Synthesis of structural analogues of hexadecylphosphocholine and their antineoplastic, antimicrobial and amoebicidal activity

Article abstract of DOI:10.1016/j.ejmech.2015.02.014

Twelve derivatives of hexadecylphosphocholine (miltefosine) were synthesized to determine how the position and length of the alkyl chain within the molecule influence their biological activities. The prepared alkylphosphocholines have the same molecular formula as miltefosine. Activity of the compounds was studied against a spectrum of tumour cells, two species of protozoans, bacteria and yeast. Antitumour efficacy of some alkylphosphocholines measured up on MCF-7, A2780, HUT-78 and THP-1 cell lines was higher than that of miltefosine. The compounds showed antiprotozoal activity against Acanthamoeba lugdunensis and Acanthamoeba quina. Some of them also possess fungicidal activity against Candida albicans equal to miltefosine. No antibacterial activity was observed against Staphylococcus aureus and Escherichia coli. A difference in position of a long hydrocarbon chain within the structure with maximum efficacy was observed for antitumour, antiprotozoal and antifungal activity.

Full text of DOI:10.1016/j.ejmech.2015.02.014

European Journal of Medicinal Chemistry 93 (2015) 263e273
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of structural analogues of hexadecylphosphocholine and
their antineoplastic, antimicrobial and amoebicidal activity
a
,
*
b
c
ꢀꢁ
ꢀ
ꢀ c
Lukas Timko , Eva Fischer-Fodor , Maria Garajova , Martin Mrva ,
b
d
e
ꢁ
ꢀ
ꢁ
ꢀꢁ a
Gabriela Chereches , Frantisek Ondriska , Marian Bukovský , Milos Lukac ,
Janka Karlovska , Janka Kubincova , Ferdinand Devínsky
ꢀ f
ꢀ a
,
g
a
a
ꢁ
Department of Chemical Theory of Drugs, Faculty of Pharmacy, Comenius University, Kalinciakova 8, 832 32 Bratislava, Slovakia
b
c
d
e
f
ꢂ
The Oncology Institute “Prof. Dr. Ion Chiricut¸a”, 400015 Cluj-Napoca, Romania
ꢀ
Department of Zoology, Faculty of Natural Sciences, Comenius University, Mlynska Dolina B-1, 842 15 Bratislava, Slovakia
HPL (Ltd), Department of Parasitology, Microbiological Laboratory, Istrijska 20, 841 07 Bratislava, Slovakia
Department of Cell and Molecular Biology of Drugs, Faculty of Pharmacy, Comenius University, Kalinciakova 8, 832 32 Bratislava, Slovakia
NMR Laboratory, Faculty of Pharmacy, Comenius University, Odbojarov 10, 832 32 Bratislava, Slovakia
ꢀ
ꢁ
ꢀ
g
ꢁ
Department of Physical Chemistry of Drugs, Faculty of Pharmacy, Comenius University, Kalinciakova 8, 832 32 Bratislava, Slovakia
a r t i c l e i n f o
a b s t r a c t
Article history:
Twelve derivatives of hexadecylphosphocholine (miltefosine) were synthesized to determine how the
position and length of the alkyl chain within the molecule influence their biological activities. The
prepared alkylphosphocholines have the same molecular formula as miltefosine. Activity of the com-
pounds was studied against a spectrum of tumour cells, two species of protozoans, bacteria and yeast.
Antitumour efficacy of some alkylphosphocholines measured up on MCF-7, A2780, HUT-78 and THP-1
cell lines was higher than that of miltefosine. The compounds showed antiprotozoal activity against
Acanthamoeba lugdunensis and Acanthamoeba quina. Some of them also possess fungicidal activity
against Candida albicans equal to miltefosine. No antibacterial activity was observed against Staphylo-
coccus aureus and Escherichia coli. A difference in position of a long hydrocarbon chain within the
structure with maximum efficacy was observed for antitumour, antiprotozoal and antifungal activity.
© 2015 Elsevier Masson SAS. All rights reserved.
Received 14 August 2014
Received in revised form
9 January 2015
Accepted 10 February 2015
Available online 12 February 2015
Keywords:
Acanthamoeba
Alkylphosphocholines
Candida albicans
Cytotoxic activity
Cut-off effect
SAR
1. Introduction
chemotherapeutical regimens.
Besides antineoplastic activity alkylphosphocholines possess
Synthetic alkylphosphocholines (APCs) have the capacity to
inhibit the growth of cancer cells selectively [1,2]; their cellular
uptake is efficient, and they are capable to target some of the
important signalling pathways in tumour growth [3]. Some of them
were already tested successfully in clinical trials [4e9]. The leading
compound miltefosine (hexadecylphosphocholine, HPC) is used for
topical application in patients with skin metastases of breast cancer
[2]. These compounds exhibit inhibitory capacity against tumour
cells derived from different malignant localisations, such as breast
[10] and ovarian [11,12] cancer. HPC have a co-stimulatory effect on
granulopoiesis and thrombocytopoiesis [9], which is in contrast
also antiprotozoal properties. They show a trophocidal action
against a wide spectrum of protozoans, including Leishmania [13],
Trypanosoma [14], Entamoeba [15], Acanthamoeba [16], Trichomonas
[17], and Naegleria [18]. Miltefosine is a registered drug for the
treatment of visceral leishmaniasis [19]. Acanthamoeba spp. is a
free-living amoeba which causes granulomatous amoebic enceph-
alitis (GAE) and amoebic keratitis (AK) [20]. GAE is characterised by
high mortality and optimal therapy has not been established yet
[21]. Most cases of amoebic keratitis are related to the use of con-
tact lenses. AK is treated with combinations of different drugs:
nevertheless, an optimal therapy is also needed to be found [22].
Alkylphosphocholines are known to have antifungal activity as
well. Susceptible species are Candida, Cryptococcus, Aspergillus,
Fusarium and Scedosporium. APCs act as fungicidal agents [23].
In previous studies authors investigated antineoplastic or anti-
leishmanial activity of broad spectrum of alkylphosphocholines,
with
bone-marrow
suppression
typical
for
most
* Corresponding author.
E-mail address: timko@fpharm.uniba.sk (L. Timko).
http://dx.doi.org/10.1016/j.ejmech.2015.02.014
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

264
L. Timko et al. / European Journal of Medicinal Chemistry 93 (2015) 263e273
Scheme 1. Synthesis of alkylphosphocholines. Reagents and conditions: a) 4-methylbenzenesulphonyl chloride, pyridine, 20 ꢀC; b) 2-(N,N-dimethylamino)ethanol, CH3CN, reflux;
c) i: CH3(CH2)xOH, POCl3, Et3N, CHCl3, 25 ꢀC; ii: pyridine, H2O, 25 ꢀC.
which included molecules with cycles in the structure alone [24] or
in combination with unsaturated bonds [13,25], or ether bond in
the alkyl chain [26]. Heterocyclic substitutions on aminium nitro-
gen were also tested [26,27]. The presented compounds have
structural analogies with the known alkylphosphocholines and are
isomers of miltefosine. We evaluated their cytotoxicity and anti-
protozoal and antimicrobial activity in order to study structur-
eeactivity relationship. The aim of the study was to discover
influence of isomers upon the biological activity, i. e. the influence
of the position of the alkyl chain within the molecule on the anti-
neoplastic, antimicrobial and amoebicidal activity.
primary alcohols with 4-methylbenzenesulphonyl chloride. The
purity of the APCs was determined using 31P NMR spectroscopy,
where a single signal of the phosphate group was observed, and by
mass spectrometry. Three compounds, C7eC10, C8eC9, and C9-8,
were previously prepared by Menger and Peresypkin [28] by a
method using 2-chloro-2-oxo-1,3,2-dioxophospholane as a starting
material. Compound C10eC7 is a patented structure of Zentaris
GmbH [29].
Several attempts were made to prepare a compound with an
inverted hexadecylphosphocholine structure, with the hexadecyl
chain linked to the aminium nitrogen and the methyl group
attached to the phosphate moiety. Different synthetic approaches
were used: the synthetic procedure mentioned above and the
procedure which includes the hydrolysis of methyldi-
chlorophosphate and the reaction of the resulting methyl-
2. Results and discussion
2.1. Chemistry
phosphonic acid with
a choline 4-methylbenzenesulphonate
derivative catalysed by 2,4,6-triisopropylbenzenesulphonyl chlo-
ride [26]. None of the methods, even the one used by Peresypkin
and Menger [30], which should afford higher yields, was successful.
A series of twelve alkylphosphocholines, structural derivatives
of hexadecylphosphocholine, was synthesized according to Scheme
ꢀꢁ
1 using a procedure modified by Lukac et al. [27]. This series was
prepared to determine the effect of the position and length of the
alkyl chain linked to the phosphate or aminium moiety on the
biological activity. The synthesized APCs have the same molecular
formula as miltefosine. The APCs were prepared by phosphoryla-
tion of primary alcohols with phosphorus oxychloride and subse-
2.2. Antineoplastic activity
Alkylphosphocholines C2eC15 to C15eC2 and HPC (miltefo-
sine) showed different inhibitory effect on cell growth in the
tumour cell and normal cell cultures. IC50 values (Table 1),
reflecting the cytotoxicity of the compounds against MCF-7 breast
cancer cells, A2780 ovarian cancer cells, THP-1 and HUT-78
leukaemia cells, were determined on the basis of their dose-
eeffect relationship, in the 95% confidence interval. IC50 is the
concentration that inhibited 50% of the population growth; low
IC50 values indicate high cytotoxicities.
quent
reaction
with
derivatives
of
choline
4-
methylbenzenesulphonate. The reaction mixture was then hydro-
lysed by addition of water to get the desired alkylphosphocholine.
The products were purified by column chromatography and crys-
tallisation. Choline 4-methylbenzenesulphonate derivatives were
synthesized by a reaction of 2-(N,N-dimethylamino)ethanol and
alkyl 4-methylbenzenesulphonate in acetonitrile. Alkyl 4-
methylbenzenesulphonates were prepared by tosylation of
Normal human peripheral blood cells, mainly a lymphocyte
Table 1
Values of IC50, minimal trophocidal concentration (MTC) and minimal inhibitory concentration (MIC) of prepared compounds. Figures marked with * represent values outside
the 95% confidence interval. Figures in bold correspond to the most active compounds.
Compound
IC50 [
mM]
MTC [
mM]
MIC [mM]
MCF-7
A2780
HUT-78
THP-1
A. lugdunensis
A. quina
S. aureus
E. coli
C. albicans
C2eC15
C3eC14
C5eC12
C6eC11
C7eC10
C8eC9
14.44
20.90
25.75
64.50
26.21
19.25
33.96
62.25
111.37
66.07
50.62
43.34
35.68
7.44
9.26
18.32
25.72
4.70
35.23
29.06
68.02
30.54
29.40
71.22
62.5
125
125
62.5
125
125
250
250
125
125
125
125
250
62.5
125
125
125
125
125
250
250
250
125
125
125
125
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
11.5
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
21.14
86.30
352.27
359.53
352.23
367.14
734.29
179.77
187.54
183.57
11.71
96.05
31.22
436.90*
279.80*
146.10*
52.93
82.59
55.19
51.37
62.74
67.24
646.60*
543.85*
170.47
101.40
92.05
93.25
127.07
96.80
4.32
7.31
C9eC8
C10eC7
C11eC6
C12eC5
C14eC3
C15eC2
HPC
35.05
19.15
24.82
28.58
10.06
8.87
2.93
2.87
81.28

L. Timko et al. / European Journal of Medicinal Chemistry 93 (2015) 263e273
265
Fig. 1. Inhibitory effect of alkylphosphocholines on the proliferation of A2780 ovarian
carcinoma cells.
Fig. 3. Activity of alkylphosphocholines activity against HUT-78 leukaemia cell line.
IC50 values corresponding to C7eC10, C8eC9 and C9eC8 were approximated by the
Graph Pad Prism software outside the 95% confidence interval; one can consider those
values as extrapolated.
population, showed very good survival after 24-h treatment with
the examined compounds, the viability of control cells was well
maintained without other stimuli, and in the treated cells the in-
hibition was very weak. With these peripheral blood cells, a
maximum of 12% inhibition of growth was detected at a concen-
the highest activity, followed by C5eC12 and C7eC10; these five
compounds were more efficient than miltefosine (Fig. 2). The new
alkylphosphocholines C2eC15, C3eC14 and C8eC9 have signifi-
cantly better activity than miltefosine (one-way ANOVA, multiple
comparison test, p < 0.05), while C6eC11, C10eC7, C11eC6, and
C12eC5 show a significantly lower activity in MCF-7 cells than the
reference HPC.
In HUT-78 cells, the most active are C2eC15 and C3eC14 (Fig. 3);
except C7eC10 and C8eC9 having very high IC50 values, the ac-
tivities of the other ten compounds of the series do not differ
significantly from that of miltefosine. In THP-1 leukaemia popula-
tion, there are three compounds with low activities (significantly
lower than miltefosine); while the most efficient are C2eC15,
C3eC14 and C6eC11 (Fig. 4).
tration of 500 mM in C3eC14, followed by 10% inhibition in C2eC15.
No cytotoxicity was measured after 24 h in C7eC10, C8eC9, and
C9eC8, and this was the reason why for neither of the compounds
the IC50 values could be computed using statistic programs in the
range of concentrations used.
The synthesized alkylphosphocholines reached the best inhibi-
tory effect against A2780 cell line (Fig. 1). All the IC50 values are
below 35.05 mM, activities of the most of the compounds do not
differ significantly from that of miltefosine (multiple comparison
test, p < 0.05), except C6eC11, C10eC7, C12eC5 and C14eC3.
Nevertheless, IC50 values of these four compounds are lower in
A2780 cells in comparison with the other three cell lines.
The IC50 values of three compounds in HUT-78 and THP-1 T-
lymphocyte and monocyte leukaemia differ in an order of
In MCF-7 cell population, C2eC15, C3eC14 and C8eC9 showed
Fig. 4. Activity of alkylphosphocholines activity against THP-1 undifferentiated
monocyte leukaemia cell line.
Fig. 2. Inhibitory effect of alkylphosphocholines on the proliferation of MCF-7 breast
carcinoma cells.

266
L. Timko et al. / European Journal of Medicinal Chemistry 93 (2015) 263e273
magnitude from the IC50 pattern corresponding to the breast and
ovarian cancer cells. In the case of compounds C7eC10, C8eC9 and
C9eC8, the statistics cannot provide values in 95% confidence in-
terval. The values depicted on the graphs (Figs. 3 and 4) are
computed by extrapolation and they are very close to the highest
concentration actually used. Therefore it can be concluded that
these compounds do not exhibit any toxicity against leukaemia
cells.
carcinoma cells
membrane and its downregulation enhances the apoptosis and
inhibits cell proliferation [38]. Very high levels of -catenin are
constitutively expressed in the leukaemia cells and although inhi-
bition of the signalling leads to the decrease of cell proliferation, the
apoptotic process is regulated in a different way. Normal human
b-catenin is localised in cytoplasm and on the cell
b
lymphocytes do not contain detectable levels of
phorylation is involved in pathways regulated by
b
-catenin. Phos-
-catenin.
b
Previously tested alkylphosphocholines, such as miltefosine,
perifosine, erucylphosphocholine and erufosine, and alkylphos-
pholipid edelfosine act on cancer cells by altering the cell mem-
brane, they are capable of interfering with the cell cycle in highly
proliferative tumours [3]. They can induce apoptosis by activation
of caspases 3 and 8 and by cleavage of cell death substrates, such as
poly-ADP-ribose polymerase (PARP) [31].
Therefore the position of the phosphate moiety in the structure of
the alkylphosphocholines probably influences the biologic
outcome and it could play an important role in the selectivity of the
compounds.
2.3. Antiprotozoal activity
In the present study, the results put forward the fact that the
synthesized compounds were more active against highly prolifer-
ative cells in comparison with lymphocytes and malignant cells
with lower turnover rate. Also, they are more active against cells
which grew attached (A2780 and MCF-7), suggesting that the
compounds interfere with adhesion and migration of the malignant
cells. Alkylphosphocholines are capable of decreasing the propor-
tion of cells in the S phase and increase the proportion in the G2/M
phases of the cell cycle [12], and this could lead to a selective in-
hibition of highly proliferative cells, in this case the solid tumours
versus tumour cells with slower turnover and normal haemato-
poietic cells.
Selectivity of the synthesized alkylphosphocholines is
confirmed by their low inhibitory capacity against normal human
lymphocytes. These results are in concordance with the biologic
properties of other similar compounds with therapeutic potential
[9,31].
MCF-7 and A2780 cells derived from female genital tumours are
strongly inhibited by the alkylphosphocholines, but C2eC15 in-
fluence is probably not dependent on oestrogen receptors. MCF-7
cells express [10] the oestrogen receptor alpha, while A2780
ovarian carcinoma cells do not express this receptor [32]. Alkyl-
phosphocholines were proven to be effective against breast cancer
cells in vitro, but they exhibit toxicity against cell lines without
discriminate oestrogen receptor positive and negative [33] cell
lines and they interfere with the PI3K and Akt [24] cell signalling
The antiprotozoal activity of the prepared compounds was
tested on strains of Acanthamoeba lugdunensis and Acanthamoeba
quina, which are responsible for developing amoebic keratitis.
Values of minimal trophocidal concentration are summarised in
Table 1.
The activities of the synthesized compounds on both strains
were comparable to the activity of the reference compound, mil-
tefosine (Fig. 5). In case of A. lugdunensis, the compounds achieved a
better antiprotozoal effect than the standard except C9eC8 and
C10eC7, which displayed the same activity as miltefosine. Anti-
protozoal activities of the compounds against A. quina reached the
same value as miltefosine, only compounds C9eC8, C10eC7 and
C11eC6 were less effective. The most active compound in both
cases was C2eC15, which was four times more effective against
A. lugdunensis and twice as active as miltefosine against A. quina.
Higher antiprotozoal activity of C2eC15 is probably due to its closer
structural similarity to quaternary aminium compounds (QUATs).
QUATs are known to have better antiprotozoal effects than alkyl-
phosphocholines, on the other hand they are also more toxic to
healthy human cells [27,39]. Saran et al. observed by NMR spec-
troscopy that negatively charged phosphate group of miltefosine
interacts with positive aminium nitrogen of the polar head group of
the phospholipid in the membrane and vice versa [40]. We sup-
posed that compound C2eC15 has better solubilisation properties
than alkylphosphocholine with inverted structure. This compound
can better interact with membrane phospholipids of Acanthamoeba
cells than C15eC2. Its aminium cation, which is closer to the long
alkyl chain, can interact more easily with the phosphate anion of
pathways. The Wnt/b-catenin signalling is also affected [34] by
alkylphosphocholines and this leads to the triggering of apoptosis.
The haematopoietic cells, both normal and tumour, are mildly
affected by compounds C2eC15 to C15eC2 and HPC: tumour
lymphoblasts or monocytes are weakly inhibited. The compounds
have an insignificant effect on normal lymphocytes, which means
they probably do not interfere with the main growth factors, cy-
tokines and chemokines influencing the viability and proliferation
of haematopoietic cells. The selectivity against cells derived from
blood versus solid tumours was reported for some alkylphos-
phocholine analogues [35], being the premise of a good therapeutic
potential. The behaviour of the prepared compounds is very similar.
For example, erufosine affects the leukaemia cells selectively while
it is harmless against normal human lymphocytes [36]. A similar
pattern was observed in our experiment. On the other hand, eru-
cylphosphohomocholine exhibits cytotoxicity in acute leukaemia
with highly proliferative cells [37], by interfering with JNK-ERK
pathways. The studied leukaemia cells had a much lower prolifer-
ation rate in comparison with acute leukaemia and these cells were
of an inferior differentiation stage. The tested compounds displayed
a moderate toxicity against these cell lines.
A possible explanation of distinctive antiproliferative effect of
the synthesized compounds on carcinoma cells is their effect on the
Wnt/
b
-catenin signalling pathway. In adherent epithelial
Fig. 5. Antiprotozoal activities of alkylphosphocholines against Acanthamoeba spp.

L. Timko et al. / European Journal of Medicinal Chemistry 93 (2015) 263e273
267
the membrane phospholipid and the phosphate anion of C2eC15 is
closer to the aminium cation of the choline part of the phospho-
lipid. In this orientation the long alkyl chain is also incorporated in
the cell membrane. It means that the zwitterion of C2eC15 has a
better orientation for interaction with a membrane phospholipid
than the zwitterion of C15eC2.
chain in the molecule. The shortening of the chain causes a
decrease of the activity with minimum activity for compound
containing 11 carbons in the chain. In case of solid tumours, high
activity was also observed for compounds possessing two
approximately equal long chains on nitrogen and on the phosphate
moiety, C8eC9 and C7eC10 (Figs. 1 and 2). Whereas for activity
against leukemic cells the long alkyl chain was essential and could
not be substituted by two shorter chains (Figs. 3 and 4). The
observed behaviour of activity of the series for leukemic cell lines
copies a pattern similar to the cut-off effect [44]. Based on this, one
can assume that the lengthening of the chain on the phosphate site
or on the aminium nitrogen site could increase antitumour activity.
Comparison of two compounds with invert structure (e. g. C2eC15
and C15eC2) favours the compound with the long alkyl chain
linked to nitrogen for all four tested tumour cell lines. Above
mentioned higher similarity with quaternary aminium salts could
be possible explanation. Long chain aminium salts are more effi-
cient than alkylphosphocholines against tumour cells [27]. An
advantage of the synthesized compounds vs. QUATs is their low
toxicity for normal cells.
2.4. Antimicrobial activity
Antimicrobial activity was measured against gram-positive
bacteria Staphylococcus aureus, gram-negative bacteria Escherichia
coli and yeast Candida albicans (Table 1). The prepared APCs showed
very weak antibacterial activity against E. coli. All compounds had
MIC out of range of tested concentrations. These findings corre-
spond to results obtained by Obando et al. with different series of
alkylphosphocholines, who reported no activity against gram-
negative bacteria [41]. Similar results were observed for S. aureus.
The only active compound was miltefosine with MIC value of
11.5 mM which is twofold lower than the value measured by Obando
et al. for methicillin-resistant strain of S. aureus [41].
The antiprotozoal activity also depends on the position of the
long alkyl chain (nitrogen or phosphate). Compounds with two
shorter chains showed insufficient activity in comparison with
compounds possessing one long chain. With shortening of the alkyl
chain the critical micelle concentration (ck) of the compounds in-
creases. The study of aggregation properties of compounds
described here in this paper, namely the ck, is the subject of our
next paper which will be published elsewhere. Preliminary data
show that critical micelle concentration is in a range between ck of
miltefosine, 1.25 ꢁ 10ꢂ6 mol dmꢂ3 [45], and ck of derivative with
two eight-carbon long chains, 1.00 ꢁ 10ꢂ3 mol dmꢂ3 [30]. Solubility
of all compounds in water was satisfactory for testing of biological
activities. Compounds with approximately equally long alkyl chain
didn't show better antiprotozoal activity in comparison with mil-
tefosine, except of C8eC9 in case of A. lugdunensis. Study made by
The prepared compounds showed moderate activity against
C. albicans although none of them was more effective than milte-
fosine. MIC of miltefosine was 2.87 mM, which is similar to the value
ꢀꢁ
measured by Lukac et al. [39], Obando et al. [41] or Ravu et al. [42].
Comparable activity was observed in the case of C15eC2 with MIC
of 2.93
mM. The antifungal activity of the tested compounds
decreased with the shortening of the hydrocarbon chain attached
to the phosphate moiety (Fig. 6) with minimum activity for com-
pound C9eC8. The activity began to smoothly increase again with
extension of the alkyl chain on the aminium side. But even the
C2eC15 with the longest alkyl chain attached to nitrogen did not
achieve the MIC of miltefosine and its activity was ten times lower
than the activity of C15eC2, the compound with an inverted
structure. It could be assumed that antifungal activity depends on
long hydrocarbon chain attached to the phosphate moiety. How-
ever there is limitation in length of the chain. Exceeding the
number of carbon atoms over 18 decreases the activity [43].
ꢀꢁ
Lukac et al. [46] suggests that exchange of one of the hydrocarbon
chains for perflourinated one can increase the activity in case of
alkylphosphonatocholines with eight carbon chain linked to
phosphonate and nitrogen. However this activity is still similar to
analogical alkylphosphocholines from our series. The most active
compound possesses long alkyl chain linked to aminium moiety.
The optimal length of the alkyl chain of the alkylphosphocholine is
also important for antiprotozoal activity. The compound with
similar polar head, however, with a chain containing 20 carbon
atoms (C2eC20) was less active than C2eC15. Its minimal troph-
2.5. Structureeactivity relationship
A repeating pattern can be observed in the plots of activities of
the prepared compounds against tumour cell lines (Figs. 1e4). The
activity of the compounds depends on the presence of long alkyl
ocidal concentration is over 400 mM for A. lugdunensis [43]. This can
be explained by a relatively poor solubility of this compound in
water under normal conditions.
Better antifungal properties were observed for compounds with
long alkyl chain linked to the phosphate moiety. The lipophilicity of
alkylphosphocholine plays important role. Obando et al. [41]
observed that alkylphosphocholines that have hexadecyl or octa-
decyl alkyl chain connected to phosphate possess the best anti-
candidal activities. Further increase in alkyl chain up to 20 carbons
causes drop of anticandidal activity [43]. These observation is
another example of cut-off effect described in detail by Balgavý and
Devínsky [44]. However this observation is different to our findings
in case of antiprotozoal and antitumour activity, where opposite
analogues with long alkyl chains in the aminium moiety were the
most active.
3. Conclusions
In this study, twelve compounds of the alkylphosphocholine
Fig. 6. Activities of the prepared alkylphosphocholines against yeast Candida albicans.
group were synthesized. The main aim of the study was to find out

268
L. Timko et al. / European Journal of Medicinal Chemistry 93 (2015) 263e273
how the different position and length of alkyl chains linked to
phosphate or aminium moiety affect the biological activity. The
total sum of carbons in both chains is 17 which is equal to the
number of relevant carbon atoms in the reference compound,
miltefosine.
after his written informed consent. A. lugdunensis and A. quina were
clinical isolates of free-living amoeba isolated from corneas of pa-
tients with Acanthamoeba keratitis [48,49]. Cultures used for anti-
microbial tests were S. aureus ATCC 29/58, E. coli ATCC 377/79 and C.
albicans ATCC 8186.
The studied series of alkylphosphocholines displayed a very
promising antitumour potential in solid tumours, and two of its
representatives, C2eC15 and C3eC14, are the most active com-
pounds against all four tested tumour cell lines. Nine compounds of
the series are highly selective; i. e., their cytotoxic effect is signifi-
cantly higher in the studied tumour cells versus normal human
lymphocytes. The structures having a longer chain linked to the
phosphate moiety displayed a better cytotoxic effect and they show
similarities with the activity of miltefosine in solid tumour and
leukaemia cells.
The prepared compounds also possess antiprotozoal activity
against pathogenic strains of amoebae A. lugdunensis and A. quina.
The most active compound against both strains is C2eC15. The
antibacterial efficiency was measured against E. coli and S. aureus.
None of the synthesized alkylphosphocholines showed a significant
antibacterial activity. Yeast C. albicans was used for tests of anti-
fungal properties. Best activity was observed in the case of com-
pound C15eC2.
4.2. Chemistry
4.2.1. General procedure for preparation of alkyl 4-
methylbenzenesulphonates
4-Methylbenzenesulphonyl chloride (55 mmol) was added
slowly to a mixture of a primary alcohol (50 mmol) and 20 ml of
pyridine at 10 ꢀC. The reaction mixture was stirred for 3 h at 20 ꢀC.
After that 120 ml of 25% hydrochloric acid was slowly added. The
reaction mixture was then extracted with chloroform, organic layer
dried with Na2SO4 and evaporated to yield alkyl 4-
methylbenzenesulphonate as colourless oily liquid or white solid,
which was used without further purification. Ethyl 4-
methylbenzenesulphonate was purchased from Fluka.
4.2.1.1. Propyl 4-methylbenzenesulphonate. Yield 97.0%; 1H NMR
(CDCl3)
d
0.90 (t, 3H, J ¼ 7.5 Hz); 1.61e1.73 (m, 2H); 2.45 (s, 3H);
3.98 (t, 2H, J ¼ 6.6 Hz); 7.35 (d, 2H, J ¼ 7.9 Hz); 7.78 (d, 2H,
J ¼ 8.2 Hz); 13C NMR (CDCl3)
d 10.0; 21.6; 22.3; 72.2; 127.9; 129.8;
4. Experimental
133.2; 144.7.
4.1. Material and methods
4.2.1.2. Pentyl 4-methylbenzenesulphonate. Yield 93.3%; 1H NMR
(CDCl3) 0.82e0.93 (m, 3H); 1.18e1.33 (m, 4H); 1.59e1.68 (m, 2H);
d
Chemicals for synthesis were obtained from commercial sup-
pliers: CentralChem, Slovakia e acetone, acetonitrile, diethyl ether,
chloroform, propan-1-ol, propan-2-ol, pyridine, tetrahydrofurane,
triethylamine; Fluka, Germany e decan-1-ol, dodecan-1-ol, ethyl 4-
methylbenzenesulphonate, 4-methylbenzenesulphonyl chloride,
phosphorus oxychloride, methyl 4-methylbenzenesulphonate, N,N-
dimethyl-2-aminoethanol, nonan-1-ol, undecan-1-ol; Lachema,
Czech republic e octan-1-ol, tetradecan-1-ol; Merck, Germany e
Amberlite MB-3, pentadecan-1-ol, pentan-1-ol; Reachim, Russia e
heptan-1-ol, hexan-1-ol. Chemicals were used as supplied. Solvents
for synthesis were purified and dried before use according to Perrin
and Armarego [47]. Choline salts were dried at 61 ꢀC at diminished
pressure prior to use.
2.44 (s, 3H); 4.01 (t, 2H, J ¼ 6.5 Hz); 7.34 (d, 2H, J ¼ 7.9 Hz); 7.78 (d,
2H, J ¼ 8.2 Hz); 13C NMR (CDCl3)
d 13.5; 21.3; 21.7; 27.1; 28.2; 70.4;
127.5; 129.5; 132.8; 144.4.
4.2.1.3. Hexyl 4-methylbenzenesulphonate. Yield 86.2%; 1H NMR
(CDCl3)
0.84 (t, 3H, J ¼ 6.9 Hz); 1.17e1.34 (m, 6H); 1.58e1.67 (m,
d
2H); 2.44 (s, 3H); 4.01 (t, 2H, J ¼ 6.6 Hz); 7.34 (d, 2H, J ¼ 7.9 Hz); 7.78
(d, 2H, J ¼ 8.2 Hz); 13C NMR (CDCl3)
d 13.9; 21.6; 22.4; 25.0; 28.8;
31.1; 70.7; 127.9; 129.8; 133.2; 144.7.
4.2.1.4. Heptyl 4-methylbenzenesulphonate. Yield 95.4%; 1H NMR
(CDCl3)
0.86 (t, 3H, J ¼ 6.9 Hz); 1.19e1.31 (m, 8H); 1.33e1.69 (m,
d
2H); 2.44 (s, 3H); 4.01 (t, 2H, J ¼ 6.5 Hz); 7.34 (d, 2H, J ¼ 7.9 Hz); 7.78
1H, 13C and 31P NMR spectra were measured on a Varian MER-
CURY plus spectrometer working at frequency 300, 75 and
121.5 MHz respectively. 13C and 31P NMR spectra were decoupled
against protons. Spectra were measured in CDCl3 or DMSO-d6. TMS
was used as the internal standard for 1H and 13C NMR spectra and
85% H3PO4 was used as the external standard for 31P NMR spectra.
Mass spectra were recorded on an LTQ Orbitrap XL hybrid FTMS
spectrometer (Thermo Fisher Scientific) using electrospray Ion
Max-ESI in positive mode. Elementary analysis was carried out on
FLESCH 2000 (Thermo Fischer Scientific).
(d, 2H, J ¼ 8.2 Hz); 13C NMR (CDCl3)
d 14.0; 21.6; 22.5; 25.3; 28.6;
28.8; 31.6; 70.7; 127.9; 129.8; 133.2; 144.7.
4.2.1.5. Octyl 4-methylbenzenesulphonate. Yield 76.0%; 1H NMR
(CDCl3)
d
0.86 (t, 3H, J ¼ 6.9 Hz); 1.22e1.27 (m, 10H); 1.60e1.65 (m,
2H); 2.45 (s, 3H); 4.02 (t, 2H, J ¼ 6.6 Hz); 7.34 (d, 2H, J ¼ 8.1 Hz); 7.78
(d, 2H, J ¼ 8.2 Hz); 13C NMR (CDCl3)
d 14.1; 21.6; 22.6; 25.3; 28.8;
28.9; 29.1; 31.7; 70.7; 127.9; 129.8; 133.2; 144.7.
4.2.1.6. Nonyl 4-methylbenzenesulphonate. Yield 81.4%; 1H NMR
Cell cultures were performed in a fully equipped cell culture
laboratory, with class II laminar hoods (Lamil Plus and Heto Hol-
ten), sterile humidified incubators with 5% CO2 level and constant
37 ꢀC temperature (from Uniequip and Thermo Electron Corpora-
tion), centrifuges with swing-out rotors, for cell cultures (Hettich
320R), e80 ꢀC ultra freezer (Heto Ultra Freeze), e190 ꢀC liquid ni-
trogen tank (Cryosystem 2000 from MVE), incubator with a shaker
(Heidolph Titramax 1000) and other laboratory devices. Colori-
metric and fluorimetric measurements were assessed using Syn-
ergy 2 multiplate reader (BioTek). MCF-7 (breast tumour), A2780
(ovarian carcinoma), THP-1 and HUT-78 (leukaemia) cell lines were
acquired from European Collection of Cell Cultures (ECCAC).
Normal human lymphocytes were isolated from whole blood
collected by venipuncture from a healthy 42-years old male donor,
(CDCl3)
d
0.87 (t, 3H, J ¼ 6.9 Hz); 1.22e1.31 (m, 12H); 1.58e1.65 (m,
2H); 2.44 (s, 3H); 4.01 (t, 2H, J ¼ 6.5 Hz); 7.34 (m, 2H, J ¼ 8.8 Hz);
7.78 (d, 2H, J ¼ 8.2 Hz); 13C NMR (CDCl3)
d 14.1; 21.6; 22.6; 25.3;
29.3; 29.4; 29.6; 31.8; 31.9; 70.7; 127.8; 129.8; 133.2; 144.6.
4.2.1.7. Decyl 4-methylbenzenesulphonate. Yield 85.1%; 1H NMR
(CDCl3)
d
0.88 (t, 3H, J ¼ 6.6 Hz); 1.08e1.18 (m, 14H); 1.27e1.35 (m,
2H); 2.44 (s, 3H); 4.01 (t, 2H, J ¼ 6.6 Hz); 7.33 (d, 2H, J ¼ 8.0 Hz); 7.78
(d, 2H, J ¼ 8.4 Hz); 13C NMR (CDCl3)
d 14.1; 21.6; 22.6; 25.3; 28.8;
28.9; 29.3; 29.4; 31.9; 70.7; 127.9; 129.8; 133.2; 144.6.
4.2.1.8. Undecyl 4-methylbenzenesulphonate. Yield 88.7%; 1H NMR
(CDCl3)
2H); 2.45 (s, 3H); 4.01 (t, 2H, J ¼ 6.5 Hz); 7.35 (d, 2H, J ¼ 7.9 Hz); 7.78
d
0.88 (t, 3H, J ¼ 6.8 Hz); 1.22e1.30 (m, 16H); 1.58e1.71 (m,

L. Timko et al. / European Journal of Medicinal Chemistry 93 (2015) 263e273
269
(d, 2H, J ¼ 8.5 Hz); 13C NMR (CDCl3)
d
14.1; 21.6; 22.7; 25.3; 28.8;
4.2.2.5. N-(2-hydroxyethyl)-N,N-dimethylheptane-1-aminium 4-
methylbenzenesulphonate. Yield 82.6%; 1H NMR (DMSO-d6)
0.87
28.9; 29.3; 29.4; 29.5; 29.6; 31.9; 70.7; 127.9; 129.8; 133.3; 144.6.
d
(t, 3H, J ¼ 6.6 Hz); 1.27 (m, 8H); 1.64e1.69 (m, 2H); 2.29 (s, 3H); 3.06
(s, 6H); 3.30e3.40 (m, 4H); 3.80 (m, 2H); 5.47 (t, 1H, J ¼ 5.1 Hz); 7.12
(d, 2H, J ¼ 7.9 Hz); 7.48 (d, 2H, J ¼ 8.4 Hz); 13C NMR (DMSO-d6)
4.2.1.9. Dodecyl 4-methylbenzenesulphonate. Yield 71.7%; 1H NMR
(CDCl3)
d
0.88 (t, 3H, J ¼ 6.7 Hz); 1.08e1.18 (m, 18H); 1.27e1.35 (m,
2H); 2.44 (s, 3H); 4.01 (t, 2H, J ¼ 6.6 Hz); 7.33 (d, 2H, J ¼ 7.9 Hz); 7.78
d 13.8; 20.7; 21.7; 21.9; 25.6; 28.1; 30.9; 50.7; 54.8; 63.9; 64.4;
(d, 2H, J ¼ 8.5 Hz); 13C NMR (CDCl3)
d
14.1; 21.6; 22.6; 25.3; 28.8;
125.4; 128.0; 137.5; 145.6. HRMS calcd. for C11H26ONþ ¼ 188.2009;
28.9; 29.3; 29.4; 29.5; 31.9; 70.7; 127.9; 129.8; 133.2; 144.6.
found m/z: [M]þ 188.2007.
4.2.1.10. Tetradecyl 4-methylbenzenesulphonate. Yield 80.6%; 1H
4.2.2.6. N-(2-hydroxyethyl)-N,N-dimethyloctane-1-aminium 4-
methylbenzenesulphonate. Yield 68.1%; 1H NMR (DMSO-d6)
d 0.87
NMR (CDCl3)
d
0.88 (t, 3H, J ¼ 6.7 Hz); 1.08e1.18 (m, 22H);
1.27e1.35 (m, 2H); 2.44 (s, 3H); 4.01 (t, 2H, J ¼ 6.6 Hz); 7.33 (d, 2H,
(t, 3H, J ¼ 6.8 Hz); 1.27 (m, 10H); 1.63e1.68 (m, 2H); 2.29 (s, 3H);
3.04 (s, 6H); 3.26e3.38 (m, 4H); 3.81 (m, 2H); 5.26 (t, 1H,
J ¼ 4.8 Hz); 7.11 (d, 2H, J ¼ 7.8 Hz); 7.47 (d, 2H, J ¼ 8.1 Hz); 13C NMR
J ¼ 7.9 Hz); 7.78 (d, 2H, J ¼ 8.5 Hz); 13C NMR (CDCl3)
d 14.1; 21.6;
22.6; 25.3; 28.8; 28.9; 29.3; 29.4; 29.5; 29.6; 31.9; 70.7; 127.9;
129.8; 133.2; 144.6.
(DMSO-d6) d 14.0; 20.8; 21.8; 22.1; 25.8; 28.5; 31.2; 50.8; 54.9; 64.1;
64.6; 125.5; 128.0; 137.6; 145.8. HRMS calcd. for
C
4.2.1.11. Pentadecyl 4-methylbenzenesulphonate. Yield 59.5%; 1H
12H28ONþ ¼ 202.2165; found m/z: [M]þ 202.2163.
NMR (CDCl3)
d
0.88 (t, 3H, J ¼ 6.8 Hz); 1.21e1.30 (m, 24H);
1.54e1.60 (m, 2H); 2.45 (s, 3H); 4.01 (t, 2H, J ¼ 6.6 Hz); 7.34 (d, 2H,
4.2.2.7. N-(2-hydroxyethyl)-N,N-dimethylnonane-1-aminium 4-
methylbenzenesulphonate. Yield 76.0%; 1H NMR (DMSO-d6)
0.86
J ¼ 8.2 Hz); 7.78 (d, 2H, J ¼ 8.5 Hz); 13C NMR (CDCl3)
d 14.1; 21.7;
d
22.7; 25.3; 25.7; 28.8; 28.9; 29.4; 29.5; 29.6; 29.7; 31.9; 32.8; 70.7;
127.9; 129.8; 133.2; 144.6.
(t, 3H, J ¼ 6.6 Hz); 1.26 (m, 12H); 1.63e1.67 (m, 2H); 2.29 (s, 3H);
3.04 (s, 6H); 3.26e3.31 (m, 2H); 3.34e3.37 (m, 2H); 3.80 (m, 2H);
5.27 (t, 1H, J ¼ 5.0 Hz); 7.11 (d, 2H, J ¼ 7.9); 7.47 (d, 2H, 8.2 Hz); 13
C
4.2.2. General procedure for preparation of choline salts
NMR (DMSO-d6) d 13.9; 20.7; 21.7; 22.0; 25.7; 28.4; 28.5; 28.7; 31.1;
A solution of alkyl 4-methylbenzenesulphonate (40 mmol) in
acetonitrile (20 ml) was added to a solution of 2-(N,N-dimethyla-
mino)ethanol (40 mmol) in acetonitrile (20 ml). The reaction
mixture was refluxed for 4 h. The solvent was evaporated in vac-
uum and the crude solid was left for crystallisation from acetone
yielding a white, hygroscopic solid as the product. The product was
50.7; 54.9; 64.0; 64.5; 125.4; 128.0; 137.5; 145.6. HRMS calcd. for
C
13H30ONþ ¼ 216.2322; found m/z: [M]þ 216.2321.
4.2.2.8. N-(2-hydroxyethyl)-N,N-dimethyldecane-1-aminium 4-
methylbenzenesulphonate. Yield 79.3%; 1H NMR (DMSO-d6)
0.88
d
(t, 3H, J ¼ 6.6 Hz); 1.17e1.50 (m, 18H); 1.60e1.69 (m, 2H); 2.34 (s,
3H); 3.27 (s, 6H); 3.36e3.45 (m, 2H); 3.64e3.74 (m, 2H); 4.09e4.20
(m, 2H); 4.78 (t, 1H, J ¼ 5.7 Hz); 7.15 (d, 2H, J ¼ 8.0 Hz); 7.74 (d, 2H,
kept in vacuum desiccator over P4O10
.
4.2.2.1. N-ethyl-N,N-dimethyl-2-hydroxyethane-1-aminium 4-
methylbenzenesulphonate. Yield 77.9%; 1H NMR (DMSO-d6)
1.23
J ¼ 8.1 Hz); 13C NMR (DMSO-d6)
d 14.1; 21.3; 22.7; 22.8; 26.2; 29.1;
d
29.3; 29.4; 29.5; 29.6; 31.9; 51.8; 56.4; 65.8; 66.0; 125.8; 128.7;
139.5; 143.1. HRMS calcd. for C14H32ONþ ¼ 230.2478; found m/z:
[M]þ 230.2477.
(t, 3H, J ¼ 7.2 Hz); 2.29 (s, 3H); 3.02 (s, 6H); 3.51e3.64 (m, 2H);
3.77e3.88 (m, 2H); 5.23e5.33 (m, 2H); 7.11 (d, 2H, J ¼ 7.8 Hz); 7.48
(d, 2H, J ¼ 8.2 Hz); 13C NMR (DMSO-d6)
d 7.9; 20.8; 50.1; 54.9; 59.6;
64.0; 125.4; 128.0; 137.6; 145.7. HRMS calcd. for
C6H16ONþ ¼ 118.1226; found m/z: [M]þ 118.1224.
4.2.2.9. N-(2-hydroxyethyl)-N,N-dimethylundecane-1-aminium 4-
methylbenzenesulphonate. Yield 56.0%; 1H NMR (DMSO-d6)
d 0.86
(t, 3H, J ¼ 6.7 Hz); 1.25 (m, 16H); 1.63e1.67 (m, 2H); 2.29 (s, 3H);
3.06 (s, 6H); 3.29e3.39 (m, 4H); 3.80 (m, 2H); 5.48 (t, 1H,
J ¼ 5.1 Hz); 7.12 (d, 2H, J ¼ 7.9 Hz); 7.48 (d, 2H, J ¼ 7.9 Hz); 13C NMR
4.2.2.2. N-(2-hydroxyethyl)-N,N-dimethylpropane-1-aminium 4-
methylbenzenesulphonate. Yield 67.1%; 1H NMR (DMSO-d6)
d 0.88
(t, 3H, J ¼ 7.3 Hz); 1.65e1.73 (m, 2H); 2.29 (s, 3H); 3.06 (s, 6H);
3.26e3.34 (m, 4H); 3.80e3.81 (m, 2H); 5.46 (t, 1H, J ¼ 5.1 Hz); 7.12
(d, 2H, J ¼ 7.9 Hz); 7.49 (d, 2H, J ¼ 8.2 Hz); 13C NMR (DMSO-d6)
(DMSO-d6)
d 13.9; 20.7; 21.7; 22.0; 25.7; 28.4; 28.6; 28.7; 28.9;
31.2; 50.7; 54.8; 63.9; 64.4; 125.4; 128.0; 137.5; 145.6. HRMS calcd.
for C15H34ONþ ¼ 244.2635; found m/z: [M]þ 244.2633.
d
10.4; 15.3; 20.7; 50.7; 54.8; 64.5; 65.3; 125.4; 128.0; 137.6; 145.5.
HRMS calcd. for C7H18ONþ ¼ 132.1383; found m/z: [M]þ 132.1381.
4.2.2.10. N-(2-hydroxyethyl)-N,N-dimethyldodecane-1-aminium 4-
methylbenzenesulphonate. Yield 92.2%; 1H NMR (DMSO-d6)
d 0.86
4.2.2.3. N-(2-hydroxyethyl)-N,N-dimethylpentane-1-aminium 4-
methylbenzenesulphonate. Yield 78.4%; 1H NMR (DMSO-d6)
d 0.89
(t, 3H, J ¼ 6.8 Hz); 1.25 (m, 18H); 1.65 (m, 2H); 2.28 (s, 3H); 3.03 (s,
6H); 3.26e3.36 (m, 4H); 3.80 (m, 2H); 5.26 (m, 1H); 7.10 (d, 2H,
(t, 3H, J ¼ 7.3 Hz); 1.20e1.37 (m, 4H); 1.64e1.70 (m, 2H); 2.29 (s,
3H); 3.07 (s, 6H); 3.29e3.37 (m, 4H); 3.81 (m, 2H); 5.47 (t, 1H,
J ¼ 5.3 Hz); 7.12 (d, 2H, J ¼ 8.4 Hz); 7.48 (d, 2H, J ¼ 7.9 Hz); 13C NMR
J ¼ 7.6 Hz); 7.47 (d, 2H, J ¼ 8.2 Hz); 13C NMR (DMSO-d6)
d 13.9; 20.7;
21.7; 22.0; 25.7; 28.4; 28.6; 28.7; 28.8; 28.9; 31.2; 50.7; 54.8; 64.0;
64.5;
C
125.4;
127.9;
137.5;
145.7.
HRMS
calcd. for
(DMSO-d6)
d 13.6; 20.7; 21.4; 21.5; 27.8; 50.7; 54.8; 63.9; 64.4;
16H36ONþ ¼ 258.2791; found m/z: [M]þ 258.2787.
125.4; 128.0; 137.5; 145.5. HRMS calcd. for C9H22ONþ ¼ 160.1696;
found m/z: [M]þ 160.1693.
4.2.2.11. N-(2-hydroxyethyl)-N,N-dimethyltetradecane-1-aminium
4.2.2.4. N-(2-hydroxyetyl)-N,N-dimethylhexane-1-aminium 4-
4-methylbenzenesulphonate. Yield 84.4%; 1H NMR (DMSO-d6)
methylbenzenesulphonate. Yield 71.7%; 1H NMR (DMSO-d6)
d
0.87
d
0.88 (t, 3H, J ¼ 6.7 Hz); 1.17e1.52 (m, 22H); 1.60e1.69 (m, 2H);
(t, 3H, J ¼ 6.7 Hz); 1.22e1.30 (m, 6H); 1.63e1.68 (m, 2H); 2.29 (s,
3H); 3.04 (s, 6H); 3.26e3.32 (m, 2H); 3.34e3.43 (m, 2H); 3.81 (m,
2H); 5.27 (t, 1H, J ¼ 5.0 Hz); 7.11 (d, 2H, J ¼ 7.9); 7.47 (m, 2H); 13C
2.34 (s, 3H); 3.26 (s, 6H); 3.36e3.45 (m, 2H); 3.65e3.74 (m, 2H);
4.09e4.20 (m, 2H); 5.21 (t, 1H, J ¼ 5.7 Hz); 7.15 (d, 2H, J ¼ 8.0 Hz);
7.74 (d, 2H, J ¼ 8.0 Hz); 13C NMR (DMSO-d6)
d 14.1; 21.3; 22.7; 22.8;
NMR (DMSO-d6)
d
13.7; 20.7; 21.6; 21.8; 25.3; 30.6; 50.7; 54.8;
26.2; 29.1; 29.3; 29.4; 29.5; 29.6; 29.7; 31.9; 51.8; 56.4; 65.8; 66.0;
125.8; 128.7; 139.6; 143.0. HRMS calcd. for C18H40ONþ ¼ 286.3104;
found m/z: [M]þ 286.3099.
64.0; 64.5; 125.4; 128.0; 137.6; 145.6. HRMS calcd. for
C
10H24ONþ ¼ 174.1852; found m/z: [M]þ 174.1849.

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L. Timko et al. / European Journal of Medicinal Chemistry 93 (2015) 263e273
4.2.2.12. N-(2-hydroxyethyl)-N,N-dimethylpentadecane-1-aminium
1.25e1.33 (m, 22H); 1.57e1.71 (m, 4H); 3.34 (s, 6H); 3.42e3.48 (m,
2H); 3.79e3.86 (m, 4H); 4.29 (m, 2H); 13C NMR (CDCl3)
14.1; 22.7;
22.9; 25.6; 26.3; 29.3; 29.4; 29.5; 29.6; 31.0; 31.1; 31.7; 31.9; 51.8;
4-methylbenzenesulphonate. Yield 85.9%; 1H NMR (DMSO-d6)
d
d
0.86 (t, 3H, J ¼ 6.8 Hz); 1.24 (m, 24H); 1.63e1.67 (m, 2H); 2.29 (s,
3H); 3.04 (s, 6H); 3.26e3.30 (m, 2H); 3.33e3.37 (m, 2H); 3.80 (m,
2H); 5.25 (t, 1H, J ¼ 5.7 Hz); 7.11 (d, 2H, J ¼ 8.4 Hz); 7.47 (d, 2H,
58.9; 64.2; 65.5; 65.7; 31P NMR (CDCl3)
d
ꢂ0.11; HRMS calcd. for
C
21H46O4NPNa ¼ 430.3057; found m/z: [MþNa]þ 430.3053; Anal.
J ¼ 8.1 Hz); 13C NMR (DMSO-d6)
d
13.9; 20.8; 21.7; 22.0; 25.8; 28.5;
Calcd. for C21H46NO4P ꢁ 0.5 H2O: C, 60.55; H, 11.37; N, 3.36; found:
28.7; 28.8; 28.9; 29.0; 31.3; 50.8; 54.9; 64.0; 64.6; 125.5; 128.0;
137.5; 145.8. HRMS calcd. for C19H42ONþ ¼ 300.3261; found m/z:
[M]þ 300.3256.
C, 60.58; H, 11.40; N, 3.09.
4.2.3.5. [2-(N-decyl-N,N-dimethylaminio)ethyl]heptylphosphate
(C7eC10). Yield 7.5%; 1H NMR (CDCl3)
d
0.85e0.90 (m, 6H);
1.26e1.32 (m, 22H); 1.57e1.71 (m, 4H); 3.33 (s, 6H); 3.44 (m, 2H);
3.79e3.85 (m, 4H); 4.30 (m, 2H); 13C NMR (CDCl3)
14.1; 21.9; 22.7;
22.9; 25.9; 26.4; 29.2; 29.3; 29.5; 31.0; 31.9; 51.8; 52.4; 58.9; 64.2;
4.2.3. General procedure for preparation of alkylphosphocholines
To a stirred solution of phosphorus oxychloride (10 mmol) and
(triethyl)amine (20 mmol) in chloroform (10 ml) was added
dropwise a solution of an alcohol (9 mmol) in chloroform (20 ml) at
0 ꢀC. The reaction mixture was stirred for 2 h at room temperature.
The prepared intermediate was used immediately and without
purification. Pyridine (15 ml) was added dropwise at 0 ꢀC followed
by addition of a choline 4-methylbenzenesulphonate derivative
(12.5 mmol). The reaction mixture was stirred at room temperature
overnight. After that it was hydrolysed by addition of water (1.5 ml)
and stirred for one hour at room temperature. Solvents were
evaporated at vacuum and the crude solid was diluted in tetrahy-
drofuran and water (5:1). Exchange resin Amberlite MB3 was
added to the stirred solution until the resin stopped changing
colour. The resin was filtered off and solvents were evaporated in
vacuum. The product was purified by chromatography using silica
gel with the liquid phase containing CHCl3:CH3OH:H2O (65:25:3).
After purification, solvents were evaporated in vacuum, the product
was dried by azeotropic distillation using propan-2-ol, dissolved in
chloroform and precipitated with acetone. The product was filtered
off as white hygroscopic solid and was stored in desiccator over
phosphorus pentoxide.
d
65.7; 31P NMR (CDCl3)
C
Calcd. for C21H46NO4P ꢁ 0.8 H2O: C, 59.77; H, 11.37; N, 3.32; found:
d
ꢂ0.17; HRMS calcd. for
21H46O4NPNa ¼ 430.3057; found m/z: [MþNa]þ 430.3056; Anal.
C, 59.67; H, 11.15; N, 3.06.
4.2.3.6. [2-(N,N-dimethyl-N-nonylaminio)ethyl]octylphosphate
(C8eC9). Yield 10.2%; 1H NMR (CDCl3)
d 0.85e0.90 (m, 6H);
1.26e1.34 (m, 22H); 1.55e1.62 (m, 2H); 1.71 (m, 2H); 3.35 (s, 6H);
3.44e3.49 (m, 2H); 3.80e3.87 (m, 4H); 4.31 (m, 2H); 13C NMR
(CDCl3)
d 14.1; 22.6; 22.7; 22.9; 25.9; 26.4; 29.2; 29.3; 29.4; 29.5;
31.0; 31.1; 31.8; 31.9; 51.8; 58.9; 64.2; 65.6; 65.7; 31P NMR (CDCl3)
d
ꢂ0.20; HRMS calcd. for C21H46O4NPNa ¼ 430.3057; found m/z:
[MþNa]þ 430.3053; Anal. Calcd. for C21H46NO4P ꢁ 0.9 H2O: C,
59.77; H, 11.37; N, 3.32; found: C, 59.68; H, 10.92; N, 3.15.
4.2.3.7. [2-(N,N-dimethyl-N-octylaminio)ethyl]nonylphosphate
(C9eC8). Yield 17.5%; 1H NMR (CDCl3)
d 0.85e0.90 (m, 6H);
1.25e1.32 (m, 22H); 1.55e1.64 (m, 2H); 1.70e1.71 (m, 2H); 3.35 (s,
6H); 3.43e3.49 (m, 2H); 3.79e3.86 (m, 4H); 4.29 (m, 2H); 13C NMR
(CDCl3)
d 14.1; 22.6; 22.7; 22.9; 26.0; 26.4; 29.1; 29.3; 29.4; 29.5;
4.2.3.1. [2-(N,N-dimethyl-N-pentadecylaminio)ethyl]ethylphosphate
31.1; 31.2; 31.7; 31.9; 51.8; 58.9; 64.2; 65.5; 65.7; 31P NMR (CDCl3)
(C2eC15). Yield 7.0%; 1H NMR (CDCl3)
d
0.88 (t, 3H, J ¼ 6.8 Hz);
d
ꢂ0.06; HRMS calcd. for C21H46O4NPNa ¼ 430.3057; found m/z:
1.21e1.33 (m, 27H); 1.71 (m, 2H); 3.33 (s, 6H); 3.41e3.47 (m, 2H);
[MþNa]þ 430.3056; Anal. Calcd. for C21H46NO4P ꢁ 1.25 H2O: C,
3.79 (m, 2H); 3.92 (q, 2H, J ¼ 7.0 Hz); 4.31 (m, 2H); 13C NMR (CDCl3)
58.65; H, 11.37; N, 3.26; found: C, 58.74; H, 11.08; N, 3.13.
d
14.1; 16.7; 22.7; 22.9; 26.3; 29.3; 29.4; 29.5; 29.6; 29.7; 31.9; 51.8;
58.9; 61.2; 64.2; 65.8; 31P NMR (CDCl3)
d
ꢂ0.29; HRMS calcd. for
4.2.3.8. [2-(N-heptyl-N,N-dimethylaminio)ethyl]decylphosphate
C
21H46O4NPNa ¼ 430.3057; found m/z: [MþNa]þ 430.3053; Anal.
(C10eC7). Yield 23.0%; 1H NMR (CDCl3)
d 0.86e0.90 (m, 6H);
Calcd. for C21H46NO4P ꢁ 0.75 H2O: C, 59.90; H,11.37; N, 3.33; found:
1.25e1.34 (m, 22H); 1.56e1.60 (m, 2H); 1.71 (m, 2H); 3.32 (s, 6H);
C, 59.89; H, 11.28; N, 3.05.
3.43e3.48 (m, 2H); 3.77e3.83 (m, 4H); 4.27 (m, 2H); 13C NMR
(CDCl3)
d 14.0; 14.1; 22.5; 22.7; 22.9; 25.9; 26.3; 29.0; 29.4; 29.6;
4.2.3.2. [2-(N,N-dimethyl-N-tetradecylaminio)ethyl]propylphosphate
29.7; 31.1; 31.2; 31.9; 51.8; 58.8; 64.0; 65.5; 65.6; 31P NMR (CDCl3)
(C3eC14). Yield 5.7%; 1H NMR (CDCl3)
d
0.86e0.93 (m, 6H);
1.25e1.32 (m, 22H); 1.56e1.71 (m, 4H); 3.33 (s, 6H); 3.41e3.47 (m,
2H); 3.76e3.83 (m, 4H); 4.30 (m, 2H); 13C NMR (CDCl3)
10.4; 14.1;
22.7; 22.9; 24.1; 24.2; 26.3; 29.3; 29.4; 29.5; 29.6; 29.7; 31.9; 51.8;
d
ꢂ0.34; HRMS calcd. for C21H46O4NPNa ¼ 430.3057; found m/z:
[MþNa]þ 430.3053; Anal. Calcd. for C21H46NO4P ꢁ 0.6 H2O: C,
d
60.29; H, 11.37; N, 3.35; found: C, 60.29; H, 11.33; N, 3.25.
58.8; 64.2; 65.7; 67.1; 31P NMR (CDCl3)
C
Calcd. for C21H46NO4P ꢁ 1.6 H2O: C, 57.80; H, 11.36; N, 3.21; found:
d
ꢂ0.18; HRMS calcd. for
4.2.3.9. [2-(N-hexyl-N,N-dimethylaminio)ethyl]undecylphosphate
21H46O4NPNa ¼ 430.3057; found m/z: [MþNa]þ 430.3056; Anal.
(C11eC6). Yield 14.7%; 1H NMR (CDCl3)
d 0.86e0.91 (m, 6H);
1.25e1.33 (m, 22H); 1.54e1.61 (m, 2H); 1.71 (m, 2H); 3.35 (s, 6H);
C, 57.72; H, 10.88; N, 3.01.
3.45e3.50 (m, 2H); 3.78e3.85 (m, 4H); 4.28 (m, 2H); 13C NMR
(CDCl3)
d 13.9; 14.1; 22.5; 22.7; 22.8; 25.9; 26.0; 29.4; 29.5; 29.7;
4.2.3.3. [2-(N-dodecyl-N,N-dimethylaminio)ethyl]pentylphosphate
31.2; 31.4; 31.9; 51.8; 58.9; 64.1; 65.5; 65.6; 31P NMR (CDCl3)
(C5eC12). Yield 7.0%; 1H NMR (CDCl3)
d
0.88 (t, 6H, J ¼ 6.89 Hz);
1.25e1.33 (m, 22H); 1.56e1.72 (m, 4H); 3.34 (s, 6H); 3.43e3.49 (m,
2H); 3.81e3.88 (m, 4H); 4.33 (m, 2H); 13C NMR (CDCl3)
14.1; 22.5;
22.7; 22.9; 26.4; 28.0; 29.3; 29.4; 29.5; 29.6; 30.6; 30.7; 31.9; 51.8;
d
ꢂ0.04; HRMS calcd. for C21H46O4NPNa ¼ 430.3057; found m/z:
[MþNa]þ 430.3053; Anal. Calcd. for C21H46NO4P ꢁ 1 H2O: C, 59.27;
d
H, 11.37; N, 3.29; found: C, 59.31; H, 11.25; N, 3.11.
60.0; 64.2; 65.8; 31P NMR (CDCl3)
C
Calcd. for C21H46NO4P ꢁ 0.75 H2O: C, 59.90; H,11.37; N, 3.33; found:
d
ꢂ0.48; HRMS calcd. for
4.2.3.10. [2-(N,N-dimethyl-N-pentylaminio)ethyl]dodecylphosphate
21H46O4NPNa ¼ 430.3057; found m/z: [MþNa]þ 430.3053; Anal.
(C12eC5). Yield 19.3%; 1H NMR (CDCl3)
d 0.86e0.94 (m, 6H);
1.25e1.37 (m, 22H); 1.54e1.64 (m, 2H); 1.72 (m, 2H); 3.35 (s, 6H);
C, 60.12; H, 11.32; N, 3.14.
3.45e3.51 (m, 2H); 3.79e3.86 (m, 4H); 4.29 (m, 2H); 13C NMR
(CDCl3)
d 13.8; 14.1; 22.3; 22.5; 22.7; 25.9; 28.4; 29.4; 29.5; 29.7;
4.2.3.4. [2-(N,N-dimethyl-N-undecylaminio)ethyl]hexylphosphate
31.1; 31.2; 51.8; 58.8; 64.1; 65.5; 65.6; 31P NMR (CDCl3)
d 0.001;
(C6eC11). Yield 13.0%; 1H NMR (CDCl3)
d
0.85e0.90 (m, 6H);
HRMS calcd. for C21H46O4NPNa ¼ 430.3057; found m/z: [MþNa]þ

L. Timko et al. / European Journal of Medicinal Chemistry 93 (2015) 263e273
271
430.3053; Anal. Calcd. for C21H46NO4P ꢁ 0.75 H2O: C, 59.90; H,
according to the protocol of Hemelen et al. Cryopreservation does
not alter the lymphocyte viability and preserves the surface
markers as well, if handled carefully [51]. The lymphocytes were
11.37; N, 3.33; found: C, 59.94; H, 11.34; N, 3.21.
4.2.3.11. [2-(N,N-dimethyl-N-propylaminio)ethyl]tetradecylphos-
seeded on 96-well plates, at a density of 30 000 cells in 95 ml RPMI-
phate (C14eC3). Yield 16.5%; 1H NMR (CDCl3)
d
0.88 (t, 3H,
1640 media having the supplements described for the THP-1 cells
line. Lymphocytes do not proliferate under these conditions,
differently from the leukaemia cells, consequently more cells were
needed to obtain a cell density similar to the malignant populations
after 24 h.
J ¼ 6.6 Hz); 1.02 (t, 3H, J ¼ 7.2); 1.25 (m, 22H); 1.54e1.61 (m, 2H);
1.78e1.81 (m, 2H); 3.36 (m, 6H); 3.46e3.51 (m, 2H); 3.78e3.84 (m,
4H); 4.28 (m, 2H); 13C NMR (CDCl3)
d
10.7; 14.1; 16.3; 22.7; 25.9;
29.4; 29.5; 29.7; 31.1; 31.2; 31.9; 51.7; 58.8; 58.9; 64.0; 65.4; 65.5;
66.8; 31P NMR (CDCl3)
C
d
ꢂ0.09; HRMS calcd. for
The compounds were solubilised directly in the RPMI-1640
culture media. For each compound 7 concentrations were pre-
21H46O4NPNa ¼ 430.3057; found m/z: [MþNa]þ 430.3055; Anal.
Calcd. for C21H46NO4P ꢁ 0.75 H2O: C, 59.90; H,11.37; N, 3.33; found:
pared, in the range from 20 mM to 10
adding 10 l solution of the alkylphosphocholine into each well
containing 190 l media in adherent cells, or 5 l in 95 l cellular
suspension. The final concentration of the alkylphosphocholines
mM. Cells were treated by
C, 59.96; H, 11.23; N, 3.17.
m
m
m
m
4.2.3.12. [2-(N-ethyl-N,N-dimethylaminio)ethyl]pentadecylphos-
phate (C15eC2). Yield 18.9%; 1H NMR (CDCl3)
d
0.88 (t, 3H,
varied between 1 mM and 1
reference.
mM. Untreated cells were used as a
J ¼ 6.8 Hz); 1.25e1.41 (m, 27H); 1.56e1.60 (m, 2H); 3.33 (s, 6H);
3.66e3.68 (m, 2H); 3.77e3.84 (m, 4H); 4.28 (m, 2H); 13C NMR
After 24-h treatment, cytotoxicity assessment was performed
on the cells. The microplates were stained with MTS dye (CellTiter
96 Aqueous Proliferation Assay from Promega), when cell lines
were in suspension, or with MTT dye (Thiazolyl Blue Tetrazolium
Bromide from Sigma Aldrich) for adherent cells, according to a
method described earlier [52]. The two methods are convergent;
they are based on the same principle: the capacity of living cells
mitochondria to transform the dye into coloured formazan form.
The only difference between the two methods is the solubility of
the crystals yielded by the viable cells. In adherent cell lines, the
MTT is a convenient method, because the supernatants can be
easily removed and then solubilise the formazan products in
dimethyl sulfoxide, while the MTS formazan form is water-soluble
and requires no further processing. The microplates were measured
at 492 nm/520 nm using the multiplate reader and the extinctions
were analysed with Gen5 software. The IC50 values for each com-
pound were calculated with the Graph Prism5 biostatistics soft-
ware, using the best-fit values of the log inhibitor versus
normalized response sigmoidal curve. Significance of values was
examined using the column statistics, one-way ANOVA test and
Bonferroni post-test.
(CDCl3) d 8.5; 14.1; 22.7; 26.0; 29.4; 29.5; 29.7; 31.1; 31.9; 51.1; 58.8;
58.9; 60.8; 63.5; 65.5; 65.6; 31P NMR (CDCl3)
d
ꢂ0.19; HRMS calcd.
for C21H46O4NPNa ¼ 430.3057; found m/z: [MþNa]þ 430.3054;
Anal. Calcd. for C21H46NO4P ꢁ 0.8 H2O: C, 59.77; H, 11.37; N, 3.32;
found: C, 59.82; H, 11.33; N, 3.10.
4.3. Cytotoxicity assay
The biological model used for the in vitro study was a selection
of tumour cell lines and normal human peripheral blood cells as a
reference. Two cell lines derived from solid human tumours were
used: MCF-7 human breast adenocarcinoma and A2780 ovarian
carcinoma cell lines, both adherent immortalized cell lines. Two
leukaemia cell lines were also used, namely the THP-1 and HUT-78
cells.
The THP-1 human leukemic monocyte cell line grew in sus-
pension, in cell RPMI-1640 culture media enriched with foetal
bovine serum (FCS), glutamine, penicillin-streptomycin, non-
essential amino acid solution (NEA), 2-sulfanylethanol and sodium
pyruvate. Cells were thawed, carefully; they grew in culture flasks,
in a sterile incubator at 37 ꢀC, 5% CO2, in humidified atmosphere.
For cells growing in suspension passages were made by centrifu-
gation of the cell culture flask content and redistribution in sub-
cultures. After expansion and two passages cells were placed on 96-
4.4. Antiprotozoal assay
The antiprotozoal assay was carried out according to the method
modified by Mrva et al. [22]. Amoebas used for testing were A.
lugdunensis and A. quina. From the 2-day monoxenic cultures on
agar plates, the trophozoites were axenized by inoculation into the
Bacto-Casitone/Serum medium (BCS) with penicillin and ampi-
cillin. 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 consecutively cultivated in
this medium. Cytotoxicity measurements were performed in sterile
well microplates, in 95
ml of media, at a concentration of 20
000 cells per well. Plates were kept in incubator and after
approximately 20 h they were treated with the solution of the
compounds.
HUT-78 is a T-cell lymphoma human malignant cell line: the
cells have lymphoblast morphology. They were cultivated and
prepared for experiments similarly with THP-1 cells.
MCF-7 cells were grown in MEM cell culture media, supple-
mented with NEA, FCS and glutamine. The passages were made
enzymatically with a Trypsine-EDTA solution. The appropriate cell
culture media for the A2780 cell line is RPMI-1640 supplemented
with FCS and glutamine. The two adherent cell lines were seeded
onto a microplate at a concentration of 20 000 cells per well, in
96-well microtiter plates. Each well was seeded with 100
ml
(2 ꢁ 105 cells mlꢂ1) of a trophozoite suspension. Then, 100
ml of a
freshly prepared medium containing APC at 6 concentrations was
added to all wells except untreated control wells, which received
190
ml cell culture media, and after about 20 h they were treated
100
ml of a pure medium. The APCs were tested at final concen-
with 10
ml of solution of the studied compounds.
trations of 500, 250, 125, 62.5, 31.25, and 15.6
mM. The reduction of
Separation of lymphocytes from whole blood was made using a
method improved by our laboratory [50], by density gradient
centrifugation, using Histopaque solution. The lymphocyte ring
separated between plasma and the separation solution was har-
vested using a pipette. Cells were washed twice in Hank's balanced
salt solution.
trophozoites was recorded after 1, 24 and 48 h by counting the
surviving cells in a Bürker-Türk haemocytometer. Viability of tro-
phozoites was determined by trypan blue exclusion; 100% eradi-
cation was confirmed by transferring 50 ml of the suspension to a
PYG medium and recording the amoeba growth for 14 days. The
lowest concentration of the APC supporting 100% eradication of the
trophozoites was defined as the minimal trophocidal concentration
(MTC). The cultivations and the cytotoxicity measurements were
The lymphocytes were preserved in an ultra freezer at ꢂ70 ꢀC
and they were defrosted gently in order to preserve their viability,

272
L. Timko et al. / European Journal of Medicinal Chemistry 93 (2015) 263e273
carried out at 37 ꢀC for A. quina as well as A. lugdunensis. The ex-
periments were repeated 4 times for each concentration.
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bacteria S. aureus ATCC 29/58, Gram-negative bacteria E. coli ATCC
377/79 and yeast C. albicans ATCC 8186. Testing was performed by a
ꢀꢁ
method used previously by Lukac et al. [53]. Solutions of studied
compounds were prepared in water. Stock solution had the con-
centration of 1000 mg/ml. Cultures of bacteria and yeast grew 24 h
in blood agar and in the Sabouraud agar respectively. From these
cultures suspensions of microorganisms were prepared with con-
centration of 5 ꢁ 107 CFU/ml of bacteria and 5 ꢁ 105 CFU/ml of
Candida. The suspensions were prepared in physiological solution
(pH 7.2). Concentrations of microorganisms were determined
spectrophotometrically and the suspensions were adjusted to
€
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absorbance A ¼ 0.35 at
(5 l) was added to solutions containing the tested compound
(100 l) and to double concentrated peptone broth medium (8%) for
bacteria or Sabouraud medium (12%) for Candida (100 l). For a
l
¼ 540 nm. The microorganism suspension
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m
m
m
testing assay the solutions were serially diluted by half (concen-
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15.63, 7.81, 3.91 and 1.95
96-well microtiter plates. The microorganisms were incubated for
24 h at 37 ꢀC. Then from each well, 5
l of tested suspension was
mg/ml) and used. Tests were performed in
^
m
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taken and cultured on blood agar (bacteria) or on Sabouraud agar
(yeast). The Petri dishes were incubated for 24 h at 37 ꢀC. The
lowest concentration of APC that prevented colony formation was
defined as the minimal inhibition concentration (MIC).
^
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Acknowledgement
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This work was supported by the Slovak Research and Develop-
ment Agency under the contract No. APVV-0516-12 and grants UK/
123/2014, UK/263/2014 and VEGA 1/0796/12. This publication is
the result of the project implementation: Centre of Excellency in
Security Research supported by the Research and Development
Operational Programme funded by the ERDF Grant number: ITMS
26240120034. This publication is the result of the project imple-
mentation: Comenius University in Bratislava Science Park sup-
ported by the Research and Development Operational Programme
funded by the ERDF Grant number: ITMS 26240220086. Testing of
cytotoxicity was supported by the Romanian UEFISCDI Grant for
Exploratory Research Projects code PN-II-ID-PCE-2011-3-1057 No.
250/2011. Authors from “Prof.Dr.I.Chiricuta” Oncology Institute
express their gratitude to Dr. Tibor L. Krausz from “Iuliu Hatieganu”
University of Medicine and Pharmacy in Cluj-Napoca for his
generous help in providing the cell lines.
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