Phosphonium-ammonium-based di-cationic ionic liquids as antibacterial over the ESKAPE group

Article abstract of DOI:10.1016/j.bmcl.2020.127389

Emergence of antibioresistance is currently a major threat of public health worldwide. Hence there is an urge need of finding new antibacterial material. Herein, we report a simple and eco-friendly method to synthesize homo and heterodicationic ionic liquids based on quaternary phosphonium and ammonium salt. In order to investigate the structure activity relationship (SAR) we measured the MICs of a series of 16 derivatives with structural variations (nature of cations and counter-ions, size of linker and alkyl side chains as well as structural symmetry) over a range of Gram-positive and Gram-negative bacterial strains from the ESKAPE group. Some of the tested structures exhibit high antimicrobial activities (MIC = 0.5 mg/L) and are active over a wide range of bacteria from Gram-positive to Gram-negative. Overall, these results reveal the strong potential of di-cationic derivatives as antibacterial agents and the determination of activities from structural features gives decisive information for future synthesis of such di-cationic structures for biocidal purpose.

Full text of DOI:10.1016/j.bmcl.2020.127389

Journal Pre-proofs
Phosphonium-ammonium-based di-cationic ionic liquids as antibacterial over
the ESKAPE group
Frédéric Brunel, Christelle Lautard, Frédéric Garzino, Jean-Manuel
Raimundo, Jean-Michel Bolla, Michel Camplo
PII:
DOI:
Reference:
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https://doi.org/10.1016/j.bmcl.2020.127389
BMCL 127389
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14 June 2020
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3 July 2020
Please cite this article as: Brunel, F., Lautard, C., Garzino, F., Raimundo, J-M., Bolla, J-M., Camplo, M.,
Phosphonium-ammonium-based di-cationic ionic liquids as antibacterial over the ESKAPE group, Bioorganic &
Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127389
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Phosphonium-ammonium-based di-cationic ionic liquids as antibacterial over the
ESKAPE group.
Frédéric Brunel,a Christelle Lautard,b Frédéric Garzino,a Jean-Manuel Raimundo,a, * Jean-Michel Bolla,b, * and Michel Camploa, *
a Aix Marseille Univ., CNRS, CINaM UMR 7325, Campus de Luminy, Case 913, 13288 Marseille Cedex 09, France.
bAix-Marseille Univ., INSERM, SSA, MCT, 27 boulevard Jean Moulin 13385 Marseille cedex 05, France.
Abstract
Emergence of antibioresistance is currently a major threat of public health worldwide. Hence there is an urge need of finding new
antibacterial material. Herein, we report a simple and eco-friendly method to synthesize homo and heterodicationic ionic liquids based on
quaternary phosphonium and ammonium salt. In order to investigate the structure activity relationship (SAR) we measured the MICs of a
series of 16 derivatives with structural variations (nature of cations and counter-ions, size of linker and alkyl side chains as well as
structural symmetry) over a range of Gram-positive and Gram-negative bacterial strains from the ESKAPE group. Some of the tested
structures exhibit high antimicrobial activities (MIC = 0.5 mg/L) and are active over a wide range of bacteria from Gram-positive to
Gram-negative. Overall, these results reveal the strong potential of di-cationic derivatives as antibacterial agents and the determination of
activities from structural features gives decisive information for future synthesis of such di-cationic structures for biocidal purpose.
gemini analogues in high yields through straightforward or two-
steps reaction. The iterative process allows the possibility to
Introduction
vary structurally some key parameters such as the length of the
Nosocomial infections constitute a major issue for the public
linker and side-chains, the nature of the cation (ammonium or
health worldwide. In US alone, the number of deaths attributed
phosphonium) or the halogen counter-ion (chloride, bromide or
to Healthcare Associated Infections (HCAIs) is about 98 000
iodide) as well as the structural and cationic symmetry
per year while 1.7 million patients hospitalized will contract a
HCAI over the same period.1 The increasing difficulties to treat
(different pattern on both side of the linker). Thus, antibacterial
activities were investigated by the determination of the
bacterial infections are directly linked to the antimicrobial
minimum inhibition concentrations (MIC) against several
resistance phenomena (AMR) caused for instance by over-used
bacterial strains from the ESKAPE group for the new
or inadequate antibiotics treatments. To overcome this
resistance threat there is an urgent need to design new
antibiotics or find novel strategies and new antibacterial
materials. For these purposes researchers have striven several
synthesized compounds to study the Structure-Activity
Relationship (SAR). ESKAPE is an acronym that stands for the
major human pathogens with multi-drug resistance16 including
both Gram-positive and Gram-negative bacteria.
innovative alternatives based on the development of
antimicrobial peptides2, bacteriophages3, cationic organic
Synthesis
compounds and polymers4 that have attracted much attention
lately for their high efficiency as biocidal materials. Besides,
ionic liquids have recently attracted lot of interest for
biocompatible biomedical5 and antimicrobial applications6 due
to their versatility and ability to easily fine-tune their properties
in various morphologies, sizes, and surface charges. Those
Homo and heterodicationic structures were designed and
synthesized during the course of this study following a
straightforward and eco-friendly protocol depicted in Scheme 1
and 2. Firstly, the symmetric homodicationic compounds were
obtained in one step by reacting all commercially available
reactants together. Hence, the selected dihalogenated aliphatic
linkers having a C10 or C6 length underwent a nucleophilic
substitution reaction with the appropriate trialkylphosphine or
trialkylamine nucleophiles affording the desired gemini
dicationic derivatives 1-10 in a yield from moderate to high.
The reactions were performed under neat conditions using a
micro-wave irradiation at high temperature for 3 hours (200 W,
130 °C) for the gemini diphosphoniums compounds (Scheme 1,
a) while for diammonium analogues conventional heating at
110°C with prolongated reaction time (48 hours) are required
(Scheme 1, b). These harsh conditions can explain the lowered
yield (20-30%) obtained for the gemini diammoniums
derivatives since ammonium is more susceptible to undergo
undesired competitive Hofmann β-eliminations in the presence
of base and/or high temperature.17
include
imidazolium,7
ammonium,8
pyridinium9
or
phosphonium salts.10 Interestingly, some studies have recently
evidenced a net benefit in the bactericidal activity when the
charge density of the ionic liquids increases. For example, di-
cationic ionic liquids have exhibited improved efficiency11,12
associated to broaden spectrum of activity13 compared to their
monomer counterparts. In addition, gemini-type ionic liquids
constitute a new class of amphiphilic structures possessing two
polar head groups (identical or not) linked either by a flexible
or rigid spacer in a symmetric or asymmetric fashion.
Compared to the classical monocationic structures the gemini
ionic liquids have demonstrated superior physical properties14
and enlarge the scope of applications with an exclusive way to
fine-tune their biological and antimicrobial properties.15
Although these gemini derivatives have shown unprecedented
advantages in several applications, they are still
underdeveloped in particular for biological and antimicrobial
applications. Among them, phosphonium gemini-based
molecules have been only scarcely reported and still largely
underexploited opening the way to innovative structures
associated with novel biological properties. In that context we
will report herein the synthesis of novel phosphonium-
ammonium-based di-cationic compounds as well as their

Scheme 2: Synthetic pathway of the heterodicationic derivatives 11-16. (a)
PR3 or NR3, CH2Cl2, reflux, 12h; (b) PR’3 or NR’3, MW (200W), 130°C,
3h.
All synthesized dicationic derivatives have been characterized
1
by mass spectroscopy and H, 13C and 31P NMR spectroscopy
(See SI).
Scheme 1: Synthetic pathway of the homodicationic derivatives 1-10. (a)
PR3, MW (200W), 130°C, 3h; (b) NR’3, 110°C, 48h.
Table 1: Structural characteristics of the homo and heterodicationic
derivatives 1-16.
Secondly, the heterodicationic derivatives were obtained using
an iterative pathway. The first step consists in a mono-
substitution reaction in dilute CH2Cl2 under reflux conditions
affording the reaction intermediaries A, B, C and D in 70, 88,
25 and 31% yield respectively after column chromatography
purifications (Scheme 2 a, see SI). Subsequent nucleophilic
substitution reactions with these intermediates (Scheme 2 b, see
SI), using the aforementioned micro-wave irradiation protocol,
led to the heterodicationic compounds 11-16 in good to
excellent yields (Table 1 and see SI).
Yield
Compound
M
M’
R
R’
N
X-
(%)
95
85
96
20
87
30
91
93
90
96
88
70
95
57
93
82
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
P
P
P
N
P
N
P
P
P
P
P
P
P
N
N
N
P
P
P
N
P
N
P
P
P
P
P
P
P
P
P
P
Hex
Hex
Hex
Hex
Hex
Hex
Oct
Oct
But
But
Hex
Hex
Hex
Hex
Hex
Hex
Hex
Hex
Hex
Hex
Hex
Hex
Oct
Oct
But
But
But
Oct
But
Hex
But
But
8
8
8
8
4
4
4
8
4
8
8
4
4
8
8
4
Cl
Br
I
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Table 2: Minimal Inhibition Concentration (MIC) (mg/L) of compounds 1-16 on ESKAPE bacteria
Compounds
Bacterial strain
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
E. faecium 20,477
0.5
1
0.5
0.5
0.5
1
2
32
>64 >64
2
2
8
1
2
64
S. aureus CIP 7625
0.5
2
0.5
4
0.5
2
0.5
4
0.5
8
1
1
32
8
16
>64
16
0.5
16
8
1
4
4
8
8
8
2
0.5
2
1
32
K. pneumonia CIP 82.91
A. Baumannii ATCC 19606
P. aeruginosa 100720
E. aerogenes ATCC 13048
E. coli CIP 54.8
>64
>64
>64 >64 >64
>64 >64 >64
>64
>64
>64
>64
>64
64
>64
2
4
2
16
8
16
8
8
>64 >64
>64 >64
>64 >64
4
4
2
>64 >64 >64 >64 >64
64
32
4
8
4
8
4
8
8
>64
32
64
32
>64 >64 >64
>64 >64 >64
8
2
2
2
2
4
2
16
>64
all, we have examined the influence of the halogen counter-ion
as compounds 1, 2 and 3 only differ from their halide,
respectively chloride, bromide and iodide. Analogously to
reported results, similar activities were obtained for compounds
1, 2 and 3 highlighting the low impact of the halide on the
efficiency whatever the strain tested. Comparison of
compounds 2 (di-P+) and 4 (di-N+) can give some insights about
the effect of pnictogen atom on the antimicrobial properties.
Indeed, 2 and 4 present identical hydrocarbon scaffolds (a C10
linker and hexyl peripheral chains) and differ only by the nature
of the pnictogen, nevertheless no clear conclusion can be done
as the bactericidal effect behave similarly in both cases
regardless the strain. Curiously, if the linker length is changed
from C10 to C6 (compounds 25 (di-P+) and 46 (di-N+)) clear
changes appear in favor to the di-phosphonium derivative 5 that
became more efficient than its nitrogen analogue 6, particularly
over Gram-negative strains while its efficiency remains similar
to dicationic 2. Furthermore, the replacement of the hexyl
peripheral chains to octyl around the phosphonium core (28
(di-P+) and 57 (di-P+)) have led to drastic changes associated
to a clear decrease in activity in both cases. The effects are
more predominant for 8 which biocidal efficiency was
annihilated for all the ESKAPE group strains while 7 remained
Biological assays and SAR analyses
The biological assays clearly demonstrate the antibacterial
effectiveness for some of the newly synthetized homo- and
heterodicationic ionic liquids against Gram-positive and Gram-
negative strains but associated with a certain variability (Table
2). For example, the compound 2 exhibits a MIC of 0.5 mg/L
over S. aureus which is comparable to the best efficiency
attained with commercially available antibiotics.18 Moreover,
from a general point of view even if the effectiveness is
broaden to the ESKAPE group the dicationic compounds
display a better activity toward Gram-positive strains (E.
faecium and S. aureus) than on Gram-negative but with some
disparities in the tested series. At the first glance, these trends
can be explained by the nature and structure of the membrane
that differs in these two bacteria families.19 However, some di-
cationic structures namely 1 to 5, 12 and 14 interestingly
disclose a wider spectrum of activity for the different
considered strains which can be attributed of particular
structural changes on the di-cationic compounds. In that
respect, we have decided to take a closer look over which kind
of structural variations tend to modify those activities in order
to settle a SAR study by doing a rational comparison. First of

slightly active on Gram-positive strains. On the other hand,
similar behaviors were observed when shorter peripheral chains
have been used (59 (di-P+) and 210 (di-P+)) associated with
a detrimental antimicrobial effect leading to inactive dicationic
compounds in these series. Thus, for symmetric homodicationic
compounds the sizes of the peripheral chains and linker appear
to be interdependent factors in those series. From this study, it
was also highlighted that the predominant structural parameters
that dramatically affect the activity are the side chains. Indeed,
comparison of the efficiency of 2, 8 and 10, has unequivocally
evidenced that compound 2 bearing hexyl side chains exhibits
the highest efficiency proving the central role of this chain
length.
70
60
50
40
30
20
10
0
symmetric di-P+
symmetric di-N+
dissymmetric di-P+
P+/N+
The heterodicationic structures 11-16 have been also analyzed
in order to identify further structural features that could affect
the antimicrobial efficiencies. During the course of the study
several heterostructures have been envisioned either based on a
dissymmetry at the level of the peripheral chains, or at the level
of the central pnictogen or a combination of both. Moreover,
the linker length has a significant impact on the activity (vide
supra) and has been concomitantly considered. Comparison of
compounds 5 (C6H13/C6H13, C6), 12 (C6H13/C8H17, C6) and 13
(C6H13/C6H9, C6), evidenced slight effect on the antimicrobial
properties that combine the effects previously observed from
the homodicationic structures. Once again, the replacement of
the hexyl peripheral chains, even from only one side, by longer
or shorter ones led to a detrimental effect on the efficiency. The
detrimental effect is more obvious in the case of shorter chains
as heterodicationic compound 12 exhibits almost unaffected
efficiency compared to the homodicationic parent compound 5
while 13 remains only active on Gram-positive strains. This
behavior attests again that hexyl might correspond to the
optimal chain length in this series. In addition, substitution of
one pnictogen from 2 (di-P+)14 (N+-P+) or 4 (di-N+)14 (N+-
P+) doesn’t change the efficiency compared to the parent
homodicationic derivatives 2 and 4. However, although the
activity remains almost identical it is noticeable to point out
that the difference could come from the substitution of
phosphonium by an ammonium, as the decrease of the
antimicrobial effect is of the same order than the one observed
between 2 and 4. This behavior will suggest a greater
importance of phosphonium compared to ammonium in such
series. This behavior is also supported by the MICs obtained on
the heterodicationic derivatives 15 and 16. Therefore, the
activity could not be strictly linked to an isolated factor such as
the nature of the cation or the anion, the symmetry or the linker
length, it seems that the main parameter that significantly
influences the antimicrobial activity of these structures is the
number of carbon, and it appears that this balance is specific for
each bacterial species. To illustrate that we decided to only
consider the S. aureus strain, and we graphically compare the
MICs with the number of carbons for each dicationic structure
it gives the Figure 1.
30
35
40
45
50
55
60
Number of carbon
Figure 1: Effect of the total number of carbons and symmetry of
compounds 1-16 on their bactericidal efficiency for S. aureus
Herein, the parabola curve obtained is representative of a
square function, and we can easily release an optimal number
of carbons independently of the nature and structure of the
studied DCILs. In the case of S. aureus this optimum is located
between 40 and 48 carbons. Similar results have already been
demonstrated by Luczak et al.20 In their study, they evaluate
antimicrobial activity of 1-alkyl-3-imidazolium and defined a
“cut-off” effect, where those ILs exhibit an optimum of activity
for alkyl chain with a number of carbon between 16 and 18,
above and below the activity decrease. Same phenomenon has
also been observed for pyridinium21 and quinolinium22 ionic
liquids.
To date, the exact bactericidal mechanism of action remains
unknown but the main hypothesis meeting consensus for such
structures with cationic and hydrophobic parts involves the
preferential adsorption of the cationic compound onto the
negatively charged cell wall followed by the diffusion of the
hydrophobic alkyl chains through the lipid bilayer causing
disruption and irreversible damages to the cell membranes.4
This two steps mechanism supposes an accurate
hydrophilic/lipophilic balance (HLB), and corroborates the
obtained results and the demonstrated “cut-off” effect. Another
approach specifies that the improved antibacterial efficiency of
di-cationic ionic liquids compared to their mono-cationic
counterparts, could be conferred by their “bolaamphiphiles”
structures, typically two ionic heads linked together via an alkyl
linker.12 Indeed, this specific structure is well known for its
ability to auto-assemble into lipid monolayer, form stable
micelles and cross cell membrane.23 However this hypothesis is
difficult to confirm with data collected herein. Additional
studies currently on going such as Diffraction Light Scattering
(DLS), Isothermal Titration Calorimetry (ITC), or microscopy
will help us to have a better understanding of this mechanism.
From the strong antibacterial activities of some of the
synthesized compounds we decided to measure the cytotoxicity
to evaluate if they can be considered as new infection
treatments or antibiotics. Cytotoxicity has been determined over
Human hepatocytes (HepG2) for the structures 2, 4, 8, 10, 11
and 14. As expected, all the tested DCILs show strong
cytotoxicity (see SI). Indeed some alkyl-phosphonium are
known to be membrane lysing agents (MLA)24.
In summary, thanks to an innovative and eco-friendly MW
based synthetic methodology, we have been able to design a
series of 16 DCILs by varying several structural parameters.
The measure of MICs over the bacterial strains from the
ESKAPE group allowed us to study the SAR. Some of our
compounds exhibit very low MICs along with a broad spectrum
of activities (from Gram-positive to Gram-negative) but
because they demonstrate high cytotoxicity they cannot be

3770-3773.
considered as candidates for new antibiotic treatments. These
phosphonium derivatives seem to be slightly more active than
their ammonium counterparts, those phosphonium based DCILs
have proven to be easier to synthetize and more chemically
stable over time. Overall, the SAR study reveals a clear
dependence to the HLB and we hope that the “cut-off” effect
that we have been able to define will help to design new DCIL
structures for antimicrobial purpose.
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Acknowledgement
This work was supported by the Centre National de la
Recherche Scientifique (CNRS) and the Ministère de
l’Enseignement Supérieur et de la Recherche. F. B. thanks also
the Ministère de l’Enseignement Supérieur et de la Recherche
for its doctoral financial support.
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70
60
50
40
30
20
10
0
symmetric di-P+
symmetric di-N+
dissymmetric di-P+
P+/N+
30
35
40
45
50
55
60
Number of carbon
Figure 1: Effect of the total number of carbons and symmetry of compounds 1-16 on their bactericidal efficiency for S. aureus
Scheme 3: Synthetic pathway of the homodicationic derivatives 1-10. (a) PR3, MW (200W), 130°C, 3h; (b) NR’3, 110°C, 48h.

Scheme 4: Synthetic pathway of the heterodicationic derivatives 11-16. (a) PR3 or NR3, CH2Cl2, reflux, 12h; (b) PR’3 or NR’3, MW (200W), 130°C, 3h.
Table 3: Structural characteristics of the homo and heterodicationic derivatives 1-16.
Yield
Compound
M
M’
R
R’
N
X-
(%)
95
85
96
20
87
30
91
93
90
96
88
70
95
57
93
82
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
P
P
P
N
P
N
P
P
P
P
P
P
P
N
N
N
P
P
P
N
P
N
P
P
P
P
P
P
P
P
P
P
Hex
Hex
Hex
Hex
Hex
Hex
Oct
Oct
But
But
Hex
Hex
Hex
Hex
Hex
Hex
Hex
Hex
Hex
Hex
Hex
Hex
Oct
Oct
But
But
But
Oct
But
Hex
But
But
8
8
8
8
4
4
4
8
4
8
8
4
4
8
8
4
Cl
Br
I
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Table 2: Minimal Inhibition Concentration (MIC) (mg/L) of compounds 1-16 on ESKAPE bacteria
Compounds
Bacterial strain
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
E. faecium 20,477
0.5
1
0.5
0.5
0.5
1
2
32
>64 >64
2
0.5
16
8
2
8
1
2
64
S. aureus CIP 7625
0.5
2
0.5
4
0.5
2
0.5
4
0.5
8
1
1
32
8
16
>64
16
1
4
4
8
8
8
2
0.5
2
1
32
K. pneumonia CIP 82.91
A. Baumannii ATCC 19606
P. aeruginosa 100720
E. aerogenes ATCC 13048
E. coli CIP 54.8
>64
>64
>64 >64 >64
>64 >64 >64
>64
>64
>64
>64
>64
64
>64
2
4
2
16
8
16
8
8
>64 >64
>64 >64
>64 >64
4
4
2
>64 >64 >64 >64 >64
64
32
4
8
4
8
4
8
8
>64
32
64
32
>64 >64 >64
>64 >64 >64
8
2
2
2
2
4
2
16
>64
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that
could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential
competing interests: