Synthesis, Aggregation Properties, and Antiprotozoal Activity of Heterocyclic Heterogemini Surfactants

Article abstract of DOI:10.1002/hc.20587

A series of four heterogemini surfactants have been synthesized: 2-decyl [do- decyl(dimethyl)ammonio]ethyl phosphate, decyl 2-(1-dodecylpyrrolidinio-1- yl)ethyl phosphate, decyl 2-(1-dodecylpiperidinio-1-yl)ethyl phosphate, and decyl 2-(1-dodecylmorpholinio-1-yl)ethyl phosphate. Antiprotozoal activity ofprepared compounds against Acanthamoeba lugdunensis was investigated. Decyl 2-(1-dodecylpyrrolidinio-1-yl)ethyl phosphate exhibited the best trophocidal activity. The activities of heterogemini surfactants were compared with their aggregation properties.

Full text of DOI:10.1002/hc.20587

Heteroatom Chemistry
Volume 21, Number 3, 2010
Synthesis, Aggregation Properties, and
Antiprotozoal Activity of Heterocyclic
Heterogemini Surfactants
M. Luka´cˇ,1,2 L. Timko,1 M. Mrva,3 F. Ondriska,4 J. Karlovska´,2,5
J. Valentova´,1 and I. Lacko1
1Department of Chemical Theory of Drugs, Faculty of Pharmacy, Comenius University,
Kalincˇiakova 8, 832 32 Bratislava, Slovakia
2NMR Laboratory, Faculty of Pharmacy, Comenius University, Odboja´rov 10, 832 32
Bratislava, Slovakia
3Department of Zoology, Faculty of Natural Sciences, Comenius University, Mlynska´ Dolina B-1,
842 15 Bratislava, Slovakia
4Department of Parasitology, Microbiological Laboratory, HPL (Ltd), Istrijska´ 20, 841 07
Bratislava, Slovakia
5Department of Physical Chemistry of Drugs, Faculty of Pharmacy, Comenius University,
Kalincˇiakova 8, 832 32 Bratislava, Slovakia
Received 27 May 2009; revised 15 February 2010
INTRODUCTION
ABSTRACT:
surfactants have been synthesized: 2-decyl [do-
decyl(dimethyl)ammonio]ethyl phosphate, decyl
A
series of four heterogemini
The term gemini surfactant was coined by
Menger and Littau in 1991 [1]. The vast major-
ity of work on gemini surfactants has been made
with symmetrical geminis, i.e., gemini surfactants,
which contain identical polar head groups and iden-
tical hydrophobic tails [2]. Geminis can be divided
into anionic [3,4], cationic [5–7], or nonionic surfac-
tants [8,9]. Unsymmetrical geminis, the surfactants
that have different head groups, such as anionic—
nonionic or anionic—cationic, have been referred
to as heterogemini surfactants [10]. Probably, the
first example of a true heterogemini surfactant is an
anionic–cationic species reported in 1996 by Jaeger
et al. [11]. One of the hydrophilic groups is a car-
boxylate anion, and the other one is a quaternary
ammonium cation. Other types of anionic–cationic
gemini surfactants (zwitterionic gemini surfactants)
are represented by the phosphate-quaternary ammo-
nium heterogemini surfactants with branched, un-
branched, or fluorinated alkyl chains [12–15]. These
2-(1-dodecylpyrrolidinio-1-yl)ethyl phosphate, decyl
2-(1-dodecylpiperidinio-1-yl)ethyl phosphate, and
decyl 2-(1-dodecylmorpholinio-1-yl)ethyl phosphate.
Antiprotozoal activity of prepared compounds against
Acanthamoeba lugdunensis was investigated. Decyl
2-(1-dodecylpyrrolidinio-1-yl)ethyl phosphate exhib-
ited the best trophocidal activity. The activities of
heterogemini surfactants were compared with their
ꢀ
C
aggregation properties.
2010 Wiley Periodicals, Inc.
Heteroatom Chem 21:203–209, 2010; Published online in
Wiley InterScience (www.interscience.wiley.com). DOI
10.1002/hc.20587
Correspondence to: M. Luka´cˇ; e-mail: lukac@fpharm.uniba.sk.
Contract grant sponsor: VEGA.
Contract grant numbers: 1/3416/06, 1/0164/08, 1/4341/07.
2010 Wiley Periodicals, Inc.
c
ꢀ
203

204 Luka´cˇ et al.
geminis have interesting physicochemical properties
[16–19]. Depending on the length of alkyl chains,
they form gels, micelles, vesicles, tubules, or coacer-
vates in water [12,13]. These zwitterionic gemini sur-
factants are a special type of alkylphosphocholines
(APC), where one methyl group of choline is replaced
with a long alkyl chain.
RESULTS AND DISCUSSION
Four heterogemini surfactants based on various
types of cations were synthesized. The com-
pounds were designed as analogues of de-
cyl 2-[dodecyl(dimethyl)ammonio]ethyl phosphate
(C10PC2NC12) with different polar head groups. The
dimethylamino group was replaced with hetero-
cyclic rings, containing nitrogen as a heteroatom (in
the case of morpholine analogue, an oxygen atom
was incorporated into the heterocyclic ring) to estab-
lish the influence of chemical structure on antipro-
tozoal activities and physicochemical properties.
The synthetic strategy for the preparation
of the heterogemini surfactants is depicted in
Scheme 1. Representative phosphate analogues were
prepared to determine the importance of the phos-
phocholine moiety for bioactivity according to
modified procedures [37]. The strategy involves
phosphorylation of decanol using POCl3 and sub-
sequent attachment of the desired choline analogue.
C10PC2NC12, C10PC2N(Pyr)C12, C10PC2N(Pip)C12, and
C10PC2N(Morph)C12 were prepared by reaction of de-
cyl dichloridophosphate with choline tosylate ana-
logue and subsequently hydrolyzed with H2O. Het-
erogemini surfactants were prepared as hydrates.
APCs possess a wide range of biological activ-
ities. Miltefosine (hexadecylphosphocholine, HPC)
is the main representative of APCs. HPC exhibits
antineoplastic [20], antiprotozoal [21], antibacte-
rial, antimycotic [22], and antivirotic activities [23].
Antiprotozoal activity of HPC and other APCs is
the most studied in recent time. HPC affects many
types of protists such as Leishmania spp. [24], Try-
panosoma spp. [25], Balamuthia mandrillaris, Naeg-
leria fowleri [26], Trichomonas vaginalis [27], Enta-
moeba histolytica [28], and Acanthamoeba spp. [29].
The last named genus is the causative agent of
keratitis, granulomatous amoebic encephalitis, or
nasopharyngeal and cutaneous infections. No effi-
cient and easily manageable treatments are available
for these diseases. Often, the same drugs that have
been successfully used for treatment in one patient
have a little or no effect in the treatment of other
cases of acanthamoebiasis [30]. Therefore, the sus-
ceptibility of several Acanthamoeba spp. to APCs was
investigated [26,29,31–37].
1
The amount of crystal water was estimated by H
NMR. A single 31P NMR signal indicated the purity
of the compound. The choline analogues were ob-
tained by quaternization of 1a, 1b, 1c, and 1d with
dodecyl p-toluenesulfonate in acetonitrile. Tertiary
2-aminoethanols 1b and 1d were prepared by the
reaction of a secondary amine with 2-chloroethanol
and potassium carbonate in the presence of a cat-
alytic amount of KI [38]. Dodecyl p-toluenesulfonate
was prepared according to the procedure described
The aim of the study was to prepare the novel
heterocyclic heterogemini surfactants and to eval-
uate their potential efficacies against free-living
amoeba of the genus Acanthamoeba. The trophoci-
dal activities of four different APCs against Acan-
thamoeba lugdunensis were evaluated in vitro. The
activity of heterogemini surfactants was compared
with their aggregation properties.
SCHEME 1 Preparation of heterogemini surfactants.
Heteroatom Chemistry DOI 10.1002/hc

Synthesis, Aggregation Properties, and Antiprotozoal Activity of Heterocyclic Heterogemini Surfactants 205
TABLE 1 Characterization of Prepared Compounds, Their
Trophocidal Activity, and Diameter of Aggregates Formed in
Aqueous Suspensions
of Acanthamoeba keratitis. Recently, we reported
[36] trophocidal activities of a series of 15 similar
heterogemini surfactants against A. lugdunensis.
Decyl 3-[dodecyl(dimethyl)ammonio]propyl phos-
phate (C10PC3NC12, MTC = 100 μM) and decyl
MTC
(μM)
d
(μm)
Formula
Abbreviation
4-[dodecyl(dimethyl)ammonio]butyl
phosphate
O-
O
N+
(C10PC4NC12, MTC = 200 μM) possess compara-
ble activities as C10PC2N(Pyr)C12 but compounds
with longer spacers between the phosphate group
and ammonium cation (decyl 6-[dodecyl(dime-
H33C16
P
× 1.5 H2O
O
O
HPC
400 [37]
–
O-
O
12H25
H21C10
P
N+C
O
O
H2O
×
C10PC2NC12
>400 [36] <15
O-
H21C10
O
N+
C12H25
thyl)ammonio]hexyl
MTC > 400 μM; and decyl 10-[dodecyl(dimethyl)
ammonio]decyl phosphate (C10PC10NC12)
phosphate
(C10PC6NC12),
H2O
×
P
O
O
C10PC2N(Pyr)C12
C10PC2N(Pip)C12
200
<128
<20
O-
×
1.5 H2O
1.5 H2O
O
H21C10
P
N+
C12H25
MTC > 400 μM) were practically inactive. This
observation indicates that changes in the polar
group of C10PC2NC12 can increase the activity, but
such modifications have limitations. Incorporation
of a five-membered ring to the ammonium cation
and extension of the spacer between the phosphate
group and the ammonium cation from two carbon
atoms to three or four carbon atoms proved useful.
We assume that the different activity of
C10PC2N(Pyr)C12 in comparison with other hetero-
gemini surfactants can be partly explained by their
physicochemical properties. HPC formed clear aque-
ous solution at the concentration c = 1 × 10−3 mol
L−1. No aggregates were observed under an opti-
cal microscope. Heterogemini surfactants formed
cloudy aqueous suspensions at the same concen-
trations. Clouds were caused by the presence of
self-assembled vesicles; solid insoluble particles of
the compounds were not observed. C10PC2NC12,
C10PC2N(Pip)C12, and C10PC2N(Morph)C12 form ag-
gregates in water suspensions with diameters up to
20 μm (Figure 1). C10PC2N(Pyr)C12 formed similar
aggregates but also giant aggregates with diameters
O
O
>800
O
O-
×
O
H21C10
P
N+
C12H25
O
O
C10PC2N(Morph)C12 >800 <12.5
MTC: Minimal trophocidal concentration; d: diameter of aggregates.
by Marvel and Sekera [39]. The synthesis of choline
p-toluenesulfonate analogues 2a, 2b, 2c, and 2d is
depicted in Scheme 1.
The antiprotozoal efficiencies of the prepared
compounds were evaluated against a strain of Acan-
thamoeba lugdunensis (Table 1), which was isolated
from a patient suffering from amoebic keratitis
[40]. The trophocidal activities of heterogemini
surfactants were compared with the activity of HPC.
HPC is considered a standard in the series of APCs.
Its minimal trophocidal concentration (MTC) was
400 μM [37]. This strain of Acanthamoeba lugdunen-
sis is relatively resistant to the action of APCs. Other
species of Acanthamoeba seem to be more sensitive
to HPC. Walochnik et al. [29] studied three species
of Acanthamoeba: A. castellanii, A. polyphaga, A.
lenticulata, and they observed lower values of MTC
(MTC = 20–40 μM). Lower values of MTC were also
obtained by McBride et al. [31]. They studied the
influence of HPC on two strains of Acanthamoeba:
A. polyphaga (MTC = 31.25 μM), A. castellanii
(MTC = 31.25 μM). In our case, HPC was about
10 times less active against A. lugdunensis [37] in
comparison with its activity against A. castellanii, A.
polyphaga, and A. lenticulata, which was observed by
other authors [29,31]. C10PC2NC12, C10PC2N(Pip)C12,
and C10PC2N(Morph)C12 exhibited lower trophoci-
dal activity than HPC. Their MTCs were higher than
400 μM. The amoebae were not affected by these het-
erogemini surfactants. The trophozoites were viable,
and their cells were of typical shapes without any
pathological signs. Only C10PC2N(Pyr)C12 was about
twice as active as HPC (MTC = 200 μM). It may be a
promising new candidate for the topical treatment
FIGURE 1 Aggregate of an aqueous suspension of com-
pound C10PC2NC12 (bar is 5 μm).
Heteroatom Chemistry DOI 10.1002/hc

206 Luka´cˇ et al.
some artifacts, which can be considered as parts of
destroyed cells of amoebae. We suppose that the gi-
ant aggregates affect the trophozoites more than the
vesicles with smaller diameters.
MATERIALS AND METHODS
Materials
All chemicals used for synthesis were purchased
1
from commercial suppliers. H, 13C, and 31P NMR
spectra were recorded on a Varian Gemini 2000
spectrometer operating at 300, 75.5, and 121.5 MHz,
respectively, with 13C and 31P spectra being recorded
with proton-decoupling. The spectra were measured
in CDCl3 relative to the internal standard TMS for 1H
and 13C NMR spectra and to external standard 85%
H3PO4 for 31P NMR spectra. Infrared spectra were
recorded on a FT-IR Impact 400 D spectrophotome-
ter as potassium bromide disks.
FIGURE 2 Aggregate of an aqueous suspension of com-
pound C10PC2N(Pyr)C12 (bar is 5 μm).
up to 128 μm (Figure 2). Smaller aggregates could
be unilamellar or multilamellar vesicles. Com-
pound C10PC2NC12 formed a lamellar phase in water
(Figure 3) (C10PC2NC12: H2O 1:2). The lamellar phase
is stable in a range of temperatures from 25◦C to
70◦C. The similarity of the chemical structures of the
prepared compounds and their behavior in aqueous
suspensions indicate that the heterocyclic analogues
might form the same phases. The occurrence of this
phase in compound/water mixtures justifies the as-
sumption that vesicles could be formed. The giant
aggregates seem to be multicomponent, multilamel-
lar vesicles. Small unilamellar and/or multilamellar
vesicles are enclosed in the larger vesicles (Figure 2).
When we tested the trophocidal activity, we observed
that the giant aggregates of C10PC2N(Pyr)C12 bound
Chemistry
Synthesis of decyl 2-[dodecyl(dimethyl)amonio]
ethyl phosphate (C10PC2NC12) and its pyrrolidinium
(C10PC2N(Pyr)C12), piperidinium (C10PC2N(Pip)C12),
and morpholinium (C10PC2N(Morph)C12) analogues
is shown in Scheme 1.
General Procedure for the Quaternization of
Aminoalcohols with Dodecyl p-Toluenesulfonate.
Aminoalcohol (22 mmol) was added to solution of
dodecyl p-toluenesulfonate (20 mmol) in acetonitrile
(20 mL). The resulting mixture was refluxed for 4 h.
After cooling down, the acetonitrile was evaporated
in vacuum. The resulting mixture was crystallized
from acetone. The quaternary salts were obtained as
white, hygroscopic solids.
N-(2-Hydroxyethyl)-N,N-dimethyldodecane-1-
ammonium Tosylate (2a). Yield 73.9%; 1H NMR
(CDCl3, TMS) δ: 0.88 (t, 3H, J = 6.6 Hz), 1.17–
1.50 (m, 18H), 1.60–1.69 (m, 2H), 2.34 (s, 3H),
3.27 (s, 6H), 3.36–3.45 (m, 2H), 3.64–3.74 (m, 2H),
4.09–4.20 (m, 2H), 5.51 (t, 1H, J = 5.7 Hz), 7.15
(d, 2H, J = 7.9 Hz), 7.74 (d, 2H, J = 8.2 Hz); 13C
NMR (CDCl3, TMS) δ: 14.1, 21.3, 22.7, 22.8, 26.2,
29.1, 29.3, 29.4, 29.5, 29.6, 31.9, 51.8, 56.4, 65.8,
66.0, 125.8, 128.7, 139.6, 143.0.
N-Dodecyl-N-(2-hydroxyethyl)pyrrolidinium To-
1
sylate (2b). Yield 35.8%; H NMR (CDCl3, TMS) δ:
0.88 (t, 3H, J = 6.7 Hz), 1.18–1.42 (m, 18H), 1.58–
1.67 (m, 2H), 1.98–2.32 (m, 4H), 2.34 (s, 3H), 3.26–
3.40 (m, 2H), 3.50–3.63 (m, 4H), 3.73–3.85 (m, 2H),
3.98–4.05 (m, 2H), 7.15 (d, 2H J = 8.1 Hz), 7.73 (d,
2H, J = 8.1 Hz); 13C NMR (CDCl3, TMS) δ: 14.1, 21.3,
21.6, 22.7, 22.9, 23.4, 26.4, 29.2, 29.3, 29.4, 29.5, 29.6,
FIGURE 3 31P NMR spectrum of C10PC2NC12 (25◦C).
Heteroatom Chemistry DOI 10.1002/hc

Synthesis, Aggregation Properties, and Antiprotozoal Activity of Heterocyclic Heterogemini Surfactants 207
31.9, 54.7, 56.5, 57.4, 57.6, 63.4, 125.8, 128.7, 139,7,
142.9.
N-Dodecyl-N-(2-hydroxyethyl)piperidinium Tosy-
Decyl 2-(1-Dodecylpyrrolidinio-1-yl)ethyl Phos-
phate × H2 O (C10PC2N(Pyr)C12). Yield 4.3%; 1H NMR
(CDCl3, TMS) δ: 0.8–0.98 (m, 6H), 1.18–1.52 (m,
32H), 1.57–1.74 (m, 4H), 1.98–2.18 (m, 2H), 2.29–
2.47 (m, 2H), 2.77 (s, 2H), 3.35–3.50 (m, 2H), 3.63–
3.91 (m, 6H), 3.94–4.07 (m, 2H), 4.20–4.31 (m, 2H);
13C NMR (CDCl3, TMS) δ: 14.1, 21.5, 22.7, 23.4, 25.9,
26.5, 29.4, 29.5, 29.6, 29.7, 31.1, 31.9, 58.7, 59.1, 60.2,
63.0, 63.5, 65.6; 31P NMR (CDCl3, H3PO4) δ: 0.26; IR
νmax (cm−1): 3416, 2921, 2851, 1645, 1468, 1260, 1101,
1069, 826.
late (2c). Yield 49.4%; 1H NMR (CDCl3, TMS) δ: 0.88
(t, 3H, J = 6.7 Hz), 1.20–1.32 (m, 18H), 1.58–2.01
(m, 8H), 2.34 (s, 3H), 3.38–3.50 (m, 4H), 3.60–3.76
(m, 2H), 4.03–4.15 (m, 2H), 5.60 (t, 1H, J = 5.8 Hz),
7.14 (d, 2H, J = 7,9 Hz), 7.75 (d, 2H, J = 8.1 Hz);
13C NMR (CDCl3, TMS) δ: 14.3, 20.0, 21.0, 21.5, 21.9,
22.8, 26.6, 29.3, 29.5, 29.6, 29.7, 29.8, 32.1, 55.9, 58.6,
60.1, 61.6, 128.0, 128.8, 139.6, 143.4.
N-Dodecyl-N-(2-hydroxyethyl)morpholinium To-
sylate (2d). Yield 24.2%; H NMR (CDCl3, TMS) δ:
Decyl 2-(1-Dodecylpiperidinio-1-yl)ethyl Phos-
1
1
phate × 1.5H2 O (C10PC2N(Pip)C12). Yield 16.3%; H
0.88 (t, 3H, J = 6.5 Hz), 1.18–1.42 (m, 18H), 1.58–
1.65 (m, 2H), 2.34 (s, 3H), 3.28–3.62 (m, 6H), 3.71–
4.17 (m, 8H), 5.51 (m, 1H), 7.16 (d, 2H, J = 7.8 Hz),
7.18 (d, 2H, J = 7.9 Hz); 13C NMR (CDCl3, TMS) δ:
14.2, 21.3, 21.7, 22.7, 26.3, 29.2, 29.4, 29.5, 29.7, 31.9,
52.7, 55.7, 56.1, 59.0, 59.4, 60.5, 61.1, 63.8, 125.8,
128.9, 139.9, 142.8.
NMR (CDCl3, TMS) δ: 0.81–0.95 (m, 6H), 1.18–1.54
(m, 32H), 1.54–2.05 (m, 10H), 2.91 (s, 3H), 3.44 (m,
4H), 3.75–3.92 (m, 6H), 4.22–4.36 (m, 2H); 13C NMR
(CDCl3, TMS) δ: 14.1, 20.0, 20.8, 21.9, 22.7, 26.0, 26.5,
29.3, 29.4, 29.5, 29.6, 29.7, 31.1, 31.2, 31.9, 57.3, 58.2,
58.3, 59.9, 60.8, 65.4, 65.5; 31P NMR (CDCl3, H3PO4)
δ: 0.78; IR νmax (cm−1): 3417, 2922, 2852, 1634, 1469,
1222, 1100, 1066, 823.
General Procedure for the Preparation of
Decyl 2-(1-Dodecylmorpholinio-1-yl)ethyl Phos-
phate × 1.5H2 O (C10PC2N(Morph)C12). Yield 1.7%;
1H NMR (CDCl3, TMS) δ: 0.84–0.96 (m, 6H), 1.18–
1.45 (m, 32H), 1.54–1.65 (m, 2H), 1.65 (m, 2H),
2.70 (s, 3H), 3.46–3.69 (m, 4H), 3.76–3.86 (m, 2H),
3.90–4.11 (m, 6H), 4.13–4.25 (m, 2H), 4.25–4.36 (m,
2H); 13C NMR (CDCl3, TMS) δ: 14.1, 21.9, 22.7, 25.9,
26.5, 29.3, 29.4, 29.5, 29.6, 29.7, 31.0, 31.1, 31.9, 58.3,
58.4, 59.1, 60.0, 60.1, 60.6, 65.6; 31P NMR (CDCl3,
H3PO4) δ: 0.15; IR νmax (cm−1): 3420, 2922, 2852,
1636, 1468, 1249, 1087, 827.
Alkylphosphocholines. Solution
of
decanol
(9 mmol) in chloroform (20 mL) was added
dropwise at 0◦C to a stirred solution of phosphorous
oxychloride (10 mmol) and triethylamine (20 mmol)
in chloroform (10 mL). The resulting mixture was
stirred at room temperature (r.t.) for 2 h. This
intermediate was used immediately without any
purification. Pyridine (15 mL) was added dropwise
at t = 0◦C to the resulting solution, followed by the
addition of choline tosylate analogue (12.5 mmol).
The reaction mixture was stirred at r.t. overnight.
After cooling, the mixture was hydrolyzed by ad-
dition of H2O (1.5 mL) and stirred for 1 h at r.t.
The solvents were evaporated in vacuum, and the
resulting crude solid was dissolved in a mixture
of tetrahydrofuran–water (5:1 V/V). To the stirred
solution, exchange resin MB3 was added sequen-
tially until the color of the resin ceased to change.
Then, the resin was filtered off and the solvents were
evaporated in vacuum. The resulting crude solid
was purified by crystallization from a mixture of
chloroform and acetone or chloroform and diethyl
ether. ALPs were dried in vacuo over P4O10.
Testing Heterogemini Surfactants for
Trophocidal Susceptibility
These procedures were carried out using the mod-
ified methods described by Walochnik et al. [29].
The cultures of amoeba were grown axenically in
100-mL Erlenmeyer flasks. Axenic culture was ob-
tained from the monoxenic culture of clinically iso-
lated Acanthamoeba lugdunensis as reported previ-
ously [40]. Trophozoites were harvested from 2-day
monoxenic plate cultures and transferred to Bacto-
Casitone/Serum (BCS) medium with penicillin and
ampicillin. Actively growing trophozoites were har-
vested by centrifugation at 500×g for 7 min, and
subsequently they were incubated at 37◦C for 3
days. Thereafter, trophozoites were transferred to
BCS medium without antibiotics and cultivated at
37◦C for 3 days. The cultivation in the BCS medium
was repeated five times, then the trophozoites were
transferred to peptone–yeast extract–glucose (PYG)
medium and afterwards cultivated in this medium.
Experiments were carried out in 96-well microtiter
Decyl 2-[Dodecyl(dimethyl)ammonio]ethyl Phos-
1
phate × H O (C10PC2NC12). Yield 22.2%; H NMR
2
(CDCl3, TMS) δ: 0.84–0.92 (m, 6H), 1.18–1.41 (m,
32H), 1.55–1.78 (m, 4H), 2.18 (s, 2H), 3.35 (s, 6H),
3.41–3.50 (m, 2H), 3.76–3.89 (m, 4H), 4.27–4.35 (m,
2H); 13C NMR (CDCl3, TMS) δ: 14.1, 22.7, 22.9, 26.0,
26.3, 29.3, 29.4, 29.5, 29.6, 29.7, 31.0, 31.1, 31.9, 51.9,
58.8, 64.2, 65.5, 65.6, 65.9; 31P NMR (CDCl3, H3PO4)
δ: 0.76; IR νmax (cm−1): 3427, 2919, 2851, 1635, 1469,
1242, 1100, 1067, 822.
Heteroatom Chemistry DOI 10.1002/hc

208 Luka´cˇ et al.
plates at 37◦C under sterile conditions. Each well was
seeded with 100 μL (2 × 105 cells) of a trophozoite
suspension. Then 100 μL of test substance solution
in PYG medium was added and mixed with the sus-
pension of trophozoites. APCs were tested at five con-
centrations (50, 100, 200, 400, and 800 μM). Viability
was determined by trypan blue exclusion. One hun-
dred percent eradication was confirmed by transfer-
ring 50 μL of the suspension to a PYG medium and
by recording the amoeba growth for 14 days.
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pounds in Water. The samples were prepared by
simple soaking of solids in deionized water. Com-
pounds were dissolved by shaking in a volumetric
flask. The concentrations of solutions/suspensions
were 1 × 10−3 mol L−1. The samples were inves-
tigated 2 weeks after preparation. The aggregates
were observed under Nikon Labophot optical mi-
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Measurement of Aggregates of C10PC2NC12 in
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NMR tube. 100 μL of deionized water was added to
50 mg of C10PC2NC12. The mixture was homogenized
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The authors wish to thank Prof. Ferdinand Dev´ınsky
for useful comments and Dr. Milan Hudecˇek for cor-
recting the English of the manuscript.
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