This document summarizes the synthesis of new imidazolium salts intended for use as vectors for siRNA transfection. It describes the step-by-step synthesis of intermediates including 1-alkyl-3-[3,4-bis(dodecyloxy)benzyl]-4H-imidazolium chloride and its deprotected form. It also discusses the inhibition of lactoperoxidase-catalyzed oxidation by an imidazole-based thione synthesized from one of the intermediates. The synthesis routes achieved good yields for the intermediates and products, which were characterized by various analytical techniques. The document concludes by discussing the different potential applications of the synthesized compounds.

1. 1 / 7
Mesomorphic Imidazolium Salts: New vectors for efficient siRNA Transfection
Jing YI
Abstract
The chloride derivatives of 1-alkyl-3-[3,4-bis(dodecyloxy)benzyl]-4H-imidazolium were
prepared by the reaction of derivates N-substituted imidazole with 1-chloromethyl-3,4-
bis(dodecycloxy)benzene in acetonitrile solvent. Such cationic amphiphiles were expected to
be candidate molecules to design a new delivery reagent for nucleic acid transfection,
particularly for short interfering RNA (siRNA). The use of an RNA interference mechanism,
by introduction into cells by transfection of chemically synthesized siRNAs, is a powerful
method for gene silencing studies. One of these ionic liquids has been further deprotected, the
other one was used to synthesize a imidazole-based thione, this in turn, was supposed to
inhibit the lactoperoxidase-catalyzed oxidation.
Key words : bioorganic chemistry, heterocycles, ionic liquid, inhibitors, liquid crystal
Introduction
Ambient temperature molten salts (ionic
liquids) which are having notable
properties such as low flammability, low
volatility and wide liquid temperature
range.1
Furthermore attractive properties
such as polarity, viscosity and melting
temperature can be changed with respect to
counter anions. They have negligible vapor
pressure, thermal stability, large
electrochemical window, insoluble in super
critical CO2 and easily soluble in large
range of organic molecules and transition
metal complexes,1
especially those
comprising mixtures of N,N’-
dialkylimidazolium are increasingly
finding a range of laboratory,
developmental, and technical applications,
for example, as media for organic and
inorganic chemical synthesis, in materials
production, in electrochemical and
separation processes, and as prototype
novel materials. 1-3
Much current research
is focused upon the possible use of ionic
liquids as media for cleaner organic
synthesis and processing 4
and upon better
understanding of liquid structure and
solvation phenomena of ionic liquids and
its relevance to reactivity.2,5,6
Recently ionic liquids have been used as
media for the preparation of some
bioactive molecules, such as new vectors
for efficient siRNA transfection7
, and
usage for inhibition of lactoperoxydase-
catalyzed oxidation (LPO). 8
(Scheme 1)
Scheme 1: synthesis of compound 7 from 6, compound 8
from 6’
Scheme 1 illustrates two final products
of our synthesis. Where compound 7
represents an imidazole-based thione,
which can inhibit LPO-catalyzed
oxidation/iodination due to its ability to
decrease the concentrations of the co-

2. 2 / 7
substrates, H2O2 and I2 by forming stable
charge-transfer complexes with oxidized
iodide species, and it can be used as anti-
thyroid drug; compound 8 can be used as
an intermediate of liquid crystal synthesis.
Results and discussion
Scheme 2: synthetic procedure of intermediates 2-6/6’.
Reagents and conditions : i) 1-bromododecane/DMSO,
K2CO3, reflux, 4h ; ii) LiAlH4, dry THF, TA, 1/4 h ; iii)
SOCl2, dry CH2Cl2, TA, 4h ; iv) CH3CN, reflux, 12h.
Synthesis of intermediates 9
The product 2 (Scheme 2) was firstly
synthesized by etherifying methyl 3,4-
dihydroxybenzoate with 1-bromododecane
in the presence of K2CO3 in refluxing
DMSO, the use of K2CO3 allows the
deprotonation of two OH groups, therefore,
the oxygen of these groups possess highly
nucleophilic character. The first OH to be
deprotonated is situated on para position of
aromatic ring, this has been proven by the
higher stability of corresponding phenolate
due to mesomeric effect. (Scheme 3).
While meta phenolate is less stable and
reacts later.
Scheme 3: stabilization of para phenolate by mesomeric
effect
The amphipathic ester 2 obtained by the
previous reaction was then reduced by
catalytic quantity of LiAlH4 in dry THF
into the corresponding benzyl alcohol 3.
The resulting alcohol 3 was further
converted into 3,4-bis(dodecycloxy)benzyl
chloride with thionyl chloride 4 in dry
CH2Cl2 for 3h, a typical SN2
mechanism is
applied for this reaction as the compound 3
is a primary alcohol. The final
intermediates, compound 6 and 6’ were
obtained by quaternalization of 1-
methylimidazole 5 or 3-imidazole-1-yl-
propionitrile 5’ with de chloride 4 under
refluxing CH3CN in inert atmosphere for a
night, an SN2
mechanism was involved due
to the steric environment of compound 4.
As compound 5’ was synthesized by
Michael Addition of imidazole and
propionitrile under refluxing for a night,
we obtained two different IL, which were
purified by crystallization in a non-polar
solvent, Et2O, where IL is immiscible; and
characterized by IR spectroscopy, melting
point, 1
H NMR and 13
C NMR. These
intermediates have been synthesized in
good yield. (Scheme 4)
Product 2 3 4 5’ 6 6’
Yield 91% 94.5% 77% 99.5% 96% 90%
Scheme 4: yields of intermediates synthesis
Distinctive signals assigned to the CH
group at the second position of the
imidazolium ring appear in the 1
H NMR
spectra of 6 and 6’ at δ = 11.019 and
10.931 ppm. While the increasing order of
acidity of IL was based on chemical shift
of these protons10
, we can assume that
compound 6 is more acid than compound
6’. However, more investigations should
be carried out in order to prove our

3. 3 / 7
hypothesis, such as its pH test in melted
liquid form. The interest of acid character
of IL, which depends on type of substituent
on imidazolium ring as well as type of
anion, is to modify acid-base properties of
IL, and thus to discover new applications
of this materiel, for example, the usage of
pyrazole-like strong acidity IL in
preparation of biodiesel.11
Besides, the melting points of these two
IL show a light dependence upon
substituents on the imidazole ring, where
Mp (6) = 68°C, Mp (6’) = 74°C. The
application of IL depends on this property,
such as electrolyte solvent in the battery,
has been developed quickly nowadays.
Finally, the IR spectra of 6 and 6’ showed
a large band at 3380 cm-1
, which indicated
the water presence in these products. While
the counteranion Cl-
is a small ion, it’s
easy to form H bond between it and water.
As a result, the dryness of these IL is hard
to achieve. However, by changing
counteranion, we could minimize its
solubility in water, such as PF6
-
etc.
Inhibition of lactoperoxydase-catalyzed
oxidation by imidazole-based thiones
and selones
N,N-disubstituted thione 7 was
synthesized by treating compound 6 by its
reaction with elemental sulfur. In this
reaction, deprotonation of the H at the
second position of imidazolium salt by a
base NaOH leads to an in situ generation
of reactive carbene, which, in turn, reacts
with elemental sulfur to afford the
corresponding thione (Scheme 1). The
carbene intermediate obtained is a Fischer
carbene due to π backdonation provided by
nitrogen and sulfur. The final product
obtained was then purified by flash
chromatography and characterized by 1
H
NMR, 13
C NMR and also IR spectroscopy.
Comparing to 13
C NMR (DEPT) and 1
H
NMR spectra of reactant 6, which possess
six aromatic/imidazolium H, and three
quaternary carbons, the product thione 7
lost one H on imidazole ring, but got one
more quaternary carbon at the second
position of imidazole ring. This, thereby,
allowed us to suppose that the formation of
C=S bond has been succeeded at the
corresponding position. However, more
analysis are necessary in order to turn out
our hypothesis to be true, such as mass
spectrometry etc. Further bio-catalytic
reaction could be carried out to prove its
inhibitory activity.
Deprotection of imidazole derivative
The compound 8 was obtained by
deprotection in a basic NaOH medium of
imidazolium salt 6’. After purification by
crystallization, the pure product was
isolated and characterized by 1
H NMR, 13
C
NMR and also IR spectroscopy.
The propionitrile group disappeared on
imidazole ring, which has been proven by
the 1
H NMR spectra. However, this allows
the liberation of a nitrogen site, which can
further react with a same chloride 4, or
another chain length type chloride, to form
a symmetric or asymmetric N,N-
disubstitued imidazolium. This
manipulation permits the synthesis of
liquid crystal.
Conclusion
price €
for 0.55g
of 6
280.4
price €
for 0.35g
of 6'
279.4
price €
for 0.41g
of 7
280.5
price €
for 0.15g
of 8
281.84
Scheme 5 : prices of synthesis

4. 4 / 7
We have prepared two imidazole
derivatives where their application is
totally different, we have also determinate
the price of each synthesis (Scheme 5). But
we are now attempting to investigate their
biological activities.
Experimental section
1-Methyl-3,4-bisdodecycloxybenzoate
(2) : The reaction was carried out under
argon atmosphere. Methyl 3,4-
dihydroxybenzoate (1g, 6mmol) and 1-
bromododecane (4.49g, 18mmol) were
dissolved in degassed suspension of
potassium carbonate (3.3g, 24mmol) in
DMF (25mL), then the mixture was heated
to 70°C. After 4h, the advancement of the
reaction was indicated by TLC method
until no more ester was observed. The
reaction mixture was cooled to room
temperature, and then poured into cold
water (200mL). The white solid was
filtrated then dissolved in a minimum hot
dichloromethane, this mixture was then
added methanol (200mL) and stirred in an
ultra-sonic machine. A white mousse was
obtained and filtrated. The residue was
then dissolved in dichloromethane and
purified by flash chromatography column
(Silica, dichloromethane/cyclohexane=1/1).
2.75g (5mmol, 91%) of a white solid was
yielded. TLC
(dichloromethane/cyclohexane=8/2) : Rf =
0.78.1
H NMR (300MHz; CDCl3) : δ :
0.875 (t, 6H); 1.258 (m, 32H); 1.468 (m,
4H); 1.807 (m,4H); 3.876 (s, 3H); 4.034
(td, 4H), 6.85 (d, 1H); 7.53 (d, 1H); 7.62
(dd, 1H). IR : vmax/cm-1
2918 (CH
aliphatic), 1709 (C=O ester), 1519 (C=C
aromatic), 1272 and 1213 (C-O aromatic
ether), 1132 and 1106 (C-O ester).
[3,4-bis(dodecyloxy)phenyl]methanol
(3) : the reaction was carried out under
argon atmosphere. 1 (1.5g, 3mmol) was
dissolved in dry THF (20mL) in a Shlenk
tube. LiAlH4 (0.0987g, 2.6mmol) was
added and the mixture was stirred during
1/4h, the advancement of the reaction was
followed by TLC. Cold water (20mL) was
added and then HCl acid was used to
adjust pH (pH<4). The product was
extracted with dichloromethane (5x15mL)
and concentrated. A white solid (1.35g,
2.8mmol, 94.5%) was obtained. TLC
(dichloromethane/cyclohexane=6/4).1
H
NMR (300MHz; CDCl3) : δ : 0.88 (t, 6H);
1.263 (m, 32H); 1.46 (m, 4H); 1.81 (m,4H);
3.99 (td, 4H); 4.6 (s, 2H); 6.853 (s, 1H);
6.855 (s, 1H); 6.923 (s, 1H). IR : vmax/cm-1
3314 (OH alcohol), 2917 (CH
aliphatic),1520 (C=C aromatic), 1264 and
1237 (C-O aromatic ether).
3,4-bis(dodecycloxy)benzyl chloride (4) :
2 (1.04g, 1.5mmol) was added in dry
dichloromethane (10mL), the reaction
allowed to stir for 3h at rt under argon after
which was added thionyl chloride (0.25g,
2.1mmol). TLC allowed monitoring the
advancement of the reaction. A neutral
mixture was obtained by adding a K2CO3
solution. The product was washed with
water (3x10mL), then dried over
anhydrous MgSO4 and concentrated. A
white solid (0.85g, 1.6mmol, 77%) was
given. TLC (dichloromethane/methanol =
98/2). 1
H NMR (300MHz; CDCl3) : δ :
0.881 (t, 6H); 1.264 (m, 32H); 1.43 (m,
4H); 1.78 (m,4H); 3.99 (td, 4H); 4.545 (s,
2H); 6.806 (s, 1H); 6.833 (s, 1H); 6.89 (dd,
1H). IR : vmax/cm-1
2917 (CH aliphatic),
1467 (C-Cl), 1518 (C=C aromatic), 1273
and 1236 (C-O aromatic ether).

5. 5 / 7
1-Methyl-3-[3,4-bis(dodecyloxy)benzyl]-
4H-imidazolium chloride (6) : 4 (0.5g,
1mmol) and freshly distilled 1-
methylimidazole 5 (0.123g, 1.5mmol) were
stirred in refluxing dry CH3CN (3mL) for
a night under argon. 0.55g (0.95mmol,
96%) of 6 was obtained as a white solid
after precipitation in Et2O. 1
H NMR
(300MHz; CDCl3) : δ : 0.871 (t, 6H); 1.254
(m, 32H); 1.43 (m, 4H); 1.80 (m,4H); 3.97
(td, 4H); 4.074 (s, 3H); 5.431 (s, 2H); 6.84
(d, 1H); 6.95 (dd, 1H); 6.98 (d, 1H); 7.076
(s, 1H); 7.146 (s, 1H); 11.019 (s, 1H). 13
C
NMR (500MHz; CDCl3) :
δ : 139.208, 124.888, 122.665, 122.16, 121
.144, 114.507, 113.887, 113.827, 113.082,
69.812, 69.423, 53.874, 36.87, 32.146, 29.
92, 29.855, 29.682, 29.634, 29.593, 29.473
, 29.426, 29.382, 26.257, 26.233, 22.916, 1
4.35 ppm. IR : vmax/cm-1
2917 (CH
aliphatic), 1520 (C=C aromatic), 1269 and
1243 (C-O aromatic ether).
3-Imidazole-1-yl-propionitrile (5’):
imidazole (3g, 44mmol) was distilled to
dry (Bp=40°C at 1mmHg), then added to
propionitrile (3.6mL, 55mmol) and the
mixture was heated to 80°C for a night.
The extra reactant was removed under
vacuum. The product was obtained
(43.8mmol, 99.5%) as yellow oil. 1
H
NMR (300MHz; CDCl3) : δ : 2.744 (t, 2H);
4.174 (t, 2H); 6.951 (s,1H); 7.009 (s, 1H);
7.477 (s, 1H). IR : vmax/cm-1
3113 (CH
aromatic), 2963 (CH aliphatic), 2252 (CN
nitrile), 1506 (C=C aromatic).
1-Yl-propionitril-3-[3,4-
bis(dodecyloxy)benzyl]-4H-imidazolium
chloride (6’): this compound was
synthesized by the same procedure
described for the synthesis of compound 6.
From 4 (0.3g, 0.6mmol) and 5’ (0,12mL,
0.9mmol) in CH3CN (3mL), compound 6’
(0.35g, 0.6mmol, 90%) was obtained as
white solid. 1
H NMR (300MHz;
CDCl3) :δ :0.873 (t, 6H); 1.256 (m, 32H);
1.432 (m, 4H); 1.81 (m,4H); 3.346 (t, 2H);
3.97 (td, 4H); 4.874 (t, 2H); 5.333 (s, 2H);
6.9 (m, 3H); 7.079 (m, 1H); 7.647 (d, 1H);
10.931 (s,1H). IR : vmax/cm-1
2917 (CH
aliphatic), 2253 (CN nitrile), 1521 (C=C
aromatic), 1269 and 1243 (C-O aromatic
ether).
1-Methyl-3-[3,4-bis(dodecyloxy)benzyl]-
4H-imidazole-2-thione (7) : a 25mL two-
necked flask that was fitted with a reflux
condenser and septum was charged with
methylimidazolium 6 (0.55g, 0.95mmol),
sulfur (0.03g, 0.95mmol), K2CO3 ( 0.125g,
0.9mmol) and dry methanol (6mL). The
reaction was heated at 86°C for 24h to
produce a light yellow solid. The crude
product was purified by flash column
chromatography (thin silica) using
CH2Cl2/cyclohexane = 85/15 as eluent to
give thione 7 as light yellow solid (0.41g,
0.7mmol, 75%). 1
H NMR (300MHz;
CDCl3) : δ : 0.871 (t, 6H); 1.254 (m, 32H);
1.43 (m, 4H); 1.80 (m,4H); 3.97 (td, 4H);
13
C NMR (500MHz; CDCl3) :
δ : 162.672, 149.581, 149.318, 128.347, 12
1.381, 117.944, 116.357, 114.368, 113.814
, 69.483, 69.436, 51.55, 35.354, 32.12, 29.
894, 29.831, 29.646, 29.619, 29.565, 29.44
4, 26.227, 22.889, 14.325 ppm. IR :
vmax/cm-1
2918 (CH aliphatic), 1515 (C=C
aromatic), 1262 and 1233 (C-O aromatic
ether).
3-[3,4-bis(dodecyloxy)benzyl]-4H-
imidazole (8) : imidazolium 6’ (0.2g,
0.32mmol) and NaOH (0.03g, 0.64mmol)
were added in H2O (10mL) and the
mixture was stirred for 1h at rt. The
product was extracted with ether (3x20mL),
dried over anhydrous MgSO4 and

6. 6 / 7
concentrated. Compound 8 (0.29mmol,
91%) was obtained as white solid. 1
H
NMR (300MHz; CDCl3) :δ :0.878 (t, 6H);
1.26 (m, 32H); 1.43 (m, 4H); 1.7875
(m,4H); 3.942 (td, 4H); 5.016 (s, 2H); 6.65
(d, 1H); 6.7 (dd, 1H); 6.83 (d, 1H); 6.882
(s, 1H); 7.071 (s, 1H); 7.521 (s, 1H). IR :
vmax/cm-1
2917 (CH aliphatic), 1519 (C=C
aromatic), 1267 and 1240 (C-O aromatic
ether).
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(11) Qu, Guangfei; Zhang, Jun; Ning, Ping;
Shi, Yukun. Faming Zhuanli Shenqing.
2012