Synthesis, Surface Activity, and Biological Activities of Phosphonium and Metronidazole Salts

Article abstract of DOI:10.1002/jsde.12439

A series of phosphonium amphiphilic compounds was synthesized. Cationic parts of molecules contain triphenylphosphonium moieties. Lipophilic parts of compounds are represented by straight alkyl chain or the alkyl chains which are ornamented by benzyl or metronidazole. The physicochemical properties of phosphonium amphiphilic compounds were investigated by the measurements of surface tension and conductivity. The critical micelle concentration (cmc), the surface tension value at the cmc (γ_cmc), the surface area at the surface saturation per head group (A_cmc) were determined. The lowest cmc value was determined for phosphonium salts with straight dodecyl alkyl chain. Its value was 1.5 × 10^?3 mol dm^?3. Surface tension at the cmc decreases with the addition of bulky moieties (benzyl, radical from metronidazol) at the end of alkyl chains. Biological activities of compounds were studied on human erythrocytes and strains of Acanthamoeba lugdunensis and Acanthamoeba quina. Dodecyltriphenylphosphonium bromide showed the highest activity against Acanthamoeba. To the best of our knowledge, it is the first compound of the group of phosphonium amphiphiles, which exhibited high activity against Acanthamoeba. The determined structure–activity relationship indicated nonspecific trophocidal and hemolytic activity that depends on physicochemical properties of the studied compounds.

Full text of DOI:10.1002/jsde.12439

J Surfact Deterg (2020)
DOI 10.1002/jsde.12439
ORIGINAL ARTICLE
Synthesis, Surface Activity, and Biological Activities of
Phosphonium and Metronidazole Salts
Miloš Lukácˇ1 · Martin Pisárcˇik1 · Mária Garajová2 · Martin Mrva2 · Aneta Dušeková2 ·
Andrej Vrták1 · Renáta Horáková1 · Branislav Horváth3 · Ferdinand Devínsky4
Received: 14 August 2019 / Revised: 20 February 2020 / Accepted: 10 April 2020
© 2020 AOCS
Abstract A series of phosphonium amphiphilic com-
pounds was synthesized. Cationic parts of molecules con-
tain triphenylphosphonium moieties. Lipophilic parts of
compounds are represented by straight alkyl chain or the
alkyl chains which are ornamented by benzyl or metronida-
zole. The physicochemical properties of phosphonium
amphiphilic compounds were investigated by the measure-
ments of surface tension and conductivity. The critical
micelle concentration (cmc), the surface tension value at
the cmc (γcmc), the surface area at the surface saturation per
head group (Acmc) were determined. The lowest cmc value
was determined for phosphonium salts with straight dode-
cyl alkyl chain. Its value was 1.5 × 10−3 mol dm−3. Surface
tension at the cmc decreases with the addition of bulky
moieties (benzyl, radical from metronidazol) at the end of
alkyl chains. Biological activities of compounds were stud-
ied on human erythrocytes and strains of Acanthamoeba
Acanthamoeba. The determined structure–activity relation-
ship indicated nonspecific trophocidal and hemolytic activ-
ity that depends on physicochemical properties of the
studied compounds.
Keywords Acanthamoeba Á Critical micelle
concentration Á Erythrocytes Á Metronidazole Á
Phosphonium amphiphilic compounds
J Surfact Deterg (2020).
Introduction
Metronidazole is an antibiotic used in therapy of infection
caused by bacteria and parasites growing in anaerobic con-
ditions. It was introduced to treat chronic trichomonad
infections. Besides Trichomonas vaginalis, Entamoeba
histolytica or Gardia lamblia are sensitive to this chemo-
therapeutic drug (Samuelson, 1999). These parasites do not
have mitochondria. The oxidative-reduction processes are
performed in hydrogenosomes or mitosomes (Kamkowska
et al., 2016). Metronidazole is activated by reduction of its
nitro group. It is occurred only under strongly reducing
conditions (Samuelson, 1999).
lugdunensis
and
Acanthamoeba
quina.
Dode-
cyltriphenylphosphonium bromide showed the highest
activity against Acanthamoeba. To the best of our knowl-
edge, it is the first compound of the group of phosphonium
amphiphiles, which exhibited high activity against
* Miloš Lukácˇ
lukac@fpharm.uniba.sk
1
The genus Acanthamoeba belongs to opportunistic
amoebae, which causes amebic keratitis (AK) and granulo-
matous amoebic encephalitis (GAE) in humans and ani-
mals. Treatments of these diseases are difficult and many
times without any success. Many patients had to undergo
transplantation of cornea (Lorenzo-Morales et al., 2015) or
the GAE is fatal for them (Kot et al., 2018). Metronidazole
has been also infrequently used in therapy (Taravaud
et al., 2017) but its effect is disputable. No benefit for the
Department of Chemical Theory of Drugs, Faculty of Pharmacy,
Comenius University, Kalincˇiakova 8, Bratislava, SK-83232,
Slovakia
2
Department of Zoology, Faculty of Natural Sciences, Comenius
University, Mlynská dolina Ilkovicˇova 6, Bratislava, SK-84215,
Slovakia
3
NMR laboratory, Faculty of Pharmacy, Comenius University,
Odbojárov 10, Bratislava, SK-83232, Slovakia
4
Faculty of Pharmacy, Comenius University, Odbojárov 10,
Bratislava, SK-83232, Slovakia
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patients was observed when they were treated with combi-
nations of drugs containing metronidazole (Gunawan
et al., 2016; Rodríguez-Pérez et al., 2017). Although it was
noted that A. polyphaga is sensitive to the action of metro-
nidazole (Ondarza et al., 2006), this is not the case of A.
castellani (Taravaud et al., 2017).
Experimental
Material and Methods
Benzyl alcohol was purchased from AFT (Bratislava, Slo-
vakia) and was of p.a. purity. p-Toluenesulfonic acid was
obtained from LACHEMA (Brno, Czech Republic) and
was of p.a. purity. The chemicals 11-bromoundecanoic
acid, 4-dimethylaminopyridine, and 1-bromododecane
were purchased from Sigma Aldrich (Saint Louis, MO,
USA) and their purities were 99%, ≥ 98%, and 98%,
respectively. Triphenylphosphine and metronidazole were
obtained from Alfa Aesar (Kandel, Germany). The purities
of chemicals were 99%. Dicyclocarbodiimide with the
purity 99% was purchased from Fluka (Buchs, Switzer-
land). Sodium hydrogen carbonate, anhydrous magnesium
sulfate, and solvents were obtained from CentralChem
(Bratislava, Slovakia) and were of p.a. purity. NMR spectra
were measured on a MERCURY plus spectrometer (Var-
Cationic amphiphiles are used as antimicrobial com-
pounds (Block, 2000; Falk, 2019). Their molecules are com-
posed of lipophilic and hydrophilic parts. Lipophilic parts
are mainly represented by alkyl chains, which can be modi-
fied by various groups. Hydrophilic parts are mainly repre-
sented by aminium cations connected with alkyl/aryl groups
or they are parts of cationic heterocycles. This type of surfac-
tants is denoted as quaternary aminium compounds (QAC).
They are the most investigated cationic surfactants. The
replacement of a nitrogen by a phosphorus in the structure of
QAC leads to the formation of quaternary phosphonium
compounds (QPC) (Devínsky et al., 2017). QPC are com-
pounds with the biological activities studied only recently.
Many of them contain lipophilic triphenylphosphonium
(triarylphosphonium) cation. Their preparation is easier than
that of trialkylphosphonium salts. Trialkylphosphines are
more sensitive to oxidation than triarylphosphines (Lukácˇ
et al., 2017). As with QAC, they also show antimicrobial
activities (Bittner Fialová et al., 2019; Lukácˇ et al., 2017;
Tatarinov et al., 2018). However, they are intensively studied
for their unique ability to target compounds to mitochondria
(Zielonka et al., 2017). The difference in membrane potential
in mitochondria is greater than elsewhere in the cell, which
causes the cations to selectively accumulate in mitochondria.
Polar cations require a specific transmitter so that they can
pass the lipid bilayer into the mitochondria. However, lipo-
philic cations pass easily through the phospholipid bilayer
and may accumulate in the mitochondrial matrix (Sandoval-
Acuña et al., 2016; Subramanian et al., 2010). Many drugs
and bioactive compounds have been modified with a lipo-
philic phosphonium cation. The activities of mitochondrially
targeted bioactive compounds are higher than the activities
of drugs, which are used as templates in the synthesis.
(Devínsky et al., 2017; Zielonka et al., 2017)
ian, Palo Alto, CA, USA). H-, 13C, and 31P NMR spectra
1
were measured at frequencies of 300, 75, and 121.5 MHz,
respectively. Decoupling against protons was used in the
measurements of 13C and 31P NMR spectra. The spectra
were measured in deuterated chloroform and deuterated
methanol. The chemical shifts were referenced with respect
to an internal TMS (δ 1H = 0, δ 13C = 0) or 85% H3PO4 (δ
31P = 0 for ꢀ 31P = 40.4807420 MHz) signal. The IR spec-
tra were recorded on Nicolet FT IR 6700 spectrophotome-
ter (Thermo Scientific). Elemental analysis was performed
on Flash 2000 Elemental Analyzer (Thermo Scientific).
Melting points were determined using Kofler block and are
reported uncorrected.
Benzyl 11-Bromoundecanoate
The ester was prepared in the Dean-Stark apparatus. Benzyl
alcohol (0.14 mol, 12.26 g), 11-bromoundecanoic acid
(0.095 mol, 25.1 g), and p-toluenesulfonic acid (0.01 mol,
1.8 g) were dissolved in toluene (150 mL). The reaction
mixture was heated to 150 ꢀC for 6 h. The solution was
extracted with solution of sodium hydrogen carbonate and
brine after reaction. The solvent was evaporated under
reduced pressure after drying with anhydrous magnesium
sulfate. The product was distilled under reduced pressure,
The aim of this study was the preparation, characteriza-
tion, and investigation of the phosphonium amphiphilic
compounds. The connection of metronidazole, benzyl
group, and a straight alkyl chain with a lipophilic phospho-
nium cation was performed. The physicochemical proper-
ties, hemolytic and anti-Acanthamoeba activities of
prepared compounds were investigated. Our main goal is
the study of the influence of triphenylphosphonium cation
on the antiprotozoal activity of metronidazole. In particular,
we investigated the possible increase in antiprotozoal activ-
ity upon the transformation of metronidazole into a com-
pound with the potential mitochondria targeting.
1
b.p. 205–207ꢀC, p = 40 Pa. Yield: 31 g (92.3%). H NMR
(CDCl3, TMS) δ: 1.23–1.45 (m, 12H), 1.64 (t, J = 9 Hz,
2H), 1.80–1.87 (m, 2H), 2.36 (t, J = 9 Hz, 2H), 3.40
(t, J = 9 Hz, 2H), 5.11 (s, 2H), 7.25–7.38 (m, 5H);
13C NMR: (CDCl3, TMS) δ: 24.9, 28.1, 28.7, 29.1, 29.2,
29.3, 29.3, 32.3, 34.0, 34.3, 66.1, 128.1, 128.5, 136.1,
173.7; IR(νmax/cm−1): 2926, 2853, 1736, 1455, 1162,
735, 697.
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2-(2-methyl-5-nitro-1H-Imidazol-1-Yl)Ethyl 11-
Bromoundecanoate
J = 85.5 Hz), 130.6 (d, J = 12.4 Hz), 133.6 (d, J = 9.9 Hz),
135.1 (d, J = 2.9 Hz); 31P NMR (CDCl3) δ: 24.1. Elemental
analysis calcd (%) for C30H40BrP × 0.25 H2O: C 69.83, H
7.91; found (%): C 69.97, H 7.59.
Metronidazole (0.855 g, 5 mmol), 11-bromoundecanoic
acid (1.328 g, 5 mmol), dicyclocarbodiimide (0.611 g,
5 mmol) and 4-dimethylaminopyridine (1.135 g, 5.5 mmol)
were dissolved chloroform (40 mL). The reaction mixture
was stirred at r.t. and formation of product was monitored
by TLC. The dicyclourea formed was filtered off. The solu-
tion was extracted by water (3 × 50 mL), followed by 5%
solution of acetic acid (3 × 50 mL), water (3 × 50 mL),
dried over anhydrous Na2SO4, filtered, evaporated in
vacuo, and purified by silica gel chromatography (CHCl3/
EtOAc, 95/5, v/v) to afford ester (1.7 g, 81.3%) as slightly
yellow viscous liquid which solidified in refrigerator; m.p.
= 38–40ꢀC. 1H NMR (CDCl3, TMS) δ: 1.21–1.62 (m,
10H), 1.75–1.89 (m, 6H), 2.26 (t, J = 7.5 Hz, 2H), 2.52 (s,
3H), 3.41 (t, J = 6.9 Hz, 2H), 4.41 (t, J = 5 Hz, 2H), 4.60
(t, J = 5.3 Hz, 2H), 7.96 (s,1H); 13C NMR: (CDCl3, TMS)
δ: 14.4, 24.6, 24.9, 25.6, 28.1, 28.7, 29.0, 29.1 29.3, 29.3,
32.8, 34.0, 45.0, 133.1, 150.1, 173.0; IR(νmax/cm−1): 2915,
2849, 1730, 1522, 1462, 1369, 1266, 1186, 825, 746,
681, 547.
[11-(Benzyloxy)-11-Oxoundecyl]Triphenylphosphonium
Bromide
1
Viscous liquid; yield 1.51 g (30.8%); H NMR (CDCl3,
TMS) δ: 1.15–1.38 (m, 10H), 1.55–1.76 (m, 6H), 2.33 (t,
J = 7.8 Hz, 2H), 3.78–3.91 (m, 2H), 5.1 (s, 2H), 7.30–7.37
(m, 5H), 7.66–7.91 (m, 15H); 13C NMR: (CDCl3, TMS) δ:
22,5 (d, J = 56.9 Hz) 22,6 (d, J = 4.8 Hz), 24.9, 29.0 29.1,
29.2, 30.3, 30.5, 66.0, 118.3 (d, J = 85.5 Hz), 128.1, 128.2,
128.5, 130.5 (d, J = 12.7 Hz), 133.6 (d, J = 10.5 Hz), 135.1
(d, J = 3.0 Hz) 136.1, 173.7; 31P NMR: (CDCl3) δ: 24.49;
IR(νmax/cm−1): 2927, 2855, 1731, 1438, 1164, 1113, 746,
723, 691. Elemental analysis calcd (%) for
C36H42BrO2P × 1.5 H2O: C 67.08, H 7.04; found (%): C
67.47, H 6.64.
{11-[2-(2-methyl-5-nitro-1H-Imidazol-1-Yl)Ethoxy]-11-
Oxoundecyl}Triphenylphosphonium Bromide
General Procedure for Preparation of Phosphonium
Salts
1
Viscous liquid; yield 1.0 g (49.2%); H NMR (CD3OD,
TMS) δ: 1.16–1.35 (m, 10H), 1.45–1.71 (m, 6H), 2.24 (t,
J = 7.4 Hz, 2H), 2.51 (s, 3H), 3.32–3.38 (m, 2H), 4.43 (t,
J = 4.6 Hz, 2H), 4.68 (t, J = 5.1 Hz, 2H), 4.85 (s, 1H),
7.75–7.91(m, 15H); 13C NMR: (CD3OD, TMS) δ: 12.7,
21.3 (d, J = 57.8 Hz), 22.1 (d, J = 4.4 Hz), 24.3, 28.4, 28.5,
28.7, 28.8, 30.0, 30.2, 33.3, 44.8, 61.9, 118.6 (d,
J = 85.7 Hz), 130.1 (d, J = 12.6 Hz), 131.3, 133.4 (d,
J = 9.8 Hz), 134.9 (d, J = 3.0 Hz), 151.2, 173.2; 31P NMR:
(CD3OD) δ: 23.8; IR(νmax/cm−1): 2928, 2855, 1735, 1528,
1464, 1437, 1363, 1262, 1188, 1113, 824, 744, 723, 691.
Elemental analysis calcd (%) for C35H43BrN3O4P × 1.5
H2O: C 59.41, H 6.55, N 5.94; found (%): C 59.49, H 6.20,
N 5.82.
Bromoalkanes (1-bromododecane 24.9 g, 0.1 mol; benzyl
11-bromoundecanoate 2.84 g, 8 mmol or 2-(2-methyl-5-
nitro-1H-imidazol-1-yl)ethyl 11-bromoundecanoate 1.25 g,
3 mmol) and triphenylphosphine (26.2 g, 0.1 mol; 2.36 g,
9 mmol resp. 0.918 g, 3.5 mmol) were dissolved in aceto-
nitrile (50 mL resp. 15 mL). The mixture was heated at
81 ꢀC and stirred for 48 h. The reaction mixture was
cooled down to the room temperature. Solvent was evapo-
rated in rotary evaporator. Dodecyltriphenylphosphonium
bromide was purified by crystallization from mixture
aceton/diethyl
ether.
[11-(benzyloxy)-11-oxoundecyl]
triphenylphosphonium bromide was purified by column
chromatography over silica gel (ethyl acetate/petrolether,
95/5, v/v). {11-[2-(2-methyl-5-nitro-1H-imidazol-1-yl)eth-
oxy]-11-oxoundecyl}triphenylphosphonium bromide was
purified by column chromatography over silica gel (CHCl3/
MeOH, 95/5, v/v).
Measurements of Physicochemical Properties
The measurements of equilibrium surface tension and elec-
trical conductivity were carried out according to the previ-
ously described procedure (Lukácˇ et al., 2017).
Dodecyltriphenylphosphonium Bromide
In vitro Amoebicidal Activity Assay
1
White powder; yield 26.6 g (52%); m. p. = 96–97ꢀC; H
NMR (CDCl3, TMS) δ: 0.86 (t, J = 7.5 Hz, 3H); 1.20–1.28
(m, 16H); 1.63–1.64 (m, 4H); 3.63–3.76 (m, 2H); 7.69–
7.88 (m, 15H); 13C NMR (CDCl3, TMS) δ: 14.1, 22.6(0)
(d, J = 5,9 Hz), 22.6(3), 22.7 (d, J = 49.6 Hz), 29.2, 29.3,
29.5, 29.6, 30.4 (d, J = 15.4 Hz), 31.8, 118.2 (d,
The trophocidal activities against acanthamoebae were
tested according to the previously described procedure
(Garajová et al., 2014). Acanthamoeba lugdunensis (strain
AcaVNAK02, T4 genotype) and A. quina (strain
AcaVNAK03, T4 genotype) represented clinical isolates of
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free-living amoebae, which were isolated from the corneas
of two patients who suffered from Acanthamoeba keratitis.
metronidazole derivatives using the Mitsunobu reaction,
however, with no success.
Physicochemical properties of the compounds were
investigated by the measurements of surface tension and
conductivity of their aqueous solutions. The high critical
micelle concentration of MTZC11P(Ph)3Br allowed to
determine its physiochemical properties only by the surface
tension method. (The conductivity measurement would
have required high amount of phosphonium compound.).
Figure 1 depicts the surface tension vs log surfactant con-
centration dependence. The results of the measurements are
summarized in Table 1. The cmc values were obtained
from the breakpoints of the curves. The values for each
compound obtained from the surface tension and conduc-
tivity are in a good agreement (Fig. 2, Table 1). However,
the cmc values obtained from the conductivity measure-
ments are somewhat higher than the cmcs obtained from
surface tension measurements.
The cmc of C12P(Ph)3Br corresponds well with the pub-
lished data (Prasad et al., 2004). This compound has cmc
smaller than BnC11P(Ph)3Br and MTZC11P(Ph)3Br. The
insertion of ester moiety at the end of alkyl chains
decreases hydrophobicity of the lipophylic part of mole-
cules. A similar behavior was observed by De et al. (2010).
The insertion of 1,4-dioxyphenylene unit into the hydro-
phobic part of QAC resulted in the cmc increase. The posi-
tion of the units also had the influence on the lipophilicity
of surfactants. Increasing the distances between the polar
head group and 1,4-dioxyphenylene unit resulted in the
cmc increase. The replacement of benzyl in BnC11P
(Ph)3Br with metronidazole in MTZC11P(Ph)3Br caused
an additional increase in cmc. He et al., 2019 observed a
similar behavior when phenyl was replaced with a hetero-
cycle (thiazole, pyrimidine) in the polar head group of the
investigated surfactants. Heterocyclic surfactants had
higher values of cmc than the hydrocarbon analogues.
Surface tension at cmc (γcmc) decreases with the addition
of bulky moieties (benzyl, MTZ) at the end of alkyl chains.
The density increase of surfactant-tail layer at the air/water
interface resulted in the γcmc decrease (Czajka et al., 2015).
We observed a similar influence on the surface activity in
the case of alkylphosphocholines. Branching of alkyl
chains or replacing hydrogens with fluorine atoms in alkyl
chains decreased the value of γcmc (Lukácˇ
et al., 2012, 2014).
In vitro Hemolytic Activity Assay
Human blood was collected into plastic tubes with volume
10 mL which contained di-potassium EDTA as anticoagu-
lant. The blood was added to glass centrifuge tube and the
cells were washed three times with phosphate-buffer saline
(PBS—Na2HPO4 c = 22.2 × 10−3 mol dm−3, KH2PO4
c = 5.6 × 10−3 mol dm−3, NaCl c = 123.3 × 10−3 mol dm−3).
Erythrocytes were collected by centrifugation at 3000g for
10 min. The cells were then suspended in the PBS. The
density of erythrocytes in the stock suspension was
7.6 × 108 cells mL−1. The erythrocyte suspension was
stored at 4ꢀC for 48 h. The cell suspension (500 μL) was
pipetted to 500 μL of buffer containing different concentra-
tions of the tested compounds. Final erythrocyte concentra-
tions were 3.8 × 108 cells mL−1. The mixtures were
incubated at 37 ꢀC for 1 h. They were gently shaken during
the incubation. The mixtures were then centrifugated at
3000 g for 10 min. The degree of hemolysis was deter-
mined by comparing the absorbance (542 nm) of dilutions
of the supernatant 400 μL to 10 mL deionized water with
that of control samples. The control samples were prepared
by hemolysis of erythrocytes in deionized water (the stock
solution of erythrocytes 25–200 μL were added to 10 mL
of deionized water).
Results and Discussion
Phosphonium salts were prepared according the Scheme 1.
One compound contains straight alkyl chain C12P(Ph)3Br.
Two compounds are represented by phosphonium salts
which alkyl chains contain ester groups. The connection of
benzyl alcohol (BnC11P(Ph)3Br) and metronidazole
(MTZC11P(Ph)3Br) on the alkyl chain was performed by
this group. Ester is easily cleavable. The cleavable groups
on the alkyl chain can be released after having been
targeted to the place of action. Metronidazol represents
antiinfective drugs and benzyl is a group without specific
activities. BnC11P(Ph)3Br was prepared as a compound
for the comparison with the metronidazole derivative. All
phosphonium salts were prepared by quarternization of
triphenylphosphine with 1-bromoalkanes. Substituted 1-
bromoalkanes needed for preparation of compounds
BnC11P(Ph)3Br and MTZC11P(Ph)3Br were prepared by
the esterification of 11-bromoundecanoic acid with benzyl
alcohol and metronidazole, respectively. The first esterifica-
tion was performed as the Fischer esterification, the second
one as the Steglich esterification. We also tried to prepare
The values of area per head group (Acmc) show that
C12P(Ph)3Br and MTZC11P(Ph)3Br molecules are packed
more densely at the air/water interface than BnC11P
(Ph)3Br molecules. The Acmc value of MTZC11P(Ph)3Br
is lower than that of BnC11P(Ph)3Br. This can be caused
by stronger π-stacking of aromatic parts of molecules in the
case of the metronidazole derivative. Imidazole ring is
substituted with two types of groups. One is represented by
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Scheme 1 Synthesis of amphiphilic phosphonium salts: (a) preparation of esters, (b) quarternisation of triphenylphospine with bromoalkanes
Denser packing of molecules of MTZC11P(Ph)3Br in com-
parison with BnC11P(Ph)3Br can be supported by the pres-
ence of hydrogen bonds utilizing nitrogen and oxygen
atoms as acceptors. These two van der Waals interactions
were considered as the factors, which have influenced the
crystal packing of 1-alkyl-2-methyl-4-nitroimidazoles
(Kubicki, 2005).
Biological activities of the compounds were tested
against two strains of Acanthamoeba. The most active com-
pound was C12P(Ph)3Br, a phosphonium salt with straight
alkyl chain (Table 2). After 24 h of compounds activity,
EC50 (effective concentration of tested compound that
reduces the survival of amoebae by 50%) and MTC (mini-
mal trophocidal concentration) values against both strains
(A. lugdunensis, A. quina) were very low. These activities
are comparable with those of previously tested CTAB,
Fig. 1 Dependence of surface tension on log surfactant concentration
for phosphonium salts
benzalkoniumbromide with hexadecyl alkyl chain, and
cetylpyridinium bromide. Their values of MTC are the
same (Lukácˇ et al., 2013). Other phosphonium salts with
alkyls. They are bonded in positions 1 and 2 of the hetero-
cycle. Especially, the methyl group represents a weak elec-
tron-donating group. The second type of the substituent is
represented by the nitro group. It is a strong electron-with-
drawing group. The presence of electron-donating and
withdrawing substituents and electron-withdrawing nitro-
gen atoms in the ring causes the polarization of imidazole
skeleton. Unequal distribution of electrons in the heterocy-
cle causes stronger π-π interactions between the aromates.
benzyl and metronidazol moiety indicate lower anti-
Acanthamoeba activities. Figure 3 shows the dependence
of anti-Acanthamoeba activity on critical micelle concen-
tration. The increase in cmc resulted in the decrease of bio-
logical activities. BnC11P(Ph)3 show higher activity than
MTZC11P(Ph)3 which contains the drug moiety. This is
the proof of a nonspecific action of the prepared phospho-
nium salt. The activities of salts depend on physicochemi-
cal properties.
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Table 1 Structure and physicochemical properties of phosphonium salts
No.
Compound
Structure
cmc (mol dm−3) surface
tension
cmc (mol dm−3
conductivity
)
γcmc
Acmc
(mN m−1
)
(nm2)
1
2
3
C12P(Ph)3Br
1.5 × 10−3
2.5 × 10−3
6.0 × 10−3
1.8 × 10−3
3.3 × 10−3
—
41.9
34.8
34.9
0.83
+
P
H
C
3
-
Br
BnC11P(Ph)3Br
1.77
0.75
+
P
O
-
Br
O
-
O
O
N
MTZC11P
(Ph)3Br
+
N
+
P
O
-
Br
N
O
CH
3
Interesting dependences of the aggregation properties
of phosphonium amphiphilic compounds on biological
activities of unicellular organisms were observed
by Ryzhkina et al. (2019). They investigated the influ-
ence of tributylhexadecylphosphonium bromide and
hexadecyltriphenylphosphonium bromide on the biologi-
cal activity of infusoria (Parametium caudatum) and one
single-celled algae (Chlorella vulgaris). Protozoa Para-
metium caudatum was sensitive to phosphonium salt in a
similar way to our case of Acanthamoeba. The minimum
protocidal concentration was 1 × 10−5 mol dm−3. An
interesting influence of phosphonium salts was observed
in the case of algae. Micromolar concentrations have no
effect on Chlorella vulgaris. However, the concentra-
tions of salts in range 1 × 10−15 to 1 × 10−6 mol dm−3
have a harmful or even toxic effect. The authors
explained it by the formation of the nanoassociates at
low concentrations of phosphonium salts in aqueous
solutions.
Metronidazol, a compound without amphiphilic charac-
ter, showed no biological activity against Acanthamoeba.
Both strains are insusceptible to the action of metronida-
zole. This observation corresponds with the investigations
of Taravaud et al. (2017) who used the strain of A.
castellanii. This species is included in the group II
according to the classification of Pussard and Pons (1977)
which is similar to our investigated strains of A.
lugdunensis and A. quina. It is interesting to note that A.
polyphaga also belongs in group II but it was noted suscep-
tible to the action of metronidazole (Ondarza et al., 2006).
Fig. 2 Dependence of electrical conductivity on surfactant concentra-
tion for phosphonium salts
Table 2 Biological activities of phosphonium compounds and metronidazole
Compound
A. lugdunensis
A. quina
Haemolytic activity EC50 (μM)
EC50 (μM)
MTC (μM)
EC50 (μM)
MTC (μM)
C12P(Ph)3Br
BnC11P(Ph)3
MTZC11P(Ph)3
MTZ
5 Æ 1.6
44 Æ 4
15.6
62.5
250
7.9 Æ 1.4
39 Æ 5
72 Æ 7
> 500
15.6
62.5
250
51
72
144 Æ 12
> 500
205
> 500
> 500
> 1000
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J Surfact Deterg
application of phosphonium salts as promising candidates
for the drugs against Acanthamoeba infections, the therapy
of which remains still difficult.
Acknowledgements The authors thank to Andrej Slivka, MSc. for
assistance in obtaining of human blood. This research was funded by
the Grant Agency of Ministry of Education and Academy of Science
of Slovak republic (VEGA), Project No. VEGA 1/0298/16 and VEGA
1/0054/19, by the Slovak Research and Development Agency Grant
No. APVV-17-0373. This work utilizes 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. This
work was supported by the Slovak Research and Development
Agency under the contract No. APVV-15-0123. Further, it was
supported with grants VEGA 1/0365/16, VEGA 1/0389/19 and
KEGA 013TTU-4/2019.
Conflict of Interest The authors declare that they have no conflict
of interest.
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