinical use strongly reduced their clinical efficacy through the selection of resistant
bacteria.7,8 Originally, AAGs have been developed in the search for new AGs which are less
susceptible to resistance. In this approach, Hanessian, Westhof, and coworkers have reported
the first example of in vivo antibacterial lipophilic ether-modified AG paromomycin targeting
rRNA with a novel mode of binding.9,10
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Figure 1. Structure of the natural antibiotic aminoglycoside neomycin B 1, of neamine 2 and
of some amphiphilic neamines previously prepared 3-1218-20 and of new neamine derivatives
13-25 described here.
More lipophilic AAGs resulting from the introduction of one to four lipophilic groups on AG
cores were shown to target bacterial membranes.11-24 The corresponding strong increase in the
AG lipophilicity results in a bacterial target shift from rRNA to membranes and significantly
improves activity against bacterial strains resistant to the parent AG drugs and to antibiotic
drugs of other classes. AAGs are active against resistant bacteria expressing different modes
of resistance e.g. (i) reduction in the intracellular concentration of the antibiotics by efflux
pump proteins, (ii) deactivation by AG-modifying enzymes, AG nucleotidyltransferases
(ANTs), AG phosphotransferases (APHs), and AG acetyltransferases (AACs), and (iii)
structural modifications of the 16S ribosomal RNA binding site that lead to reduced target
affinity.7,8
AAGs have appeared to be active mainly against susceptible and resistant Gram-positive
bacteria to most antibiotic drugs or, more rarely, against susceptible and resistant Gram-
positive and Gram-negative bacteria. The most active AAGs against Gram-negative bacteria
are polyalkyl derivatives of neamine 2 (Figure 1),18-20,22 neosamine20 and nebramine.16
Antibacterial AAGs can also present immunomodulatory effects like tobramycin
derivatives.25 Furthermore, efficient antifungal AAGs exhibiting or lacking antibacterial
activity have been also identified.26-29
Interestingly, more recently, amphiphilic AG conjugates showed other antibacterial and
antiviral properties. Amphiphilic tobramycin conjugates to lysine appeared to be able to
sensitize multidrug resistant Gram-negative bacteria to antibiotic drugs,30,31 and conjugates to
efflux pump inhibitors32 or to a fluoroquinolone33 were shown to overcome resistance in P.
aeruginosa by enhancing bacterial outer membrane penetration and reducing efflux.
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Amphiphilic neamine conjugates bearing a metal binding motif active against MDR E.
aerogenes Gram-negative bacteria34 have also been obtained using the chemistry previously
developed in the search for antiviral AGs and AAGs.35-36 AAGs also display in vitro antiviral
activity like neamine- and neosamine-peptide nucleic acid (PNA) conjugates, the neamine and
neosamine cores allowing cell penetration of PNA targeting viral RNA.37-39
In the synthesis of AAGs, several strategies have been developed including partial conversion
of (i) the AG amine groups into alkyl- or aryl-amide(s), reducing the number of positive
charges carried at physiological pH, or (ii) the hydroxyl groups in carbamate, ether and
thioether groups, respectively.1,2 We have previously synthesized different mono-, di- and tri-
O-alkyl derivatives of the small aminoglycoside neamine 2 prepared from the natural
antibiotic drug neomycin B 1 and identified three series of antibacterial agents acting on
susceptible and resistant Gram-positive and Gram-negative bacteria to AGs and other
antibiotic drugs, the series of 3´,4´,6-trialkyl derivatives and the 3´,4´- and 3´,6-dialkyl
derivative series (Figure 1).18-20
The 3´,6-dialkyl derivatives appeared to be the most promising in regard to their broad-
spectrum antibacterial activity and their lowest eukaryotic cytotoxicity.19 In these series, we
have identified three broad-spectrum antibacterial neamines, two 2-naphthylalkyl derivatives,
the 3´,6-di-(2-naphthyl)propyl (3´,6-di2NP, 4), and 3´,6-(2-naphthyl)butyl (3´,6-di2NB, 5)
neamines and, only, one alkyl derivative, the 3´,6-dinonyl neamine 9 (3´,6-diNn), the latter
appearing to be the most active derivative against Gram-negative bacteria (Figure 1).19 The
3´,6-dinaphthylmethyl (3), -dinaphthylhexyl (6), -dibutyl (7), -dihexyl (8) and -dioctadecyl
(10) neamines have been found to be inactive or weakly active against Gram-negative
bacteria. 3´,6-DiNn neamine 9 has appeared to inhibit P. aeruginosa biofilm growth and also
shown antibacterial activity on P. aeruginosa strains resistant to the polycationic lipopeptide
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colistin targeting lipopolysaccharides (LPS),22 colistin being one of the drug of last resort to
combat multidrug-resistant (MDR) P. aeruginosa infections.40
A window of lipophilicity has been delineated to fit to the optimal antibacterial activities of
both 3´,6-dialkyl and 3´,6-di-2-naphthylalkyl neamines.19 Such a window has been also
observed in the 3´,4´,6-trialkyl series.19 In regard to the unique antibacterial properties of 3´,6-
diNn neamine 9, here, we report on the synthesis, antibacterial activity and eukaryotic
cytotoxicity of new 3´,6-dialkyl neamines carrying linear alkyl chains, the 3´,6-diheptyl
(diHp, 13), -dioctyl (diOc, 14), -didecyl (diDe, 15) and -diundecyl (diUd, 16) (Figure 1). The
studied dialkyl neamines were designed in order to finely tune the previously delineated SAR
relating their activity expressed as 1/MIC values to clogP values that corresponds to a
previously delineated activity window against MRSA and susceptible P. aeruginosa located
between clogP= -12.7 and -9.0.19 The present study allows to split this window into two
windows differentiating the alkyl derivatives and the previously synthesized naphthylalkyl
derivatives and to identify new broad-spectrum antibacterial 3´,6-dialkylneamine, the dioctyl
(14) and didecyl (15) derivatives. We also demonstrate here that dialkyl neamines carrying
branched alkyl chains can have antibacterial activity through the synthesis and study of the
3´,6-dialkyl neamine derivative and its 3´,4´-isomer carrying two branched dimethyloctyl
(DiMOc) chains, respectively 17 and 18 (Figure 1).
In order to investigate the importance of the respective length and position of both linear alkyl
groups grafted on the neamine core at positions 3´ and 6, we also report on the synthesis and
antibacterial properties of two 3´,6-dialkyl neamines carrying two linear alkyl chains of
different lengths, the 3´-heptyl-6-undecyl (3´-Hp-6-Ud, 19) and 3´-undecyl-6-heptyl (3´-Ud-
6-Hp, 20) which are isomers having lipophilicities close to the lipophilicity of the
antibacterial 3´,6-diNn neamine 9 (Figure 1). Both isomers were found to be broad-spectrum
antibacterial agents but more cytotoxic on eukaryotic cells than 9. From the delineated SAR,
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two fluorescent antibacterial 3´,6-dialkyl neamine isomers 21 and 22, carrying a heptyl and a
pyrenylbutyl groups were identified, their activity making them useful for further mechanistic
study. The antibacterial properties of these 3´,6-heterodialkyl neamines were compared to
those of the corresponding 3´- or 6-monoalkyl derivatives 23-26.
Antibacterial AAGs such as dialkyl neamines may use differences in the membrane
composition to interact selectively with bacterial cells.3,41,42 Our previous works have
characterized the mechanism of action against P. aeruginosa.18,20-24 By using membrane
model systems, we demonstrated the interaction of 3´,6-diNn neamine 9 with outer membrane
lipopolysaccharides22 and inner membrane anionic phospholipids mostly cardiolipin leading
to membrane permeabilization and depolarization.23 By targeting cardiolipin-bacterial
microdomains mainly located at the cell poles, 3′,6-diNn neamine leads to disassembly these
microdomains into cardiolipin clusters and relocation of cardiolipin domains as confirmed by
using membrane model systems mimicking inner membrane of P. aeruginosa.24 Importantly,
targeting cardiolipin-lipid domains results in bacterial morphological changes. These changes
are characterized by a severe length decrease probably as a result from the decrease of the
inner membrane fluidity/hydration induced by 9 which could impair the dynamics of cell-
shape determining proteins like MreB.24 Targeting cardiolipin could also explain the observed
inhibition of the redox chain accompanied with an intracellular ATP concentration and pH
decrease, and inhibition of cell growth induced by the amphiphilic neamine derivative.24
Thus, several key membrane components present in multiple copies in the bacterial
membranes are the targets of antibacterial amphiphilic neamines. Biochemical modifications
of such multiple targets should have a high cost for the bacteria resulting in a slow
development of resistances, in a rapid reversibility of the emerging resistances and in a high
sensitivity to conventional antibiotic drugs of other classes.43
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In order to demonstrate the expected difficulty of resistance emergence to AAGs, we report
here on the Minimum Inhibitory Concentration (MIC) changes against susceptible P.
aeruginosa upon exposure over one month to half-MIC of one of the most active derivative,
3´,6-diNn neamine 9, in comparison to the antibiotic drug ciprofloxacin.
SYNTHESIS
The chemistry was developed from tetra-N-trityl neamine 2735 (Scheme 1) that was mono-
and di-alkylated under phase transfer conditions for obtaining concomitantly tetratritylated
3´,6-dialkyl neamines and tetratritylated 3´- and 6-monoalkyl neamines intermediates.
Tetra-N-trityl neamine 27 was alkylated at 50 °C with the corresponding 1-bromoalkane,
concentrated aqueous sodium hydroxide (30%), toluene and TBAI as a phase transfer agent.
In the presence of TBAI, no tri- and tetra-alkylations were observed whereas using at room
temperature TBAF as a phase transfer agent the 3´,4´,6-alkylated derivatives are formed.44
After purification, the tritylated 3´,6-alkylated neamines were deprotected in the presence of
TFA and anisole to afford the potential antibacterial amphiphilic neamines.
Scheme 1: Synthesis of the studied 3´,6-dialkyl neamines and of their related 3´-mono- and 6-
mono-alkyl compoundsa
+
NHTr
NHTr
-
NH3
4 CF3CO2
O
O
O
HO
R2O
HO
R2O
HO
HO
+
NHTr
NH3
NHTr
HO
a, RX
b
H3N+
TrHN
TrHN
+
O
O
O
NHTr
NH3
NHTr
HO
HO
OR1
OR1
OH
28: R1= R2= Hp
29: R1= R2= Oc
30: R1= R2= De
31: R1= R2= Ud
27
13: R1= R2= Hp
14: R1= R2= Oc
15: R1= R2= De
16: R1= R2= Ud
32: R1= R2= diMOc
33: R1= H, R2= Hp
34: R1= Hp, R2= H
35: R1= H, R2= Ud
36: R1= Ud, R2= H
17: R1= R2= diMOc
23: R1= H, R2= Hp
24: R1= Hp, R2= H
25: R1= H, R2= Ud
26: R1= Ud, R2= H
aReagents and conditions: (a) 1-bromoalkane (RX), toluene, TBAI, aqueous NaOH (30%),
50°C, 24 h; (b) TFA/CH2Cl2, anisole, rt, 2 h.
8

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The previously determined SAR window frame relating the antibacterial activity (1/MIC) and
the calculated lipophilicity (clogP, Table 1)45 of 3´,6-dialkyl neamine derivatives suggests that
the clogP of antibacterial amphiphilic neamines should have a lipophilicity value higher than
3´,6-dihexyl (3´,6-diHx) neamine 8 (clogP= -14.2) and lower than 3´,6-di-2-naphthylhexyl
(3´,6-di2NH) neamine 6 (clogP = -9.0) that are inactive against most of the selected
bacteria.19 In these studies, 3´,6-di-2-naphthylmethyl 3 (3´,6-di2NM) neamine (clogP= -12.7)
showed unique properties being mainly active against susceptible and resistant Gram-positive
bacteria. The clogP values of the tetraprotonated amphiphilic neamines calculated using the
MarvinSketch software 5.11.4 have been used to define the window frame.45 In this study, in
order to define more accurately the window frame of SAR and to identify new strongly
antibacterial amphiphiphilic neamines, 3´,6-diHp, (13), -diOc (14), -diDe (15), -diUd, (16)
neamines carrying the same linear alkyl chains and the 3´,6-(3,7-dimethyl)octyl (diDiMOc,
17) neamine derivative carrying the same branched alkyl chains were prepared (Scheme 1,
Table 1). During this synthesis, the formed tetratritylated 3´- and 6-monoheptyl (33 and 34)
and 3´- and 6-monoundecyl neamines (35 and 36) were isolated for a potential second
alkylation and/or for deprotection (Scheme 1).
After deprotection, 3´- and 6-monoheptyl (23 and 24, respectively) and 3´- and 6-
monoundecyl neamines (25 and 26, respectively) were isolated and evaluated for comparison
of their potential antibacterial activity to those of their parent 3´,6-dialkyl derivatives.
Scheme 2. Synthetic pathway used for the preparation of 3´,6-dialkyl neamines derivatives
bearing two different alkyl chainsa
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-
+
4 CF3CO2
NHTr
O
NHTr
NH3
O
O
HO
R2O
HO
R2O
HO
R2O
+
NHTr
NHTr
NH3
b
a, RX
H3N+
TrHN
TrHN
O
+
O
O
NHTr
NHTr
NH3
HO
HO
HO
OR1
OR1
37: R1= Ud, R2= Hp
38: R1= Hp, R2= Ud
OR1
19: R1= Ud, R2= Hp
20: R1= Hp, R2= Ud
33: R1= H, R2= Hp
34: R1= Hp, R2= H
35: R1= H, R2= Ud
39: R1= 1PyBu, R2= Hp
40: R1= Hp, R2= 1PyBu
21: R1= 1PyBu, R2= Hp
22: R1= Hp, R2= 1PyBu
OH
OH
OMs
MsCl
O
LiAlH4
41
42
aReagents and conditions: (a) RX= 1-bromoalkane or 4-(1-pyrenyl)butyl mesylate 42
(synthesized from the corresponding acid via the alcohol 41), toluene, TBAI, aqueous NaOH
(30%), 50 °C, 24 h (b) TFA/CH2Cl2, anisole, rt, 2 h.
The second alkylation of the isolated tetratrityl 3´-monoHp (33) and 3´-monoUd (35)
derivatives (Scheme 2) was performed under the same conditions in order to introduce a
second undecyl and a heptyl group, respectively, and to obtain two isomeric 3´,6-dialkyl
neamines having same clogP= -11.9 than the antibacterial 3´,6-diNn neamine 9 previously
identified. After deprotection, the isomeric 3´-heptyl-6-undecyl (19) and 3´-undecyl-6-heptyl
(20) neamines were isolated for antibacterial evaluation and comparison with 9.
We also synthesized fluorescent amphiphilic neamines having clogP= -10.7, value
corresponding to a potential antibacterial activity (Scheme 2). The pyrenyl ring was chosen as
fluorophore and the lipophilic 4-(1-pyrenyl)butyl group (1PyBu) was selected to be
introduced at the 3´- or the 6-position of the neamine core after introduction of one heptyl
group at the 6- or 3´-position in order to maintain a lipophilicity close to the one of 3´,6-
dinonyl neamine 9 This procedure could allow the obtention of fluorescent antibacterial
amphiphilic neamines useful for mechanistic studies. 4-(1-pyrenyl)butyl methanesulfonate 42
was prepared in two steps from commercially available 4-(1-pyrenyl)butanoic acid: (i)
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reduction with LiAlH4 to lead to the corresponding alcohol (41) and (ii) mesylation of the
alcohol with methanesulfonyl chloride (Scheme 2).
The isolated tetratrityl 3´-monoheptyl (33) and 6-monoheptyl (34) neamines were alkylated
with pyrenylbutyl sulfonate 42 under phase transfer conditions in the presence of TBAI. The
tetratritylated 3´,6-dialkyl neamines formed were purified and deprotected to lead to 3´-
heptyl-6-(4-(1-pyrenyl)butyl) (21) and 3´-(4-(1-pyrenyl)butyl)-6-heptyl (22) isomeric
neamines.
The 3´,4´-di-2-naphthylpropyl (2NP) (11) and 3´,4´-dinonyl (Nn) (12) neamine derivatives
have shown interesting antibacterial activity against susceptible and resistant bacteria with
MIC mostly slightly higher than those of their 3´,6-isomers 4 and 9, respectively. Thus, we
synthesized the 3´,4´-isomer 18 of compound 17 (3´,6-diDiMOc neamine) from the N-
tetratrityl neamine derivative 4344 in which the 6-hydroxyl group is selectively protected by
the p–methoxybenzyl group (Scheme 3). It was obtained in 17% yield in two steps: (i)
alkylation of 43 in DMF with NaH and 1-bromo-3,7-dimethyloctane and (ii) deprotection
with TFA/anisole.
Scheme 3. Synthesis of 3´,4´-di-O,O´-(3,7-dimethyl)octyl neamine 18 (3´,4´-diDiMOc) from
the N-tetratritylated p-methoxybenzyl neamine derivative 4344 a
NHTr
O
NH3+
O
-
4 CF3CO2
NH3+
HO
HO
RO
RO
NHTr
HO
a, b
TrHN
H N+
3
O
O
NHTr
OPMB
NH3+
HO
OH
43
18
O
OR = ODiMOc =
aReagents and conditions: (a) NaH, DMF, 1-bromo-3,7-dimethyloctane, rt, 31%. (b)
TFA/CH2Cl2, anisole, rt, 55%.
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Table 1. clogP values calculated using the MarvinSketch software 5.11.4 for the
tetraprotonated amphiphilic neamine derivatives used in this study and for some antibacterial
agents. 45
Reference antibacterials
Neomycin B 1
Dialkyl neamines
Dihexyl (diHx) 819
-29.9
-29.7
-19.4
-14.2
-13.5
-12.7
-11.9
Colistin
Diheptyl (diHp) 13
Dioctyl (diOc) 14
Neamine 2
Monoalkyl neamines
Dinonyl (diNn) 919 and 12,20 heptyl-undecyl
19 and 20
Heptyl (MonoHp) 23 and 24
Undecyl (MonoUd) 25 and 26
-16.4
-14.8
Di(3,7-dimethyl)octyl (diDiMOc) 17 and 18
Didecyl (diDe) 15
-11.3
-11.1
-10.3
Diundecyl (diUd) 16
Di-naphthylalkyl neamines
Di(2-naphthyl)methyl (di2NM) 334
Di(2-naphthyl)propyl (di2NP) 436
and 1135
-12.7 Di(2-naphthyl)butyl (di2NB) 535
-11.4 Di(2-naphthyl)hexyl (di2NH) 635
-10.6
-9.0
(1-Pyrenyl)butyl-heptyl (1PyBu-Hp) 21 and 22
-10.7
ANTIBACTERIAL ACTIVITY, RESULTS AND DISCUSSION
The synthesized amphiphilic neamines were evaluated against a large panel of susceptible and
resistant Gram-positive and Gram-negative bacteria. In the former class of bacteria, the
minimum inhibitory concentrations (MIC) were measured against susceptible S. aureus
ATCC 25923 and two resistant strains, the SA-1 strain overexpressing resistance pump
(NorA) and the Methicillin-resistant strains ATCC 33592 HA-MRSA (Table 2).46 In the
Gram-negative class of bacteria, the effects were evaluated against susceptible and resistant
strains of P. aeruginosa (Table 2), A. lwoffii and E. coli overexpressing aminoglycoside-
modifying enzymes or efflux pumps and against the susceptible ATCC 700603 K. pneumonia
strain (Table 3).
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Table 2. Minimum Inhibitory Concentrations (MIC) of the neamine derivatives synthesized
and some representative AG against susceptible and resistant Staphylococcus aureus and
Pseudomonas aeruginosa strains.
MIC µg/mL
S. aureus
P. aeruginosa
AGs
ATCC
33592
SA-1
Pump
NorA
ATCC
25923
ATCC
27853
Psa.
PA22b
PA406c
1
FO3a
HA-MRSA
8-16
1
Amikacin
4
2
0.5
0.5
0.5-1
8
2-4
1
8
8
4
Gentamicin
Tobramycin
Neomycin B 1
Neamine 2
0.25-0.5
0.25-0.5
1-2
>128
128
< 0.25
64
< 0.25
64
0.5
< 0.25
2-4
>128
>128
128
32-64
>128
16-32
>128
>128
64
Dialkyl neamines incorporating same alkyl groups
3´,6-di2NP 419
3´,6-di2NB 519
3´,6-diHp 13
2
2
8
1
2
2-4
8
2
2
8-16
4
16
8
16
8
2-4
8
8
32
2
8
16
8
4
1
2
3´,6-diOc 14
2
8
3´,6-diNn 919
3´,4´-diNn 1220
3´,6-diDe 15
1
4
1
8
1
4
1
4
1
8
1
4
2-4
4
2-4
8-16
4
4-8
8-16
4
4
8-16
4
2-4
8-16
8
1-2
64
1
3´,6-diUd 16
16
4
16
8
16
4-8
4
16
3´,6-diDiMOc 17
3´,4´-diDiMOc 18
2-4
4
4
4
4
Dialkyl neamines incorporating two different alkyl groups
3´-Hp-6-Ud 19
1
1
2
1
1
32
2
1
2
2
4
8
4
4
4
4
4
4
2
2
4
8
3´-Ud-6-Hp 20
1
3´-Hp-6-1PyBu 21
3´-1PyBu-6-Hp 22
1
16
16
4
2
Monoalkyl neamines
3´-monoHp 23
6-monoHp 24
3´-monoUd 25
6-monoUd 26
>128
>128
16
>128
>128
16
>128
>128
16
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
4-8
32
8
8
16
a: Psa.F03 AAC6´-IIA; b: surexp MexXY; c: PAO509.5 ∆triABC.
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Table 3. Minimum Inhibitory Concentrations (MIC) of the neamine derivatives synthesized
and some representative AG against selected bacterial susceptible and resistant Acinetobacter
lwoffii, Escherichia coli and Klebsiella pneumonia strains.
MIC µg/mL
K.
A. lwoffii
E. coli
AGs
pneumonia
ATCC
AI.88-483a
ATCC
25922
PAZ505H81
01b
ATCC
700603
L8058.1c
17925
< 0.25
< 0.25
< 0.25
< 0.25
0.5
Amikacin
128
2
4
0.5
1
64
1
8
1
Gentamicin
Tobramycin
Neomycin B 1
Neamine 2
64
64
1
4-8
4-8
4-8
32
0.5-1
64-128
>128
32
2
4
32
>128
32
Dialkyl neamines incorporating same alkyl groups
3´,6-di2NP 419
3´,6-di2NB 519
3´,6-diHp 13
4
2
32-64
>128
>128
128
8
8
4-8
4
16
8
32
32
64
8
32
2-4
2
32
4
16
32
4
3´,6-diOc 14
2-4
2-4
3´,6-diNn 919
3´,4´-diNn 1220
3´,6-diDe 15
2-4
4-8
2
2-4
8
16
4
4-8
2
32
64
32
4-8
8
4-8
8
16-32
16
32
2
128
16
2-4
4
3´,6-diUd 16
32
1-2
4
4
16
1-2
4
3´,6-diDiMOc 17
3´,4´-diDiMOc 18
2-4
8
8
Dialkyl neamines incorporating two different alkyl groups
2
>128
8
3´-Hp-6-Ud 19
16
2
4
2
4
4
4
4
3´-Ud-6-Hp 20
2
4
8
>128
>64
4
8
4
3´-Hp-6-1PyBu 21
3´-1PyBu-6-Hp 22
4
16
8
>128
4
Monoalkyl neamines
3´-monoHp 23
6-monoHp 24
3´-monoUd 25
6-monoUd 26
64
64
32
64
>128
>128
>128
64
>128
>128
32
>128
>128
32
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
a: APH3´-VIA; b: AAC6´-IB; c: ANT2´´-IA.
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Antibacterial activity of the 3´,6-homodialkyl neamines diHp 13 to diUd 16 made of
linear alkyl chains
In order to finely tune the antibacterial activity-lipophilicity relationship, we evaluated the
synthesized 3´,6-dialkyl neamines carrying two linear alkyl chains heptyl (7 carbon atoms in
both alkyl chains, Hp/C7, 13), octyl (Oc/C8, 14), decyl (De/C10, 15) and undecyl (Ud/C11,
16). The clogP of these derivatives (Table 1) have been selected to match with the -12.7 to -
9.0 clogP window corresponding to derivatives having broad-spectrum activity. This window
has been delineated from Figure 2A on HA-MRSA and Figure 3A on susceptible P.
aeruginosa ATCC 27853, Figures in which the values of (1/MIC) are plotted versus clogP
values.19
Against S. aureus strains, the diOc (14), diNn (9) and diDe (15) neamines showed MIC values
from 1 to 2-4 µg/mL (Table 2) with clogP values ranging from -12.7 to -11.1 (Table 1). The
antibacterial activity decreases for the diHp (13) and diUd (16) neamines with MIC against
MRSA of 8 and 64 µg/mL, respectively. The clog P values of the diHp and diUd neamines
are -13.5 and -10.3, respectively, and correspond to the limits of the clogP windows (Figure 2
A).19
Against P. aeruginosa strains (Table 2), the diOc (14), diNn (9) and diDe (15) neamines also
showed the lower MICs (from 2 to 8 µg/mL). The antibacterial effect decreased for the diHp
and diUd derivatives (MICs from 4 to 32 µg/mL) with a better effect of the diHp derivative
against PA406. The diNn derivative 9 appeared to be the most active derivative in the series.
Against susceptible A. lwoffii and K. pneumonia (Table 3), the most active derivatives are
again the diOc (14), diNn (9) and diDe (15) neamines with MIC= 2-4 µg/mL and 4-16
µg/mL, respectively to the strains. Once again, the diNn derivative appeared to be the most
active derivative in the series especially against resistant A. lwoffii overexpressing
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aminoglycoside-modifying enzymes (MIC= 16 µg/mL), the diOc (14) and diDe (15)
derivatives showing high MIC values (128 and 64 µg/mL, respectively).
Against the susceptible E. coli strain (Table 3), the four diOc (14), diNn (9), diDe (15) and
diUd (16) neamines showed similar good antibacterial activity (MIC= 2 to 4 µg/mL). The
most active compounds are the diNn (9) and diDe (15) with close MIC from 2 to 4 µg/mL
and, against resistant E. coli strains, the activity decreased mainly for the diDe (15) and diUd
(16) derivatives (MIC from 8 to 128 µg/mL) whereas the activity of the diOc (14) and diNn
(9) remained unchanged (MIC= 2-4 µg/mL).
The difference in the antibacterial activity of the 3´,6-dialkyl neamines appears in the graphs
of (1/MIC) versus clogP plotted for HA-MRSA (Figure 2) and susceptible P. aeruginosa
ATCC 27853 (Figure 3).
In Figure 2B, the previously obtained graph plotted for MRSA (Figure 2A)19 is decomposed
into two graphs, one for the neamine derivatives carrying linear alkyl chains 7 (diBu), 8
(diHx), 9 (diNn) and 10 (diOcD) and the other for derivatives carrying 2-naphthylalkyl groups
3 (di2NM), 4 (di2NP), 5 (di2NB) and 6 (di2NH). Figure 2B shows clearly the previous lack
of data for linear alkyl chains. In Figure 2C, the graph incorporating the data obtained with
the new synthesized derivatives carrying diHp (diC7) to diUd (diC11) alkyl chains (neamines
13, 14, 9, 15, 16) is compared to the graph obtained with the previously synthesized 2-
naphthylalkyl derivatives. Clearly, two different windows of clogP values can be delineated
for both series, one for the dialkyl derivatives from -13.5 to -10.5 and the other for the
naphthylalkyl derivatives from -13.0 to -9.5. Figure 2C also shows clearly that both dialkyl
and dinaphthylalkyl derivatives can have similar good antibacterial activity.
In Figure 3, the same window-splitting can be observed for the susceptible P. aeruginosa
strain with delineation of two new windows of clogP values, one for the dialkyl derivatives
from -13.5 to -10.5 and second for the naphthylalkyl derivatives from -13.0 to -9.5 (Figure
16

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3C). Figure 3C also shows the higher antibacterial activity of the dialkyl neamines in
comparison to the naphthylalkyl derivatives on susceptible P. aeruginosa that is also observed
in Table 2 against E. coli and K. pneumonia strains.
Figure 2. Values of 1/(MIC (µg/mL)) as a function of clogP values for 3′,6-dialkyl neamines
against ATCC 33592 HA-MRSA. A: Graph resulting from the previous study;19 B: Previous
graph decomposed into two graphs, one for neamines carrying linear alkyl chains (7 (diBu), 8
(diHx), 9 (diNn) and 10 (diOcD): blue diamonds) and, the other one, for neamines carrying 2-
naphthylalkyl groups (3 (di2NM), 4 (di2NP), 5 (di2NB) and 6 (di2NH): red squares); C:
Graphs obtained with 3´,6-dialkyl neamines carrying linear alkyl chains including the new
synthesized derivatives 13-16 (blue diamonds) in comparison to the naphthylalkyl derivatives
3-6 (red squares).
17

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Figure 3. Values of 1/(MIC (µg/mL)) as a function of clogP values for 3′,6-dialkyl neamines
against susceptible P. aeruginosa ATCC 27853. A: Graph resulting from the previous study;19
B: Previous graph decomposed into two graphs, one for neamines carrying linear alkyl chains
(7 (diBu), 8 (diHx), 9 (diNn) and 10 (diOcD): blue diamonds) and the other one for neamines
carrying 2-naphthylalkyl groups (3 (di2NM), 4 (di2NP), 5 (di2NB) and 6 (di2NH): red
squares); C: Graphs obtained with 3´,6-dialkyl neamines carrying linear alkyl chains
including the new synthesized derivatives 13-16 (blue diamonds) in comparison to the
naphthylalkyl derivatives 3-6 (red squares).
Antibacterial activity of the 3´,6-diDiMOc (17) and 3´,4´-diDiMOc (18) neamines made
of branched alkyl chains.
The 3´,6-diDiMOc neamine 17 having a -11.3 clogP value (Table 1) included in the
delineated lipophilicity window showed antibacterial activity against susceptible and resistant
18

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S. aureus strains (MIC= 1 µg/mL, Table 2) similar to those of the diOc (14), diNn (9) and
diDe (15) neamines (MIC= 1 to 2-4 µg/mL). 17 also revealed activity against susceptible and
resistant Gram-negative bacteria similar to those measured with the previously identified best
antibacterial diNn neamine 9 (MIC= 1-8 and 2-16 µg/mL, respectively, Table 3) with a
potential interest for resistant A. lwoffii (MIC= 8 µg/mL as compared to 16 µg/mL for diNn
neamine).
Its 3´,4´-diDiMOc isomer 18 showed a slightly weaker antibacterial activity against
susceptible and resistant Gram-negative and Gram-positive bacteria with MICs values ranging
from 4 to 8 µg/mL, Table 2). This activity appeared to be close to the activity of the
previously studied 3´,4´-diNn neamine 12 (Table 2).20
Antibacterial activity of the 3´,6-heteroalkyl neamines, mixed n-heptyl/n-dodecyl
derivatives (19 and 20) and fluorescent mixed n-heptyl/(1-pyrenyl)butyl (21 and 22)
In order to evaluate the importance of the hydrophobic chain length in the antibacterial
activity maintaining the lipophilicity in the delineated windows of clogP values, 3´-Hp-6-Ud
(19) and 3´-Ud-6-Hp (20) neamines carrying mixed linear C7 and C11 alkyl groups and two
fluorescent derivatives 21 and 22 incorporating Hp (C7) and (1-pyrenyl)butyl (1PyBu) groups
(clogP= -10.7) were synthesized. Their structures were designed to lead, for the dialkyl
isomers, to clogP= -11.9 corresponding to the calculated value for the diNn derivative 9 and
to clogP= -10.7 for the pyrenyl derivatives (Table 1). As expected, MIC values of both Hp/Ud
neamines 19 and 20 appeared to be mainly from 1 to 4 µg/mL except for compound 19
against E. coli (MIC 16 µg/mL) and compound 20 against S. aureus NorA (32 µg/mL). They
are inactive (MIC> 128 µg/mL) against resistant A. lwoffii (Tables 2 and 3).
Both fluorescent pyrenyl derivatives 21 and 22 designed and synthesized for mechanistic
study showed similar antibacterial activity (Tables 2 and 3) slightly lower as compared to
19

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those of both mixed Hp/Ud derivatives 19 and 20 against all selected bacteria strains. Again,
they are inactive on resistant A. lwoffii and weakly active on P. aeruginosa PA22.
The corresponding 3´-mono- and 6-mono-heptyl neamines (23 and 24, respectively) were
found inactive or very weakly active (MIC> 64 µg/mL) (Tables 2 and 3). The 3´-mono- and
6-mono-C11 neamines (25 and 26 respectively) also appeared to be mainly inactive (MIC= 8
to >128 µg/mL) (Tables 2 and 3). However, some MIC values were found to be relatively low
(4 to 32 µg/mL against S. aureus strains and against PA406) but they are clearly higher than
those observed with the derivatives 19-22 carrying two different alkyl chains.
CYTOTOXICITY
Using the MTT assay, the viability of murine J774 macrophages were evaluated in the
presence of 10 and 30 µM of the antibacterial 3´,6-dialkyl neamines (13-26) described here
and are compared in Table 4 to those of antibacterial neamines (3, 4, 9, 11 and 12) previously
described.
At 10 µM, the viability values measured in the presence of the 3´,6-homodialkyl neamines 13,
14, 9, 15, 16 carrying from Hp (C7) to Ud (C11) linear alkyl chains or the branched DiMOc
chain (17) decreased slightly with the linear chain length and remains close to 80-90%. At 30
µM, the corresponding viability values decreased more drastically with the increase of the
chain length and thus clogP, the values are near to 80% for the diHp (diC7, 13), to 70% for
the diNn (diC9, 9) and to 65% for the diOc (diC8, 14) neamines and much smaller (15%
viability) for the diDe (C10, 15) and diUd (diC11, 16) neamines. The 3´,6-diDiMOc
derivative 17 showed a viability of 59%, smaller than the 67% value measured in the presence
of its 3´,6-diNn analogue 9 of close lipophilicity (clogP= -11.3 and -11.9, respectively).
20

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Viability %
Dialkyl neamines
incorporating same alkyl
groups
Viability %
Reference AGs
Amikacin
Gentamicin
Tobramycin
Neomycin B 1
Neamine 2
10 µM
30 µM
10 µM
30 µM
81.2 (3)
88.4 (11)
99.7 (4)
87.3 (10)
94.8 (9)
92.0 (2)
82.9 (4)
83.2 (2)
69.8 (2)
84.4 (2)
3´,6-di2NM 318
3´,6-di2NP 419
86.0 (7)
75.7 (2)
91.1 (13) 89.5 (2)
96.3 (3)
91.3 (4)
86.7 (9)
79.9 (3)
65.1 (3)
67.4 (3)
3´,6-diHp 13
3´,6-diOc 14
3´,6-diNn 919
Dialkyl neamines incorporating different
alkyl groups
3´-Hp-6-Ud 19
3´-Ud-6-Hp 20
3´-Hp-6-1PyBu 21
3´-1PyBu-6-Hp 22
82.9 (3)
88.4 (3)
77.7 (3)
55.9 (3)
38.8 (2)
39.1 (2)
23.8 (2)
45.3 (2)
3´,6-diDe 15
3´,6-diUd 16
3´,6-diDiMOc 17
85.5 (4)
81.7 (4)
88.4 (8)
14.5 (3)
14.1 (3)
58.7 (9)
Monoalkyl neamines
3´,4´-diDiMOc 18
62.6 (2)
87.7 (6)
82.7 (4)
75.4 (2)
66.9 (2)
48.6 (2)
3´-monoHp 23
77.5 (4)
84.3 (3)
81.8 (4)
74.7 (3)
70.2 (3)
71.3 (2)
77.1 (3)
83.4 (2)
3´,4´-di2NP 1120
3´,4´-diNn 1220
6-monoHp 24
3´-monoUd 25
6-monoUd 26
Table 4. Viability (%) of murine J774 macrophages determined using the MTT assay in the
presence of 10 and 30 µM of the prepared neamine derivatives, the numbers of independent
experiences are mentioned after the viability values in brackets. *Measurements from two
different samples isolated and characterized separately.
A strong decrease of the viability at 30 µM in comparison to 10 µM was also observed for the
3´,6-dialkyl neamines 19 and 20 carrying mixed heptyl/undecyl groups (C7/C11) (around
85% at 10 µM and 39% at 30 µM, respectively). The fluorescent heptyl/pyrenylbutyl
neamines 21 and 22 showed as expected reduced viability values (78 and 56% at 10 µM and
24 and 45% at 30 µM, respectively). However, the 3´-Hp-6-PyBu neamine 19 appeared to be
strongly less cytotoxic at 10 µM than its 3´-PyBu-6-Hp isomer 20. At 10 µM, the antibacterial
3´,4´-diDiMOc derivative 18 showed a reduced viability in comparison to its 3´,6-isomer 17
21

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(63 and 88%, respectively). All the monoalkyl neamines 23-26 showed similar effects with
viability roughly close to 70-80% both at 10 and 30 µM.
MIC CHANGES AGAINST P. AERUGINOSA UPON LONG EXPOSURE TO AAGs
We have previously studied the MIC changes against susceptible P. aeruginosa ATCC 27853
upon 12 days exposure to half-MIC of 3´,6-di2NP neamine (4) and its 3´,4´-isomer (11) in
comparison to ciprofloxacin. 20 Exposure of P. aeruginosa to subinhibitory concentrations of
ciprofloxacin, or di2NP neamines caused a decrease in susceptibility that has appeared later
for the di2NP derivatives. We repeated this type of experiment with the 3´,6-diNn neamine
derivative 9, one of the most active neamine identified against susceptible and resistant P.
aeruginosa strains. MIC of 9 against susceptible P. aeruginosa ATCC 27853, after exposure
to half-MIC at different times over more than one month, appeared to be slightly increased
from 1 to 4 µg/mL at day 15, 30 and 38 as compared to MIC values measured with
ciprofloxacin which increased faster from 0.5 to 16 µg/mL at days 6, 21 and 24 (Figure 4).
22

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Figure 4. Evolution of the MIC against P. aeruginosa of 3´,6-diNn neamine 9 (orange line) in
comparison to the evolution of the MIC of ciprofloxacin (blue line) after exposure to half-
MIC concentrations for the indicated times. The concentration of the antibiotic was re-
adjusted each day to remain equivalent to half the MIC. Results are expressed in changes in
MICs over initial value.
DISCUSSION AND CONCLUSION
Previously, we have identified three broad-spectrum antibacterial amphiphilic neamines
carrying two lipophilic groups grafted at positions 3´ and 6 of the neamine core, two 3´,6-
dinaphtylalkyl neamines 3, 4 and 5 and one 3´,6-dialkyl neamine, the dinonyl derivative 9. In
the search for new antibacterial neamines, new 3´,6-dialkyl neamines 13-17 carrying two
identical linear alkyl chains made of 7 to 11 carbon atoms (C7 to C11) were synthesized and
evaluated in comparison to the previously active 3´,6-diNn derivative 9. The best antibacterial
activity against S. aureus strains was observed with the octyl, nonyl and decyl chains
23

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(compounds 14, 9 and 15, respectively; 3 strains among which MRSA). These derivatives
also showed the best activity against the majority of susceptible and resistant Gram-negative
bacteria. In comparison, the antibacterial activity of the diheptyl (diC7, 13) and diundecyl
(diC11, 16) derivatives decreases against most bacteria strains and more strongly against
Gram-negative bacteria.
These results well confirm the key role of the lipophilicity in the antibacterial activity and
lead to differentiate the lipophilicity/activity window corresponding to linear 3´,6-dialkyl
neamine (-13.5 to -10.5 clogP values) from the window corresponding to naphthylalkyl
derivatives (-13.0 to -9.5 clogP values) (Figure 2 against MRSA and Figure 3 against
susceptible P. aeruginosa). From this study, we may conclude that the 3´,6-dialkyl neamines
are more active against most of Gram-negative bacteria than 3´,6-naphthylalkyl neamines
(Figure 3). The presence on the neamine core of linear alkyl chains more flexible than
arylalkyl groups is probably more favorable to insertion of the antibacterial derivatives into
Gram-negative bacteria membranes. Into the bacterial membranes, the linear alkyl chains can
pack to the linear saturated alkyl chains carried by the targeted anionic lipids,
lipopolysaccharides and cardiolipin.
The delineated values of clogP corresponding to good antibacterial activities can be used for
the design of new antibacterial amphiphilic neamines but have no biological significance
since we have shown previously that (i) the antibacterial activity is correlated directly to the
lipophilicity of the grafted chains19 and (ii) smaller amphiphilic neosamines having weaker
clogP exibit similar antibacterial effects.20 As demonstrated with di2NM (3)18,21 and diNn (9)
neamines22-24 and neosamines derivatives,20 the antibacterial activity of the amphiphilic
aminoglycosides results from outer effects on bacterial membranes and on different targets
and, thus, the delineated clogP values cannot be easily related to these effects also depending
on the size and shape of the active compounds. The number of positive charges carried by the
24

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amphiphilic neamines is also a critical parameter in the antibacterial activity and several
protonated forms are present at physiological pH.
In order to evaluate the importance in the antibacterial effect of the alkyl chain linearity, the
3´,6-dialkyl neamine 17 carrying two branched dimethyloctyl (DiMOc) chains and having a
lipophilicity close to the one of the most active diNn derivative 9 (clogP= -11.3 and -11.9,
respectively) was synthesized and evaluated. 17 showed a broad-spectrum antibacterial
activity similar to the one of diNn derivative 9 and, thus, branched chains can be used for
obtaining antibacterial AAGs. Its 3´,4´-isomer (18) appeared to be mainly less active as well
as the 3´,4´-diNn derivative 12 in comparison to their respective 3´,6-isomers. The difference
of the molecular shapes and volumes of the 3´,4´- and 3´,6-dialkyl isomers involved in the
membrane effects explains probably this result.
In order to investigate the importance of the respective length and position of both chains in
the antibacterial activity, we synthesized 3´,6-heterodialkyl isomeric neamines carrying heptyl
and undecyl chains selected for maintaining a lipophilicity (clogP) close to the lipophilicity of
the antibacterial diNn derivative 9 (clogP= -11.9). Both Hp/Ud (C7/C11) isomers 19 and 20
prepared revealed broad-spectrum antibacterial activity similar to the one of the best
antibacterial 3´,6-homodialkyl neamines 9, 14 and 15, excepted for their lack of activity
against the resistant A. lwoffii strain. A critical nonyl chain length at both positions is
necessary for a significant antibacterial activity against this strain.
Both designed antibacterial fluorescent 3´,6-heteroalkyl neamines carrying a pyrenyl
fluorophore and a heptyl chain 21 and 22 matching with the delineated lipophilicity window
of activity were also found to be broad-spectrum antibacterial agents and will be used for
mechanistic study.
Regarding the viability of murine J774 macrophages in the presence of the antibacterial 3´,6-
homodialkyl derivatives, the cell viability decreased slightly at 10 µM from the diHp (13) to
25

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the diUd (16) derivatives increasing the linear chain lengths and for the 3´,6-diDiMOc
derivative 17 (viability values from 96 to 82%). At 30 µM, the viability decreased strongly,
especially for the most lipophilic diDe (15) and diUd (16) neamines and for the 3´,6-diDiMOc
derivative. A similar strong decrease in mammalian cell viability at 30 µM was observed for
the less lipophilic 3´,6-heterodialkyl neamines 19 and 20 carrying Hp/Ud chains and the
fluorescent pyrenyl neamines 21 and 22.
In conclusion, the fine tuning of the previously delineated window of lipophilicity
corresponding to broad-spectrum activity of 3´,6-dialkyl neamines performed through a focus
on the structure of their lipophilic groups led to the identification of new antibacterial
derivatives carrying two linear octyl and undecyl chains or two branched dimethyloctyl
chains.
This study led to the delineation of new structure-activity and structure-eukaryotic viability
relationships showing that (i) 3´,6-homodialkyl neamines are more active against Gram-
negative bacteria than 3´,6-homodinaphthylalkyl neamines but more cytotoxic on mammalian
cells, (ii) the grafting of branched alkyl chains of similar lipophilicities preserves or increases
the antibacterial activity (examples in the 3´,6- and the 3´,4´-dialkyl series) and, also,
increases the eukaryotic cytotoxicity and (iii) 3´,6-heterodialkyl neamines can have good
antibacterial activity and the dissymmetry of their chains increases their cytotoxicity.
Recently, Fridman and coworkers reported that an increased degree of unsaturation in the
lipophilic chain of antifungal cationic amphiphiles derived from the aminoglycoside
tobramycin significantly reduced immediate toxicity to mammalian cells in comparison to
saturated derivatives.29 A similar conclusion can be made here from the decreased eukaryotic
cytotoxicity of the dinaphthylalkyl neamines in comparison to the one of the corresponding
dialkyl neamines having close lipophilicity. The incorporation of small aromatic rings in the
26

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amphiphilic aminoglycoside chains decreases their cytotoxicity and reduced their antibacterial
effects against Gram-negative bacteria but interestingly not against Gram-positive bacteria.
The previously identified 3´,6-dinonyl neamine 9 appeared to be the most active amphiphilic
neamine against P. aeruginosa strains and the less cytotoxic derivative on mammalian cells.
The exposure of susceptible P. aeruginosa to half-MIC of 9 at different times over one month
demonstrated the expected high difficulty of resistance emergence to amphiphilic
aminoglycosides with a highly weaker and slower increase of MIC in comparison to
ciprofloxacin.
SUPPLEMENTARY DATA
1
Supporting Information Available: General information for the synthesis, H and 13C NMR
spectra and purities of the evaluated amphiphilic derivatives. This material is available free of
charge via the Internet at….
AUTHOR INFORMATION
*Phone +32 2 764 73 74; Fax: +32 2 764 73 63; E-mail: Marie-Paule.Mingeot@uclouvain.be.
*Phone +33 4 76 63 53 17; Fax +33 4 76 63 52 98; E-mail: Jean-Luc.Decout@ujf-grenoble.fr.
ACKNOWLEDGMENTS
This work was supported by « Région Rhône-Alpes » (ARC1: 12 00887201, post-doctoral
grant for LZ), « Fondation pour la Recherche Médicale » (post-doctoral grant for JK,
DBF20161136768), Agence Nationale de Recherche « Programme Labex » (ARCANE,
ANR-11-LABX-003), SATT Linksium Grenoble Alpes, and « Fonds de la Recherche
Scientifique Médicale » (grants T.1003.14 and J.0205.16). We thank for providing us with
strains R. Vanhoof (E. coli PAZ505H8101 and L58058.1, P. aeruginosa ATCC 27853,
Psa.F03, A. lwoffii Al.88-483), P. Plésiat (P. aeruginosa PA22) and the Network on
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Antimicrobial Resistance in S. aureus (MRSA and VRSA; Eurofins Medinet Inc., Hendon,
VA). V. Mohymont provided dedicated technical assistance for cell studies.
ABBREVIATIONS USED
AAC, aminoglycoside N-acetyltransferase; AAG, amphiphilic aminoglycoside; AG,
aminoglycoside; ANT, aminoglycoside O-nucleotidyltransferase; APH, aminoglycoside O-
phosphoryltransferase; CA, cationic amphiphiles; cfu, colony forming unit; De, decyl;
DiMOc, 3,7-(dimethyl)octyl; Hx, Hexyl; Hp, heptyl; MRSA, methicillin resistant S. aureus;
2NB, 2-naphthylbutyl; 2NH, 2-naphthylhexyl; 2NM, 2-naphthylmethyl; Nn, nonyl; 2NP, 2-
naphthylpropyl; Oc, octyl; OM, outer membrane; Ud, undecyl.
EXPERIMENTAL SECTION
Calculation of clogP Values.45 The lipophilicity character of the neamine derivatives
prepared was estimated through the calculation of clogP values (octanol/water partition
coefficients) using the MarvinSketch software [Marvin 5.11.4, 2012, ChemAxon
(http://www.chemaxon.com)]. The clogP plug-in in this software calculates the octanol/water
partition coefficient, which is used in QSAR analysis and rational drug design as a measure of
molecular hydrophobicity. The calculation method used here is based on a modification of the
method published by Viswanadhan and Ghose et al. (VG method).45
Synthesis. General procedures.
Procedure I: General procedure used for the 3´-mono-, 6-mono- and 3´,6-di-O-alkylation of
the tetra-N-tritylated neamine 27 alkylation under phase transfer conditions:44
To a solution of compound 2735 (1.0 eq, 3.0 g) in toluene (150 mL) were added N-tetrabutyl
ammonium iodide TBAI (1.5 eq), the halide (1.6 to 3.6 eq) and an aqueous solution of NaOH
(30%, w/w, 100 mL). The mixture was vigorously stirred at 50 °C during 5 h. Another portion
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of halide (0.6 eq) was added and the reaction was heated again at 50 °C for 24 h. The mixture
was cooled to room temperature and the organic phase was washed with water (3 x 50 mL)
and brine (1 x 50 mL) before drying over MgSO4. The solution was filtered off and the
solvent evaporated to dryness under reduced pressure. The solid was chromatographed on
silica gel eluting with mixtures toluene/ethyl acetate /Et3N (0.25%) from 100:0 to 80:20 to
give the mono and dialkyl products.
Procedure II: General procedure used for the deprotection of the alkylated N-tetratritylated
neamine derivatives.
The tritylated compound was dissolved in DCM/TFA (4/1, v/v) and anisole was added (0.1
mL/mL of solvent mixture). The mixture was stirred at room temperature during 2 h and
methanol was added (10 mL). The solvents and TFA were co-evaporated under reduced
pressure. A second evaporation after addition of methanol (10 mL) led to the crude product
which was dissolved in a mixture of H2O/DCM (1/1). The aqueous phase was washed twice
with DCM before evaporation under reduced pressure. The residue was purified by C18
reversed phase chromatography (gradient H2O/MeOH) to give the desired product as the pure
tetratrifluoroacetate.
Purification: The aminosugar purity of the evaluated compounds was ≥95%. Before the final
deprotection step under acidic conditions, careful purifications of the tritylated derivatives
allowing minor isomers or impurities removal were performed by chromatography on silica
1
gel. The purity of the deprotected neamine derivatives was controlled by H NMR
spectrometry and TLC on silica gel (eluent, EtOH/H2O/(NH3, H2O; 20%) 80:10:10; TLC
visualization: ninhydrin spray (0.3 g, 3 mL AcOH, 100 mL of EtOH).
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