Synthesis, physicochemical properties and biological activities of novel alkylphosphocholines with foscarnet moiety

Article abstract of DOI:10.1016/j.bioorg.2020.104224

A series of alkylphosphocholines with foscarnet moiety was synthesized. The structure of these zwitterionic amphiphiles was modified in both polar and non-polar parts of surfactant molecule. Investigations of physicochemical properties are represented by the determination of critical micelle concentration, the surface tension value at the cmc and the surface area per surfactant head group utilising surface tension measurements. Hydrodynamic diameter of surfactant micelles was determined using the dynamic light scattering technique. Alkylphosphocholines exhibit significant cytotoxic, anticandidal (Candida albicans) and antiamoebal (Acanthamoeba spp. T4 genotype) activity. The relationship between the structure, physicochemical properties and biological activity of the tested compounds revealed that lipophilicity has a significant influence on biological activity of the investigated surfactants. More lipophilic alkylphosphocholines with octadecyl chains show cytotoxic activity against cancer cells which is higher than that of the compounds with shorter alkyl chains. The opposite situation was observed in case of anticandidal and antiamoebal activity of these surfactants. The most active compounds were found to have pentadecyl chains. The foscarnet analogue of miltefosine C15-PFA-C showed the highest anticandidal activity. The minimum value of anticandidal activity of this compound is 1,4 μM thus representing the highest anticandidal activity found within the group of alkylphosphocholines.

Full text of DOI:10.1016/j.bioorg.2020.104224

BioorganicChemistry104(2020)104224
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, physicochemical properties and biological activities of novel
alkylphosphocholines with foscarnet moiety
Lukáš Timkoa, Martin Pisárčika, Martin Mrvab, Mária Garajováb, Anna Juhásováb, Ján Mojžišc,
⁎
Gabriela Mojžišovád, Marián Bukovskýe, Ferdinand Devínskyf, Miloš Lukáča,
a Department of Chemical Theory of Drugs, Faculty of Pharmacy, Comenius University, Kalinčiakova 8, 832 32 Bratislava, Slovakia
b Department of Zoology, Faculty of Natural Sciences, Comenius University, Mlynská Dolina, Ilkovičova 6, 842 15 Bratislava, Slovakia
c Department of Pharmacology, Faculty of Medicine, P.J. Šafárik University, SNP 1, 040 66 Košice, Slovakia
d Department of Experimental Medicine, Faculty of Medicine, P.J. Šafárik University, SNP 1, 040 66 Košice, Slovakia
e Department of Cell and Molecular Biology of Drugs, Faculty of Pharmacy, Comenius University, Kalinčiakova 8, 832 32 Bratislava, Slovakia
f Faculty of Pharmacy, Comenius University, Odbojárov 10, 832 32 Bratislava, Slovakia
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of alkylphosphocholines with foscarnet moiety was synthesized. The structure of these zwitterionic
amphiphiles was modified in both polar and non-polar parts of surfactant molecule. Investigations of physico-
chemical properties are represented by the determination of critical micelle concentration, the surface tension
value at the cmc and the surface area per surfactant head group utilising surface tension measurements.
Hydrodynamic diameter of surfactant micelles was determined using the dynamic light scattering technique.
Alkylphosphocholines exhibit significant cytotoxic, anticandidal (Candida albicans) and antiamoebal
(Acanthamoeba spp. T4 genotype) activity. The relationship between the structure, physicochemical properties
and biological activity of the tested compounds revealed that lipophilicity has a significant influence on bio-
logical activity of the investigated surfactants. More lipophilic alkylphosphocholines with octadecyl chains show
cytotoxic activity against cancer cells which is higher than that of the compounds with shorter alkyl chains. The
opposite situation was observed in case of anticandidal and antiamoebal activity of these surfactants. The most
active compounds were found to have pentadecyl chains. The foscarnet analogue of miltefosine C15-PFA-C
showed the highest anticandidal activity. The minimum value of anticandidal activity of this compound is 1,4
μM thus representing the highest anticandidal activity found within the group of alkylphosphocholines.
Acanthamoeba
Candida albicans
Critical micelle concentration
Foscarnet
Miltefosine
Zwitterionic amphiphilic compounds
1. Introduction
the numerous examples indicate, aminium cation can be closed to a
heterocycle [1,9,13,14], methyl group of choline can be exchanged for
Alkylphosphocholines (APCs) are amphiphilic compounds with a
characteristic molecular structure composed of an alkyl chain and a
phosphocholine moiety. Hydrophobic and hydrophilic molecular parts
can have diverse structures. The alkyl groups are mostly represented by
straight hydrocarbon chains. The best known APCs are miltefosine and
perifosine with their molecular structure shown in Fig. 1 [1]. The
straight alkyl chains can have double bonds in their structure as well.
The APCs with unsaturated docos-13-enyl chain are represented by
erucylphosphocholine and erufosine (Fig. 1) [1]. The alkyl chains can
be also branched [2,3], with fluorocarbon parts [3,4], cycles [5–10] or
bonded to bioactive compounds [11,12]. Phosphocholine moiety is
mostly composed of choline group bonded to a phosphate group.
However, this part of molecule often undergoes structural changes. As
a longer alkyl chain [4,15,16] or phosphate group can be replaced with
a phosphonate group [4].
The phosphocholine moiety in APCs can be prepared by several
synthetic approaches. Precursors for phosphate groups can be re-
presented by phosphoryl chloride, dimethylchlorophosphite, 2-bro-
moethyldichlorophosphate, 2-chloro-1,3,2-dioxaphospholane or 2-
chloro-2-oxo-1,3,2-dioxaphospholane. Aminium cations in phos-
phocholine moiety are provided by quaternization of tertiary amines
with phosphate precursors (2-bromoethyl phosphate, 2-alkyloxy-2-oxo-
1,3,2-dioxolane) or aminium cations are introduced to molecules of
APCs as choline salts [17]. If a phosphate group is replaced with a
phosphonate group, the bond between carbon and phosphorus atoms is
conventionally synthesized by the Michaelis-Arbuzov reaction. Heating
⁎ Corresponding author.
E-mail address: lukac@fpharm.uniba.sk (M. Lukáč).
https://doi.org/10.1016/j.bioorg.2020.104224
Received 1 June 2020; Received in revised form 20 August 2020; Accepted 25 August 2020
Availableonline26August2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

L. Timko, et al.
BioorganicChemistry104(2020)104224
miltefosine
yeast and fungi. Widmer et al. [23] showed that miltefosine has a broad
antimycotic spectrum, as well. This APC inhibits growth of several
species of the genera Candida, Cryptococcus, Aspergillus, Fusarium or
Scedosporium. The mixture of miltefosine with amphotericin B represent
a promising combination of drugs with a potential application in the
treatment of multi-drug resistant candidiasis [24].
O–
O
O
P
N+
O
erucylphosphocholine
Antiprotozoal activity belongs to one of the most studied properties
of APCs. Miltefosine is a drug which is used in the treatment of severe
diseases caused by Leishmania. These protozoans are causative agents of
cutaneous or visceral leishmaniasis (kala-azar). Leishmaniasis results in
700 000 to 1 million new cases annually worldwide [25]. Miltefosine is
the only oral drug approved for the treatment of this disease [26–28].
Its activity is not limited to Leishmania, the spectrum of sensitive pro-
tozoans is broader. Miltefosine was used in the treatment of dis-
seminated Acanthamoeba sp. infection [29], Acanthamoeba keratitis
[30], and primary amoebic meningoencephalitis caused by Naegleria
fowleri [31]. Many other genera of protozoans show sensitivity to mil-
tefosine and other APCs. They are represented by Entamoeba [32],
Trichomonas [33–35], Balamuthia [36] and Trypanosoma [37,38].
Foscarnet, phosphonoformic acid, is an organic analogue of in-
organic pyrophosphate. It inhibits viral DNA-polymerases and reverse
transcriptases. Foscarnet trisodium salt is used in the treatment of
several viral diseases. This compound is active against herpes viruses,
cytomegalovirus, hepatitis B viruses or human immunodeficiency
viruses [39]. Biological activity of foscarnet is not limited only to
viruses but it is active against cancer cells. Rose [40] documented an-
tineoplastic activity of this drug against human anaplastic thyroid
carcinoma cells. The main disadvantage of foscarnet is its low oral
bioavailability. It can be increased by the preparation of lipid prodrugs.
Foscarnet binding to alkylglycerols or alkyltioglycerols not only in-
creases its bioavailability but also enhances its antiviral activity
[41–43].
O–
O
O
P
P
N+
O
erufosine
O–
O
O
N+
O
perifosine
O–
O
P
O
N+
O
Fig. 1. Structure of well-known APCs.
of trialkyl phosphites with alkyl halogenides results in the formation of
dialkyl alkylphosphonates that can be hydrolysed to free phosphonate
acids [4]. The most versatile method of the preparation of APCs is de-
picted in Scheme 1. It provides the synthesis of compounds with the
different types of alkyl chains bonded to phosphate/phosphonate
groups, with the different length of the spacer between anionic and
cationic part of the molecule and with various types of alkyl chains
bonded to the cationic part of surfactant molecule.
In the past, APCs were investigated as the compounds with cytotoxic
activities against cancer cells [18,19]. Edelfosine, miltefosine, erucyl-
phosphocholine and perifosine were the first candidates that were in-
tensively studied. APCs caused cell death by either non-specific or
specific action. They caused lysis of plasmatic membrane at the high
concentration as indicate haemolytic activity of APCs [13]. The specific
mode of action can be explained at several levels. APCs target plasmatic
membranes, especially lipid rafts. They alter phospholipase C and D
signalling cascades. These cascades modulate the PI3K/Akt/mTOR and
RAS/RAF/MEK/ERK pathways. These changes at the subcellular levels
lead to the arrest of cell cycle at G2/M-phase and result in the pro-
grammed cell death [1,20].
The present study is focused on the preparation, characterisation
and investigation of the alkylphosphocholines that contain a foscarnet
moiety in their molecular structure. The insertion of a phosphono-
fromate group between an alkyl chain and a choline results in the
formation of a novel structural type of APCs. The main intention of our
study is to investigate the relationship between structural changes,
physicochemical properties and biological activity of the prepared
compounds. The structural changes in the molecule of the model APC
(miltefosine) are represented by the alkyl chain extension, the addition
of phosphonofromate group and the modification of choline moiety.
Physicochemical properties of APCs in aqueous solutions were studied
by determining the surface activity and the micelle size utilising surface
tension and dynamic light scattering experimental techniques.
Biological activity was tested on cancer cells, bacteria, yeast and
Acanthamoeba. Diseases caused by cancer cells or by the microorgan-
isms mentioned above often lack efficient therapy or they show re-
sistance to conventionally used drugs. Therefore, there is a demand for
Antimicrobial activities of APCs showed that several bacteria are
sensitive to their action, such as Staphylococcus aureus, Enterococcus sp.,
Streptococcus pneumoniae [21] or Acinetobacter baumannii [22]. How-
ever, Escherichia coli or Pseudomonas aeruginosa were resistant to mil-
tefosine [21]. The highest antimicrobial activity was observed against
R2
R3
Cl
OH
O
HO
O
O
a
c
b
d
O–
xN+ R4
e
R OH
R Br
R
P
R
P
+
+
( )
O
O
P
R2
( )
R5
Cl
HO
R4
R6
O
R2
+
5
xN R
R3
R3
+
O
P
O
e
OH
OH
O
HO
P
O
R1
4
( )
xN R
R
R
R5
R1
Scheme 1. Synthetic approach of preparation of alkylphosphocholines. Abbreviations: R, R1, R2, R3, R4, R5 are alkyl chains; R2 and R3can be connected to alkane-
α,ω-diyls (formed heterocycles); R6 is alkyloxy or alkyl; x represented different number of methylene groups. Reagents: a) POCl3; b) H2O; c) trialkyl phosphite; d) HBr
or (CH3)3SiBr/CH3OH; e) (i) pyridine, (ii) 1-(methylsulfonyl)3-nitro-1H-1,2,4-tirazole or 2,4,6-triisopropylbenzenesulfonyl chloride.
2

L. Timko, et al.
BioorganicChemistry104(2020)104224
Fig. 2. Structural differences between alkylpho-
sphocholines and alkylphosphocholines with fos-
carnet moiety.
O
P
O
O
O–
O
O–
N+
O
N+
R
P
R
O
O
alkylphosphocholine
alkylphosphocholine with foscarnet moiety
new bioactive compounds. Some of the presented APCs show very at-
tractive levels of antimicrobial activity against microorganisms causing
severe illnesses and can provide a promising solution in the search for
novel drugs in the treatment of yeast and protozoal infections.
plots of surface tension vs. log concentration curves of APCs are shown
in Fig. 3. The critical micelle concentration (cmc), the surface tension
value at the cmc (γcmc) and, the surface area per head group at the
surface saturation (Acmc) were determined. The compounds C18-PFA-A
and C18-PFA-4P have low solubility in water and could not be mea-
sured in aqueous solutions. The results are summarized in the Table 1.
The comparison of cmc values of HPC and its structural analogues
C15-PFA-C and C16-PFA-C shows that the values are very similar. C15-
PFA-C is slightly more hydrophilic than HPC. Its increased hydro-
philicity is caused by the presence of an extra oxygen atom in the
structure of C15-PFA-C in comparison with HPC. C16-PFA-C is more
hydrophobic than HPC. The extension of the alkyl chain of C16-PFA-C
by single methylene group in comparison with C15-PFA-C results in the
cmc decrease. The cmc of C16-PFA-C is half the value of cmc of C15-
PFA-C. Another insertion of two methylene groups in the alkyl chain of
C16-PFA-C results in the cmc decrease, however, not so significantly.
The difference between the cmc of C18-PFA-C and C16-PFA-C is lower
than the difference between cmc values C16-PFA-C and C15-PFA-C.
This complies with the published data [46]. The extension of the alkyl
chain above 16 carbon atoms shows only marginal influence on the cmc
values. The lipophilicity of alkyl chains has the same influence on cmc
in the series of APCs with heterocycle moiety in the polar part of mo-
lecules.
2. Results and discussion
2.1. Chemistry
Prepared alkylphosphocholines (APCs) represent zwitterionic am-
phiphilic compounds with the structure similar to miltefosine (HPC). All
prepared compounds contain foscarnet (phosphonoformic) moiety in the
molecular structure. Structural comparison of both types of compounds is
depicted in Fig. 2. Phosphonoformate group (the respective molecule
part in Fig. 2 is marked bold) was inserted between the alkyl chain and
the choline moiety. The compound C15-PFA-C (C21H44NO5P; Scheme 1)
shows the closest structural resemblance to HPC (C21H46NO4P; Fig. 1)
within the tested compounds. It contains 16 carbon atoms in the lipo-
philic part of molecule. However, one carbon atom is isolated from
pentadecyl chain by oxygen and this carbon atom is bonded to another
oxygen by a double bond. This connection of atoms (ReOeC(]O)e)
represents the ester group (Fig. 2). C16-PFA-C can be also considered as
an analogue to HPC because the structures are very similar to each other.
They have hexadecyl chains, but C16-PFA-C has a carbonyl group in-
serted between oxygen and phosphorus of the phosphate group. C16-
PFA-C contains only one more carbonyl group in comparison with HPC.
We have prepared a perifosine analogue, as well. The relationship be-
tween C18-PFA-4P and perifosine is the same as the relationship be-
tween C16-PFA-C and HPC. These compounds differ in the presence or
absence of carbonyl group in the structures. C18-PFA-4P represents an
APC with the heterocyclic moiety. The piperidinium skeleton replaced
the choline in the polar part of amphiphile. Other types of heterocyclic
APCs are represented by compounds where trimethylaminium of the
choline group is replaced with N-methylpiperidinium, N-methylazepa-
nium or N-methylmorpholinium groups (Scheme 1).
Heterocyclic substitution on aminium nitrogen of APCs with pen-
tadecyl chain results in the cmc decrease in comparison with the cho-
line derivative. The size of rings affects the cmc values. The insertion of
single methylene group into a heterocycle results in the cmc decrease.
C15-PFA-A with azepane has cmc smaller than that of C15-PFA-P with
piperidine. On the other hand, the insertion of hydrophilic oxygen into
the heterocycle causes the cmc increase. The morpholine derivative
C15-PFA-M shows the cmc larger than that of the piperidine derivative
C15-PFA-P. We observed the same influence of the heterocycle pre-
sence on cmc values in the case of HPC derivatives [45]. The com-
parison of two piperidine derivatives C15-PFA-P and C15-PFA-4P
shows that the removal of ethylene bridge between phosphonate and
heterocycle results in the cmc increase. C15-PFA-4P is less lipophilic
than C15-PFA-P. The cmc values of the series of heterocyclic APCs with
hexadecyl and octadecyl chains are very low. Therefore, it is difficult to
corelate the structural changes with the cmc values. Their values are in
the range 4–4.6 × 10−6 mol·dm−3 for the derivatives with hexadecyl
chains and 2.6–2.7 × 10−6 mol·dm−3 for derivatives with octadecyl
chains, respectively.
The synthetic strategy in the preparation of alkylphosphocholines
(APCs) is depicted in Scheme 2. The compounds were synthesized in
several steps. Alkoxycarbonylphosphonic acids 3 were prepared from
the respective fatty alcohols. The alcohols reacted with triphosgene to
produce alkoxycarbonylchlorides 1. The next step was Michaelis-Ar-
buzov reaction. The carbonylchlorides are more reactive than alkyl-
halides. Therefore, the formation of phosphonates 2 required lower
temperature than generally needed. Phosphonic acids 3 were released
from diesters by the reaction with trimethylsilylbromide and methanol.
The alkoxycarbonylphosphonic acids were prepared according to the
modified methods published by Rosowsky et al. [44]. The last step of
synthesis was the reaction of alkoxycarbonylphosphonic acid 3 with
choline tosylates in the presence of 2,4,6-triisopropylbenzenesulfo-
nylchloride (TIPS-Cl). Cholines contain trimethylaminium, piper-
idinium, morpholinium or azepanium cations. The synthesis was per-
formed according to the procedure described for dialkyphosphocholines
[4]. The synthesized APCs are illustrated in Scheme 2.
The surface tension value at the cmc (γcmc) is found to be between
30.2 mN·m−1 and 36.0 mN·m−1. The compounds with octadecyl chains
have γcmc slightly higher than APCs with shorter alkyl chains. The in-
sertion of an oxygen atom into heterocyclic ring also causes a slight
increase in γcmc. The same influence was observed in the case of het-
erocyclic derivatives of HPC [45].
The surface area values per head group at the surface saturation
(Acmc) of choline derivatives C15-PFA-C and C16-PFA-C correlate well
with the previously published data of HPC [45]. However, C18-PFA-C
is densely packed at the water/air interface which results in its value of
A
cmc being only 0.37 nm2. The same results were observed in the case of
2.2. Physicochemical properties of APCs
heterocyclic APCs with the longest alkyl chains. The lipophilicity in-
crease may result in the increased intensity of Van der Waals interac-
tions between molecules of amphiphiles. These interactions lead to the
decrease in Acmc. Piperidine and azepane derivatives of C15-PFA-C and
Physicochemical properties of the compounds were investigated by
the measurements of surface tension of their aqueous solutions. The
3

L. Timko, et al.
BioorganicChemistry104(2020)104224
O
P
C15-PFA-C x = 14
C16-PFA-C x = 15
C18-PFA-C x = 17
O
O–
O
N+
( )x
O
O
O
O
OH
O
O
O
P
a
b
c
d
( )x
O
( )x
O
OH
( )x
O
O
P
P
O–
( )x
N+
( )x
O
OH
O
Cl
O
O
C15-PFA-4P x = 14
C18-PFA-4P x = 17
1
2
3
O
P
O
O–
O
N+
( )x
O
Y
C15-PFA-P x = 14, Y = CH2; C15-PFA-A x = 14, Y = (CH2)2; C15-PFA-M x = 14, Y = O
C16-PFA-P x = 15, Y = CH2; C16-PFA-A x = 15, Y = (CH2)2; C16-PFA-M x = 15, Y = O
C18-PFA-P x = 17, Y = CH2; C18-PFA-A x = 17, Y = (CH2)2; C18-PFA-M x = 17, Y = O
Scheme 2. Synthesis of alkylphosphocholines derived from foscarnet. Reagents: (a) triphosgene; (b) trimethyl phosphite, 80 °C; (c) i: trimethylsilylbromide, ii:
methanol; (d) TIPS-Cl, pyridine, choline tosylates.
C16-PFA-C have Acmc larger than that of choline derivatives. Hetero-
cyclic cations are bulkier than trimethylaminium cations and require
more space at the water/air interface. On the other hand, morpholine
derivatives are packed very tightly. Their Acmc values are smaller than
the values of C15-PFA-C and C16-PFA-C. We assume that the interac-
tion of oxygen atom of the morpholine ring with water molecules
through hydrogen bonds is responsible for the dense arrangement of
APC molecules as compared with other heterocyclic derivatives.
Hydrodynamic diameter (dH) of APCs was determined at the con-
centrations that were 200-times higher than cmc. The data are sum-
marized in the Table 2. The compounds with pentadecyl and hexadecyl
chains formed small micelles. Their diameter values are in the range
from 5.5 nm to 7.6 nm. The values correspond with the data published
and human breast adenocarcinoma cells, estrogen receptor positive
(MCF-7). Cytotoxic activity of the compounds is compared with the
activity against non-cancer cells fibroblasts 3T3. The values of the half
maximal inhibitory concentration (IC50) are summarized in Table 3.
The results show that the APCs with pentadecyl chains are practi-
cally inactive against the tested cells. Cytotoxic activity is only observed
in the case of C15-PFA-C and C15-PFA-A. The activity of C15-PFA-C is
practically identical with that of the standard HPC. Both compounds
show cytotoxic activity against HeLa. MCF-7 was resistant to the action
of the compounds. A different situation is observed in the case of C15-
PFA-A. The compound is inactive against HeLa, but it shows cytotoxic
activity against MCF-7. However, the activity is very low.
The compounds with hexadecyl and octadecyl chains have higher
cytotoxic activity against cancer cells than APCs with a shorter alkyl
chain. The activity increases with the increasing alkyl chain length.
Generally, APCs with octadecyl chains are more active than the com-
pounds with hexadecyl chains. We have made a similar observation in
the case of dialkylphosphocholines. The compounds with the longest
alkyl chain (eicosanyl) showed the highest cytotoxicity [15]. The cy-
totoxicity increase with the increasing lipophilicity of APCs was also
observed in other series of compounds [47,48]. Lipophilicity is one of
the most important factors that affects cytotoxic activity of APCs. The
cmc is a parameter that has a strong relationship to lipophilicity. Higher
cytotoxic activity of compounds is observed for APCs with smaller cmc.
Another reason of high cytotoxic activity of these compounds is that
they can form different types of aggregates. Large intermicellar clusters
or vesicles can be expected as showed the measurement of the hydro-
dynamic diameters of APCs with octadecyl chain. They may interact
more easily with plasmatic membrane of cancer cells than micelles. An
example of the compound with high lipophilicity is erufosine that has
been widely tested as potential chemotherapeutic [49–53].
for HPC (dH = 5.8
1.0 nm) and similar nitrogen heterocyclic APCs
published previously [45]. A different situation is observed in the case
of APCs with octadecyl chains. Two compounds (C18-PFA-A, C18-PFA-
4P) were poorly soluble in water. Therefore, their hydrodynamic dia-
meter could not be measured. C18-PFA-P shows a bimodal particle size
spectrum with one peak assigned to small micelles (dH = 7.5 nm) and
the second assigned to large aggregates (dH = 299 nm). Large ag-
gregates, bigger than 240 nm in size, are also formed by C18-PFA-C and
C18-PFA-M. The aggregates are > 50 times larger than the aggregates
of structurally similar compounds with shorter alkyl chains. Higher li-
pophilicity of these compounds may result in the aggregation of mi-
celles into large intermicellar clusters or in the formation of vesicles.
We observed a similar behaviour in case of APCs with branched alkyl
chains [2]. The increased molecular lipophilicity may be responsible for
the formation of large aggregates along with the presence of small
micelles in aqueous solution of the studied compounds.
2.3. Biological activity
The insertion of the foscarnet moiety into the structure of APCs has
no effect on cytotoxic activity. C15-PFA-C, C16-PFA-C and HPC have
approximately the same activity. Their cytotoxicity also correlates with
their cmc values. All three compounds have similar lipophilicity and
2.3.1. Cytotoxic activity
Cytotoxic activity of APCs was determined against cancer cells.
They are represented by human cervical adenocarcinoma cells (HeLa)
4

L. Timko, et al.
BioorganicChemistry104(2020)104224
Fig. 3. Plots of surface tension vs. log concentration of APCs.
their cytotoxic activity has been changed minimally, as well.
The investigations of the activity of APCs against cancer and non-
cancer cells revealed that the APCs with hexadecyl chains and C18-
PFA-C are more active against HeLa and MCF-7 than against 3T3. In
contrast, heterocyclic APCs with the longest alkyl chain are more toxic
to non-cancer cells in comparison with cancer cells. The compound
5

L. Timko, et al.
BioorganicChemistry104(2020)104224
Table 1
same results in the case of E. coli. The modification of the lipophilic part
of APC molecule had no effect on antimicrobial activity. However, HPC
and APCs with higher lipophilicity than HPC were active against S.
aureus and its methicilline-resistant strain.
Surface tension data of APCs.
Compound
cmc
A
γcmc
[mol.dm−3
]
[Å2]
[mN.m−1
]
On the other hand, the antifungal activity values of APCs were
found to be significant. The MIC values of all compounds were lower
than 40 μM. The activity of compounds with pentadecyl and hexadecyl
chains was comparable with the HPC standard. These compounds have
similar lipophilicity. The carbonyl group of foscarnet moiety affects
physico-chemical property only marginally. The structural changes in
polar head groups have only small influence on the activity. The highest
activity was determined for C15-PFA-C. It is twice as active as the HPC
standard and other APCs tested. The MIC value is 1.4 μM. The same
value was obtained by Obando [21] for octadecylphosphocholine. To
the best of our knowledge, this is the highest anticandidal activity at-
tained within the group of APCs. The extension of the alkyl chain of the
investigated APCs to the length of 18 carbon atoms results in the de-
crease of anticandidal activity. The compounds with heterocyclic
groups in the structure are twelve times less active than their analogues
with shorter alkyl chains. Only C18-PFA-C shows the activity that is
comparable with other APCs of pentadecyl or hexadecyl chain length.
These findings are in accordance with similar observations published
previously [2,15,21]. The compounds with the highest lipophilicity (i.e.
with the lower cmc values) show decreased activity against Candida
albicans. The relationship between anticandidal activity and the alkyl
chain length was studied in case of APCs with branched alkyl chains [2]
where the cut-off effect was observed. The cut-off effect is typical for
biological activity of a series of amphiphilic compounds with different
alkyl chain length [54]. In other words, the highest activity is observed
for a compound with specific lipophilicity. The compounds with very
short or very long alkyl chains were less active.
C15-PFA-C
C15-PFA-P
C15-PFA-A
C15-PFA-M
C15-PFA-4P
C16-PFA-C
C16-PFA-P
C16-PFA-A
C16-PFA-M
C18-PFA-C
C18-PFA-P
C18-PFA-M
HPC [45]
(1.45
(1.17
(8.71
(1.36
(1.29
(6.41
(4.01
(4.58
(4.26
(3.83
(2.63
(2.69
(1.25
0.03) × 10−5
0.02) × 10−5
0.27) × 10−6
0.01) × 10−5
0.04) × 10−5
0.45) × 10−6
0.14) × 10−6
0.24) × 10−6
0.23) × 10−6
0.23) × 10−6
0.28) × 10−6
0.02) × 10−6
0.05) × 10−5
51
65
73
49
54
56
72
62
52
37
26
47
57
2
2
4
1
3
8
5
6
5
4
5
0
3
32.3
31.0
30.2
34.9
30.7
32.2
31.8
31.3
32.6
35.0
35.9
36.0
38.3
0.2
0.1
0.2
0.1
0.3
0.5
0.1
0.4
0.4
0.6
1.6
0.1
0.1
Table 2
Micelle sizes of APCs.
Compound
Micelle size (dH)
Compound
Micelle size (dH)
[nm]
[nm]
C15-PFA-C
C15-PFA-P
C15-PFA-A
C15-PFA-M
5.5
5.5
5.5
5.7
0.4
C16-PFA-A
C16-PFA-M
C18-PFA-C
C18-PFA-P
5.7
0.3
0.7
0.0
0.3
0.3
7.6
286.6
299.3
7.5
7.2
9.8
0.6
C15-PFA-4P
C16-PFA-C
C16-PFA-P
6.3
6.7
6.2
0.2
0.7
0.6
C18-PFA-A
C18-PFA-M
C18-PFA-4P
−
245.1
−
1.7
C18-PFA-C shows the highest selectivity. This compound has the lowest
values of IC50 against HeLa and MCF-7 among all tested APCs and it is
approximately two times less active against non-cancer cells 3T3.
2.3.3. Acanthamoeba spp.
Antiprotozoal activity of the APCs with foscarnet moiety was de-
termined against two clinical isolates of A. lugdunensis and A. quina of
T4 genotype, which were causative agents of Acanthamoeba keratitis.
The values of minimal trophocidal concentrations (MTC) and half
maximal effective concentration (EC50) are summarized in Table 3.
The MTC values of APCs with foscarnet moiety show that only two
compounds with pentadecyl chains have demonstrable trophocidal
activity. C15-PFA-A shows the activity against both A. lugdunensis and
A. quina. C15-PFA-P is active only against A. lugdunensis isolate. These
two compounds reach or exceed the level of trophocidal activity of the
standard HPC. On the basis of MTC values, the other tested compounds
can be classified as trophocidally inactive. However, this does not mean
2.3.2. Antimicrobial activity
Antimicrobial activity was determined against Gram-positive bac-
teria Staphylococcus aureus, Gram-negative bacteria Escherichia coli and
yeast Candida albicans. Minimal inhibition concentration (MIC) values
are summarized in Table 3.
The results show that all prepared compounds do not demonstrate
significant antimicrobial activity against S. aureus and E. coli. The
standard (HPC) is antimicrobially active only against Gram-positive
bacteria. In our previous study, we have made a similar observation in
case of dialkyl derivatives of HPC [16]. Obando et al. [21] obtained the
Table 3
Cytotoxic, antimicrobial and antiprotozoal activity of APCs.
Compound
IC50 (µM)
MIC (µM)
MTC (µM)
EC50 (µM)
HeLa
MCF-7
3T3
S.a.
E.c.
C.a.
A.l.
A.q.
A.l.
A.q.
C15-PFA-C
C15-PFA-P
C15-PFA-A
C15-PFA-M
C15-PFA-4P
C16-PFA-C
C16-PFA-P
C16-PFA-A
C16-PFA-M
C18-PFA-C
C18-PFA-P
C18-PFA-A
C18-PFA-M
C18-PFA-4P
HPC
72.5
> 100
> 100
94.3
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
91.4
> 1000
> 1000
> 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
> 1000
> 1000
1.39
2.59
5.05
2.56
2.65
2.71
2.49
2.44
2.49
5.05
38.1
36.8
n.d.
> 500
500
> 500
> 500
500
96
95
> 100
> 100
> 100
> 100
86.4
34
104
500
25
9.6
> 100
> 100
> 100
> 100
88.2
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
500
75
59
84
61
371
273
85.0
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
42
419
64.7
> 500
356
77.5
> 100
50.6
46.9
57.3
> 500
> 500
> 500
> 500
469
55.0
52.6
44.8
50.9
40.8
88.5
72.7
57.5
50.4
58.4
40.7
n.d.
77.8
> 100
> 100
2.87
45
A.l. – Acanthamoeba lugdunensis, A. q. – Acanthamoeba quina, C.a. – Candida albicans, E.c. – Escherichia coli, n.d. – not determined, S.a. – Staphylococcus aureus.
6

L. Timko, et al.
BioorganicChemistry104(2020)104224
that they are inactive against acanthamoebae. All investigated com-
pounds inhibit the growth of Acanthamoeba trophozoites. The half
maximal effective concentration shows that the compounds with pen-
tadecyl chains and several compounds with longer alkyl chains are
strongly trophostatic. The concentration of these APCs lower than
500 μM is capable of reducing the cell populations by 50%. The values
of EC50 show that lipophilicity is crucial for a good activity. All APCs
with alkyl chains with 15 carbon atoms have EC50 lower than 100 μM.
Only C15-PFA-P has EC50 slightly higher in the case of A. quina. This
value was 104 μM. APCs with the alkyl chain length above 15 carbon
atoms were less active. The values of EC50 lower than 500 μM were
observed in the case of C16-PFA-C, C16-PFA-P, C16-PFA-M and C18-
PFA-4P. The first compound is active against both strains. The other
compounds show activity only against A. quina. However, their values
of EC50 are > 200 μM.
observed for C18-PFA-C. Its value was approximately twice the value of
HPC. APCs with foscarnet moiety were antibacterially inactive.
However, anticandidal activity was determined in case of all tested
compounds. The highest activity was found for the compound C15-
PFA-C with the MIC = 1.4 μM. It is the highest anticandidal activity
ever determined within the group of APCs. The highest antiprotozoal
activity was found in the case of C15-PFA-A. This compound is more
active against both pathogenic strains of Acanthamoeba (T4 genotype)
than HPC. One can conclude that more lipophilic APCs with octadecyl
chain have higher cytotoxic activity against cancer cells than the
compounds with shorter alkyl chains. The opposite situation was ob-
served for anticandidal and antiprotozoal activity. The most active
compounds are those with pentadecyl chains.
Increasing resistance of Candida spp to antimycotics and inadequate
treatment of acanthamoebiasis raise demand for new antimicrobial
compounds. As the results obtained within our study indicate, two of
our novel compounds show promising potential. The compound C15-
PFA-C might be efficient in the treatment of candidiasis whereas the
compound C15-PFA-A represents a potential high efficient APC in the
treatment of Acanthamoeba infections.
Among all compounds investigated, the compound C15-PFA-A
shows the highest activity. It has the lowest MTC and EC50 values as
well. These values are approximately two times (for A. lugdunensis) and
five times (for A. quina) lower than those of the HPC standard. It can be
assumed that the heterocycle presence may have the influence on
biological antiprotozoal activity. We obtained the same results for
heterocyclic APCs which were published previously [13]. Azepane and
azocane analogues of HPC were the most active compounds against A.
lugdunensis. However, heterocyclic analogues of APCs with a branched
alkyl chain do not support this assumption [14]. The antiprotozoal
activity of APCs is non-specific and therefore, it depends on the ability
to penetrate the plasmatic membrane of trophozoites. Lipophilicity of
molecules is more important for antiprotozoal activity than the pre-
sence of a heterocycle in the molecule polar part. The changes in the
activity are more pronounced as a result of the alkyl chain extension
than the structural modification of polar head group [2,14]. The pre-
sence or absence of carbonyl group in the molecules of APCs affects
antiprotozoal activity only minimally. The most active compounds have
4. Experimental part
Chemicals for synthesis were obtained from the following com-
mercial suppliers: CentralChem, Slovakia – acetone, acetonitrile, die-
thyl ether, chloroform, propan-2-ol, pyridine, tetrahydrofurane, trie-
thylamine; Fluka, Germany – phosphorus oxychloride, methyl 4-
methylbenzenesulphonate,
N,N-dimethyl-2-aminoethanol;
Merck,
Germany – pentadecane-1-ol, hexadecane-1-ol, octadecane-1-ol, N-
methylpiperidine-4-ol, Amberlite MB-3; Chemicals were used as sup-
plied. Solvents for synthesis were purified and dried before use ac-
cording to Perrin and Armarego [55]. 2-(piperidine-1-yl)ethanol, 2-
(azepane-1-yl)ethanol, 2-(morpholine-1-yl)ethanol were prepared by
hydroxyethylation [56]. Choline salts were dried at 61 °C at diminished
pressure prior to use.
cmc approximately 1 × 10−5 mol·dm−3
.
3. Conclusions
1H, 13C and 31P NMR spectra were measured on a Varian MERCURY
plus spectrometer working at frequency 300, 75 and 121.5 MHz re-
spectively. 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 re-
corded on an LTQ Orbitrap XL hybrid FTMS spectrometer (Thermo
Fisher Scientific) using electrospray Ion Max-ESI in positive mode.
Elemental analysis was carried out using FLESCH 2000 (Thermo Fischer
Scientific).
Within the framework of this study, fourteen novel APCs were
synthesized and investigated. The novel compounds represent zwitter-
ionic amphiphiles with phosphocholine moiety where the phosphate
group is replaced with phosphonometanoate group. The synthesized
compounds represent a new type of APCs that link the structure of
miltefosine and its analogues to the antiviral drug foscarnet. We in-
vestigated the effect of polar head group and carbonyl group bonded to
phosphonate on physicochemical properties, cytotoxic, antimicrobial
and anti-Acanthamoeba activity. The physicochemical properties of the
novel compounds were studied by determining the surface activity and
the micelle size utilising surface tension and dynamic light scattering
experimental techniques. The most important parameter obtained from
the measurements was the critical micelle concentration. The cmc can
be regarded as the measure of lipophilicity [17]. The insertion of car-
bonyl group between oxygen and phosphorus atoms of the phosphate
group of HPC resulted in the cmc decrease. However, the compound
C15-PFA-C that has the number of carbon atoms identical with HPC
and the only structural difference is the presence of two hydrogen
atoms and one oxygen atom in its molecular structure, has cmc larger
than that of HPC. The presence of hydrophilic oxygen atom decreases
the lipophilicity of APC with foscarnet moiety. The size of the ag-
gregates formed in aqueous solution indicates that APCs with penta-
decyl and hexadecyl chain formed small micelles. However, complex
aggregates large in size are typical for the most lipophilic APCs among
the investigated compounds.
A. lugdunensis and A. quina were clinical isolates of free-living
amoebae isolated from corneas of patients with Acanthamoeba keratitis
[57].
4.1. Synthesis
4.1.1. General procedure for preparation of choline salts
A solution of methyl 4-methylbenzenesulfonate (50 mmol) was
added to a solution of tertiary amine (2-(N,N-dimethylamino)ethanol,
2-(piperidine-1-yl)ethanol, 2-(azepane-1-yl)ethanol, 2-(morpholine-1-
yl)ethanol or N-methylpiperidine-4-ol; 50 mmol) in acetonitrile
(30 mL). The reaction mixture was refluxed for 4 h. The solvent was
evaporated in vacuum and the crude solid was left for crystallisation
from the mixture of acetone and methanol yielding a white, hygro-
scopic solid as the product. The product was kept in vacuum desiccator
over P4O10
.
Biological activity of APCs was examined on two cancer cell lines
(HeLa, MCF-7), non-cancer cells (3T3), two bacteria (S. aureus, E. coli),
one yeast (C. albicans) and two protozoan parasites (A. lugdunensis and
A. quina). The highest cytotoxic activity against cancer cells was
4.1.1.1. 2-hydroxy-(N,N,N-trimethyl)ethane-1-aminium 4-methylbenzen-
esulfonate. Yield: 93.8%; 1H NMR (DMSO‑d6) δ 2.29 (s, 3H); 3.10 (s,
9H); 3.37–3.41 (m, 2H); 3.81–3.84 (m, 2H); 5.28 (t, 1H, J = 5.3 Hz);
7.12 (d, 2H, J = 8.4 Hz); 7.48 (d, 2H, J = 6.6 Hz); 13C NMR (DMSO‑d6)
7

L. Timko, et al.
BioorganicChemistry104(2020)104224
δ 20.8; 53.1; 53.2; 55.1; 66.9; 125.5; 128.1; 137.6; 145.7; HRMS calcd.
for C5H14ON+ = 104.1070; found m/z: [M]+ 104.1069.
4.1.2.3. Dimethyl
octadecyloxycarbonylphosphonate. Yield:
88.7%;
m.p. = 41–42 °C; 1H NMR (CDCl3) δ 0.88 (t, 3H, J = 6.8 Hz);
1.26–1.33 (m, 30H); 1.72 (qi, 2H, J = 6.8 Hz); 3.93 (d, 6H,
J = 11.1 Hz); 4.27 (t, 3H, J = 6.8 Hz); 13C NMR (CDCl3) δ 14.1;
22.7; 25.8; 28.4; 29.1; 29.4; 29.5; 29.6; 29.7; 31.9; 54.5; 54.6; 66.3;
164.6; 168.1; 31P NMR (CDCl3) δ –2.27.
4.1.1.2. N-(2-hydroxyethyl)-N-methylpiperidinium 4-methylbenzenesulf-
onate. Yield: 86.0%; 1H NMR (DMSO‑d6) δ 1.49–1.56 (m, 2H);
1.75–1.79 (m, 4H); 2.29 (s, 3H); 3.06 (s, 3H); 3.30–3.44 (m, 6H);
3.83–3.84 (m, 2H); 5.27 (t, 1H, J = 4.8 Hz); 7.12 (d, 2H, J = 7.8 Hz);
7.48 (d, 2H, J = 7.8 Hz); 13C NMR (DMSO‑d6) δ 19.3; 20.6; 20.8; 48.2;
54.6; 60.8; 63.7; 125.5; 128.1; 137.6; 145.7; HRMS calcd. for
C8H18ON+ = 144.1383; found m/z: [M]+ 144.1381.
4.1.3. General procedure for preparation of alkyloxycarbonylphosphonic
acids
Dimethyl alkyloxycarbonylphosphonate (10 mmol) was dissolved in
20 mL tetrachloromethane and bromotrimethylsilane (30 mmol) was
added. The reaction mixture was stirred at r.t. for 20 h. After that, the
volatile compounds were evaporated and methanol was added. The
mixture was heated up to 65 °C for 3 h and methyl trimethylsilyl ether
was distilled off during the reaction. The unreacted methanol was
evaporated, and the crude acid was crystalized from hexane. The
compounds were obtained as white amorphous powders.
4.1.1.3. N-(2-hydroxyethyl)-N-methylazepanium
4-methylbenzenesulf-
onate. Yield: 89.0%; 1H NMR (DMSO‑d6) δ 1.56–1.60 (m, 4H); 1.81
(m, 4H); 2.29 (s, 3H); 3.05 (s, 3H); 3.32–3.40 (m, 4H); 3.51–3.59 (m,
2H); 3.84 (m, 2H); 5.29 (t, 1H, J = 4.8 Hz); 7.11 (d, 2H, J = 7.9 Hz);
7.47 (d, 2H, J = 7.9 Hz); 13C NMR (DMSO‑d6) δ 20.7; 20.7; 27.1; 50.8;
55.0; 63.9; 65.4; 125.4; 127.9; 137.5; 145.7; HRMS calcd. for
C9H20ON+ = 158.1539; found m/z: [M]+ 158.1536.
4.1.3.1. Pentadecyloxycarbonylphosphonic
acid. Yield:
62.0%;
1
m.p. = 31–32 °C; H NMR (CDCl3) δ 0.88 (t, 3H, J = 6.8 Hz); 1.26
(m, 24H); 1.67–1.70 (m, 2H); 3.71 (t, 2H, J = 6.6 Hz); 9.53 (m, 2H);
13C NMR (CDCl3) δ 14.1; 22.7; 25.6; 28.3; 29.3; 29.4; 29.6; 29.7; 31.9;
63.4.
4.1.1.4. N-(2-hydroxyethyl)-N-methylmorpholinium 4-methylbenzenesulf-
1
onate. Yield: 94.4%; H NMR (DMSO‑d6) δ 2.29 (s, 3H); 3.20 (s, 3H);
3.38–3.58 (m, 6H); 3.86–3.91 (m, 6H); 5.33 (t, 1H, J = 4.8 Hz); 7.12
(d, 2H, J = 8.2 Hz); 7.48 (d, 2H, J = 7.9 Hz); 13C NMR (DMSO‑d6) δ
21.3; 48.1; 54.9; 55.0; 60.2; 60.3; 65.2; 126.0; 128.5; 138.1; 146.2;
HRMS calcd. for C7H16O2N+ = 146.1176; found m/z: [M]+ 146.1174.
4.1.3.2. Hexadecyloxycarbonylphosphonic
acid. Yield:
64.0%;
m.p. = 42–44 °C; 1H NMR (CDCl3) δ 0.88 (t, 3H, J = 6.7 Hz);
1.26–1.30 (m, 26H); 1.52–1.61 (m, 2H); 3.64 (t, 2H, J = 6.6 Hz); 9.45
(m, 2H); 13C NMR (CDCl3) δ 14.1; 22.7; 25.7; 29.4; 29.6; 29.7; 32.8;
63.1.
4.1.1.5. 4-hydroxy-N,N-dimethylpiperidinium
4-methylbenzenesulfo-
nate. Yield: 86.5%; 1H NMR (DMSO‑d6)
δ
1.71–1.72 (m, 2H);
1.92–2.00 (m, 2H); 2.29 (s, 3H); 3.04 (s, 3H); 3.08 (s, 3H); 3.24–3.31
(m, 2H); 3.37–3.46 (m, 2H); 3.74–3.78 (m, 1H); 5.08 (d, 1H,
J = 3.8 Hz); 7.11 (d, 2H, J = 8.5 Hz); 7.46 (d, 2H, J = 7.9 Hz); 13C
NMR (DMSO‑d6) δ 20.7; 27.6; 49.7; 51.3; 58.1; 60.8; 125.4; 127.9;
137.5; 145.7; HRMS calcd. for C7H16ON+ = 130.1226; found m/z:
[M]+ 130.1225.
4.1.3.3. Octadecyloxycarbonylphosphonic
acid. Yield:
78.3%;
m.p. = 81–82 °C; 1H NMR (CDCl3) δ 0.88 (t, 3H, J = 6.8 Hz);
1.25–1.30 (m, 30H); 1.67–1.72 (m, 2H); 3.61 (t, 3H, J = 6.6 Hz); 9.52
(m, 2H); 13C NMR (CDCl3) δ 14.1; 22.7; 25.8; 29.4; 29.5; 29.7; 32.0;
32.6; 62.8.
4.1.2. General
procedure
for
preparation
of
dimethyl
4.1.4. General procedure for preparation of alkyloxycarbonylphosphonato-
cholines
alkyloxycarbonylphosphonates
Primary alcohol (pentadecane-1-ol, hexadecane-1-ol, octadecane-1-
ol; 10 mmol) was dissolved in 30 mL of tetrachloromethane and pyr-
idine was added (11 mmol). The solution was cooled to −20 °C and
triphosgene (5 mmol) was added at vigorous stirring. The mixture was
stirred another 20 min at −20 °C. Then, the solution was warmed to
room temperature and stirred overnight. The precipitate was filtered
and the solution was evaporated. The obtained alkylox-
ycarbonylchloride was used in the next reaction step without further
purification.
Alkyloxycarbonylphosphonic acid (5 mmol) was dissolved in pyr-
idine (15 mL) and the solution was stirred at 50 °C for 2 h. After that,
pyridine was evaporated, and another 20 mL of pyridine was added.
The appropriate choline derivative (7.5 mmol) and 2,4,6-triisopro-
pylbenzenesulfonyl chloride (10 mmol) in pyridine (20 mL) were added
to the solution of pyridinium salt of alkyloxycarbonylphosphonic acid.
The solution was stirred at 50 °C overnight. After cooling, the mixture
was hydrolysed by the addition of H2O (1.5 mL) and stirred at r.t. for
1 h. Solvents were evaporated at vacuum and the crude solid was di-
luted in tetrahydrofuran 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 va-
cuum. The product was purified by chromatography using silica gel
with the liquid phase containing CHCl3:CH3OH:H2O (65:25:1 →
65:25:3). After purification, solvents were evaporated in vacuum, the
product was dried by azeotropic distillation using propan-2-ol, dis-
solved in chloroform and precipitated with acetone. The product was
filtered off as white hygroscopic solid and was stored in desiccator over
phosphorus pentoxide.
Alkyloxycarbonylchloride (10 mmol) was mixed with trimethyl
phosphite (10 mL) and heated at 80 °C overnight. After cooling, the
excess of trimethyl phopshite was evaporated and the raw product was
crystalized from hexane. The esters were obtained as white crystalline
compounds.
4.1.2.1. Dimethyl pentadecyloxycarbonylphosphonate. Yield: 51.4%;
m.p. = 26–28 °C; 1H NMR (CDCl3) δ 0.88 (t, 3H, J = 6.6 Hz);
1.26–1.36 (m, 24H); 1.72 (qi, 2H, J = 6.9 Hz); 3.93 (d, 6H,
J = 11.1 Hz); 4.27 (t, 2H, J = 6.8 Hz); 13C NMR (CDCl3) δ 14.1;
22.7; 25.8; 28.4; 29.1; 29.4; 29.5; 29.6; 29.7; 31.9; 54.5; 54.6; 66.3;
164.6; 168.2; 31P NMR (CDCl3) δ –2.26.
4.1.4.1. 2-(trimethylaminio)ethyl
pentadecyloxycarbonylphosphonate
(C15-PFA-C). Yield: 18.8%; 1H NMR (CDCl3)
δ 0.88 (t, 3H,
4.1.2.2. Dimethyl hexadecyloxycarbonylphosphonate. Yield: 57.5%;
m.p. = 32–33 °C; 1H NMR (CDCl3, TMS) 0.88 (t, 3H, J = 6.7 Hz);
1.26–1.36 (m, 26H); 1.72 (qi, 2H, J = 6.7 Hz); 3.93 (d, 6H,
J = 11.1 Hz); 4.27 (t, 2H, J = 6.7 Hz); 13C NMR (CDCl3, TMS) δ
14.1; 22.7; 28.4; 29.1; 29.4; 29.5; 29.6; 29.7; 31.9; 53.7; 63.1; 164.4;
168.0; 31P NMR (CDCl3, H3PO4) δ –2.24.
J = 6.7 Hz); 1.25–1.30 (m, 24H); 1.68 (qi, 2H, J = 7.0); 3.33 (s,
9H); 3.79–3.82 (m, 2H); 4.12 (t, 2H, J = 7.0 Hz); 4.44 (m, 2H); 13C
NMR (CDCl3) δ 14.2; 22.7; 25.9; 28.6; 29.3; 29.4; 29.6; 29.7; 32.0; 54.6;
59.8; 64.5; 66.6; 173.5 (d, J = 240 Hz); 31P NMR (CDCl3) δ –5.39;
HRMS calcd. for C21H45O5NP = 422.3030; found m/z: [M + H]+
422.3037; Anal. calcd. for C21H44O5NP × 0.9 H2O: C: 57.62; H: 10.55;
8

L. Timko, et al.
BioorganicChemistry104(2020)104224
N: 3.20; found: C: 57.72; H: 10.22; N: 2.98.
4.1.4.8. 2-(N-methylazepanio-1-yl)ethyl
hexadecyloxycarbonylphosphonato (C16-PFA-A). Yield: 16.8%; 1H NMR
(CDCl3) δ 0.88 (t, 3H, J = 6.8 Hz); 1.25 (m, 26H); 1.65–1.72 (m, 6H);
1.91 (m, 4H); 3.35 (s, 3H); 3.49–3.54 (m, 2H); 3.76–3.87 (m, 4H); 4.10
(t, 2H, J = 6.9 Hz); 4.49 (m, 2H); 13C NMR (CDCl3) δ 14.1; 21.8; 22.1;
26.0; 27.8; 28.7; 29.4; 29.6; 29.7; 51.8; 59.6; 64.2; 65.5; 174.4 (d,
4.1.4.2. 2-(N-methylpiperidinio-1-yl)ethyl
pentadecyloxycarbonylphosphonate (C15-PFA-P). Yield: 23.6%; 1H NMR
(CDCl3) δ 0.88 (t, 3H, J = 6.7 Hz); 1.25–1.33 (m, 26H); 1.63–1.75 (m,
2H); 1.89–1.91 (m, 4H); 3.36 (s, 3H); 3.51–3.56 (m, 2H); 3.69–3.77 (m,
2H); 3.92 (m, 2H); 4.10 (t, 2H, J = 6.9 Hz); 4.48 (m, 2H); 13C NMR
(CDCl3) δ 14.1; 20.2; 21.0; 22.7; 26.0; 28.6; 29.4; 29.6; 29.7; 31.9; 48.6;
59.1; 62.2; 64.2; 69.6; 174.2 (d, J = 238 Hz); 31P NMR (CDCl3) δ –4.84;
HRMS calcd. for C24H49O5NP = 462.3342; found m/z: [M + H]+
462.3342; Anal. calcd. for C24H48O5NP × 0.5 H2O: C: 61.25; H: 10.50;
N: 2.98; found: C: 61.16; H: 10.23; N: 2.73.
J
=
233 Hz); 31P NMR (CDCl3)
δ –5.76; HRMS calcd. for
C
26H53O5NP = 490.3656; found m/z: [M + H]+ 490.3660; Anal.
calcd. for C26H52O5NP × 0.6 H2O: C: 62.40; H: 10.71; N: 2.80; found:
C: 62.39; H: 10.56; N: 2.71.
4.1.4.9. 2-(N-methylmorpholinio-1-yl)ethyl
hexadecyloxycarbonylphosphonate (C16-PFA-M). Yield: 25.5%; 1H NMR
(CDCl3) δ 0.88 (t, 3H, J = 6.8 Hz); 1.19–1.25 (m, 26H); 1.63–1.68 (m,
2H); 3.48 (s, 3H); 3.65–3.79 (m, 4H); 4.02–4.11 (m, 8H); 4.47 (m, 2H);
13C NMR (CDCl3) δ 14.1; 22.7; 25.9; 28.7; 29.4; 29.6; 29.7; 31.9; 48.2;
59.2; 60.7; 60.8; 64.4; 173.7 (d, J = 237 Hz); 31P NMR (CDCl3) δ –5.99;
HRMS calcd. for C24H49O6NP = 478.3292; found m/z: [M + H]+
478.3293; Anal. calcd. for C24H48O6NP × 0.7 H2O: C: 58.80; H: 10.16;
N: 2.86; found: C: 58.76; H: 10.05; N: 2.70.
4.1.4.3. N,N-dimethylpiperidinio-4-yl Pentadecyloxycarbonylphosphonate
(C15-PFA-4P). Yield: 14.9%; 1H NMR (CDCl3)
δ 0.88 (t, 3H,
J = 6.7 Hz); 1.25 (m, 24H); 2.20 (m, 4H); 3.22 (s, 3H); 3.29 (s, 3H);
3.48–3.52 (m, 2H); 3.66–3.70 (m, 2H); 4.09 (t, 2H, J = 7.0); 4.62–4.65
(m, 1H); 13C NMR (CDCl3) δ 14.1; 22.7; 25.9; 27.2; 28.7; 29.4; 29.6;
29.7; 31.9; 48.9; 55.2; 58.8; 64.1; 64.8; 174.1 (d, J = 235 Hz); 31P NMR
(CDCl3) δ –5.73; HRMS calcd. for C23H47O5NP = 448.3186; found m/z:
[M + H]+ 448.3189; Anal calcd. for C23H46O5NP × 0.75 H2O: C:
59.91; H: 10.38; N: 3.04; found: C: 60.00; H: 10.17; N: 2.80.
4.1.4.10. 2-(trimethylaminio)ethyl
octadecyloxycarbonylphosphonate
(C18-PFA-C). Yield: 21.2%; 1H NMR (CDCl3)
δ 0.88 (t, 3H,
J = 6.6 Hz); 1.25 (m, 30H); 1.65–1.70 (m, 2H); 3.32 (s, 9H);
3.77–3.80 (m, 2H); 4.12 (t, 2H, J = 7.0 Hz); 4.44 (m, 2H); 13C NMR
(CDCl3) δ 14.2; 22.7; 25.9; 29.4; 29.6; 29.7; 54.6; 59.8; 64.6; 66.6;
173.3 (d, J = 241 Hz); 31P NMR (CDCl3) δ –5.49; HRMS calcd. for
4.1.4.4. 2-(N-methylazepanio-1-yl)ethyl
pentadecyloxycarbonylphosphonate (C15-PFA-A). Yield: 39.1%; 1H
NMR (CDCl3) δ 0.88 (t, 3H, J = 6.8 Hz); 1.21–1.33 (m, 24H);
1.65–1.71 (m, 6H); 1.91 (m, 4H); 3.35 (s, 3H); 3.48–3.55 (m, 2H);
3.77–3.89 (m, 4H); 4.09 (t, 2H, J = 7.1 Hz); 4.48 (m, 2H); 13C NMR
(CDCl3) δ 14.1; 21.8; 22.7; 26.0; 27.9; 28.7; 29.3; 29.6; 29.7; 31.9; 51.6;
59.7; 64.1; 65.5; 174.2 (d, J = 234 Hz); 31P NMR (CDCl3) δ –5.64;
HRMS calcd. for C25H51O5NP = 476.3499; found m/z: [M + H]+
476.3501; Anal. calcd. for C25H50O5NP × 0.4 H2O: C: 62.19; H: 10.60;
N: 2.90; found: C: 62.10; H: 10.56; N: 2.80.
C
24H51O5NP = 464.3499; found m/z: [M + H]+ 464.3499; Anal calcd.
for C24H50O5NP × 1.1 H2O: C: 59.63; H: 10.88; N: 2.90; found: C:
59.56; H: 10.64; N: 2.66.
4.1.4.11. 2-(N-methylpiperidinio-1-yl)ethyl
1
octadecyloxycarbonylphosphonate (C18-PFA-P). Yield: 15.6%; H NMR
(CDCl3) δ 0.88 (t, 3H, J = 6.7 Hz); 1.25 (m, 36H); 1.65–1.73 (m, 4H);
3.29 (s, 3H); 3.45–3.50 (m, 2H); 3.60–3.64 (m, 2H); 3.82 (m, 2H); 4.11
(t, 2H, J = 7.0 Hz); 4.46 (m, 2H); 13C NMR (CDCl3) δ 14.1; 20.1; 21.0;
22.7; 25.9; 26.8; 28.6; 29.4; 29.6; 29.7; 31.9; 48.6; 59.1; 62.3; 64.4;
174.3 (d, J = 236 Hz); 31P NMR (CDCl3) δ –5.40; HRMS calcd. for
C27H55O5NP = 504.3812; found m/z: [M + H]+ 504.3814; Anal.
calcd. for C27H54O5NP × 0.5 H2O: C: 63.25; H: 10.81; N: 2.73; found:
C: 63.19; H: 10.55; N: 2.50.
4.1.4.5. 2-(N-methylmorpholinio-1-yl)ethyl
pentadecyloxycarbonylphosphonate (C15-PFA-M). Yield: 27.9%; 1H
NMR (CDCl3) δ 0.88 (t, 3H, J = 7.1 Hz); 1.21–1.25 (m, 24H);
1.63–1.68 (m, 2H); 3.50 (s, 3H); 3.66–3.81 (m, 4H); 4.02–4.11 (m,
8H); 4.47 (m, 2H); 13C NMR (CDCl3) δ 14.1; 22.7; 25.9; 28.6; 29.4;
29.6; 29.7; 31.9; 48.1; 59.2; 60.7; 60.8; 64.3; 64.7; 173.9 (d,
J
=
236 Hz); 31P NMR (CDCl3)
δ –5.84; HRMS calcd. for
C
23H47O6NP = 464.3136; found m/z: [M + H]+ 464.3135; Anal
4.1.4.12. N,N-dimethylpiperidinio-4-yl octadecyloxycarbonylphosphonate
(C18-PFA-4P). Yield: 25.5%; 1H NMR (CDCl3)
δ 0.88 (t, 3H,
calcd. for C23H46O6NP × 0.75 H2O: C: 57.90; H: 10.04; N: 2.94; found:
C: 57.97; H: 9.87; N: 2.81.
J = 6.6 Hz); 1.26–1.31 (m, 30H); 1.63–1.70 (m, 2H); 2.05–2.25 (m,
4H); 3.09 (s, 3H); 3.19 (s, 3H); 3.25–3.29 (m, 2H); 3.70–3.73 (m, 2H);
4.13 (t, 2H, J = 6.9 Hz); 4.63 (m, 1H); 13C NMR (CDCl3) δ 14.2; 22.8;
26.0; 26.9; 27.0; 28.7; 29.4; 29.5; 29.7; 29.8; 32.0; 48.1; 55.9; 58.6;
64.6; 173.2 (d, J = 244 Hz); 31P NMR (CDCl3) δ –6.19; HRMS calcd. for
4.1.4.6. 2-(trimethylaminio)ethyl
hexadecyloxycarbonylphosphonate
(C16-PFA-C). Yield: 26.9%; 1H NMR (CDCl3)
δ 0.88 (t, 3H,
J = 6.7 Hz); 1.25–1.30 (m, 26H); 1.68 (qi, 2H, J = 7.0 Hz); 3.32 (s,
9H); 3.78 (m, 2H); 4.12 (t, 2H, J = 7.0 Hz); 4.44 (m, 2H); 13C NMR
(CDCl3) δ 14.2; 22.7; 25.9; 28.6; 29.3; 29.4; 29.6; 29.7; 54.6; 59.7; 64.5;
66.6; 173.4 (d, J = 238 Hz); 31P NMR (CDCl3) δ –5.32; HRMS calcd. for
C
26H53O5NP = 490.3656 found m/z: [M + H]+ 490.3647; Anal. calcd.
for C26H52O5NP × 0.75 H2O: C: 62.06; H: 10.72; N: 2.78; found: C:
62.07; H: 10.61; N: 2.67.
C
22H47O5NP = 436.3186; found m/z: [M + H]+ 436.3192; Anal.
calcd. for C22H46O5NP × 0.8 H2O: C: 58.72; H: 10.66; N: 3.11; found:
C: 58.70; H: 10.35; N: 2.93.
4.1.4.13. 2-(N-methylazepanio-1-yl)ethyl
octadecyloxycarbonylphosphonate (C18-PFA-A). Yield: 7.2%; 1H NMR
(CDCl3) δ 0.88 (t, 3H, J = 6.8 Hz); 1.26 (m, 30H); 1.65–1.72 (m, 6H);
1.91 (m, 4H); 3.36 (s, 3H); 3.50–3.55 (m, 2H); 3.77–3.88 (m, 4H); 4.10
(t, 2H, J = 7.1 Hz); 4.49 (m, 2H); 13C NMR (CDCl3) δ 14.1; 21.8; 22.7;
26.0; 27.9; 28.7; 29.4; 29.6; 29.7; 31.9; 51.7; 59.6; 64.2; 65.5; 174.1 (d,
4.1.4.7. 2-(N-methylpiperidinio-1-yl)ethyl
hexadecyloxycarbonylphosphonate (C16-PFA-P). Yield: 21.2%; 1H NMR
(CDCl3) δ 0.88 (t, 3H, J = 6.8 Hz); 1.25–1.33 (m, 26H); 1.65–1.75 (m,
4H); 1.89–1.91 (m, 4H); 3.36 (s, 3H); 3.50–3.57 (m, 2H); 3.69–3.77 (m,
2H); 3.90–3.93 (m, 2H); 4.09 (t, 2H, J = 7.0 Hz); 4.48 (m, 2H); 13C
NMR (CDCl3) δ 14.1; 20.2; 21.0; 22.7; 26.0; 28.6; 29.4; 29.6; 29.7; 31.9;
48.6; 59.1; 62.1; 64.2; 174.1 (d, J = 235 Hz); 31P NMR (CDCl3) δ –4.37;
HRMS calcd. for C25H51O5NP = 476.3499; found m/z: [M + H]+
476.3503; Anal calcd. for C25H50O5NP × 0.75 H2O: C: 61.39; H: 10.61;
N: 2.86; found: C: 61.51; H: 10.27; N: 2.66.
J
=
235 Hz); 31P NMR (CDCl3)
δ –5.54; HRMS calcd. for
C28H57O5NP = 518.3969; found m/z: [M + H]+ 518.3968; Anal.
calcd. for C28H56O5NP × 0.7 H2O: C: 63.41; H: 10.91; N: 2.64; found:
C: 63.45; H: 10.71; N: 2.55.
4.1.4.14. 2-(N-methylmorpholinio-1-yl)ethyl
octadecyloxycarbonylphosphonate (C18-PFA-M). Yield: 20,9%; 1H NMR
9

L. Timko, et al.
BioorganicChemistry104(2020)104224
(CDCl3) δ 0.88 (t, 3H, J = 6.6 Hz); 1.25–1.30 (m, 30H); 1.65–1.68 (m,
2H); 3.41 (s, 3H); 3.61–3.71 (m, 4H); 3.94–4.01 (m, 6H); 4.12 (t, 2H,
J = 6.9 Hz); 4.46 (m, 2H); 13C NMR (CDCl3) δ 14.1; 22.7; 25.9; 28.6;
29.3; 29.4; 29.6; 29.7; 32.0; 48.3; 59.0; 60.7; 61.0; 64.6; 65.0; 172.8 (d,
function. Size of the aggregates was evaluated from the partition size
distributions utilizing the CONTIN algorithm [60] with the rejection
probability parameter set to 0.5. Due to the large sets of processed data,
a custom application software written in Visual Basic was used for
automated data format conversion from the measurement files.
J
=
241 Hz); 31P NMR (CDCl3)
δ –5.81; HRMS calcd. for
C26H53O6NP = 506.3605; found m/z: [M + H]+ 506.3599; Anal.
calcd. for C26H52O6NP × 0.9 H2O: C: 59.84; H: 10.39; N: 2.68; zistená:
C: 59.90; H: 10.44; N: 2.61.
4.3. Determination of biological activity
4.3.1. Cytotoxicity assay
Hela cells (human cervical carcinoma) were cultured in RPMI 1640
medium (Biosera, Kansas City, MO, United States). MCF-7 (human
breast adenocarcinoma) and 3T3 (murine fibroblasts) cell lines were
maintained in a growth medium consisting of high glucose Dulbecco's
Modified Eagle Medium with sodium pyruvate (GE Healthcare,
Piscataway, NJ, United States). The growth medium was supplemented
with a 10% fetal bovine serum, 1X HyClone™ Antibiotic/ Antimycotic
Solution (GE Healthcare, Little Chalfont, UK). Cells were cultured in an
atmosphere containing 5% CO2 in humidified air at 37 °C. Cell viabi-
lity, estimated by trypan exclusion, was > 95% before each experiment.
The cytotoxic effect of novel alkylphosphocholines was determined
by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide
(MTT) assay. Cells were seeded at a density of 5 × 103cells/well in 96-
well polystyrene microplates (SARSTEDT, Nümbrecht, Germany). 24 h
after cell seeding, tested compounds in various concentrations
(1 × 10−4 to 1 × 10−8 mol·l−1) were added. After 72 h, cells in each
well were incubated with 10 μL of MTT (5 mg·mL−1, Sigma-Aldrich
Chemie, Steinheim, Germany) at 37 °C. After an additional 4 h, during
which insoluble formazan was produced, 100 μL of a 10% sodium do-
decyl sulphate (SDS) was added in each well and another 12 h were
allowed for the formazan to dissolve. The absorbance was measured at
540 nm using the automated Cytation™ 3 Cell Imaging Multi-Mode
Reader (Biotek, Winooski, VT, United States). Three independent ex-
periments were performed for each test. Results obtained from MTT
assay were used to determine a half maximal inhibitory concentration
(IC50) of each tested compound.
4.2. Measurements of physicochemical properties
4.2.1. Equilibrium surface tension measurements
The equilibrium surface tension measurements were carried out
according to the procedure described previously [58]. The Wilhelmy
plate technique was used for the determination of the solvent surface
tension values. Measurements were performed with a Krüss 100 MK2
tensiometer. All samples were prepared by dissolving amphiphilic
compounds in deionized water. The stock solutions were prepared in
volumetric flask. The measurements were performed at 25
0.1 °C.
The measurements of equilibrium surface tension were taken re-
peatedly. The values were recorded every 360 s. The measurement was
stopped if the difference between the values of two successive mea-
surements was less than 5 × 10−5N·m−1. The break point of the linear
parts of the surface tension vs. log c curve was used for the determi-
nation of the critical micelle concentration (cmc) and surface tension at
the cmc (γcmc). The adsorbed amount of surfactant Γcmc was also de-
termined from surface tension data. The value was calculated using the
Gibbs adsorption isotherm:
=
[d /dlogc]T /(2, 303iRT)
cmc
where γ represents the surface tension (mN/m), c is the concentration
of surfactant, i represents the prefactor (APCs have i = 1), R is the gas
constant, and T represents the absolute temperature. The slope below
the cmc in the surface tension vs. log c plots was used for the de-
termination of the surface excess. The values of area per head group at
the water/air interface (Acmc) was obtained from the following equa-
tion:
4.3.2. Antimicrobial assay
The antimicrobial activity was measured against Gram-positive
bacteria S. aureus ATCC 29/58, Gram-negative bacteria E. coli ATCC
377/79 and yeast C. albicans ATCC 8186. The testing was performed by
a method used previously by Lukáč et al. [61]. Solutions of studied
compounds were prepared in water. The stock solution concentration
was 1000 μg/mL. The cultures of bacteria and yeast grew 24 h in blood
agar (S. aureus), in Endo agar (E. coli) and in the Sabouraud agar (C.
albicans). From these cultures suspensions, of microorganisms were
prepared with the concentration of 5 × 107 CFU/mL of bacteria and
5 × 105 CFU/mL of Candida albicans. The suspensions were prepared in
physiological solution (pH 7.2). The concentration of microorganisms
was determined spectrophotometrically and the suspensions were ad-
justed to the absorbance value 0.35 at 540 nm. The microorganisms
suspension (5 mL) was added to the solutions containing the tested
compound (100 mL), to double concentrated peptone broth medium
(8%) for bacteria and to the Sabouraud medium (12%) for Candida
albicans (100 mL). For a testing assay the solutions were serially diluted
by half (concentrations of the solutions were: 1000, 500, 250, 125,
62.5, 31.25, 15.63, 7.81, 3.91 and 1.95 µg/mL). The tests were per-
formed in 96-well microtiter plates. The microorganisms were in-
cubated for 24 h at 37 °C. From each well, 5 mL of tested suspension
was taken and cultured on blood agar (S. aureus), on Endo agar (E. coli)
or on Sabouraud agar (yeast). The Petri dishes were incubated for 24 h
at 37 °C. The lowest concentration of APC that prevented colony for-
mation was defined as the minimum inhibitory concentration (MIC).
Acmc = 1018/NA
cmc
where NA represents the Avogadrós constant.
4.2.2. Dynamic light scattering
The hydrodynamic diameter of APCs was determined according the
modified procedure described previously [59] using a dynamic light
scattering equipment (Brookhaven BI 9000 digital correlator, Broo-
khaven SM 200 goniometer, Lexel argon laser). The argon laser at
514.5 nm wavelength was used as the incident light source. The in-
tensity time fluctuations of the scattered light were detected at a scat-
tering angle of 90° and a temperature 25 °C and autocorrelated in the BI
9000 correlator card where the time correlation function was built. The
translation diffusion coefficient was calculated from the time correla-
tion function using the method of cumulants. The method of cumulants
was used for the calculation of the mean particle diameter from the
expansion of logarithm of time correlation function into a series up to
the second quadratic term. The diffusion coefficient was determined
from the correlation function decay rate and the hydrodynamic dia-
meter dH was calculated from the diffusion coefficient D using the
Stokes–Einstein formula dH = kT / (3πηD). η is solvent viscosity, k is
the Boltzmann constant, and T is absolute temperature. Five in-
dependent measurements and calculations of time correlation function
were carried out for each APC. The mean value and the standard de-
viation of dH for each sample were calculated. The concentration of
APCs in deionized water was 200-times higher than cmc. To determine
particle size distributions and hence, the population of small micelles
and large aggregates of APCs, a numerical algorithm based on the in-
verse Laplace transformation was applied to the time correlation
4.3.3. In vitro amoebicidal activity assay
The stock solutions of the investigated compounds were prepared by
dissolving APCs in sterile distilled water. The trophocidal activity
10

L. Timko, et al.
BioorganicChemistry104(2020)104224
against acanthamoebae was tested according to the previously de-
scribed procedure [14]. Acanthamoeba lugdunensis (strain AcaVNAK02,
T4 genotype) and A. quina (strain AcaVNAK03, T4 genotype) re-
presented clinical isolates of free-living amoebae, which were isolated
from the corneas of two patients who suffered from Acanthamoeba
keratitis. In brief, from the 2-day monoxenic cultures on agar, the
amoebae were axenized by inoculation into the Bacto-Casitone/Serum
medium (BCS) with ampicillin and penicillin. Incubation was per-
formed for 72 h. The active trophozoites were then inoculated into
peptone-yeast extract-glucose medium (PYG) which contained ampi-
cillin and penicillin. The cultures were passaged five times. PYG
medium without antibiotics was then used for axenic cultivation of
trophozoites. The measurements of cytotoxicity were performed in 96-
well microtiter plates. The cultivations were carried out under sterile
conditions and at 37 °C. Trophozoite suspension of the volume of
100 µL (2 × 105 cells·mL−1) was added to each well. Afterwards,
100 µL of the solutions with the tested compounds in PYG medium was
added to all wells, except for the untreated control. In this case, the
wells were filled with 100 µL of pure medium. Each compound was
tested at six concentrations. The final concentrations were 500, 250,
125, 62.5, 31.25 and 15.6 µM. The reduction of trophozoites was in-
vestigated after 24 h. The surviving cells were counted in a Bürker-Türk
hemocytometer. A method of the trypan blue exclusion was used for the
detection of viability of trophozoites. 100% trophocidal activity of the
APCs was confirmed by transferring 50 µL of the suspension to a PYG
medium. The amoeba growth was investigated afterwards for 14 days.
The absence of amoeba in cultures indicated 100% eradication. The
linear regression analysis using Microsoft Office Excel 2010 (Microsoft
Corporation, Redmond, WA, USA) was used for the calculation of the
EC50 (effective concentration of the tested compound that reduces the
survival of amoebae by 50%). All experiments were performed four
times for each concentration.
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Declaration of Competing Interest
[13] M. Lukáč, J. Mojžiš, G. Mojžišová, M. Mrva, F. Ondriska, J. Valentová, I. Lacko,
M. Bukovský, F. Devínsky, J. Karlovská, Dialkylamino and nitrogen heterocyclic
analogues of hexadecylphosphocholine and cetyltrimetylammonium bromide:
Effect of phosphate group and environment of the ammonium cation on their
biological activity, Eur. J. Med. Chem. 44 (2009) 4970–4977, https://doi.org/10.
1016/j.ejmech.2009.08.011.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
[14] M. Garajová, M. Mrva, L. Timko, M. Lukáč, F. Ondriska, Cytomorphological changes
and susceptibility of clinical isolates of Acanthamoeba spp. to heterocyclic alkyl-
phosphocholines, Exp. Parasitol. 145 (2014) S102–S110, https://doi.org/10.1016/
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Acknowledgement
[15] M. Lukáč, M. Mrva, E. Fischer-Fodor, I. Lacko, M. Bukovský, N. Miklášová,
F. Ondriska, F. Devínsky, Synthesis and biological activity of dialkylpho-
sphocholines, Bioorg. Med. Chem. Lett. 19 (2009) 6346–6349, https://doi.org/10.
1016/j.bmcl.2009.09.079.
We would like to thank to Janka Karlovská and Branislav Horváth,
for the measurement of NMR spectra; Janka Kubincová, for the mea-
surement of HRMS and Adrián Nedorost, for the measurement of ele-
mental analysis. This research was funded by the Grant Agency of
Ministry of Education and Academy of Science of Slovak republic
(VEGA), Projects No. VEGA 1/0753/17, VEGA 1/0054/19 and VEGA 1/
0389/19. This work was supported by the Slovak Research and
Development Agency under the contracts No. APVV-15-0123, APVV-
16-0446, APVV-17-0373 and APVV-19-0056. This work utilises the
results of the research funded by the Slovak Research and Development
Agency Grant No. APVV-0516-12 and by the CEBV project, ITMS:
26240120034. Moreover, this publication is the result of the project
implementation: “Medicínsky univerzitný vedecký park v Košiciach
(MediPark, Košice-Fáza II.)”, kód ITMS2014 + 313011D103, supported
by the Operational Programme Research and Innovation, funded by the
ERDF.
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