Preparation and antimicrobial behaviour of quaternary ammonium thiol derivatives able to be grafted on metal surfaces

Article abstract of DOI:10.1016/j.ejmech.2008.05.007

New thiol derivatives containing a quaternary ammonium group bearing variable hydrocarbon chains via an amide connector or not between the sulphur and nitrogen atoms were synthesised with the future aim to be grafted on metal surfaces for obtaining contact-active auto-bactericidal surfaces. Their biostatic and bactericidal activities were evaluated against four microbial strains (Pseudomonas aeruginosa, Staphylococcus aureus, Aspergillus niger and Candida albicans). The presence of the thiol and amide functions in these surfactants was discussed in relation with the antimicrobial activity along with the influence of the length of alkyl chains in order to determine which molecular parameters are 'critical' for biological activity, and consequently what molecules must be chosen for grafting on metal surface.

Full text of DOI:10.1016/j.ejmech.2008.05.007

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European Journal of Medicinal Chemistry 44 (2009) 717e724
http://www.elsevier.com/locate/ejmech
Original article
Preparation and antimicrobial behaviour of quaternary ammonium
thiol derivatives able to be grafted on metal surfaces
Pascal Thebault a, Elisabeth Taffin de Givenchy a, Richard Levy b, Yves Vandenberghe b,
a
Frederic Guittard , Serge Geribaldi
a,
*
´ ´
´
a Institut de Chimie de Nice FR 3037 CNRS, Laboratoire de Chimie des Mate´riaux Organiques et Me´talliques, CMOM, UFR Sciences,
Universite´ de Nice-Sophia Antipolis, Parc Valrose, 06108 Nice Cedex 2, France
b Rohm and Haas France, Laboratoires Europe´ens, De´partement Process Chemicals and Biocides, 371,
rue Beethoven, Sophia Antipolis, 06565 Valbonne, France
Received 6 February 2008; accepted 6 May 2008
Available online 16 May 2008
Abstract
New thiol derivatives containing a quaternary ammonium group bearing variable hydrocarbon chains via an amide connector or not between
the sulphur and nitrogen atoms were synthesised with the future aim to be grafted on metal surfaces for obtaining contact-active auto-bactericidal
surfaces. Their biostatic and bactericidal activities were evaluated against four microbial strains (Pseudomonas aeruginosa, Staphylococcus
aureus, Aspergillus niger and Candida albicans). The presence of the thiol and amide functions in these surfactants was discussed in relation
with the antimicrobial activity along with the influence of the length of alkyl chains in order to determine which molecular parameters are
‘critical’ for biological activity, and consequently what molecules must be chosen for grafting on metal surface.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Preservatives; Quaternary ammonium salts; Surfactants; Thiols; Minimal inhibitory concentration; Minimal lethal concentration
1. Introduction
important drawback since the potentially active agents are
released gradually into the surrounding environment. Conse-
quently, metal surfaces that could kill harmful microorganisms
on contact without releasing antimicrobial agents are in a grow-
ing field of research from a long time [5,6].
Because of the ever-growing demand for healthy living, there
is a keen interest in materials capable of killing harmful micro-
organisms [1]. Concerning metallic material, the usual strategy
consists in coating the metal surfaces with natural antibacterial
polymers or synthetic polymers impregnated with antimicrobial
agents or in applicating organic or aqueous dispersion of antimi-
crobial agents [2e4]. However, these strategies suffer from an
With this aim, the concept of self-assembled monolayers
(SAMs), introduced by Nuzzo and Allara [7], of thiol and
disulfide molecules covalently bound to metals and bearing
potentially biocide moieties could be considered. These metal
grafted molecules must bear potentially biocide moieties in
their structures. These moieties could be quaternary ammo-
nium salts for which the antimicrobial activity is known for
a long time [1,8e12]. So, the final purpose of our research
project is to associate this SAMs’ concept and the well-known
bactericidal properties of quaternary ammonium compounds
in order to obtain contact-active antimicrobial metal surfaces.
This strategy can be summarized as shown in Scheme 1.
First and foremost it was necessary to demonstrate that
molecules bearing a thiol function at one end or a disulfide
Abbreviations: CMCs, Critical micellar concentrations; MIC, Minimal in-
hibitory concentration; MLC, Minimal lethal concentration; SAMs, Self-as-
sembled monolayers.
* Corresponding author. Tel.: þ33 (0) 4 92 07 61 12; fax: þ33 (0) 4 92 07 61
56.
E-mail addresses: Pascal.Thebault@unice.fr (P. Thebault), Elisabeth.
Taffin-de-Givenchy@unice.fr (E. Taffin de Givenchy), RLevy@rohmhaas.
com (R. Levy), Yvandenberghe@rohmhaas.com (Y. Vandenberghe),
Frederic.Guittard@unice.fr (S. Guittard), Serge.Geribaldi@unice.fr (S.
´
Geribaldi).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.05.007

718
P. Thebault et al. / European Journal of Medicinal Chemistry 44 (2009) 717e724
metal surface
possible that the presence of a thiol function at the end of
a tail decreases this surfactant activity.
So, the aim of this intermediate step is to test the bactericidal
efficiencies of two series (series C and CQ) of thiols compar-
atively to the commercially available preservative BAK50
(50% w/w aqueous solution of BAK) taken as reference in
this work, in order, first to check the bactericidal and surfactant
properties of these thiols and, secondly, to select the best ones
to be grafted on various metal surfaces. The two series synthes-
ised are represented in Scheme 2 along with BAK50.
O O O O O O O
Self-Assembled Monolayer
= contact-active auto-bactericidal
surface
Me
+ Cl-
N
solution of functionalized
quaternary ammonium salts
Q
=
O
SH
R
Me
2. Results and discussion
R = Alkyl or Benzyl chain
Q = Connector
2.1. Chemical synthesis
Scheme 1. Formation of contact-active auto-bactericidal metal surface via
SAMs formation with functionalized thiols.
The synthetic pathway for the preparations of compounds
of series C is described in Fig. 1.
group into their structure and a quaternary ammonium group
at the other end are able to be grafted on a metal surface to
give an SAM of good quality. In a recent work, we showed
that thiols and disulfides bearing internal quaternary ammo-
nium in their structure can absorb on clean gold surface and
form organized self-assembled monolayers providing that
the hydrocarbon spacer between the charged nitrogen and
the grafted sulphur atom is long enough to minimize the repul-
sive interactions between the charged groups when the struc-
ture is grafted [13]. Also, we showed that the presence of
the H-donor amido group as connector between the two parts
of the structures can also compensate the repulsive interaction
of neighbouring charged structures on the surface, thanks to
the formation of hydrogen bonding. These results suggested
that potential contact-active antimicrobial metal surfaces
could be obtained, at least with gold, using appropriate quater-
nary ammonium thiol or disulfide derivatives [13]. However,
one question remains: does the presence of a moderately polar
thiol function at one of the ends of these double tailed surfac-
tants that are the dialkyldimethylammonium chlorides sche-
matized in Scheme 1 preserve, avoid or, may enhance the
antimicrobial properties of these surfactant homologues to
one of the most used commercially available ammonium pre-
servatives, benzylalkonium chloride or BAKÒ? Indeed, it is
well known for a long time that the first effect used to explain
the preservative effect of these quaternary ammonium salts is
due to their surfactant properties [14e19], and it would be
The starting material for the synthesis of all the compounds
of series C is 11-bromo-undec-1-ene (Aldrich, 95%, 7766-50-
9) on which a simple nucleophilic substitution is performed us-
ing trimethylamine ethanolic solution (Fluka, no. 92260) for C1
and dimethylamine ethanolic solution (Fluka, no. 38950) for the
other compounds of series C. Excepted for C1, the Menschutkin
reaction leading to the ethylenic quaternary ammonium bro-
mides is performed at high temperature without solvent using
benzyl bromide (Aldrich, 98%, no. B17905), 1-bromodecane
(Aldrich, 98%, no. 145785), 1-bromododecane (Aldrich, 97%,
no. B65551), and tetradecyl bromide (Aldrich, 97%, no.
195332), respectively for CBz, C10, C12 and C14. For all com-
pounds of series C, the formation of the thiol function and the
halogen exchange are achieved from the addition of thioacetic
acid (Aldrich, 96%, no. 507095) under light irradiation fol-
lowed by stirring in a methanolic solution of hydrogen chloride.
The synthetic pathway for the preparation of compounds of
series CQ is described in Fig. 2.
11-Thioacetylundecanoic acid is obtained from 11-bro-
moundecanoic acid (Fluka, no. 18628) and potassium thioace-
tate (Fluka, no. 60595) [20,21]. The amide formation is
performed using 3-N,N-dimethylaminopropylamine (Aldrich,
no. D145009), N-(3-dimethylaminopropyl)-N0-ethyl-carbodiimide
hydrochloride (Aldrich, EDC, no. E7750), and 4-dimethylami-
nopyridine (Aldrich, DMAP, no. 107700). Generally, the
quaternization is achieved using the alkyl bromides previously
described, excepted for CQ1 for which methyl iodide
O
Cl-
Cl-
+
N
Cl-
+
N
R
HS
R
+
11
n
N
3
10
HS
N
H
H
Series C
R = CH2C6H5 CBz
Series CQ
R = CH2C6H5 CQBz
BAK
mixture with n = 12 (40
n = 14 (50
)
)
)
=
CH3
C1
= CH3
CQ1
n = 16 (10
= C10H21
= C12H25
= C14H29
C10
C12
C14
= C10H21
= C12H25
= C14H29
CQ10
CQ12
CQ14
Scheme 2. Quaternized ammonium chlorides used in this work.

P. Thebault et al. / European Journal of Medicinal Chemistry 44 (2009) 717e724
719
Br
The comparison of the CMC values of compounds C1 and
CBz with their homologues bearing a methyl group instead of
the thiol function [23] seems to show that, effectively, this
later decreases the surfactant efficiency of these molecules;
however, compounds C1 and CBz could not be representative
of the series C. Nevertheless, the comparison of the MIC of
BAK 50 evaluated in the same conditions with those of com-
pounds C10eC14 confirms a decrease of the microbiostatic
activity of these surfactants with the presence of the thiol
function.
9
+
+
NH
or
N
EtOH
r.t.
24 h
EtOH
r.t;
48 h
R
N+
Br-
Br-
RBr
N+
N
9
80°C / 48 h
9
9
All the compounds of series C are active towards the four
microorganisms studied with the exception of C14 which
shows only an acceptable activity towards A. niger. Concern-
ing the sub-series formed by C1 and C10eC14, a non-linear
dependence of the microbiostatic activity with the length of
the variable tail is observed (Fig. 3).
1) thioacetic acid
hν / MeOH / 12 h
2) MeOH / HCl/ 2 h
1) thioacetic acid
hν / MeOH / 12 h
2) MeOH / HCl/ 2 h
R
N+
Cl-
N+
11
Cl-
HS
HS
11
A parabolic dependence of MIC with the length of the tail
can be observed with a maximum for C10. This type of depen-
dence was previously observed in different studies [24e26].
This phenomenon is called ‘‘cut-off effect’’. The origin of
this effect is not well explained. Among the various assump-
tions proposed by Balgavy and Devinsky [25], the concept
of free volume could be applied to the quaternary ammonium
salts. In solution, the polar ammonium heads interact with
those of phospholipids and the hydrocarbon chains are parallel
to those of phopholipids of the cell. At this level, the density of
the hydrophobic area of the bilayer is necessarily modified and
a free volume is formed. When the length of the hydrocarbon
chain of ammonium salts is smaller than that of phospholipids,
the total free volume created in the bilayer is small. When the
length of the surfactant tail becomes comparable to that of
phospholipids, the free volume decreases and tends towards
zero. Molecules bearing chains between these two extremes
lead to the most important free volume inside the bilayer.
More the free volume is large; more the decrease of hydropho-
bicity of the membrane is large. Consequently, a largest desta-
bilization of the membrane is expected and the bactericidal
activity increases. Moreover, the smallest CMI value is
observed for the most bioactive surfactant C10.
C1
CBz, C10, C12, C14
Fig. 1. Synthetic pathways for the series C.
(Aldrich, no. I8507) is used. Halogen exchange is performed
as previously described for series C.
2.2. Antimicrobial activity
Antibacterial and antifungal evaluations of salts of the
series C and CQ were run using MIC (Minimal Inhibitory
Concentration) and MLC (Minimal Lethal Concentration)
measurements versus four microorganisms: Staphylococcus
aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC
9027), Aspergillus niger (ATCC 6275) and Candida albicans
(ATCC 10231). Three independent experiments were under-
taken for each compound. The strains used are ubiquitous, op-
portunistic and commonly encountered in biodeterioration
material problems or fouling of the industrial processes, in ad-
dition these types of strains could be encountered in nosoco-
mial infections.
2.2.1. Surfactant and microbiostatic activities
Table 1 summarizes the MIC values for all the molecules
tested along with their Critical Micellar Concentrations
(CMCs) and their surface tensions (gS).
As a whole, a large difference is observed between the re-
sults obtained with P. aeruginosa and those obtained with the
three other strains. It is well known that, due to the structure of
O
O
H
N
OH
OH
N
NH2(CH2)3N(CH3)2
EDC/DMAP/12h / r.t.
KSC(O)CH3
Br
S
S
10
10
10
3
CH3CN/12h / 85°C
O
O
O
RBr
80°C/ 48h
+Br-
O
+Cl-
HCl/MeOH
H
N
H
N
N
R
2h /100°C
N
R
S
10
3
HS
10
3
O
O
CQ1, CQ Bz, CQ10, CQ12, CQ14
Fig. 2. Synthetic pathway for the series CQ.

720
P. Thebault et al. / European Journal of Medicinal Chemistry 44 (2009) 717e724
Table 1
MIC values of the molecules of series C and CQ along with their CMC and gS
Molecules
MIC (mmol/L)
CMC
(mmol/L)
gS
(mN/m)
Staphylococcus
aureus
Pseudomonas
aeruginosa
Candida
albicans
Aspergillus
niger
BAK 50
3.2
93
8.36
4.74
C1
56.6
681.6
11.6
556.9
1055.9
308.9
1124.8
471.6
31.6
56.6
351.0
31.6
40 (11.6)a
6.7 (2.7)b
1.6
44.1
30.3
28.3
31.0
33.2
CBz
C10
C12
C14
34.7
1306.3
326.2
1121.5
39.5
1524.6
67.2
90.0
0.53
0.13
CQ1
CQBz
CQ10
CQ12
CQ14
a
105.4
592.2
17.5
594.6
979.8
171.4
183.5
441.8
>1065.1
>1057.3
>676.8
>687.6
>741.6
105.3
772.6
44.1
49
45.9
34.1
33.7
36.1
38.4
5.6
1.4
18.7
187.7
47.02
56.3
0.61
0.14
CMC of the homologue dodecyltrimethylammonium chloride at 25 ꢀC from Ref. [22].
CMC of the homologue benzyldodecyldimethylammonium chloride at 25 ꢀC from Ref. [22].
b
its outer membrane, this organism possesses the ability to
obtained for fungi, and only C10 shows an MLC for A. niger
in the range of concentrations used. These MLCs are weak for
S. aureus and they increase for P. aeruginosa. Similar trends
are observed for MIC and MLC. In agreement with the litera-
ture, the MLC values are generally higher than the MIC
values. Indeed, the frontier between microbiostatic activity
and microbicidal activity is sometimes a question of concen-
trations used, and a same product can be microbiostatic at
low concentration and microbicidal at higher concentration
[14]. For the most active compounds with 10 and 12 carbon
atoms, the analogues with the amide connector seem to be
slightly active than the compounds of series C.
metabolize quaternary ammonium compounds as a source of
carbon and nitrogen [27]; therefore, the result observed here
is not surprising.
In comparison with compound CBz bearing the benzyl sub-
stituent largely met in this type of preservatives, compound
C10 demonstrates always a larger microbiostatic activity for
all the strains studied.
The results obtained with the series CQ confirm those ob-
tained with series C with the exception of C. albicans for which
the MIC values are higher than the detection threshold (Fig. 4).
For this series, the most important feature is the fact that the
presence of the amide group as connector between the charged
nitrogen and the hydrocarbon chain bearing the thiol function
does not largely disturb the microbiostatic activities of these
surfactants even if their surface properties (CMC and gS) are
slightly decreased. So comparatively with the SAMs formed
with the compounds of the series C, we can expect SAMs of
best quality (higher density) with the compounds of series CQ
due to the presence of hydrogen bonds, and consequently an in-
crease of the microbiostatic activity of the treated metal surface.
3. Experimental section
3.1. Chemical synthesis
3.1.1. General
1H and 13C Nuclear Magnetic Resonance (NMR) spectra
were recorded on a Bruker AC 200 spectrometer. Signal attri-
¨
butions were confirmed by HMQC (2D NMR) experiments.
The mass spectra were recorded on a Finnigan Mat LCQ Clas-
sic Electrospray source API1. Elemental analyses were per-
formed using a Flash EA1112 automatic elemental analyzer
from Thermo Scientific equipped with Eager 300 software.
2.2.2. Microbicidal activity
Table 2 summarizes the values of MLC evaluated for all the
compounds of series C and CQ. Only three values can be
1600
700
Staphylococcus aureus
Pseudomonas aeruginosa
Candida albicans
1400
1200
1000
800
600
400
200
0
Staphylococcus aureus
Pseudomonas aeruginosa
Aspergillus niger
600
500
400
300
200
100
0
Aspergillus niger
0
2
4
6
8
10
12
14
16
0
2
4
6
8
10
12
14
16
length of hydrocarbon chain
length of hydrocarbon chain
Fig. 4. Variation of the MIC of series CQ as a function of the length of the
hydrocarbon chain.
Fig. 3. Variation of the MIC of series C as a function of the length of the hy-
drocarbon chain.

P. Thebault et al. / European Journal of Medicinal Chemistry 44 (2009) 717e724
721
Table 2
MLC values of the molecules of series C and CQ
ethanolic solution). The mixture is stirred at room temperature
for 48 h. After concentration, the residue is dissolved in di-
chloromethane (20 mL), and then precipitated by addition of
hexane (300 mL) to afford, after filtration, N,N,N-trimethylun-
dec-10-enamonnium bromide (yield: 91%). The corresponding
thiol C1 is obtained according to the same pathway as previ-
ously described (cf. Section 3.1.2.3) with a yield of 85%.
3.1.2.4.1. Compound C1. Yield: 77%. 1H NMR (200 MHz,
MeOD): d/ppm ¼ 1.32 (m, 14H [eCH2e]7), 1.59 (m, 2H
[SCH2eCH2]), 1.81 (m, 2H [NeCH2eCH2]), 2.42 (t, 2H
[SeCH2]), 3.19 (s, 9H [Nþ(CH3)3]), 3.44 (m, 2H [NeCH2]).
13C NMR (MeOD): d/ppm ¼ 24.2 (NeCeC ), 25.6 (SeC ),
30.0 (SeCeCeC7), 35.3 (SeCeC ), 53.1 (NeCH3), 68.6
(NeCH2). MS, [MeCl]þ, m/z (%): 246 (100). Anal. Calcd
for C14H32NSCl: C 59.64, H 11.44, N 4.97, S 11.37; found:
C 59.64, H 11.51, N 4.99, S 11.06.
Molecules
MLC (mmol/L)
Staphylococcus
aureus
Pseudomonas
aeruginosa
Candida
albicans
Aspergillus
niger
C1
56.6
681.6
47.95
>1116.88
>1528.65
308.9
>1904.81
>896.2
>61.29
>63.36
>1524.6
>131.10
681.6
47.95
CBz
C10
C12
C14
73.3
>1524.6
326.2
>1524.6
>92.22
>123.72
CQ1
>395.2
946.17
32.50
38.2
>864.49
>1057.32
249.88
>1065.06
>1057.32
>676.80
>687.60
>741.60
>395.2
>946.2
>60.74
>61.71
>74.16
CQBz
CQ10
CQ12
CQ14
183.5
598.67
187.7
3.1.2.4.2. Compound CBz. Yield: 67%. 1H NMR
(200 MHz, MeOD): d/ppm ¼ 1.30 (m, 14H [eCH2e]7), 1.64
(m, 2H [SCH2eCH2]), 1.70 (m, 2H [NeCH2eCH2]), 2.42
(t, 2H [SeCH2]), 3.11 (s, 6H [Nþ(CH3)2]), 3.40 (m, 4H
[CH2eNeCH2]), 4.56 (s, 2H, [CH2eC6H5]), 7.60 (m, 5H
[C6H5]). 13C NMR (MeOD): d/ppm ¼ 24.0 (NeCeC ), 25.2
(SeC ), 30.0 (SeCeCeC7), 35.5 (SeCeC ), 51.6 (NeCH3),
65.0 (NeCH2), 69.8 (NeCH2eC6H5), 129.2e131.2e132.4e
135.4 (C6H5). MS, [MeCl]þ, m/z (%): 322.6 (100). Anal.
Calcd for C19H36NSCl: C 65.65, H 10.49, N 4.05, S 9.26;
found: C 66.09, H 10.82, N 4.07, S 9.41.
3.1.2. General procedures for preparation of thiols without
amido connector, C1, CBz, C10, C12 and C14
3.1.2.1. Formation of N,N-dimethylundec-10-enamine. A total
of 21 millimoles (mmol) of 11-bromoundec-1-ene is added to
63 mmol of dimethylamine (5.6 M ethanolic solution). The
mixture is stirred at room temperature for 24 h, then, a 10%
aqueous solution of sodium hydroxide is added and the mix-
ture is extracted three times with diethyl ether. The organic
layers were washed, dried and then concentrated. The residue
is rectified on Kugelrohr apparatus to afford N,N-dimethylun-
dec-10-enamine (yield: 93%).
3.1.2.4.3. Compound C10. Yield: 59%. 1H NMR
(200 MHz, MeOD): d/ppm ¼ 0.79 (t, 3H [CH2eCH3]), 1.30
(m, 28H [eCH2e]7 and [eCH2e]7), 1.64 (m, 2H [SCH2e
CH2]), 1.70 (m, 4H [CH2eCH2eNeCH2eCH2]), 2.42 (t,
2H [SeCH2]), 3.11 (s, 6H [Nþ(CH3)2]), 3.40 (m, 4H [CH2e
NeCH2]). 13C NMR (MeOD): d/ppm ¼ 15.3 (CH2eCH3),
24.0 (CeCeNeCeC and CeCH3), 25.2 (SeC ), 30.0 (Se
CeCeC7 and NeCeCeC5), 33.1 (CeCeCH3), 35.5 (Se
CeC ), 51.6 (NeCH3), 65.0 (NeCH2). MS, [MeCl]þ, m/z
(%): 372.4 (100). Anal. Calcd for C23H50NSCl: C 67.68, H
12.35, N 3.43, S 7.85; found: C 67.75, H 12.57, N 3.87, S 7.69.
3.1.2.4.4. Compound C12. Yield: 62%. 1H NMR
(200 MHz, MeOD): d/ppm ¼ 0.82 (t, 3H [CH2eCH3]), 1.30
(m, 32H [eCH2e]7 and [eCH2e]9), 1.64 (m, 2H [SCH2e
CH2]), 1.70 (m, 4H [CH2eCH2eNeCH2eCH2]), 2.42 (t,
2H [SeCH2]), 3.11 (s, 6H [Nþ(CH3)2]), 3.40 (m, 4H [CH2e
NeCH2]). 13C NMR (MeOD): d/ppm ¼ 15.1 (CH2eCH3),
24.0 (CeCeNeCeC and CeCH3), 25.2 (SeC ), 30.0 (Se
CeCeC7 and NeCeCeC7), 33.5 (CeCeCH3), 35.5 (Se
CeC ), 51.6 (NeCH3), 65.0 (NeCH2). MS, [MeCl]þ, m/z
(%): 400.4 (100). Anal. Calcd for C25H54NSCl: C 68.83, H
12.48, N 3.21, S 7.35; found: C 68.98, H 12.60, N 4.99, S 7.16.
3.1.2.4.5. Compound C14. Yield: 61%. 1H NMR
(200 MHz, MeOD): d/ppm ¼ 0.78 (t, 3H [CH2eCH3]), 1.30
(m, 36H [eCH2e]7 and [eCH2e]11), 1.64 (m, 2H [SCH2e
CH2]), 1.70 (m, 4H [CH2eCH2eNeCH2eCH2]), 2.42 (t,
2H [SeCH2]), 3.11 (s, 6H [Nþ(CH3)2]), 3.40 (m, 4H [CH2e
NeCH2]). 13C NMR (MeOD): d/ppm ¼ 15.6 (CH2eCH3),
24.0 (CeCeNeCeC and CeCH3), 25.2 (SeC ), 30.0
(SeCeCeC7 and NeCeCeC9), 33.1 (CeCeCH3), 35.5
3.1.2.2. Formation of the unsaturated quaternary ammonium
bromides. A total of 5 mmol of ethylenic amine previously
prepared is added to 6 mmol of the selected 1-bromoalcane,
RBr. The mixture is stirred at 80 ꢀC for 48 h. After cooling,
20 mL of diethyl ether are added at room temperature. The
precipitate obtained is filtered and washed several times with
diethyl ether to give the expected ethylenic quaternary ammo-
nium bromide with yields of 89%, 83%, 83% and 87% for
R ¼ CH2C6H5, C10H21, C12H25 and C14H29, respectively.
3.1.2.3. Formation of thiols CBz, C10, C12 and C14. A total
of 10 mmol of the selected unsaturated quaternary ammonium
bromide is mixed with 30 mmol of thioacetic acid in a little
amount of methanol. The mixture is irradiated for 12 h using
a 300-W ultraviolet lamp (high pressure quartzemercury
arc). After solvent and residual thioacetic acid evaporation,
the crude product is triturated several times with diethyl ether.
Small quantities of methanol and 10% aqueous solution of hy-
drochloric acid are added. The mixture is stirred at 105 ꢀC for
2 h. Methanol and water are then evaporated under reduced
pressure and a suspension of charcoal in methanol is added.
The mixture is stirred at room temperature for 4 h. After filtra-
tion on celite, the filtrate is concentrated and the residue is
lyophilised to give the expected thiol with yields of 81%,
76%, 80% and 76% for CBz, C10, C12 and C14, respectively.
3.1.2.4. Formation of thiol C1. 11-Bromoundec-1-ene
(25 mmol) is added to 82 mmol of trimethylamine (4.2 M

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P. Thebault et al. / European Journal of Medicinal Chemistry 44 (2009) 717e724
(SeCeC ), 51.6 (NeCH3), 65.0 (NeCH2). MS, [MeCl]þ, m/z
(%): 428.4 (100). Anal. Calcd for C27H58NSCl: C 69.85, H
12.59, N 3.02, S 6.90; found: C 69.77, H 12.71, N 3.09, S 6.85.
concentrated and the residue is lyophilised to give the ex-
pected thiol with yields of 94%, 90%, 92%, 91% and 94%
for CQ1, CQBz, CQ10, CQ12 and CQ14, respectively.
3.1.3.4.1. Compound CQ1. Yield: 44%. 1H NMR
(200 MHz, MeOD): d/ppm ¼ 1.3 (m, 12H [SeCeCe
(CH2)6]), 1.62 (m, 4H [SeCeCH2 and CH2eCeC(O)]),
1.96 (m, 2H [CH2eCeNþ]), 2.08 (t, J ¼ 7.5 Hz, 2H [CH2e
C(O)]), 2.51 (t, J ¼ 7.1 Hz, 2H [SeCH2]), 3.07 (s, 9H
[N(CH3)3]), 3.35 (m, 2H [NHeCH2]), 3.43 (m, 2H [CH2e
Nþ]). 13C NMR (200 MHz, MeOD): d/ppm ¼ 24.2 (CeCe
Nþ), 25.1 (SeC ), 27.2 (CeCeCO), 30.0 (SeCeCeC6),
34.9 (SeCeC ), 35.3 (CeCO), 37.8 (NHeC ), 53.4
[N(CH3)3], 66.9 (CeNþ), 177.4 (CO). MS, [MeCl]þ, m/z
(%): 317.3 (100). Anal. Calcd for C17H35N2OSCl: C 58.17,
H 10.05, N 7.98, S 9.13; found: C 58.96, H 9.99, N 7.96, S
9.03.
3.1.3.4.2. Compound CQBz. Yield: 41%. 1H NMR
(200 MHz, MeOD): d/ppm ¼ 1.3 (m, 12H [SeCeCe
(CH2)6]), 1.60 (m, 4H [SeCeCH2 and CH2eCeC(O)]),
1.91 (m, 2H [CH2eCeNþ]), 2.12 (t, J ¼ 7.5 Hz, 2H [CH2e
C(O)]), 2.48 (t, J ¼ 7.1 Hz, 2H [SeCH2]), 3.06 (s, 6H
[N(CH3)2]), 3.30 (m, 4H [NHeCH2 and CH2eNþ]), 4.60
(m, 2H [CH2eC6H5]), 7.60 (m, 5H [C6H5]). 13C NMR
(200 MHz, MeOD): d/ppm ¼ 24.0 (CeCeNþ), 25.2 (SeC ),
27.2 (CeCeCO), 30.0 (SeCeCeC6), 35.4 (SeCeC and
CeCO), 37.4 (NHeC ), 51.2 [N(CH3)2], 63.6 (CeNþ), 66.9
(CeC6H5), 129.3e134.8e130.1e132.6 (C6H5), 177.8 (CO).
MS, [MeCl]þ, m/z (%): 393.3 (100). Anal. Calcd for
C22H39N2OSCl: C 63.66, H 9.47, N 6.75, S 7.56; found: C
63.87, H 9.70, N 6.73, S 7.56.
3.1.3. General procedures for preparation of thiols with
amido connector, CQ1, CQBz, CQ10, CQ12 and CQ14
3.1.3.1. Thiol protection. 11-Bromoundecanoic acid (5.3 g,
20 mmol) and potassium thioacetate (2.5 g, 22 mmol) are dis-
solved in acetonitrile (60 mL) under nitrogen and the mixture
is heated at 85 ꢀC for 12 h. The volatiles are removed under
vacuum and 40 mL of water are added. The solution is ex-
tracted three times with diethyl ether. The organic layers are
washed with water, dried on Na2SO4 and then concentrated
under reduced pressure. The residue is purified by column
chromatography over silica gel using hexane as the eluant.
11-Thioacetoxyundecanoic acid is obtained as a beige solid
(4.3 g, 82%).
3.1.3.2. Amide formation. Previously prepared 15 mmol
(3.9 g) of 11-thiacetoxyundecanoic acid, 15 mmol (2.87 g) of
N-(3-dimethylaminopropyl)-N0-ethyl-carbodiimide hydrochlo-
ride and 1.5 mmol (0.18 g) of 4-dimethylaminopyridine are
added to 50 mL of dichloromethane. The mixture is stirred
for 4 h at room temperature and then 15 mmol (1.53 g) of 3-
N,N-dimethylaminopropylamine are added at 0 ꢀC. After stir-
ring for 24 h at room temperature, the reaction mixture is
washed three times with water. The organic layer is separated,
dried and then concentrated under reduced pressure. The crude
product is obtained as a beige solid (3.6 g, 70%).
3.1.3.4.3. Compound CQ10. Yield: 42%. 1H NMR
(200 MHz, MeOD): d/ppm ¼ 0.79 (t, 3H [CeCH3]), 1.3 (m,
26H [SeCeCe(CH2)6 and NþeCeCe(CH2)7]), 1.60 (m,
6H [SeCeCH2, CH2eCeC(O) and NþeCeCH2]), 1.91 (m,
2H [CH2eCeNþ]), 2.12 (t, J ¼ 7.5 Hz, 2H [CH2eC(O)]),
2.48 (t, J ¼ 7.1 Hz, 2H [SeCH2]), 3.06 (s, 6H [N(CH3)2]),
3.30 (m, 6H [NHeCH2 and CH2eNþeCH2]).
13C NMR (200 MHz, MeOD): d/ppm ¼ 15.4 (CeCH3),
23.1 (NþeCeC ), 24.0 (CeCeNþ), 24.4 (CeCH3), 25.2
(SeC ), 27.2 (CeCeCO), 30.0 (SeCeCeC6), 33.2 (CeCe
CH3), 35.4 (SeCeC and CeCO), 37.4 (NHeC ), 51.2
[N(CH3)2], 63.6 (CeNþ), 65.4 (NþeC ), 177.8 (CO). MS,
[MeCl]þ, m/z (%): 443.4 (100). Anal. Calcd for
C26H53N2OSCl: C 65.44, H 11.19, N 5.87, S 6.72; found: C
65.72, H 11.2770, N 5.64, S 6.59.
3.1.3.4.4. Compound CQ12. Yield: 42%. 1H NMR
(200 MHz, MeOD): d/ppm ¼ 0.74 (t, 3H [CeCH3]), 1.3 (m,
30H [SeCeCe(CH2)6 and NþeCeCe(CH2)9]), 1.60 (m,
6H [SeCeCH2, CH2eCeC(O) and NþeCeCH2]), 1.91 (m,
2H [CH2eCeNþ]), 2.12 (t, J ¼ 7.5 Hz, 2H [CH2eC(O)]),
2.48 (t, J ¼ 7.1 Hz, 2H [SeCH2]), 3.06 (s, 6H [N(CH3)2]),
3.30 (m, 6H [NHeCH2 and CH2eNþeCH2]). 13C NMR
(200 MHz, MeOD): d/ppm ¼ 15.9 (CeCH3), 22.9 (NþeCe
C ), 24.0 (CeCeNþ), 24.2 (CeCH3), 25.2 (SeC ), 27.2
(CeCeCO), 30.0 (SeCeCeC8), 32.8 (CeCeCH3), 35.4
(SeCeC and CeCO), 37.4 (NHeC ), 51.2 [N(CH3)2], 63.6
(CeNþ), 65.8 (NþeC), 177.8 (CO). MS, [MeCl]þ, m/z (%): 471.4
3.1.3.3. Quaternization of the protected thiol. Excepted for the
trimethyl ammonium derivative, 10 mmol of the resulting ter-
tiary amine are added to 12 mmol of the selected 1-bromoal-
cane (decyl bromide, dodecyl bromide, tetradecyl bromide
or benzyl bromide). The solution is stirred and heated at
80 ꢀC for 2 days. After cooling, 20 mL of diethyl ether and
the final product precipitates. The mixture is filtered and the
salt obtained is washed several times with diethyl ether. The
crude product was obtained as beige solid [4.35 g (77%),
5 mg (84%), 5.15 g (83%) and 313 mg (80%) for
RH ¼ C10H21, C12H25, C14H29 and CH2C6H5, respectively].
For the compounds with RH ¼ CH3, 10 mmol of the precur-
sor are added to a solution of 40 mmol of methyl iodide in
30 mL of anhydrous diethyl ether. The solution was stirred
and heated at 40 ꢀC for 2 days. The final product precipitates.
The mixture was filtered and the salt obtained was washed sev-
eral times with diethyl ether. The product 3 was obtained as
beige solid (3.93 g, 81%).
3.1.3.4. Thiol deprotection. A total of 7 mmol of the selected
protected thiol is dissolved in a minimal volume of methanol
and 10% aqueous solution of hydrochloric acid. The solution
is stirred at 100 ꢀC for 1 h. After removal of methanol and wa-
ter under vacuum, a suspension of charcoal in methanol is
added to the crude product. The mixture is stirred at room tem-
perature for 4 h. After filtration on celite, the filtrate is

P. Thebault et al. / European Journal of Medicinal Chemistry 44 (2009) 717e724
723
(100). Anal. Calcd for C28H57N2OSCl: C 66.56, H 11.37, N
5.54, S 6.34; found: C 66.70, H 11.49, N 5.30, S 6.27.
exponential phase. Bacteria were grown overnight on a TSA
slant and inoculated into 20 mL of M9GY at pH 7.0 ꢁ 0.2
culture medium and incubated overnight at 30 ꢀC. The optical
density of the suspension was then measured at 660 nm. If
necessary, additional M9GY medium was added to the
suspension to adjust the optical density at 660 nm to 0.05
corresponding approximately to between 5.106 and 5.107 cfu/
mL.
Fungi were incubated on SDA slants for 5 days at 25 ꢀC.
MIC determinations were run with the third generation of
fungi; culture samples were taken during the exponential
phase of fungal growth. Each fungal strain was cultivated
during 5e7 days on SDA at 25 ꢁ 2 ꢀC until sufficient spores
were formed. Then fungal spores were harvested by adding
5 mL of M9GY at pH 5.0 ꢁ 0.2 to the SDA slant, which
were then gently scraped to suspend the microorganisms.
The fungal solution is then filtered with sterile cheesecloth
under aseptic conditions to eliminate the residual mycelium.
The suspension was adjusted using a counting cell under
microscope, to 5.106e5.107 spores/mL by adding M9GY if
necessary.
3.1.3.4.5. Compound CQ14. Yield: 43%. 1H NMR
(200 MHz, MeOD): d/ppm ¼ 0.82 (t, 3H [CeCH3]), 1.3 (m,
34H [SeCeCe(CH2)6) and NþeCeCe(CH2)11], 1.60 (m,
6H [SeCeCH2, CH2eCeC(O) and NþeCeCH2]), 1.91 (m,
2H [CH2eCeNþ]), 2.12 (t, J ¼ 7.5 Hz, 2H [CH2eC(O)]),
2.48 (t, J ¼ 7.1 Hz, 2H [SeCH2]), 3.06 (s, 6H [N(CH3)2]),
3.30 (m, 6H [NHeCH2 and CH2eNþeCH2]). 13C NMR
(200 MHz, MeOD): d/ppm ¼ 13.1 (CeCH3), 22.6 (NþeCe
C ), 24.0 (CeCeNþ), 24.4 (CeCH3), 25.2 (SeC ), 27.2
(CeCeCO), 30.0 (SeCeCeC10), 33.0 (CeCeCH3), 35.4
(SeCeC and CeCO), 37.4 (NHeC ), 51.2 [N(CH3)2], 63.6
(CeNþ), 65.7 (NþeC ), 177.8 (CO). MS, [MeCl]þ, m/z (%):
499.4 (100). Anal. Calcd for C30H61N2OSCl: C 67.56, H
11.53, N 5.25, S 6.01; found: C 67.75, H 11.35, N 5.37, S 5.99.
3.2. Surfactant property evaluation
The Critical Micellar Concentrations (CMCs) were ob-
tained by electrical conductivity measurements [28] at 25 ꢀC
using a conductimeter apparatus Consort C831. The surface
tensions (gs) were determined at 25 ꢀC by the Wilhelmy plate
Initial solution concentration of each antimicrobial agent
was prepared in a mixture of methanol/sterile deionised water
(ratio 1/1).
method using a tensiometer Kruss K100 [22,29,30]. The sub-
¨
strates being insoluble in pure water, a 10% methanolic aque-
ous solution was used. It was demonstrated that the CMCs
varies only slightly with 10% of methanol in water [31].
4. Conclusion
The two series, C and CQ, of double tailed cationic sur-
factants synthesised in this work show antimicrobial
activities against the four classical strains studied here.
For both series, the best efficiency is observed when the
length of the hydrocarbon chain is 10 carbon atoms. It is
the ‘‘cut-off effect’’ of this type of preservative. So, the fu-
ture elaboration of contact-active auto-bactericidal metal
surfaces via SAMs formation on metal surfaces using these
dialkyldimethylammonium chlorides (particularly, C10e
C14 and CQ10eCQ14) bearing a thiol function at the
end of one of the alkyl groups can be successfully
envisaged. Although the presence of a thiol function at the
end of one of the tails slightly decreases the tensioactive
properties of these quaternary ammonium salts in compari-
son with the unsulfurated homologues, their microbiostatic
and microbicidal activities against the four strains studied
remain. Moreover, the presence of an amido connector
between the charged nitrogen and the thiol function (series
CQ versus series C) does not largely affect the antibacterial
properties of the most efficient of these preservatives. This
is an important point concerning the future formation of
SAMs, since one of our previous works showed that the
presence of this amido connector, and consequently of
possible hydrogen bond formations, seem to be necessary
to compensate the repulsive electric force between the
charged nitrogen atoms of two neighbouring monomers in
the SAMs and consequently to obtain the best SAMs.
The next step of this study is the SAMs formation on var-
ious metals (particularly, gold and iron) and the antibacterial
evaluation of the treated surfaces obtained.
3.3. Antimicrobial characteristics
3.3.1. MIC determination
MIC is defined as the lowest concentration of an antimicro-
bial activity that will inhibit the visible growth of a microor-
ganism after overnight incubation. The MICs were taken as
the minimal concentration showing no growth (absence of tur-
bidity) after 24 h of incubation at 30 ꢀC for bacteria and after
72 h of incubation at 25 ꢀC for fungi. MICs were determined
using a serial dilution method. The automat BiomekÒ 1000
(BeckmanÒ) apparatus used for our experiments performed
automatically dilutions of the tested antimicrobial agents solu-
tions in 96 well micro-titration plates.
3.3.2. MLC determination
The MLC is defined as the lowest concentration of antimi-
crobial activity that will prevent the growth of a microorganism
after subculture onto antibiotic-free media. The MLC levels
were determined by taking (after 5 days incubation for bacte-
ria or 7 days for fungi) from MIC microtiter plates, 100 ml
from the three sub-doses wells where no growth was observed,
and subculture by streaking onto trypticase soy agar (TSA) or
Sabouraud Dextrose Agar (SDA). Plates are incubated as de-
scribed below and checked after incubation period to deter-
mine whether or not viable microorganisms remain.
3.3.3. Cultivation
MIC determinations were run with the third generation of
bacteria or fungi. For bacteria, samples were taken during the

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P. Thebault et al. / European Journal of Medicinal Chemistry 44 (2009) 717e724
[13] P. Thebault, E. Taffin de Givenchy, C. Guimon, F. Guittard, S. Geribaldi,
Thin Solid Films 516 (2008) 1765e1772.
Acknowledgments
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261e268.
This work was supported by the ‘‘Region PACA’’ Council,
the Society ARECO (Grasse), and Rohm and Haas France.
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