Novel fluorinated dialkylphosphonatocholines: Synthesis, physicochemical properties and antiprotozoal activities against Acanthamoeba spp.

Article abstract of DOI:10.1016/j.jfluchem.2014.04.008

The synthesis of four new organophosphorous gemini surfactants is presented. They belong to the class of dialkylphosphonatocholines. Tails are represented with two octyl groups both non or partially fluorinated. The synthesis was performed by reaction of alkylphosphonic acids with choline derivatives in the presence of 2,4,6-triisopropylbenzenesulfonyl chloride. The micellar, surface active, and solubilization properties of new surfactants were studied. Antiprotozoal activities were tested against Acanthamoeba lugdunensis and Acanthamoeba quina.

Full text of DOI:10.1016/j.jfluchem.2014.04.008

Accepted Manuscript
Title: Novel fluorinated dialkylphosphonatocholines:
synthesis, physicochemical properties and antiprotozoal
activities against Acanthamoeba spp.
ˇ
´ˇ
´
´
Author: Milos Lukac Maria Garajova Martin Mrva Ferdinand
´
ˇ
´
Devınsky Frantisek Ondriska Janka Kubincova
PII:
S0022-1139(14)00099-2
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jfluchem.2014.04.008
FLUOR 8304
To appear in:
FLUOR
Received date:
Revised date:
Accepted date:
18-12-2013
31-3-2014
15-4-2014
´
´ˇ
´
Please cite this article as: M. Lukac, M. Garajova, M. Mrva, F. Devinsky, F. Ondriska, J.
´
Kubincova, Novel fluorinated dialkylphosphonatocholines: synthesis, physicochemical
properties and antiprotozoal activities against Acanthamoeba spp., Journal of Fluorine
Chemistry (2014), http://dx.doi.org/10.1016/j.jfluchem.2014.04.008
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Highlights
-
-
-
synthesis of fluorinated gemini zwitterionic surfactants
the physicochemical properties expressed as micellar and solubilization properties
biological activities of prepared compounds tested against Acanthamoeba spp.
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Novel fluorinated dialkylphosphonatocholines: synthesis, physicochemical properties
and antiprotozoal activities against Acanthamoeba spp.
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Miloš Lukáča*, Mária Garajováb, Martin Mrvab, Ferdinand Devínskya, František Ondriskac,
and Janka Kubincováa
aDepartment of Chemical Theory of Drugs, Faculty of Pharmacy, Comenius University, Kalinčiakova 8, 832 32
Bratislava, Slovakia
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11
bDepartment of Zoology, Faculty of Natural Sciences, Comenius University, Mlynská Dolina B-1, 842 15
Bratislava, Slovakia
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cHPL (Ltd) Department of Parasitology, Microbiological Laboratory, Istrijská 20, 841 07 Bratislava, Slovakia
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Graphical abstract
HH - X = H, Y = H
C6Y13
O
HF - X = H, Y = F
FH - X = F, Y = H
FF - X = F, Y = F
C6X13
P
N+
O
-O
15
16
17
18
19
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21
22
23
24
Abstract – The synthesis of four new organophosphorous gemini surfactants is presented.
They belong to the class of dialkylphosphonatocholines. Tails are represented with two octyl
groups both non or partially fluorinated. The synthesis was performed by reaction of
alkylphosphonic acids with choline derivatives in the presence of 2,4,6-
triisopropylbenzenesulfonyl chloride. The micellar, surface active, and solubilization
properties of new surfactants were studied. Antiprotozoal activities were tested against
Acanthamoeba lugdunensis and Acanthamoeba quina.
Keywords: critical micelle concentration, fluorinated surfactants, miltefosine, solubilization,
trophocidal activity
* Corresponding author. Tel.: +421250117323; Fax.: +421250117357; e-mail:
lukac@fpharm.uniba.sk
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1. Introduction
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Alkylphosphocholines are compounds with interesting physicochemical [1-5] and biological
properties [6,7]. These amphiphilic compounds are classified as pH non-sensitive zwitterionic
surfactants. They contain two charges: the negative one, located at the phosphate anion, and
the positive one represented by the ammonium cation. Ions are connected with a spacer,
mainly ethylene. Critical micelle concentration (CMC) is one of the most important
physicochemical parameters describing the properties of surfactants. Alkylphosphocholines as
representatives of zwiterionic amphiphilic compounds have lower values of CMC than ionic
surfactants with similar structure [8]. They are more related to non-ionic surfactants than to
ionic ones. Antiprotozoal and anticancer activities of alkylphosphocholines are the most
interesting and the most studied biological properties of this class of compounds.
Hexadecylphosphocholine (HPC, miltefosine) is the main representative of
alkylphosphocholines. It is used in treatment of skin metastases of breast cancer and visceral
leishmaniosis [6].
Fluorinated surfactants have several advantages in comparison with surfactants containing
only hydrocarbon alkyl chains. Their physicochemical and biological activities are improved
in many cases in comparison with conventional surfactants [9]. They posses lower values of
CMCs [10,11], decreasing of surface tension of surfactants solution is more effective, the
values are lower in many cases than 20 mN.m-1 [12-14] or they form interesting aggregates
[12,15-17]. Fluorinated surfactants also received attention for their interesting biological
activities. They are used e.g. as stabilizers of fluorocarbon-in-water emulsions for the
preparation of blood substitutes [18]. They have lower haemolytic activities [18,19] or
enhanced activities against some microorganisms [20,21] compared to classical hydrocarbon
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surfactants. Furthermore, they are also studied as antibody binding [22] or nucleic acids
transfer agents [23].
The main goal of this work is the preparation and the study of physicochemical and biological
properties of dialkylphosphonatocholines with hydrocarbon and highly fluorinated tails. The
compounds are representatives of new types of organophosphorus heterogemini surfactants.
Optimization of the synthesis of the compounds was also studied.
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2.
Results and discussion
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2.1. Chemistry
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Altogether four new non-fluorinated and partially fluorinated dialkyphosphonatocholines
were synthesised. The common structural motif of the prepared compounds is the presence of
a phosphonate anion connected with an ammonium cation by an ethylene spacer and two
eight carbon atom length alkyl chains (C8), which are attached to the phosphonate anion and
to the quaternary nitrogen. Differences in the molecules are in alkyl chains. Compounds are
possessing an octyl and/or 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-tridecafluorooctyl groups. One
dialkylphosphonatocholine was prepared with two octyl groups (HH), two others with one
partially fluorinated alkyl chain and one octyl group (HF, FH) and one with two partially
fluorinated alkyl chains (FF).
Page 4 of 30

O
P
O
P
O
O
O
O
130°C
KOH
EtOH
AIBN
Br
+
P
O
+
F13C6
I
Na2S2O5
Br
Br
O
O
O
1
2
O
O
O
O
P
P
P
Bu3SnH
AIBN
OH
HBr
F13C6
F13C6
O
O
F13C6
I
OH
3
4
5
O
Br2
OH
Br
P
O
+
F13C6
F13C6
Ph3P
O
6
O
P
O
O
P
150°C
HBr
H17C8 Br
+
O
H17C8
O
H17C8
P
OH
O
O
OH
7
8
71
72
73
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75
76
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80
81
82
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84
85
86
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88
Scheme 1. Preparation of alkylphosphonic acids
The synthesis started with the preparation of alkylphosphonic acids. 1H,1H,2H,2H-
Perfluorooctylphosphonic acid (5) was prepared with two synthetic approaches (Scheme 1).
The main step in the first approach was the coupling of diethyl vinylphosphonate (2) with
perfluorohexyl iodide. Diethyl vinylphosphonate was prepared by a general procedure
[24,25]. The Michaelis-Arbuzov reaction of 1,2-dibromoethane with triethyl phosphite
yielded diethyl 2-bromophosphonate (1). This reaction was performed with excess of 1,2-
dibromoethane because tetraethyl ethylene-1,2-bisphosphonate was concurrently formed. 1,2-
Dibromoethane was in 4 fold excess to triethylphosphite. However, the yield of 1 was only
62% in spite of the excess of dibromide used. Higher yields can be obtained using microwave
synthesis. Jansa et al. [26] described microwave synthesis of dialkyl ω-
bromoalkylphosphonates using an equivalent amount of dibromide to phosphite and with
higher yields and shorter reaction times. Hydrogen bromine was eliminated from 1 with
potassium hydroxide and diethyl vinylphosphonate (2) was obtained in high yield after
distillation.
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The addition of perfluorohexyl iodide to vinylphosphonate (2) was catalyzed by aza-bis-iso-
butyronitrile (AIBN). 3 was used in the next synthetic step without purification. Iodine in its
molecule was reduced by tri-n-butyltin hydride. The yield of 4 was very low (13%). This
could be caused by a competitive reaction of 3 with diethyl vinylphosphonate. Ester 4 was
hydrolyzed by concentrated HBr. The syntheses were performed according adopted
procedures [27,28]. Similar addition reaction of perfluorohexyl chloride to diethyl
vinylphosphonate was performed by Huang and Chen [29]. The reaction was carried out in
the presence of zinc, nickel dichloride and triphenylphosphine. Finally the compound 4 was
obtained. The advantage of this synthetic approach is in good yield of the product (71%) and
in the preparation of 4 in one synthetic step.
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99
In view of the fact that 5 was prepared in low yield by the previously described procedure, a
second approach was performed. 1H,1H,2H,2H-perfluorooctanol was firstly brominated.
Triphenylphosphine dibromide, which was generated in situ in the reaction mixture from
bromine and triphenylphosphine, was used as brominating agent [30]. Bromide 6 was used in
the following Michaelis-Arbuzov reaction. Without isolation the product 4 was hydrolyzed
with HBr.
100
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103
104
105
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107
108
109
110
111
The comparison of the overall yields of both synthetic approaches favored the second one. 5
was prepared in 24% overall yield when the second approach was used instead of 5% overall
yield in the first approach.
The octylphosphonic acid (8) was obtained in two steps (Scheme 1): its dietyl ester 7 was
synthesized by Michaelis-Arbuzov reaction of 1-bromooctane with triethyl phosphite and
hydrolyzed by hydrobromic acid.
Ts-
N+
OH
OH
OH
TsCl
Et3N
O
80°C
CH3CN X13C6
X13C6
+
Ts
N
X13C6
9
- X = H
11 - X = H
12 - X = F
10 - X = F
Ts = tosylate
112
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Scheme 2. Preparation of choline derivatives
The choline derivatives 11 and 12 needed in the last step of the synthesis of
dialkylphosphonatocholines were prepared by quaternization of 2-(dimethylamino)ethanol
with alkyl tosylates 9 or 10, respectively (Scheme 2). An attempt was made to prepare
fluorinated derivatives of choline by quaternization of a tertiary amine with bromide 6,
however, we were unsuccessful to isolate the product in solid state. Therefore, we prepared
tosylate 10 from 1H,1H,2H,2H-perfluorooctanol and tosylchloride according to the procedure
described by Elshani et al. [31] and then 2-(dimethylamino)ethanol was quaternised with
tosylate 10.
Ts-
O
P
O
OH
OH
TIPSCl
pyridine
X13C6
C6Y13
N+
+
N+
P
X13C6
O-
Y13C6
O
OH
HH - X = H, Y = H
HF - X = H, Y = F
FH - X = F, Y = H
FF - X = F, Y = F
5
8
- X = F
- X = H
11 - X = H
12 - X = F
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133
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135
136
Scheme 3. Preparation of dialkylphosphonatocholines
The last step, the synthesis of non-fluorinated and fluorinated dialkylphosphonatocholines
(Scheme 3), was performed by the general method used in the preparation of
alkylphosphocholines [32,33] or dialkylphosphocholines [34]. Alkylphosphonic acids 5, 8 and
choline derivatives 11, 12 were coupled in the presence of 2,4,6-triisopropylbenzenesulfonyl
chloride (TIPSCl) in anhydrous pyridine. Compounds HH, HF, FH and FF were obtained in
moderate or good yields.
The influence of fluorine on chemical shifts of some atomic groups of
1
dialkylphosphonatocholines in H, 13C and 31P NMR is shown in Table 1. Substitution of
Page 7 of 30

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hydrogen by fluorine in the alkyl chains of dialkylphosphonatocholines shifted the signals of
methylene and methyl groups of partially fluorinated dialkylphosphonatocholines (except of
methylene groups of alkyl chain bounded to phosphate group of HF) observed in the 1H NMR
to higher values in comparison with nonfluorinated dialkylphosphonatocholine. However,
HF, FH and FF had the signals of carbon atoms of methylene groups present at partially
fluorinated alkyl chains observed in 13C NMR at lower values in comparison with values of
the same methylene groups present at nonfluorinated alkyl chains. Similar situation was
observed in 31P NMR spectra. The chemical shifts of phosphorus of FH and FF with partially
fluorinated alkyl chain connected to phosphonate group were shifted to lower values in
comparison with dialkylphosphonatocholines which contained hydrocarbon alkyl chain
bounded to the phosphate group.
1
Table 1. Chemical shifts of the signal of some groups of dialkylphosphonatocholines in H,
13C, 19F, 31P NMR
151
152
t
b
d
f
h
k
m
o
r
O
H3C
CX2
CX2
CX2
i
CY2
CY2
CY2
CX3
a
CX2
c
CX2
e
P
N+
CY2
n
CY2
p
CY3
s
O
O-
CH3
t
g
l
j
HH - X = H, Y = H
HF - X = H, Y = F
FH - X = F, Y = H
FF - X = F, Y = F
153
[a]
Comp
Hg
Hh
Hi
Hj
Hk
Hl
Ht
Cg[a]
Ch
Ck
Cl[a]
Ff
Fm
P
HH
1.55
1.55
4.19
4.22
4.23
4.24
3.32
3.37
3.33
3.63
3.50
3.76
3.54
3.77
1.73
2.79
1.75
2.79
3.14
31.4 d
J=17.1
31.3 d
J=17.3
26.1 t
J=22.1
26.1 t
J=22.4
27.6 d
J=136.0
27.5 d
J=136.3
18.1 d
J=138.8
18.1 d
J=139.3
66.5
26.4
‐
‐
28.0
HF
FH
FF
1.55
2.38
2.37
1.55
1.81
1.81
3.24
3.15
3.24
58.2
66.5
58.0
25.1 t
J=21.6
26.4
‐
‐113.9
‐
29.0
23.5
24.0
‐115.7
‐115.9
25.1 t
J=21.8
‐114.0
154
155
[a] d = doublet, t = triplet; values of interaction constant are expressed in Hz
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2.2. Surface active properties
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The micellar and the surface-active properties of the investigated compounds HH, HF, FH
and FF were studied by measurements of surface tension of surfactant aqueous solutions by
the Wilhelmy plate technique. The plots of surface tension vs. log concentration curves of the
surfactants are shown in Figure 1. Critical micelle concentration (CMC), surface tension at the
CMC (γCMC), and the surface area at the surface saturation per head group (ACMC) of the
surfactants are shown in Table 2.
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Figure 1. Plots of surface tension vs. log concentration curves of dialkylphosphonatocholines
Table 2. Physicochemical properties of dialkylphosphonatocholines
γCMC
[mN.m‐1]
30.3
16.1
16.1
ACMC
[Å2]
66 ± 3
65 ± 1
65 ± 3
–
Comp
c1 [mol.dm‐3]
CMC [mol.dm‐3]
HH
HF
FH
FF
(3.7 ± 0.4) × 10‐4
(1.2 ± 0.1) × 10‐5
(6.9 ± 0.3) × 10‐6
–
(1.9 ± 0.1) × 10‐3
(8.5 ± 0.1) × 10‐5
(4.4 ± 0.1) × 10‐5
< 1 × 10‐6
> 21.2
170
171
172
173
The CMC value of HH is comparable with similar compound (octyl 2-
[dimethyl(octyl)ammonio]ethyl phosphate) which was investigated by Peresypkin and
Menger [35]. They obtained the CMC value for it equal to 1 × 10-3 mol.dm-3. The replacement
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of octyl group with partially fluorinated one led to a decrease of the CMC. The compounds
with one partially fluorinated carbon chain possessed the CMC of about 1.5 orders of
magnitude lower than HH and the compound with both partially fluorinated carbon chains
had the CMC lower than 1 × 10-6 mol.dm-3, only the postmicellar part of the curve was
obtained (Figure 1).
A very interesting feature of the compounds investigated is the multilinear relationship
between the surface tension and the concentration of the surfactant showing clearly two
breaks in the curve (Figure 1). We assume that the first break (the break in the premicellar
region of the curve, Figure 1) indicated premicellar aggregation. The formation of small
aggregates from surfactant molecules is expected. We estimated that the values of surfactant
concentrations when premicellar aggregates start to form (c1) are 5 to 7 times lower than the
CMCs. This unusual phenomenon was previously observed in the case of some types of
surfactants [36-38].
The measured values of surface tension at the CMC showed that the compounds with fluorine
in the alkyl chains had lower values than the compound with hydrocarbon alkyl chains.
However, it is interesting that the replacement of one hydrocarbon alkyl chain with partially
fluorinated alkyl chains caused a decrease of the γCMC but the compound with two partially
fluorinated alkyl chains (FF) had the γCMC higher (γCMC > 21.2 mN.m-1) than HF and FH. The
most fluorinated surfactant did not have the lowest γCMC. A similar effect of increasing the
γCMC with decreasing lipophilicity (decrease of the CMC) in the ranges of partially fluorinated
surfactants was also described in the literature [12,39]. However, when the value of surface
tension (γ = 17.2 mN.m-1) of FF at a concentration (c = 9.7 × 10-5 mol.dm-3) similar to CMCs
of HF (γCMC = 16.1 mN.m-1) and FH (γCMC = 16.1 mN.m-1) is compared, the differences are
not high.
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The values of ACMC showed that partial fluorination of one alkyl chain of HH did not have an
influence on the surface area of the surfactant at the water/air interface.
2.3. Solubilization properties
Solubilization properties of the prepared surfactants were investigated by the solubilization of
a poorly soluble drug, griseofulvin. This compound is a representative solute because it is
solubilized almost exclusively in the hydrophobic micelle core [40]. Its solubility in water
was estimated at 3.34 × 10-5 mol.dm-3. The changes in the solubilities of griseofulvin with
varying concentrations of surfactants solutions are shown in Figure 2. It is apparent that HH
had the best solubilization properties. It has a molar solubilization ratio (MSR) of 6.4 × 10-3.
Surfactants with one partially fluorinated alkyl chain were poorer solubilizers than HH
(MSRHF = 2.0 × 10-3, MSRFH = 4.2 × 10-3) and FF was capable of solubilizing the lowest
amount of griseofulvin (MSRFF = 1.2 × 10-3). One can say that increasing the number of
fluorine atoms in the tails decreased the solubility of the solute. Fluorinated alkyl chains are
more hydrophobic than hydrogenated alkyl chains and griseovulvin with many of its polar
groups is more difficult to incorporate into the more hydrophobic perfluorinated micelle core.
Matsuoka et al. [41] obtained similar results. Alkylbenzenes were better solubilized in
hydrogenated surfactant micelles than the fluorinated ones. Solubilization of pyrene with
tetraalkylammonium perfluorooctanoates also showed that the solute interacted better with
hydrocarbon chains than with fluorinated chains. Pyrene was localised in the counterion layer
of the micelle containing tetraalkylammonium ions and not in the micelle core containing
perfluorinated alkyl chains of the perfluorooctanoates [42].
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Figure 2. Plots of solubility of griseofulvin vs. concentration of dialkylphosphonatocholines
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2.4. Antiprotozoal activities
The biological activities of dialkylphosphonatocholines were tested against opportunistic
parasites of the genus Acanthamoeba which are the causative agents of frequently fatal
granulomatous amoebic encephalitis and a serious sight threatening amoebic keratitis. Strains
of A. lugdunensis and A. quina were chosen because of their higher resistance to action of a
standard, HPC, than the other species, e.g., A. polyphaga or A. lenticulata [43,44]. The values
of minimal trophocidal concentrations (MTC) of the compounds against parasites after 24
hours of incubation are shown in Table 3. HF was the most active compound, as its MTCs
were four and two times lower than the MTCs of HPC against A. lugdunensis and A. quina,
respectively. The same value of activity against A. quina was reached also by FH. HF and FH
have optimal physicochemical properties (CMC), which is also a measure of the lipophilicity
of the surfactant, for the antiprotozoal activity. The activities of alkylphosphocholines could
be mainly caused by a non-specific mode of action, namely the biophysical destruction of the
cell membrane [45]. Their optimal CMCs caused that they are capable to solubilize cell
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248
membranes
of
the
parasites
most
efficiently
among
the
investigated
dialkyphosphonatocholines. As we discussed previously [46], the bilinear type of cut-off
effect, well known in the case of surfactants [47], was observed in the amoebicidal activities
of alkylphosphocholines and therefore HH with high CMC and FF with low CMC were
inactive against parasites.
Table 3. Minimal trophocidal concentration of dialkylphosphonatocholines after 24 hours of
incubation
Compound
HH
HF
A. lugdunensis [μM]
> 500
125
A. quina [μM]
> 500
125
FH
> 500
> 500
500
125
> 500
250
FF
HPC[43]
249
250
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252
253
254
255
256
257
258
259
260
261
262
263
3. Conclusion
In summary, we synthesised four new dialkylphosphonatocholines with two octyl groups, one
with hydrocarbon alkyl chains (HH) and three with partially fluorinated tails (HF, FH, FF).
The compounds were prepared by coupling the alkylphosphonic acids with choline
derivatives in the presence of 2,4,6-triisopropylbenzenesulfonyl chloride. Partially fluorinated
alkylphosphonic acids were prepared by two methods. The first approach used radical
addition of perfluorohexyl iodide to diethyl vinylphosphonate as a key step of synthesis and
the second one employed the Michaelis-Arbusov reaction of partially fluorinated
octylbromide with triethyl phosphite. Concerning the yields the second approach was more
effective. The physicochemical properties were expressed as micellar, surface-active, and
solubilization properties. Increasing numbers of fluorine atoms in the molecules caused a
decrease of the values of CMC. The second break in the curve of surface tension vs. log c of
HH, HF and FH was also observed. One can suppose that the break indicated the formation
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of small aggregates from surfactant molecules. The lowest values of the surface tension at the
CMC were shown by compounds with one partially fluorinated tail. The best solubilization
properties were obtained with HH. The reason is that the fluorinated alkyl chains of HF, FH
and FF are more hydrophobic than the hydrogenated alkyl chains of HH and griseovulvin
with many of its polar groups is more difficult to incorporate into the more hydrophobic
perfluorinated micelle core. The antiprotozoal activities were tested against A. lugdunensis
and A. quina. HF was the most effective compound. The cut-off effect was observed in
antiprotozoal activities of series of dialkylphosphonatocholines. According to expectations the
HH with high CMC and FF with low CMC were inactive against parasites.
273
274
4.
Experimental part
275
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289
4.1. Chemistry
4.1.1. Materials
1
13
19
All chemicals used for the synthesis were purchased from commercial suppliers. H, C, F
and 31P NMR spectra were recorded on a Varian Mercury plus spectrometer operating at 300,
75.5, 282 and 121.5 MHz, respectively, 13C, 19F and 31P spectra being recorded with proton-
decoupling. The spectra were measured in CDCl3, CD3COCD3, CD3SOCD3, or a mixture of
CDCl3/CD3OD relative to the internal standard TMS for 1H and C NMR spectra, CCl3F for
13
19F NMR spectra and to the external standard 85% H3PO4 for 31P NMR spectra. Infrared
spectra were recorded on a FT-IR Impact 400 D spectrophotometer as potassium bromide
discs or liquid films on potasium bromide discs. Molecular masses of final compounds were
measured by high resolution spectrometer ESI-LTQ Orbitrap XL Thermo Scientific. Melting
points were measured using a Koffler hot-stage apparatus and are uncorrected.
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4.1.2. Preparation of diethyl 2-bromoethylphosphonate
Triethylphosphite (35 ml, 0.2 mol) was mixed with 1,2-dibromoethane (69 ml, 0.8 mol) in a
round-bottom flask equipped with a reflux condenser, heated at 160°C and stirred for 4 h. The
reaction mixture was cooled and purified by distillation at reduced pressure.
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Diethyl 2-bromoethylphosphonate (1): colourless liquid, b.p. 133–135°C at p = 2 kPa, nD20
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= 1,4596, yield = 30.39 g (62%). FT-IR (liquid film): υmax = 2981, 1443, 1392, 1368, 1285,
1243, 1163, 1054, 1020, 961, 787 cm-1. 1H NMR (300 MHz, CDCl3): δ = 4.20–4.05 (m, 4H),
3.53 (q, J = 8.5 Hz, 2H), 2.47–2.32 (m, 2H), 1.34 (t, J = 7.1 Hz, 6H). 13C NMR (75 MHz,
CDCl3): δ = 62.1 (d, J = 6.1 Hz), 30.8 (d, J = 133.5 Hz), 23.9, 16.4 (d, J = 6.1 Hz). 31P NMR
(125.5 MHz, CDCl3): δ = 26.3.
4.1.3. Preparation of diethyl vinylphosphonate
Diethyl 2-bromoethylphosphonate (24.5 g, 0.1 mol) was slowly added to a cold solution of
KOH (5.6 g, 0.1 mol) in 150 ml of ethanol. The reaction mixture was stirred at room
temperature for 1 h then heated to reflux for 15 min. The formed solid was filtered off and
washed with ethanol. Ethanol was removed under vacuum and the remaining oil was distilled
under reduced pressure.
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Diethyl vinylphosphonate (2): colourless liquid, b.p. 94–96°C at p = 2.4 kPa, nD20 = 1,4283,
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yield = 14.07 g (86%). FT-IR (liquid film): υmax = 1445, 1393, 1243, 1164, 1050, 1022, 958,
785 cm-1. 1H NMR (300 MHz, CDCl3): δ = 6.38–5.97 (m, 3H), 4.18 – 4.03 (m, 4H), 1.34 (t, J
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= 7.3 Hz, 6H). 13C NMR (75 MHz, CDCl3): δ = 135.6 (d, J = 2.4 Hz), 126.0 (d, J = 183.6 Hz),
61.9 (d, J = 6 Hz), 16.4. 31P NMR (125.5 MHz, CDCl3): δ = 18.0.
4.1.4. Preparation of diethyl 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctylphosphonate
Diethyl vinylphosphonate (8.2 g, 50 mmol) was mixed with 1-iodo-1,1,2,2,3,3,4,4,5,5,6,6,6-
tridecafluorohexane (22.3 g, 50 mmol) and heated at 60°C. AIBN (0.33 g, 2 mmol) was then
added and the mixture was stirred for 4 h at 80°C. Diethyl 1-iodo-3,3,4,4,5,5,6,6,7,7,8,8,8-
tridecafluorooctylphosphonate (3) was not isolated, but immediately used in the next reaction.
Replacement of iodine with hydrogen was performed by tri-n-butyltin hydride. AIBN (1.5
mmol)
was
added
to
crude,
cold
diethyl
1-iodo-3,3,4,4,5,5,6,6,7,7,8,8,8-
tridecafluorooctylphosphonate, followed by drop wise addition of tri-n-butyltin hydride (17.5
g, 60 mmol). The reaction mixture was stirred in argon atmosphere for 12 h at 80°C. After
reaction, the lower phase was separated and distilled at reduced pressure.
diethyl 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctylphosphonate (4): colourless liquid, b.p.
86–89°C at p = 20 Pa, yield = 3.23 g (13%). 1H NMR (300 MHz, CDCl3): δ = 4.21–4.05 (m,
4H), 2.50–2.28 (m, 2H), 2.08–1.92 (m, 2H), 1.35 (t, J = 7.1 Hz, 6H). 13C NMR (75 MHz,
CDCl3): δ = 125–105 (m, 6 × C), 62.2, 25.2 (t, J = 23.6 Hz), 17.2 (d, J = 146.7 Hz), 16.4. 19F
NMR (282 MHz, CDCl3): δ = -80.73 (t, J = 9.6 Hz, 3F), 115.32 (m, 2F), 121.88 (m, 2F),
122.81 (m, 2F), 123.30 (m, 2F), 126.12 (m, 2F). 31P NMR (125.5 MHz, CDCl3): δ = 28.9.
4.1.5. Preparation of 1-bromo-3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctane
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3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctane-1-ol (20 g, 55 mmol) and triphenylphosphine
(15.7 g, 60 mmol) were dissolved in 110 mL of anhydrous acetonitrile and deoxygenated with
argon. The mixture was heated at 60°C and bromine (9.6 g, 60 mmol) was added drop wise.
The mixture was stirred for 5 h at this temperature. After cooling down to room temperature,
the reaction mixture was extracted with diethyl ether and the ether layers were washed with
brine. After drying with Na2SO4, the ether was evaporated and the crude product was
dissolved in 100 mL of dichloromethane. This solution was stirred with 50 g of silica gel for 2
h and then filtered and evaporated. The crude product was distilled at reduced pressure.
1-bromo-3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctane (6): colourless liquid, b.p. 93–94°C at
12 kPa, nD20 = 1,3312, yield = 16.4 g (70%). FT-IR (liquid film): υmax = 1453, 1361, 1236,
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1196, 1144, 1123, 1079, 951, 845, 812, 745, 734, 702, 629 cm-1. 1H NMR (300 MHz, CDCl3):
δ = 3.51 (t, J = 8.2 Hz, 2H), 2.80–2.60 (m, 2H). 13C NMR (75 MHz, CDCl3): δ = 125–105 (m,
19
6 × C), 35.0 (t, J = 21.8 Hz), 20.2. F NMR (282 MHz, CDCl3): δ = -81.27 (t, J = 10.0 Hz,
3F), -114.72 (m, 2F), -122.39 (m, 2F), -123.35 (m, 2F), -123.97 (m, 2F), -126.63 (m, 2F).
4.1.6. General procedure for the preparation of phosphonic acid derivatives by
Michaelis-Arbuzov reaction
Alkylbromide (4.27 g of 1-bromo-3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctane or 1.93 g of 1-
bromooctane, 10 mmol) was heated with triethyl phosphite (1.83 g, 11 mmol) for 4h at
150°C. After cooling, conc. HBr (12 mL) was added and heated for 4 h at 120°C. The excess
HBr and bromoethane was then distilled off and the crude products were crystallised. The
same procedure of hydrolysis of diester 4 obtained by first synthetic approach was also used.
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3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctylphosphonic acid (5): crystallised from a mixture
of acetone : CHCl3 1 : 2, white powder, m.p. = 166–168°C, yield = 1.87 g (66%) for 1st
synthetic approach (1 step), yield = 1.24 g (29%) for 2nd synthetic approach (2 steps). FT-IR
(KBr): υmax = 1445, 1367, 1302, 1229, 1212, 1186, 1141, 1074, 1012, 954, 778, 741, 697, 653
1
cm-1. H NMR (300 MHz, CD3COCD3): δ = 4.33 (s, 2H), 2.60–2.38 (m, 2H), 2.10–2.02 (m,
2H). 13C NMR (75 MHz, CD3COCD3): δ = 125–105 (m, 6 × C), 26.0 (t, J = 22.9 Hz), 18.9 (d,
J = 144.3 Hz). 19F NMR (282 MHz, CD3COCD3): δ = -80.62 (t, J = 10.4 Hz, 3F), -114.56 (m,
31
2F), -121.40 (m, 2F), -122.37 (m, 2F), -122.83 (m, 2F), -125.70 (m, 2F). P NMR (125.5
MHz, CD3COCD3): δ = 28.7. HRMS calcd. for C8H5F13PO3 = 426.9763; found m/z: 426.9762
[M-H]-
Octylphosphonic acid (8): crystallised from hexane, white powder, m.p. = 99–101°C, yield =
1.44 g (74%). FT-IR (KBr): υmax = 2958, 2929, 2853, 1468, 1260, 1230, 1173, 1107, 1006,
1
995, 947, 931, 780, 716 cm-1. H NMR (300 MHz, CD3SOCD3): δ = 5.36 (s, 2H), 1.58–1.40
(m, 4H), 1.40 – 1.18 (m, 10H), 0.86 (t, J = 6.6 Hz, 3H). 13C NMR (75 MHz, CD3SOCD3): δ =
31.3, 30.1 (d, J = 16.2 Hz), 28.6, 28.5, 27.5 (d, J = 137.0 Hz), 22.6 (d, J = 4.8 Hz), 22.1, 13.9.
31P NMR (125.5 MHz, CD3SOCD3): δ = 28.4. HRMS calcd. for C8H18PO3 = 193.0990; found
m/z: 193.0995 [M-H]-
4.1.7. General procedure for preparation of alkyl tosylates
Alcohol (8.74 g of 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctanol or 3.13 g of octanol, 24
mmol) and tertiary amine (30 mmol, triethylamine (4.2 mL) in the case of fluorinated alcohol
and pyridine (2.4 mL) in the case of octanol) were dissolved in 50 mL of dichloromethane and
p-toluenesulfonyl chloride (4.77 g, 25 mmol) was added in small portions. The reaction
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mixture was stirred overnight. The dichloromethane solution was then washed with water,
dried over anhydrous MgSO4 and evaporated in vacuo. The crude product was crystallised
from methanol in the case of 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl tosylate. Crude octyl
tosylate was used in the next step without further purification.
Octyl tosylate (9): colourless liquid, yield = 76%. 1H NMR (300 MHz, CDCl3): δ = 7.78 (d, J
= 8.2 Hz, 2H), 7.33 (d, J = 8.2 Hz, 2H), 4.01 (t, J = 6.5 Hz, 2H), 2.45 (s, 3H), 1.68–1.57 (m,
2H), 1.36–1.16 (m, 10H), 0,88 (t, J = 6.7 Hz, 3H).
3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl tosylate (10): colourless crystals, m.p. = 54.5–
55°C, yield = 11.4 g (92%). FT-IR (KBr): υmax = 1597, 1495, 1424, 1399, 1363, 1318, 1250,
1
1202, 1173, 1137, 1079, 987, 972, 933, 892, 815, 778, 697, 663, 646 cm-1. H NMR (300
MHz, CDCl3): δ = 7.80 (d, J = 8.2 Hz, 2H), 7.36 (d, J = 8.2 Hz, 2H), 4.30 (t, J = 6.7 Hz, 2H),
2.60–2.43 (m, 2H), 2.46 (s, 3H). 13C NMR (75 MHz, CDCl3): δ = 145.4, 132.3, 130.1, 128.0,
125–105 (m, 6 × C), 61.6, 31.0 (t, J = 21.7 Hz), 21.7. 19F NMR (282 MHz, CDCl3): δ = -
81.29 (t, J = 10.0 Hz, 3F), -114.01 (m, 2F), -122.38 (m, 2F), -123.36 (m, 2F), -124.08 (m, 2F),
-126.65 (m, 2F).
4.1.8. General procedure for the preparation of choline tosylates
2-(N,N-Dimethylamino)ethanol (1.07 g, 12 mmol) and an alkyl tosylate (5.18 g of
3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl tosylate or 2.84 g of octyl tosylate, 10 mmol) were
dissolved in 25 mL of acetonitrile and refluxed for 24 h. After cooling down the acetonitrile
was evaporated in vacuum. The resulting mixture was crystallised from acetone.
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N-(2-hydroxyethyl)-N,N-dimethyloctane-1-ammonium tosylate (11): white powder, m.p. =
169–170°C, yield = 2.54 g (68%). FT-IR (KBr): υmax = 2956, 2923, 2856, 1492, 1467, 1219,
1
1184, 1171, 1120, 1033, 1010, 819, 681 cm-1. H NMR (300 MHz, CDCl3): δ = 7.71 (d, J =
7.9 Hz, 2H), 7.15 (d, J = 7.9 Hz, 2H), 5.43 (t, J = 5.3 Hz, 1H), 4.10–4.00 (m, 2H), 3.64–3.55
(m, 2H), 3.39–3.28 (m, 2H), 3.20 (s, 6H), 2.34 (s, 3H), 1.64–1.54 (m, 2H), 1.37–1.12 (m,
10H), 0.88 (t, J = 6.7 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ = 143.3, 139.8, 128.9, 125.8,
65.8, 65.6, 56.3, 51.6, 31.8, 29.2, 26.3, 22.8, 22.7, 21.4, 14.1. HRMS calcd. for C12H28ON =
202.2165; found m/z: 202.2165 [M-Ts]+
N-(2-hydroxyethyl)-N,N-dimethyl-3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctane-1-
ammonium tosylate (12): white powder, m.p. = 219–221°C, yield = 1.64 g (27%). FT-IR
(KBr): υmax = 1495, 1362, 1320, 1296, 1231, 1203, 1189, 1144, 1121, 1108, 1009, 820, 723,
683, 653 cm-1. 1H NMR (300 MHz, CD3SOCD3): δ = 7.48 (d, J = 7.9 Hz, 2H), 7.10 (d, J = 7.9
Hz, 2H), 5.37 (t, J = 4.7 Hz, 1H), 3.95–3.68 (m, 4H), 3.58–3.48 (m, 2H), 3.17 (s, 6H), 3.06–
2.82 (m, 2H), 2.29 (s, 3H). 13C NMR (75 MHz, CD3SOCD3): δ = 145.7, 137.6, 128.0, 125.4,
19
125–105 (m, 6 × C), 64.9, 55.7, 54.9, 50.9, 24.4 (t, J = 21.0 Hz), 20.7. F NMR (282 MHz,
CD3SOCD3): δ = -79.8 (t, J = 9.0 Hz, 3F), -111.97 (m, 2F), -121.21 (m, 2F), -122.15 (m, 4F),
-125.32 (m, 2F). HRMS calcd. for C12H15ONF13 = 436.0941; found m/z: 436.0938 [M-Ts]+
4.1.9. General procedure for the preparation of dialkylphosphonatocholines
Pyridinium salts of alkyl dihydrogen phosphonates were prepared by the treatment of
alkylphosphonic acids (0.428 g of 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctylphosphonic acid
or 0.194 g of octylphosphonic acid, 1 mmol) with pyridine (8 mL) for 2 h at 50°C. The excess
pyridine was evaporated and the rest was dissolved in 5 mL of anhydrous pyridine. The
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appropriate choline (0.911 g of N-(2-hydroxyethyl)-N,N-dimethyl-3,3,4,4,5,5,6,6,7,7,8,8,8-
tridecafluorooctane-1-ammonium tosylate or 0.374 of N-(2-hydroxyethyl)-N,N-
g
dimethyloctane-1-ammonium tosylate, 1.5 mmol) and 2,4,6-triisopropylbenzenesulfonyl
chloride (0.45 g, 2 mmol) in pyridine (15 mL) were added to the solution of pyridinium
alkylphosphonate. The solution was stirred at 40 °C overnight. After cooling, the mixture was
hydrolyzed by addition of H2O (1.5 mL) and stirred for 1 h at room temperature. The solvents
were evaporated in vacuum, and the resulting crude solid was dissolved in a mixture of
tetrahydrofuran and water (5:1, V/V). An exchange resin, Amberlite MB3, was added
sequentially to the stirred solution until the colour of the resin ceased to change. Then the
resin was filtered off and the solvents were evaporated in vacuum. The remaining mixture was
coevaporated with propan-2-ol several times. The residue was dissolved in chloroform and
precipitated with acetone. Dialkylphosphonatocholines were filtered off and crystallised from
a mixture of chloroform and acetone. Products were dried in a vacuum dessicator over P4O10.
2-[dimetyl(octyl)ammonio]ethyl octylphosphonate (HH): white powder, m.p. = 227–
227.5°C, yield = 0.131 g (33%). FT-IR (KBr): υmax = 3297, 2918, 2851, 1467, 1195, 1091,
1057, 962, 936, 781, 717, 704 cm-1. 1H NMR (300 MHz, CDCl3 500 μL + CD3OD 100 μL): δ
= 4.24–4.16 (m, 2H), 3.55–3.45 (m, 2H), 3.37–3.26 (m, 2H), 3.14 (s, 6H), 1.80–1.66 (m, 2H),
1.65–1.47 (m, 4H), 1.45–1.19 (m, 20H), 0.95–0.82 (m, 6H). 13C NMR (75 MHz, CDCl3 500
μl + CD3OD 100 μl): δ = 66.5, 64.9, 56.7, 51.6, 32.0, 31.8, 31.4 (d, J = 17.1 Hz), 29.4(3),
29.3(5), 29.2, 29.1, 27.6 (d, J = 136.0 Hz), 26.4, 23.8, 23.7, 22.8, 22.7, 14.1(2), 14.0(6). 31P
NMR (125.5 MHz, CDCl3 500 μl + CD3OD 100 μl): δ = 28.0. HRMS calcd. for
C20H44O3NPNa = 400.2951; found m/z: 400.2950 [M+Na]+
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2-[dimetyl(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)ammonio]ethyl octylphosphonate
(HF): white powder, m.p. = 188–189°C, yield = 0.136 g (22%). FT-IR (KBr): υmax = 3274,
2933, 2860, 1240, 1222, 1139, 1097, 1060, 964, 920, 780, 738, 710 cm-1. 1H NMR (300 MHz,
CDCl3 500 μL + CD3OD 100 μL): δ = 4.26–4.17 (m, 2H), 3.82–3.70 (m, 2H), 3.65–3.57 (m,
2H), 3.24 (s, 6H), 2.89–2.69 (m 2H), 1.67–1.46 (m, 4H), 1.41–1.18 (m, 10H), 0.87 (t, J = 6.6
Hz, 3H). 13C NMR (75 MHz, CDCl3 500 μl + CD3OD 100 μl): δ = 125–115 (m, 6 × C), 64.9,
58.2, 56.7, 51.8, 32.0, 31.3 (d, J = 17.3 Hz), 29.4, 29.3, 27.5 (d, J = 136.3 Hz), 25.1 (t, J =
19
21.6 Hz), 23.6, 22.8, 14.1. F NMR (282 MHz, CDCl3 500 μl + CD3OD 100 μl): δ = -81.20
(t, J = 10.0 Hz, 3F), -113.94 (m, 2F), -122.25 (m, 2F), -123.27 (m, 4F), -126.54 (m, 2F). 31P
NMR (125.5 MHz, CDCl3 500 μl + CD3OD 100 μl): δ = 29.0. HRMS calcd. for
C20H31O3NF13PNa = 634.1726; found m/z: 634.1725 [M+Na]+
2-[dimetyl(octyl)ammonio]ethyl 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctylphosphonate
(FH): white powder, m.p. = 214–216°C, yield = 0.312 g (50%). FT-IR (KBr): υmax = 3365,
2932, 2857, 1212, 1143, 1061, 972, 928, 822, 775, 745, 732 cm-1. 1H NMR (300 MHz, CDCl3
500 μL + CD3OD 100 μL): δ = 4.29–4.18 (m, 2H), 3.62–3.49 (m, 2H), 3.42–3.28 (m, 2H),
3.14 (s, 6H), 2.50–2.26 (m, 2H), 1.91–1.58 (m, 4H), 1.45–1.20 (m, 10H), 0.89 (t, J = 6.7 Hz,
3H). 13C NMR (75 MHz, CDCl3 500 μl + CD3OD 100 μl): δ = 125–115 (m, 6 × C), 66.5,
64.9, 57.1, 51.6, 31.8, 29.2, 26.4, 26.1 (t, J = 22.1 Hz), 22.8, 22.7, 18.1 (d, J = 138.8 Hz),
14.0. 19F NMR (282 MHz, CDCl3 500 μl + CD3OD 100 μl): δ = -81.27 (t, J = 10.0 Hz, 3F), -
115.74 (m, 2F), -122.38 (m, 2F), -123.31 (m, 2F), -123.84 (m, 2F), -126.60 (m, 2F). 31P NMR
(125.5 MHz, CDCl3 500 μl + CD3OD 100 μl): δ = 23.5. HRMS calcd. for C20H31O3NF13PNa
= 634.1726; found m/z: 634.1727 [M+Na]+
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2-[dimetyl(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)ammonio]ethyl
3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl-phosphonate (FF): white powder, m.p. = 205.5–
207°C, yield = 0.211 g (25%). FT-IR (KBr): υmax = 3409, 1230, 1185, 1139, 1058, 963, 776,
1
710 cm-1. H NMR (300 MHz, CDCl3 500 μL + CD3OD 100 μL): δ = 4.32–4.16 (m, 2H),
3.84–3.70 (m, 2H), 3.68–3.58 (m, 2H), 3.23 (s, 6H), 2.90–2.68 (m, 2H), 2.50–2.24 (m, 2H),
1.90–1.72 (m, 2H). 13C NMR (75 MHz, CDCl3 500 μl + CD3OD 100 μl): δ = 125–115 (m, 12
× C), 65.0, 58.0, 56.9, 51.8, 26.1 (t, J = 22.4 Hz), 25.1 (t, J = 21.8 Hz), 18.1 (d, J = 139.3 Hz).
19F NMR (282 MHz, CDCl3 500 μl + CD3OD 100 μl): δ = -81.33 (m, 6F), -114.03 (m, 2F), -
115.92 (m, 2F), -122.37 (m, 4F), -123.32 (m, 6F), -123.91 (m, 2F), -126.62 (m, 4F). 31P NMR
(125.5 MHz, CDCl3 500 μl + CD3OD 100 μl): δ = 24.0. HRMS calcd. for C20H18O3NF26PNa
= 868.0501; found m/z: 868.0500 [M+Na]+
4.2. Equilibrium surface tension measurements
The critical micelle concentration values of the surfactants were determined from the surface
tension isotherm. The solvent surface tension values were measured by the Wilhelmy plate
technique using a Krüss 100 MK2 tensiometer. Deionised water was used in the preparation
of all samples. The temperature of the measurements was kept at 25 ± 0.1°C. Measurements
of equilibrium surface tension were taken repeatedly (every 6 min.) until the change in surface
tension was less than 0.05 mN.m-1. The critical micelle concentrations (CMC) and surface
tensions at the CMC (γCMC) were determined from the intersection of two lines – one
premicellar and second one postmicellar – of the surface tension vs. log c curve. The values of
surfactant concentrations when premicellar aggregates start to form (c1) were calculated in a
same way. From the surface tension data, the adsorbed amount of the surfactant γCMC is
calculated utilising the Gibbs adsorption isotherm
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ΓCMC = -[dγ/d log c]T/(2.303iRT) (1)
where γ is the surface tension (mN.m-1), c is the surfactant concentration, i is the prefactor, T
is the absolute temperature and R the gas constant. Surface excess may be determined from
the slope below the CMC in the surface tension vs. log c plots. Surface area at the surface
saturation per head group (ACMC) is obtained from the equation
ACMC = 1020/NAΓCMC (2)
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were NA is the Avogadro constant.
4.3. Solubilization of griseofulvin
Saturated griseofulvin solutions were prepared in glass vessels by mixing an excess powdered
drug (5 mg) with 2.5 mL of deionised water or surfactant solution with an appropriate
concentration (csurf) and stirring (250 rpm) at a constant temperature t = 25 ± 1 °C for 72
hours before filtering (Millipore, 0.22 μm) to remove any unsolubilized material. The extent
of dissolution was determined by UV spectroscopy. The filtered solution (1 mL) was diluted
quantitatively with methanol in a 25 mL volumetric flask. Absorbance was measured at the
optimum wavelength, λ = 292 nm, which was then compared with the appropriate Beer’s law
plot for the drug in methanol. Water content in the measured solution was low enough to
allow the calibration with methanol solutions to be used without correction. MSR was
calculated according to the equation:
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MSR = (c - cw)/(csurf - CMC)
where c is the concentration of griseofulvin in a solution of surfactant and cw is concentration
of griseofulvin in deionised water.
4.4. In vitro amoebicidal activity assay
The cytotoxic activity of dialkyphosphonatocholines was tested on two clinical isolates of
free-living amoebae, i.e., Acanthamoeba lugdunensis AcaVNAK02 and Acanthamoeba quina
AcaVNAK03, isolated from the corneas of two patients with amoebic keratitis.
Briefly, from the 2-day monoxenic cultures on agar plates, the trophozoites were axenised by
inoculation into the Bacto-Casitone/Serum medium (BCS) with penicillin and ampicillin.
After 72 h, the active trophozoites were transferred into peptone-yeast extract-glucose
medium (PYG) with penicillin and ampicillin. After 5 passages, the trophozoites were
transferred into a PYG medium without antibiotics and subsequently cultivated in this
medium. Cytotoxicity measurements were carried out in sterile 96-well microtitre plates. Each
well was seeded with 100 μL (2 × 105 cells mL-1) of a trophozoite suspension. Then, 100 μL
of a freshly prepared medium containing a tested compound at 10 concentrations was added
to all wells except untreated control wells that received 100 μL of pure medium. Each
compound was tested at final concentrations of 500, 250, 125, 62.5, 31.25, 15.6, 7.8, 3.9,
1.95, and 0.98 μM. The reduction of trophozoites was recorded after 24 h by counting the
surviving cells in a Bürker-Türk hemocytometer. Viability of trophozoites was determined by
trypan blue exclusion; 100% eradication was confirmed by transferring 50 μL of the
suspension to a PYG medium, then recording the amoeba growth for 14 days. The lowest
concentration of tested compounds leading to 100% eradication of the trophozoites was
defined as the minimal trophocidal concentration (MTC). The experiments were repeated 8
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times for each concentration. The cultivations and the cytotoxicity measurements were carried
out at 37°C.
Acknowledgments: We thank the NMR laboratory, Faculty of Pharmacy, Comenius
University in Bratislava for allowing us to conduct the NMR experiments. Further we thank
the management of HPL (Ltd.) for laboratory space for testing the amoebicidal activities. This
work was supported by Grants VEGA 1/0796/12, APVV-0325-07, FaF UK/28/2013,
UK/547/2013, ESF grant no. NFP 26240120065. This contribution/publication is the result of
the project implementation: Comenius University in Bratislava Science Park supported by the
Research and Development Operational Programme funded by the ERDF Grant number:
ITMS 26240220086
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References
[1] F.M. Menger, A.V. Peresypkin, K.L. Caran, A.L. Apkarian, Langmuir 16 (2000) 9113–
9116.
553
554
555
556
557
558
559
560
561
562
563
[2] F.M. Menger, A.V. Peresypkin, J. Am. Chem. Soc. 123 (2001) 5614–5615.
[3] C. Menez, P. Legrand, V. Rosilio, S. Lesieur, G. Barratt, G. Mol. Pharmacol. 4 (2007)
281–288.
[4] M. Lukáč, I. Prokipčák, I. Lacko, F. Devínsky, Eur. J. Pharm. Sci. 44 (2011) 194–199.
[5] S. Drescher, S. Sonnenberger, A. Meister, A. Blume, B. Dobner, Monatsh. Chem. 143
(2013) 1533–1543.
[6] W.J. van Blitterswijk, M. Verhej, Biochim. Biophys. Acta 1831 (2013) 663–674.
[7] S.L. Croft, K. Seifert, M. Duchêne, Molecular Biochem. Parasitol. 126 (2003) 165–172.
[8] M. Pisárčik, M. Polakovičová, M. Lukáč, F. Devínsky, M. Pupák, Colloids Surf. A -
Physicochem. Eng. Asp. 407 (2012) 169–176.
Page 26 of 30

564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
[9] A. Zaggia, B. Ameduri, Curr. Opin. Colloid Interface Sci. 17 (2012) 188–195.
[10]T. Asakawa, K. Ishikawa, S. Miyagishi, J. Colloid Interface Sci. 240 (2001) 365–367.
[11]P. Quagliotto, C. Barolo, N. Barbero, E. Barni, C. Compari, E. Fisicaro, G. Viscardi, Eur.
J. Org. Chem. (2009) 3167–3177.
[12]M. Uomar, E.T. de Givenchy, S.Y. Dieng, S. Amigoni, F. Guittard, Langmuir 27 (2011)
1668–1674.
[13]N. Bongartz, S.R. Patil, C. Stubenrauch, D. Blunk, Colloids Surf. A - Physicochem. Eng.
Asp. 414 (2012) 320–326.
[14]T. Schuster, S. Schellenberger, R. Friedrich, M. Klapper, K. Müllen, J. Fluorine Chem.
154 (2013) 30–36.
[15]A. Peresypkin, C. Clavel, F.M. Menger, Mendeleev Commun. 17 (2007) 82–84.
[16]P. Nitoń, A. Żywociński, J. Paczesny, M. Fiałkowski, R. Hołyst, B. Glettner, R. Kieffer,
C. Tschierske, D. Pociecha, E. Górecka, Chem. Eur. J. 17 (2011) 5861–5873.
[17]M.Dupuy, A. Pasc, E. Mayot, S. Cosgun, C. Gérardin-Charbonier, J. Fluorine Chem. 134
(2012) 115–121.
[18]J.G. Ries, M.P. Krafft, Biomaterials 19 (1998) 1529–1539.
[19]R. Kaplánek, O. Paleta, I. Ferjentsiková, M. Kodíček, J. Fluorine Chem. 130 (2009) 308–
316.
[20]L. Massi, F. Guittard, S. Géribaldi, R. Levy, Y. Duccini, Int. J. Antimicrob. Agents 21
(2003) 20–26.
[21]L. Massi, F. Guittard, R. Levy, S. Géribaldi, Eur. J. Med. Chem. 44 (2009) 1615–1622.
[22]T. Platen, T. Schüler, W. Tremel, A. Hoffmann-Röder, Eur. J. Org. Chem. (2011) 3878–
3887.
[23]E. Klein, C. Leborgne, M. Ciobanu, J. Klein, B. Frisch, F. Pons, G. Zuber, D. Scherman,
A. Kichler, L. Lebeau, Biomaterials 31 (2010) 4781–4788.
Page 27 of 30

589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
[24]N.R. Cichowicz, P. Nagorny, Org. Lett. 14 (2012) 1058–1061.
[25]P. Fourgeaud, C. Midrier, J.P. Vors, J.N. Volle, J.L. Pirat, D. Virieux, Tetrahedron 66
(2010) 758–764.
[26]P. Jansa, A. Holý, M. Dračínsky, O. Baszczyňski, M. Česnek, Z. Janeba, Green Chem. 13
(2011) 882–888.
[27]H. Shao, L. Jiang, W.D. Meng, F.L. Qing, J. Fluorine Chem. 124 (2003) 89–91.
[28]J.A. Bennett, E.G. Hope, K. Singh, A.M. Stuart, J. Fluorine Chem. 130 (2009) 615–620.
[29]X.T. Huang, Q.Y. Chen, J. Org. Chem. 66 (2001) 4651–4656.
[30]C. Naud, P. Calas, H. Blancou, A. Commeyras, J. Fluorine Chem. 104 (2000) 173–178.
[31]S. Elshani, E. Kobzar, R.A. Bartsch, Tetrahedron 56 (2000) 3291–3301.
[32]T. Calogeropoulou, P. Angelou, A. Detsi, I. Fragiadaki, E. Scoulika, J. Med. Chem. 51
(2008) 897–908.
[33]I. Papanastasiou, K.C. Prousis, K. Georgikopoulou, T. Pavlidis, E. Coulica, N.
Kolocouris, T. Calogeropoulou, Biorg. Med. Chem. Lett. 20 (2010) 5484–5487.
[34]M. Lukáč, M. Mrva, E. Fischer-Fodor, I. Lacko, M. Bukovský, N. Miklášová, F.
Ondriska, F. Devínsky, Bioorg. Med. Chem. Lett. 19 (2009) 6346–6349.
[35]A.V. Peresypkin, F.M. Menger, Org. Lett. 1 (1999) 1347–1350.
[36]L.D. Song, M.J. Rosen, Langmuir 12 (1996) 1149–1153.
[37]C. Xue, H. Zhu, T. Zhang, D. Cao, Z. Hu, Z. Colloid Surf. A - Physicochem. Eng. Asp.
375 (2011) 141–146.
[38]D. Arabadzhieva, B. Soklev, E. Mileva, E. Colloids Surf. A - Physicochem. Eng. Asp.
419 (2013) 194–200.
[39]T.H.V. Ngo, C. Damas, R. Naejus, R. Coudert, J. Colloid Interface Sci. 359 (2013) 157–
163.
Page 28 of 30

613
614
615
616
617
618
619
620
621
622
623
624
625
626
[40]M. Crothers, Z. Zhou, N.M.P.S. Ricardo, Z. Yang, P. Taboada, C. Chaibundit, D.
Attwood, C. Booth, Int. J. Pharm. 293 (2005) 91–100.
[41]K. Matsuoka, E. Takeuchi, M. Takahashi, S. Kitsugi, C. Honda, K. Endo, J. Colloid
Interface Sci. 333 (2009) 641–645.
[42]H. Xing, P. Yan, J.X. Xiao, Soft Matter 9 (2013) 1164–1171.
[43]M. Mrva, M. Garajová, M. Lukáč, F. Ondriska, J. Parasitol. 97 (2011) 538–540.
[44]J. Walochnik, M. Duchêne, K. Seifert, A. Obwaller, T. Hottkowitz, G. Wiedremann, H.
Eibl, H. Aspöck, Antimicrob. Agents Chemother. 46 (2002) 695–701.
[45]M. Lukáč, M. Mrva, M. Garajová, G. Mojžišová, L. Varínska, J. Mojžiš, M. Sabol, J.
Kubincová, H. Haragová, F. Ondriska, F. Devínsky, Eur. J. Pharm. Sci. 66 (2013) 46-55.
[46]M. Lukáč, M. Garajová, M. Mrva, M. Bukovský, F. Ondriska, E. Máriássy, F. Devínsky,
I. Lacko, Int. J. Pharm. 423 (2012) 247–256.
[47] P. Balgavý, F. Devínsky, Adv. Colloid Interface Sci. 66 (1996) 23–63.
Page 29 of 30