Dialkylamino and nitrogen heterocyclic analogues of hexadecylphosphocholine and cetyltrimetylammonium bromide: Effect of phosphate group and environment of the ammonium cation on their biological activity

Article abstract of DOI:10.1016/j.ejmech.2009.08.011

A series of dialkylamino and nitrogen heterocyclic analogues of hexadecylphosphocholine and cetyltrimethylammonium bromide have been synthesized. The prepared compounds exhibit significant cytotoxic, antifungal and antiprotozoal activities. Alkylphosphocholines possess higher antifungal activity against Candida albicans in comparison with quaternary ammonium compounds. However, quaternary ammonium compounds exhibit significant higher activity against human tumor cells and Acanthamoeba lugdunensis compared to alkylphosphocholines. In addition, their haemolytic toxicity has been investigated. The relationship between structure and biological activity of the tested compounds is discussed.

Full text of DOI:10.1016/j.ejmech.2009.08.011

European Journal of Medicinal Chemistry 44 (2009) 4970–4977
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Dialkylamino and nitrogen heterocyclic analogues of hexadecylphosphocholine
and cetyltrimetylammonium bromide: Effect of phosphate group and
environment of the ammonium cation on their biological activity
,
b
,
c
d
e
f
a
´ ˇ a
´
´
ˇ
´
´
ˇ
´
*
ꢀ
ꢀ
ˇ
ˇ
Milos Lukac
, Jan Mojzis , Gabriela Mojzisova , Martin Mrva , Frantisek Ondriska , Jindra Valentova ,
a
a
b,h
ꢁ g
´
Ivan Lacko , Marian Bukovsky , Ferdinand Devınsky , Janka Karlovska
a
ˇ
Department of Chemical Theory of Drugs, Faculty of Pharmacy, Comenius University, Kalinciakova 8, 832 32 Bratislava, Slovakia
b
c
d
e
f
´
NMR laboratory, Faculty of Pharmacy, Comenius University, Odbojarov 10, 832 32 Bratislava, Slovakia
ˇ
´
ˇ
Department of Pharmacology, Faculty of Medicine, P.J. Safarik University, SNP 1, 040 66 Kosice, Slovakia
ˇ
´
ˇ
Department of Experimental Medicine, Faculty of Medicine, P.J. Safarik University, SNP 1, 040 66 Kosice, Slovakia
´
Department of Zoology, Faculty of Natural Sciences, Comenius University, Mlynska Dolina B-1, 842 15 Bratislava, Slovakia
HPL (Ltd), Department of Parasitology, Microbiological Laboratory, Istrijska 20, 841 07 Bratislava, Slovakia
Department of Cell and Molecular Biology of Drugs, Faculty of Pharmacy, Comenius University, Kalinciakova 8, 832 32 Bratislava, Slovakia
Department of Physical Chemistry of Drugs, Faculty of Pharmacy, Comenius University, Kalinciakova 8, 832 32 Bratislava, Slovakia
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g
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a r t i c l e i n f o
a b s t r a c t
Article history:
A series of dialkylamino and nitrogen heterocyclic analogues of hexadecylphosphocholine and cetyl-
trimethylammonium bromide have been synthesized. The prepared compounds exhibit significant
cytotoxic, antifungal and antiprotozoal activities. Alkylphosphocholines possess higher antifungal
activity against Candida albicans in comparison with quaternary ammonium compounds. However,
quaternary ammonium compounds exhibit significant higher activity against human tumor cells and
Acanthamoeba lugdunensis compared to alkylphosphocholines. In addition, their haemolytic toxicity has
been investigated. The relationship between structure and biological activity of the tested compounds is
discussed.
Received 18 March 2009
Received in revised form
10 June 2009
Accepted 27 August 2009
Available online 2 September 2009
Keywords:
Alkylphosphocholines
Antifungal activity
Ó 2009 Elsevier Masson SAS. All rights reserved.
Antiprotozoal activity
Cytotoxic activity
Haemolytic activity
Quaternary ammonium compounds
1. Introduction
only to antineoplastic activity but they showed activity against
fungi and protists. Obando et al. [8] studied antifungal activity of 14
Alkylphosphocholines (APCs) represent a class of potential
agents for the clinical treatment of cancer [1–3]. The prototype of
the APCs is hexadecylphosphocholine (miltefosine, HPC) which
was initially developed as an anticancer agent [4]. The cytotoxic
effect of HPC has been demonstrated in a wide range of tumors.
MiltexÒ, 6% solution of HPC, is used as an effective palliative drug
for treatment of cutaneous metastases from breast cancer [5].
Piperidine analogues of HPC revealed potentiation in cytotoxic
activity on some cell lines [6,7]. The effect of APCs did not relate
APCs and alkylglycerophosphocholines (AGPCs). APCs exhibited
higher antifungal activity than AGPCs [8]. Up to now, no efficient
and easily manageable treatment is available for amoebic keratitis
(AK) and granulomatous amoebic encephalitis. The most important
reason for unsuccessful therapy seems to be the existence of
a double-wall cyst stage that is highly resistant to the available
treatments, causing reinfections [9]. Therefore, the susceptibility of
several Acanthamoeba spp. to APCs was investigated by Walochnik
et al. [10]. They studied the influence of the alkyl chain of APCs on
antiprotozoal activity. In this study, HPC exhibited the highest
cytotoxicity against trophozoites resulting in complete cell death at
20–40 mM, and also displayed significant cysticidal activity. The
* Corresponding author. Department of Chemical Theory of Drugs, Faculty of
HPC is active against more parasites than merely Acanthamoeba, it
exhibits also antiprotozoal activity on Leishmania [11], Plasmodium
[12], Balamuthia, Negleria [13], Trichomonas [14], Entamoeba [15] or
ˇ
Pharmacy, Comenius University, Kalinciakova 8, 832 32 Bratislava, Slovakia.
Tel.: þ421 250117322; fax: þ421 250117357.
ˇ
E-mail address: lukac@fpharm.uniba.sk (M. Luka´c).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.08.011

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M. Lukac et al. / European Journal of Medicinal Chemistry 44 (2009) 4970–4977
4971
Trypanosoma [16]. HPC (ImpavidoÒ) was registered as the first oral
examined by TLC plate technique (DC-Alufolien Kiesegel 60 F256,
drug for treatment of visceral leishmaniasis in India and Germany
and for treatment of cutaneous leishmaniasis in Colombia [17].
Some heterocyclic analogues of APCs exhibit higher antileishmanial
activity than HPC. Hexadecyl [2-(N-methylpiperidinio)ethyl]
phosphate (HPPip) is one of the most active APCs, its half maximal
Merck).
2.2. Chemistry
2.2.1. General procedure for the quaternization of aminoalcohols
effective concentration (EC50) was 0.3
mM [17].
with methyl p-toluenesulfonate
Quaternary ammonium compounds (QUATs) are widely used as
disinfectants in medicine, agriculture and industry. As more resis-
tant organisms continue to emerge in the society, identification of
additional antimicrobial agents becomes increasingly more
important [18,19]. Their antifungal activity is well known and
QUATs show high activity against fungi and yeast [20–23]. Cyto-
toxicity of surfactants has been extensively studied. Many types of
QUATs exhibit antineoplastic activity or inhibit enzymes important
for tumorigenesis [24–26]. Antiprotozoal activity of QUATs against
Acanthamoeba spp. is mainly studied in connection with the
disinfection of contact lenses. Benzalkonium chloride or poly-
quaternium 1 represents QUATs, which are used in commercial
contact lens solutions [27].
The aim of the study was to prepare the nitrogen heterocyclic
and dialkylamino analogues of HPC and cetyltrimethylammonium
bromide (CTAB) and to evaluate their potential efficacies against
human tumor cells, Candida albicans and Acanthamoeba lugdu-
nensis. Moreover, the haemolytic properties of APCs and QUATs
were studied on human red blood cells. Effect of phosphate group
and ammonium cation environment of prepared compounds on
biological activity was investigated.
Aminoalcohol (22 mmol), prepared by hydroxyethylation [29],
was added to the solution of methyl p-toluenesulfonate (20 mmol)
in acetonitrile (20 ml). The resulting mixture was refluxed for 4 h.
After cooling down, the acetonitrile was evaporated in vacuum. The
resulting mixture was crystallized from acetone. The quaternary
salts were obtained as white, hygroscopic solids.
Choline p-toluenesulfonate 2a: yield 94%; 1H NMR (DMSO-d6,
TMS)
d 2.29 (s, 3H), 3.09 (s, 9H), 3.34–3.44 (m, 2H), 3.78–3.88 (m,
2H), 5.27 (t, 1H, J ¼ 4.9 Hz), 7.04 (s, 2H, J ¼ 8.0 Hz), 7.48 (d, 2H,
J ¼ 8.0 Hz).
N,N-Diethyl-2-hydroxy-N-methylethanammonium p-toluene-
sulfonate 2b: yield 87%; 1H NMR (DMSO-d6, TMS)
d 1.20 (t, 6H,
J ¼ 7.2 Hz), 2.28 (s, 3H), 2.95 (s, 3H), 3.27–3.40 (m, 6H), 3.75–3.83
(m, 2H), 5.26 (t, 1H, J ¼ 5.1 Hz), 7.10 (d, 2H, J ¼ 7.9 Hz), 7.46 (d, 2H,
J ¼ 8.2 Hz).
N-(2-hydroxyethyl)-N-methylpyrrolidinium p-toluenesulfonate
2e: yield 89%; 1H NMR (DMSO-d6, TMS)
d 2.61–2.53 (m, 4H), 2.83 (s,
3H), 3.03 (s, 3H), 3.56–3.40 (m, 6H), 3.87–3.79 (m, 2H), 5.27 (t, 1H,
J ¼ 4.8 Hz), 7.10 (d, 2H, J ¼ 8.2 Hz), 7.45 (d, 2H, J ¼ 7.9 Hz).
N-(2-hydroxyethyl)-N-methylpiperidinium p-toluenesulfonate
2f: yield 86%; 1H NMR (DMSO-d6, TMS)
d 1.58–1.48 (m, 2H), 1.83–
1.74 (m, 4H), 2.28 (s, 3H), 3.06 (s, 3H), 3.45–3.27 (m, 6H), 3.88–3.80
(m, 2H), 5.26 (t, 1H, J ¼ 4.8 Hz), 7.10 (d, 2H, J ¼ 8.1 Hz), 7.45 (d, 2H,
J ¼ 8.0 Hz).
2. Experimental
N-(2-hydroxyethyl)-N-methylazepanium
p-toluenesulfonate
2.1. Materials and methods
2g: yield 89%; 1H NMR (DMSO-d6, TMS)
d 1.51–1.63 (m, 4H), 1.75–
1.87 (m, 4H), 2.28 (s, 3H), 3.05 (s, 3H), 3.35–3.41 (m, 4H), 3.49–3.60
(m, 2H), 3.79–3.88 (m, 2H), 5.29 (t, 1H, J ¼ 4.8 Hz), 7.10 (d, 2H,
J ¼ 7.9 Hz), 7.46 (d, 2H, J ¼ 8.0 Hz).
All chemicals used for synthesis were purchased from
commercial suppliers. Sabouraud agar and Sabouraud medium
were purchased from Imuna Pharm a.s., Slovakia. Bacto-Casitone
was obtained from E. coli s.r.o., Slovakia. C. albicans CCM 8186 was
obtained from the Czech Collection of Microorganisms. A. lugdu-
nensis was isolated from a patient suffering from amoebic ceratitis
[28]. Jurkat (human T-cell acute lymphoblastic leukemia), HeLa
(human cervical adenocarcinoma) MCF-7 (human breast adeno-
carcinoma, estrogen receptor-positive), MDA-MB-231 (human
breast adenocarcinoma, estrogen receptor-negative) and A-549 cell
lines (human lung adenocarcinoma) were kindly provided by Dr. M.
N-(2-hydroxyethyl)-N-methylmorpholinium
p-toluenesulfo-
nate 2i: yield 84%; 1H NMR (DMSO-d6, TMS)
d
2.29 (s, 3H), 3.20 (s,
3H), 3.38–3.61 (m, 10H), 3.83–3.96 (m, 2H), 5.29–5.40 (m, 1H), 7.10
(d, 2H, J ¼ 8.1 Hz), 7.47 (d, 2H, J ¼ 8.1 Hz).
2.2.2. General procedure for the preparation of APCs from choline
p-toluenesulfonates
Solution of hexadecanol (9 mmol) in chloroform (20 ml) was
added (dropwise at 0 ꢁC) to a stirred solution of phosphorous
oxychloride (10 mmol) and triethylamine (20 mmol) in chloroform
(10 ml). The resulting mixture was stirred at room temperature
(r.t.) for 2 h. This intermediate was used further without any
subsequent additional purification. Pyridine (15 ml) was added
dropwise at 0 ꢁC to the resulting solution followed by the addition
of choline p-toluenesulfonate or its diethylamino or heterocyclic
analogue (12.5 mmol). The reaction mixture was stirred at r.t. over
night. After cooling, the mixture was hydrolyzed by addition of H2O
(1.5 ml) and stirred for 1 h at r.t.. The solvents were evaporated in
vacuum, and the resulting crude solid was dissolved in a mixture of
tetrahydrofurane and water (5:1, V/V). To the stirred solution,
exchange resin MB3 was added sequentially until the color of the
resin stopped changing. Then the resin was filtered off and the
solvents were evaporated in vacuum. The remaining mixture was
dried with propan-2-ol. The resulting crude solid was purified by
flashchromatography usingCH2Cl2/MeOH/25%NH3 (60/50/5, V/V/V)
as a liquid phase. After chromatography the solvents were evapo-
rated in vacuo. The residue was dissolved in chloroform and precip-
itated with acetoneor petroleum ether. APC wasfiltered off anddried
in vacuum dessicator.
´
Hajduch (Olomouc, Czech Republic). CCRF-CEM cell line (human
T-cell acute lymphoblastic leukemia) was obtained from the
German Collection of Microorganisms and Cell Cultures
(Braunschweig, Germany). The cells were routinely maintained in
RPMI 1640 medium with L-Glutamine and 4-(2-Hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) (Jurkat, HeLa and CCR-CEM)
or Dulbecco’s modified Eagle’s medium with Glutamax-I (MCF-7,
MDA-MB-231, and A-549) supplemented with 10% fetal calf serum,
penicillin (100 IU mlꢀ1) and streptomycin (100
m
g mlꢀ1) (all from
Invitrogen, USA), in humidified air with 5% CO2 at 37 ꢁC. Before each
cytotoxicity assay, cell viability was determined by trypan blue
exclusion method and found greater than 95%. Infrared spectra
were recorded on a FT-IR Impact 400 D spectrophotometer as
potassium bromide discs. 1H, 13C and 31P NMR spectra were
recorded on a Varian Gemini 2000 spectrometer operating at 300,
75.5 and 121.5 MHz respectively, with 13C and 31P NMR spectra
being recorded using proton-decoupling. The spectra were
measured in CDCl3 and DMSO-d6 relative to internal standard TMS
for 1H and 13C NMR spectra and to external standard 85% H3PO4 for
31P NMR spectra. The purity of the prepared compounds was

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M. Lukac et al. / European Journal of Medicinal Chemistry 44 (2009) 4970–4977
4972
Hexadecyl [2-(N,N,N-trimethylammonio)ethyl] phosphate ꢂ 1.5
H2O (HPC): yield 17.2%; 1H NMR (CDCl3, TMS)
0.88 (t, 3H,
2.2.4. General procedure for the preparation of APCs from choline
bromides
d
J ¼ 6.7 Hz), 1.22–1.47 (m, 26H), 1.54–1.64 (m, 2H), 2.37 (s, 3H), 3.41
Hexadecanol (2 mmol) was treated with phosphorous oxy-
chloride (2.1 mmol) and triethylamine (3 mmol) in THF. The solu-
tion was hydrolyzed with 1.5 ml H2O for 1 h and evaporated in
vacuo. A pyridinium salt was prepared by treatment with pyridine
(16 ml) for 2 h at 50 ꢁC. Pyridinium hexadecyl phosphate was
coupled with 2c or 2h (3 mmol) in the presence of a condensing
agent 2,4,6-triisopropylbenzenesulfonyl chloride. APC was purified
by the procedure described in the previous method for preparation
of APC from choline p-toluenesulfonates.
(s, 3H), 3.78–3.86 (m, 4H), 4.25–4.34 (m, 2H); 13C NMR (CDCl3, TMS)
d
14.1, 22.7, 25.9, 29.4, 29.5, 29.7, 31.0, 31.1, 31.9, 54.5, 59.2, 65.6,
66.4; 31P NMR (CDCl3, H3PO4)
d
ꢀ0.78; IR ymax/cmꢀ1 3425, 2923,
2852, 1644, 1468, 1250, 1088, 967, 720.
Hexadecyl [2-(N,N-Diethyl-N-methylammonio)ethyl] phos-
phate ꢂ 2.5 H2O (HPDEt): yield 16.7%; 1H NMR (CDCl3, TMS)
d 0.88
(t, 3H, J ¼ 6.7 Hz), 1.18–1.41 (m, 32H), 1.55–1.67 (m, 2H), 1.82 (s, 5H),
3.28 (s, 3H), 3.53–3.86 (m, 4H), 3.70–3.77 (m, 2H), 3.85 (q, 2H,
J ¼ 6.5 Hz), 4.28–4.37 (m, 2H); 13C NMR (CDCl3, TMS) 8.1, 14.1, 22.7,
25.9, 29.4, 29.5, 29.7, 31.0, 31.9, 48.4, 56.9, 58.5, 61.4, 65.5; 31P NMR
Hexadecyl 2-[N-Methyl-N,N-dipropylammonio]ethyl phosphate
ꢂ H2O (HPDPr): yield 13.6%; 1H NMR (CDCl3, TMS)
d 0.88 (t, 3H,
(CDCl3, H3PO4)
d
ꢀ0.63; IR ymax/cmꢀ1 3440, 2922, 2852, 1645, 1467,
J ¼ 6.7 Hz), 1.03 (t, 6H, J ¼ 7.3 Hz), 1.19–1.37 (m, 26H), 1.55–1.66 (m,
2H), 1.68–1.83 (m, 4H), 2.33 (s, 2H), 3.32 (s, 3H), 3.36–3.47 (m, 4H),
3.74–3.80 (m, 2H), 3.84 (q, 2H, J ¼ 6.7 Hz), 4.28–4.36 (m, 2H); 13C
1247, 1084, 966, 766, 722.
Hexadecyl [2-(N-Methylpyrrolidinio)ethyl] phosphate ꢂ 1.5 H2O
(HPPyr): yield 23.3%; 1H NMR (CDCl3, TMS)
d
0.88 (t, 3H, J ¼ 6.6 Hz),
NMR (CDCl3, TMS) d 10.7, 14.1, 16.0, 22.7, 25.9, 29.4, 29.5, 29.7, 31.0,
1.19–1.36 (m, 26H), 1.55–1.65 (m, 2H), 2.13–2.34 (m, 7H), 3.31 (s,
31.1, 31.9, 49.6, 58.5, 62.4, 63.6, 65.5, 65.6; 31P NMR (CDCl3, H3PO4)
3H), 3.77–3.90 (m, 8H), 4.29–4.38 (m, 2H); 13C NMR (CDCl3, TMS)
d
ꢀ0.48; IR ymax/cmꢀ1 3443, 2923, 2854, 1646, 1467, 1247, 1095,
d
14.1, 22.7, 25.9, 29.4, 29.5, 29.7, 31.0, 31.1, 31.9, 48.3, 58.5, 60.8,
1075, 960, 761, 722.
60.9, 64.6, 65.6, 65.7; 31P NMR (CDCl3, H3PO4)
d
ꢀ0.86; IR ymax/cmꢀ1
Hexadecyl 2-(N-Methylazocanio)ethyl phosphate ꢂ 2 H2O
3399, 2919, 2851, 1653, 1467, 1249, 1081, 1000, 967, 768, 722.
(HPAzoc): yield 20.4%; 1H NMR (CDCl3, TMS)
d 0.88 (t, 3H,
Hexadecyl [2-(N-Methylpiperidinio)ethyl] phosphate ꢂ 2.5 H2O
J ¼ 6.7 Hz),1.22–1.38 (m, 26H),1.55–1.79 (m, 8H),1.90–2.01 (m, 4H),
(HPPip): yield 10.4%; 1H NMR (CDCl3, TMS)
d
0.88 (t, 3H,
2.08 (s, 4H), 3.37 (s, 3H), 3.54–3.73 (m, 4H), 3.80–3.90 (m, 4H),
J ¼ 6.6 Hz), 1.20–1.38 (m, 26H), 1.55–1.66 (m, 2H), 1.67–1.77 (m,
2H), 1.84–1.96 (m, 4H), 2.31 (s, 5H), 3.38 (s, 3H), 3.50–3.60 (m,
2H), 3.64–3.75 (m, 2H), 3.80–3.90 (m, 4H), 4.28–4.38 (m, 2H); 13C
4.28–4.37 (m, 2H); 13C NMR (CDCl3, TMS)
d 14.1, 22.0, 22.7, 24.5,
25.9, 26.2, 29.4, 29.5, 29.7, 31.0, 31.1, 31.9, 50.8, 58.7, 60.9, 63.8, 65.5,
65.6; 31P NMR (CDCl3, H3PO4)
2851, 1648, 1467, 1229, 1080, 1000, 962, 751, 722.
d
ꢀ0.64; IR ymax/cmꢀ1 3394, 2922,
NMR (CDCl3, TMS)
d
14.1, 20.2, 21.0, 22.7, 25.9, 29.4, 29.5, 29.7,
31.0, 31.1, 31.9, 48.8, 58.4, 62.2, 63.9, 65.5, 65.6; 31P NMR (CDCl3,
H3PO4)
d
ꢀ0.92; IR ymax/cmꢀ1 3441, 2919, 2851, 1650, 1467, 1250,
2.2.5. General method for the preparation of QUATs
1073, 971, 770, 722.
N-Methylated heterocycles or dialkylamines (0.01 mol)
prepared by Eschweiler–Clarke methylation [30], and 1-bromo-
hexadecane (0.011 mol) were added to 30 ml of dry acetonitrile,
and then heated under reflux for 12 h. After having distilled the
solvent off in vacuum the drying of the product was completed by
azeotropic distillation with toluene or benzene. The quaternary
ammonium salt was precipitated by diethyl ether. The precipitate
was then filtered off and the crude product was recrystallized
several times from a mixture of acetone/methanol.
Hexadecyl [2-(N-Methylazepanio)ethyl] phosphate ꢂ 1.5 H2O
(HPAzep): yield 16.5%; 1H NMR (CDCl3, TMS)
d 0.88 (t, 3H,
J ¼ 6.7 Hz), 1.22–1.48 (m, 26H), 1.56–1.67 (m, 2H), 1.69–1.76 (m, 4H),
1.87–1.96 (m, 4H), 2.12 (s, 3H), 3.39 (s, 3H), 3.48–3.58 (m, 2H), 3.74–
3.89 (m, 6H), 4.30–4.38 (m, 2H); 13C NMR (CDCl3, TMS)
d 14.1, 21.9,
22.7, 26.0, 27.9, 29.4, 29.5, 29.7, 31.0, 31.1, 31.9, 51.8, 58.8, 65.2, 65.3,
65.5; 31P NMR (CDCl3, H3PO4)
2851, 1648, 1467, 1264, 1082, 1002, 962, 766, 722.
d
ꢀ0.84; IR ymax/cmꢀ1 3388, 2921,
Hexadecyl [2-(N-Methylmorpholinio)ethyl] phosphate ꢂ 1.5
N,N-Dietyl-N-methylhexadecylammonium bromide (CMDEt):
H2O (HPMorph): yield 10.8%; 1H NMR (CDCl3, TMS)
d
0.88 (t, 3H,
yield 82%; 1H NMR (CDCl3, TMS)
d
0.88 (t, 3H, J ¼ 6.6 Hz), 1.2–1.54
J ¼ 6.6 Hz), 1.22–1.46 (m, 26H), 1.54–1.63 (m, 2H), 2.75 (s, 3H), 3.53
(m, 32H), 1.63–1.78 (m, 2H), 3.28 (s, 3H), 3.36–3.45 (m, 2H), 3.63 (q,
(s, 3H), 3.68–3.84 (m, 6H), 3.99–4.08 (m, 6H), 4.30–4.39 (m, 2H); 13C
4H, J ¼ 7.2 Hz); 13C NMR (CDCl3, TMS)
d 8.3, 14.1, 22.4, 22.7, 26.4,
NMR (CDCl3, TMS)
d
14.1, 22.7, 25.9, 29.4, 29.5, 29.7, 31.0, 31.1, 31.9,
29.2, 29.3, 29.4, 29.5, 29.6, 29.7, 31.9, 47.9, 56.6, 60.5; IR ymax/cmꢀ1
2919, 2849, 1469, 720.
48.3, 58.4, 60.8, 60.9, 64.6, 65.6, 65.7; 31P NMR (CDCl3, H3PO4)
d
ꢀ0.56; IR ymax/cmꢀ1 3435, 2922, 2851, 1646, 1468, 1251, 1095,
N-Methyl-N,N-dipropylhexadecylammonium bromide (CMDPr):
1079, 962, 755, 722.
yield 74%; 1H NMR (CDCl3, TMS)
d
0.88 (t, 3H, J ¼ 6.6 Hz), 1.06 (t, 6H,
J ¼ 7.2 Hz) 1.2–1.48 (m, 26H), 1.64–1.87 (m, 6H), 3.35 (s, 3H), 3.41–
3.53 (m, 6H); 13C NMR (CDCl3, TMS)
10.8, 14.1, 16.1, 22.5, 22.7, 26.4,
2.2.3. General procedure for the quaternization of N-methylazocane
or N-methyl-N,N-dipropylamine with 2-bromoethanol
d
28.7, 29.2, 29.3, 29.4, 29.5, 29.6, 29.7, 31.9, 49.0, 61.7, 63.1; IR ymax
/
N-Methyl-N,N-dipropylamine or N-methylazocane (0.01 mol),
prepared by Eschweiler-Clarke methylation [30] of dipropylamine
or azocane, was quaternized with 2-bromoethanol (0.012 mol) in
acetonitrile. The mixture was refluxed for 4 h. After cooling, the
acetonitrile was evaporated in vacuum. The resulting mixture was
triturated with ether and crystallized from acetone. White hygro-
scopic solid was obtained after crystallization.
cmꢀ1 2917, 2851, 1471, 718.
N,N-Dibutyl-N-methylhexadecylammonium bromide (CMDBu):
yield 66%; 1H NMR (CDCl3, TMS)
0.88 (t, 3H, J ¼ 6.6 Hz), 1.01 (t, 6H,
J ¼ 7.3 Hz), 1.21–1.53 (m, 30H), 1.64–1.79 (m, 6H), 3.38 (s, 3H), 3.43–
d
3.57 (m, 6H); 13C NMR (CDCl3, TMS)
d 13.7, 14.1, 19.7, 22.5, 22.7, 24.4,
26.3, 29.2, 29.4, 29.5, 29.6, 29.7, 31.9, 48.9, 61.3, 61.4; IR ymax/cmꢀ1
2920, 2851, 1468, 721.
N-(2-hydroxyethyl)-N-methyl-N-propylpropan-1-ammonium
N-Hexadecyl-N-methylpyrrolidinium bromide (CMPyr): yield
bromide (2c): yield 68%; 1H NMR (DMSO-d6, TMS)
d
1.05 (t, 6H,
76%; 1H NMR (CDCl3, TMS)
d
0.88 (t, 3H, J ¼ 6.7 Hz), 1.20–1.46 (m,
26H), 1.71–1.83 (m, 2H), 2.25–2.37 (m, 4H), 3.32 (s, 3H), 3.62–3.70
(m, 2H), 3.78–3.94 (m, 4H); 13C NMR (CDCl3, TMS)
14.1, 21.7, 22.7,
J ¼ 7.0 Hz), 1.72–2.08 (m, 4H), 3.33 (s, 3H), 3.41–3.57 (m, 4H), 3.68–
3.78 (m, 4H), 4.09–4.20 (m, 2H), 5.10 (t, 1H, J ¼ 4.9 Hz).
d
N-(2-hydroxyethyl)-N-methylazocanium bromide (2h): yield
24.1, 26.4, 29.3, 29.4, 29.5, 29.6, 29.7, 31.9, 48.6, 64.1, 64.4; IR ymax/
92%; 1H NMR (DMSO-d6, TMS)
d
1.45–1.71 (m, 6H), 1.79–1.98 (m,
cmꢀ1 2918, 2851, 1467, 721.
4H), 3.05 (s, 3H), 3.06–3.56 (m, 6H), 3.79–3.87 (m, 2H), 5.26 (t, 1H,
J ¼ 4.9 Hz).
N-Hexadecyl-N-methylpiperidinium bromide (CMPip): yield
69%; 1H NMR (CDCl3, TMS)
d
0.88 (t, 3H, J ¼ 6.7 Hz), 1.21–1.47 (m,

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M. Lukac et al. / European Journal of Medicinal Chemistry 44 (2009) 4970–4977
4973
26H), 1.66–2.02 (m, 8H), 3.37 (s, 3H), 3.60–3.71 (m, 4H), 3.79–3.88
2.5. Trophocidal susceptibility testing of APCs and QUATs
(m, 2H); 13C NMR (CDCl3, TMS)
d 14.1, 20.2, 20.7, 22.1, 22.7, 26.4,
29.3, 29.4, 29.5, 29.6, 29.7, 31.9, 48.5, 60.7, 62.6; IR ymax/cmꢀ1 2918,
2850, 1467, 721.
These procedures were carried out using the modified methods
described by [10]. The cultures of amoeba were grown axenically in
100 ml Erlenmeyr flasks. Axenic culture was obtained from the
monoxenic culture of A. lugdunensis. Trophozoites were harvested
from 2-day monoxenic plate cultures and transferred to Bacto-
Casitone/Serum (BCS) medium with penicillin and ampicillin.
Actively growing trophozoites were harvested by centrifugation at
500g for 7 min and subsequently they were incubated at 37 ꢁC for 3
days. Thereafter, trophozoites were transferred to a BCS medium
without antibiotics and cultivated at 37 ꢁC for 3 days. The cultiva-
tion in the BCS medium was repeated 5-times, then the tropho-
zoites were transferred to peptone–yeast extract–glucose (PYG)
medium and afterwards cultivated in this medium. Experiments
were carried out in 96-well microtiter plates at 37 ꢁC under sterile
N-Hexadecyl-N-methylazepanium bromide (CMAzep) yield
75%; 1H NMR (CDCl3, TMS)
d
0.88 (t, 3H, J ¼ 6.7 Hz), 1.20–1.46 (m,
26H), 1.68–1.89 (m, 6H), 1.89–2.09 (m, 4H), 3.40 (s, 3H), 3.58–3.69
(m, 4H), 3.71–3.81 (m, 2H); 13C NMR (CDCl3, TMS)
14.1, 22.1, 22.7,
d
23.0, 26.4, 27.4, 29.3, 29.4, 29.5, 29.6, 29.7, 31.9, 51.2, 64.7, 64.9; IR
ymax/cmꢀ1 2920, 2851, 1467, 721.
N-Hexadecyl-N-methylazocanium bromide (CMAzoc): yield
84%; 1H NMR (CDCl3, TMS)
d
0.88 (t, 3H, J ¼ 6.7 Hz), 1.22–1.47 (m,
26H), 1.59–1.85 (m, 8H), 1.94–2.03 (m, 4H), 3.36 (s, 3H), 3.45–3.55
(m, 2H), 3.58–3.66 (m, 2H), 3.72–3.83 (m, 2H); 13C NMR (CDCl3,
TMS)
d 14.1, 22.0, 22.7, 22.8, 24.4, 26.3, 26.4, 29.3, 29.4, 29.5, 29.6,
29.7, 31.9, 50.2, 59.6, 63.5; IR ymax/cmꢀ1 2919, 2851, 1468, 721.
N-Hexadecyl-N-methylmorpholinium bromide (CMMorph):
conditions. Each well was seeded with 100
a trophozoite suspension. Then 100 ml of test substance solution in
m
l (2 ꢂ 105 cells mlꢀ1) of
yield 58%; 1H NMR (CDCl3, TMS)
d
0.88 (t, 3H, J ¼ 6.6 Hz), 1.19–1.50
(m, 26H), 1.65–2.05 (m, 4H), 3.37 (s, 3H), 3.60–3.75 (m, 4H), 3.78–
PYG medium was added and mixed with the suspension of
trophozoites. APCs were tested at six concentrations (25, 50, 100,
3.90 (m, 2H); 13C NMR (CDCl3, TMS)
d 14.1, 22.0, 22.7, 26.3, 29.3,
29.4, 29.5, 29.6, 29.7, 31.9, 47.3, 59.7, 60.7, 65.4; IR ymax/cmꢀ1 2920,
200, 400, 800
50, 25, 12.5, 7.25
exclusion. One hundred percent eradication was confirmed by
transferring 50 l of the suspension to a PYG medium and by
mM) and QUATs were tested at 5 concentrations (100,
2851, 1473, 1120, 901, 720.
m
M). Viability was determined by trypan blue
m
2.3. Cytotoxicity assay
recording the amoeba growth for 14 days. The lowest concentration
of the APCs and the QUATs supporting for 100% eradication of
trophozoites was defined as the minimal trophocidal concentration
(MTC).
The cytotoxic effects of compounds were determined using
colorimetric microculture assay with the MTT (3[4,5-dimethylth-
iazol-2-y1]-2,5-diphenyltetrazolium bromide) end-point [31].
Briefly, 3 ꢂ103 cells mlꢀ1 (A-549, MCF-7, MDA-MB-231),
5 ꢂ103 cells mlꢀ1 (HeLa) or 1 ꢂ104 cells mlꢀ1 (Jurkat and CCRF-
CEM) cells were plated per well in 96-well polystyrene microplates
(Sarstedt, Germany) in the culture medium containing tested
2.6. Haemolytic activity assay of APCs and QUATs
Human blood was collected in 10 ml tubes containing sodium
citrate as anticoagulant. The blood was transferred to a centrifuge
tube and the cells were washed three times with calcium- and
magnesium-free phosphate-buffer saline (PBS). Cells were
collected by centrifugation at 2000g for 10 min. The cells were
then suspended in the buffer (PBS – 145 mM NaCl, 5 mM KCl,
4 mM Na2HPO4, 1 mM NaH2PO4, 1 mM MgSO4, 1 mM CaCl2, and
10 mM glucose, buffer was adjusted to pH ¼ 7.4 [32]). The
prepared cell culture could be stored at 4 ꢁC for 48 h. The cell
density of the stock suspension of the erythrocytes was 6.7–
chemicals at final concentrations of 1 ꢂ10ꢀ4 to 1 ꢂ10ꢀ8 mol lꢀ1
.
After 72 h of incubation, 10 m
L of MTT (5 mg mlꢀ1) were added in
each well. After an additional 4 h, during which insoluble formazan
was produced,100 l of 10% sodium dodecylsulphate were added in
m
each well and another 12 h were allowed for the dissolution of
formazan. Absorbance was measured at 540 nm using the auto-
mated MRX microplate reader (Dynatech Laboratories, UK). The
blank-corrected absorbance of the control wells was taken as 100%
and the results were expressed as a percentage of the control. All
experiments were performed in triplicate. IC50 values (concentra-
tions of tested agents that inhibited growth of cell cultures to 50% of
the untreated control) were determined by GraphPad Prism for
Windows version 5.01.
6.8 ꢂ 108 cells mlꢀ1
. One volume of the cell suspension was
pipetted to 9 volumes of buffer containing different concentra-
tions of the tested compounds. Final erythrocyte concentrations
were 6.7–6.8 ꢂ 107 cells mlꢀ1. The mixtures were incubated at
37 ꢁC for 1 h with gentle shaking and then centrifugated at 2000g
for 10 min. The degree of haemolysis was determined by
comparing the absorbance (542 nm) of appropriate dilutions of
the supernatant with that of control samples haemolysed in
2.4. Fungicidal susceptibility testing of APCs and QUATs
A cell suspension culture of C. albicans was prepared from 24-h
cultures of yeast in the Sabouraud agar. The concentration of
culture was 5 ꢂ105 cells mlꢀ1. The suspensions were prepared in
physiological solution (pH 7.2). The concentration of microorgan-
isms was measured spectrophotometrically and the suspensions
500 mM solution of CTAB in PBS.
3. Results and discussion
3.1. Chemistry
were concentrated to absorbance A ¼ 0.35 at
microorganism suspension (5 l) was added to solutions containing
the tested compound (100 l) and to double concentrated Sabo-
uraud medium (12%) (100 l). The cultivation was performed in 96-
well microtiter plate. The solution of the studied compounds was
prepared in water. The microorganisms were incubated for 24 h at
37 ꢁC. Then from each well, 5
ml of tested suspension was taken and
cultured on the Sabouraud agar. The Petri dishes were incubated for
24 h at 37 ꢁC. The lowest concentration of the APC and the QUATs
supporting no colony formation was defined as the minimal
fungicidal concentration (MFC).
l
¼ 540 nm. The
m
The synthetic strategy for the preparation of the APCs is
depicted in Schemes 1 and 2. Representative phosphate analogues
were prepared to determine the importance of the phosphocholine
moiety for bioactivity according to modified procedures [6,33,34].
The strategy involves phosphorylation of the hexadecanol using
POCl3 and subsequent attachment of the desired choline analogue.
HPC, HPDEt, HPPyr, HPPip, HPAzep and HPMorph were prepared
by reaction of hexadecyl dichloridophosphate with choline p-tol-
uenesulfonate and subsequently hydrolyzed. HPDPr and HPAzoc
were prepared by another strategy. Hexadecyl dichloridophosphate
m
m

´ˇ
4974
M. Lukac et al. / European Journal of Medicinal Chemistry 44 (2009) 4970–4977
( )n
( )n
Ts-
( )n
O
nN+
ii
iii
O
O-
P
N+
N
( )
H33C16
( )n
OH
( )n
O
OH
HPC n = 0
HPDEt n = 1
1a n = 0
1b n = 1
2a n = 0
2b n = 1
O-
N+
O
OH
N+
ii
iii
i
H33C16
X
NH
X
N
X
P
Ts-
O
OH
O
X
2e X = 0
X = -CH -
X = 0
X = -CH2-
1e X = 0
1f X = -CH2-
HPPyr X = 0
HPPip X = -CH2-
2f
2
X = -(CH2)2-
X = -O-
2g
X = -(CH ) -
2 2
2i X = -O-
1g X = -(CH2)2-
1i X = -O-
HPAzep X = -(CH2)2-
HPMorph X = -O-
Scheme 1. Synthesis of ALPs – HPC, HPDEt, HPPyr, HPPip, HPAzep, HPMorph. Reagents and conditions: i 2-chloroethanol, K2CO3, benzene, reflux; ii methyl p-toluenesulfonate,
CH3CN, reflux; iii POCl3, hexadecanol, Et3N, CHCl3, pyridine.
was firstly hydrolyzed by water and transformed to dipyridinium
hexadecyl phosphate using pyridine. This salt was then connected
with choline bromide in the presence of 2,4,6-triisopropylbenze-
nesulfonyl chloride. The choline analogues were obtained with two
synthetic procedures: 1. Tertiary 2-aminoethanoles 1e, 1f, 1g and 1i
were prepared by the reaction of secondary amine with 2-chlor-
oethanol and potassium carbonate in the presence of a catalytic
amount of KI. The appropriate cholines were obtained by quater-
nization of 1a, 1b, 1e, 1f, 1g and 1i with methyl p-toluenesulfonate
in acetonitrile. The synthesis of choline p-toluenesulfonate
analogues 2a, 2b, 2e, 2f, 2g, and 2i is depicted in Scheme 1. 2.
Dipropylamine and azocane were methylated by Eschweiler–Clarke
reaction, resulting in tertiary amines 3c and 3h. The choline
bromides 4c and 4h were obtained by quaternization of prepared
tertiary amines with 2-bromoethanol. Dibutyl analogue of choline
bromide or p-toluenesulfonate is very hygroscopic and it is very
hard to obtain in solid form (N,N-dibutyl-2-hydroxy-N-methyl-
ethanammonium bromide started to crystallize from the acetone at
ꢀ18 ꢁC after half a year). The prepared APCs crystallized as
hydrates. The amount of bounded water was estimated by 1H NMR
spectroscopy. The position of signal depends on the concentration
of measured substances in CDCl3.
3.2. Antineoplastic activity of APCs and QUATs
HPC was considered as a standard compound of APCs, its
cytotoxicity is very well known. HPC and other APCs exhibited the
best activity against human acute T-lymphoblastic leukemia (Table
1). The APCs with smaller heterocyclic rings or shorter alkyl chains
of dialkylamino analogues were more active than the compounds
with the biggest heterocyclic ring (HPAzoc) or dipropylamino
analogue (HPDPr). Insertion of oxygen to heterocyclic ring
increased the antileukemic activity. HPMorph (IC50 ¼ 7.7
two times active against Jurkat in comparison with its analogue
M). The antileukemic activity of
mM) was
without oxygen (HPPip, IC50 ¼ 14
m
the APCs was approximately four times higher than the antineo-
plastic activity compared to other studied carcinoma cell lines.
HPMorph again exhibited the highest activity against the
remaining investigated cancer cell lines. The least active compound
was HPPip. Its IC50s against HeLa, MDA-MB-231, MCF-7 and A-549
were higher than 50 mM. This observation is startling. Koufaki et al.
[6] tested the cytotoxicity of these two APCs against six human
tumor xenografts. HPPip was the most active compound from the
studied APCs and HPMorph exhibited only moderate activity.
HPPip was very active against small cell and non-small cell lungs
carcinoma [6] but we observed that it was inactive against lungs
QUATs CMDEt, CMDPr, CMDBu, CMPyr, CMPip, CMAzep,
CMAzoc and CMMorph were prepared by Menschutkin reaction,
tertiary amines 3b, 3c, 3d, 3e, 3f, 3g, 3h and 3i were quaternized
with 1-bromohexadecane in dry acetonitrile. The QUATs were
carcinoma A-549 (IC50 > 100 mM) in our case. Increasing of
heterocyclic rings or prolongation of alkyl chains on ammonium
cation decreased the cytotoxicity of the APCs against HeLa, MDA-
MB-231 and MCF-7 only slightly.
´
prepared by the procedure as was previously described by Devın-
sky et al. [35]. The secondary amines were methylated by
Eschweiler–Clarke reaction (Scheme 3).
CTAB was considered as a standard compound of QUATs. Its
cytotoxic activity is lower on comparison with other QUATs.
O-
Br-
OH
H33C16
i
iii
ii
O
N+
N+
NH
N
P
O
O
3c
4c
HPDPr
Br-
O-
O
N+
N+
H33C16
NH
N
P
i
ii
iii
OH
O
O
3h
4h
HPAzoc
Scheme 2. Synthesis of ALPs – HPDPr, HPAzoc. Reagents and conditions: i 35% HCHO, 85% HCOOH, reflux; ii 2-bromoethanol, CH3CN, reflux; iii hexadecanol, POCl3, Et3N, CHCl3,
2.4.6-triisopropylbenzenesulfonyl chloride, pyridine.

´ˇ
M. Lukac et al. / European Journal of Medicinal Chemistry 44 (2009) 4970–4977
4975
significantly reduces of aberration of chromosomes (clastogenicity)
without changes in proliferative activity of bone marrow cells [38].
Both the APCs and the QUATs inserted into the plasma membrane
and subsequently induce a broad range of biological effects, ulti-
mately leading to cell death [36,37].
( )n
HN
( )n
( )n
N
( )n
( )n
i
ii
H33C16
Br-
N+
( )n
n = 1
n = 2
n = 3
3b n = 1
3c n = 2
3d n = 3
n = 1
CMDEt
CMDPr n = 2
CMDBu n = 3
3.3. Antifungal activity of APCs and QUATs
Br-
N+
X
i
ii
X
NH
X
N
The MFC of HPC against C. albicans was 4
mM; this value corre-
H33C16
sponds to the same value published by Widmer at al. [39]. HPC and
HPDEt presented the best activity against C. albicans (Table 1).
Expansion of the length of head group alkyl chains from 1–2 carbon
atoms to 3 carbon atoms (HPDPr) significantly decreased the
antifungal activity. HPDPr is the least active compound from the
studied APCs. Heterocyclic analogues were less active as HPC. For
the heterocyclic APC compound series, expansion of the heterocy-
clic ring or insertion of another heteroatom (morpholine) had no
effect on antifungal activity.
X = 0
X = 0
CMPip X = -CH2-
CMAzep X = -(CH2)2-
CMAzoc X = -(CH2)3-
CMMorph X = -O-
3e X = 0
3f X = -CH2-
3g X = -(CH2)2-
3h X = -(CH2)3-
3i X = -O-
CMPyr
X = -CH2-
X = -(CH2)2-
X = -(CH2)3-
X = -O-
Scheme 3. Synthesis of QACs. Reagents and conditions: i 35% HCHO, 85% HCOOH,
reflux; ii 1-bromohexadecane, CH3CN, reflux.
CTAB as a standard compound of QUATs exhibited lower
Elongation of alkyl chains in dialkylamino groups increased the
activity in the case of cell lines: Jurkat, CCRF-CEM, Hela and MDA-
MB-231. On the other hand, the tumor cell lines MCF-7 and A-549
were not so sensitive on elongation of the alkyl chains of the dia-
lkylamino groups. The activity was affected only slightly. The
substitution of the dimethyl amino group to nitrogen heterocycle
(pyrrolidine, piperidine, azepine and azocane) in the molecule of
CTAB led to increased cytotoxic effects. Only CMAzoc exhibited
lower activity than CTAB in the case of tumor cell lines A-549 and
MCF-7. Insertion of oxygen to the heterocyclic ring decreased anti-
neoplastic activity. CMMorph was the least active QUATs. This
observation is very interesting when we compare the activity of the
APCs and the QUATs. Morpholine analogue of HPC is the most
cytotoxic active compounds from the APCs but on the other hand
morpholine analogue of CTAB was the least active compound from
the QUATs. The QUATs were about 10-times more active than the
APCs. Disadvantage of the QUATs is in theirhigh cytotoxicity notonly
on tumor cells but on health human cells. Cationic surfactants
induce apoptosis in normal and cancer cells. Benzethonium chloride
as a model QUAT increases DNA fragmentation and also accelerated
condensation of nuclei in rat thymocytes more than in Jurkat cells
[36]. Synthetic APCs are anticancer agents that in contrast to most
anticancer drugs do not target DNA [37]. Instead, HPC decreases the
destructive cytotoxic effect of epirubicine and cyclophosphamide on
mouse spermatogenic and hematopoietic cells. For example, HPC
fungicidal activity (MFC ¼ 21
stro¨m et al. observed complete elimination of Candida cells by CTAB
at concentration 100 M [40]. The higher concentration of Candida
mM) in comparison with HPC. Ahl-
m
cells in the tested culture is a possible reason of higher MFC value in
the case of fungicidal determinations provided by Ahlstro¨m et al.
[40]. Dialkylamino analogues of CTAB with an even number of
carbon atoms in the head group alkyl chains were more effective
than CTAB, however, CMDPr had similar activity as the standard
compound. Heterocyclic analogues showed the same activity as
CMDEt and CMDBu, only CMMorph was the least active from all
QUATs.
The reason of higher antifungal activity of APCs in the compar-
ison with QUATs is probably in a different mode of action. Widmer
et al. [39] suggested that the effect of HPC was due to selective
lysophospholipase-transacylase of phospholipase B1 (PLB 1) inhi-
bition rather than a nonspecific detergent-like action which is
typical for the QUATs [41]. However, the inhibition of PLB 1 is not
sufficient for the fungicidal activity of the APCs and they should also
have other biochemical targets which are responsible for antifungal
effect of APCs [8].
3.4. Antiprotozoal activity of APCs and QUATs
HPC was weakly active against Acanthamoeba lugdunensis
(Table 1). Its MTC was 400 mM. Walochnik et al. [10] studied three
Table 1
Antineoplastic, fungicidal, trophocidal and haemolytic activities of APCs and QUATs.
Compound
IC50
(m
M)
MFC (
m
M)
MTC (
m
M)
EC50 (mM)
Jurkat
CCRF-CEM
HeLa
MDA-MB-231
MCF-7
A-549
44.7
59.4
55.0
53.2
C. albicans
A. lugdunensis
Haemolytic activity
HPC
10.0
19.6
28.6
10.0
14.0
18.2
38.6
7.7
13.5
9.0
19.2
9.3
11.2
9.8
43.8
8.25
41.6
49.4
55.0
46.2
57.2
53.1
55.0
21.7
37.6
34.4
40.6
37.9
51.7
49.8
45.6
11.4
39.2
53.7
50.6
39.3
54.8
51.5
53.3
22.4
4
4
16
8
8
8
400
200
400
200
200
50
30
30
30
30
25
25
30
30
HPDEt
HPDPr
HPPyr
HPPip
HPAzep
HPAzoc
HPMorph
>100
55.1
55.0
42.0
8
8
50
200
CTAB
1.98
0.8
3.25
2.10
1.96
0.91
1.35
0.92
1.00
1.00
4.16
4.10
1.38
1.68
0.90
2.29
1.47
1.82
1.43
5.61
4.50
4.90
4.27
2.55
3.80
3.86
2.84
4.12
5.75
0.75
0.65
0.80
0.68
0.62
0.59
0.72
0.94
0.89
0.76
21
10
19
9
10
10
9
25
25
25
25
12.5
12.5
12.5
12.5
50
45
40
30
25
35
35
35
30
45
CMDEt
CMDPr
CMDBu
CMPyr
0.62
0.75
0.64
0.53
0.63
0.67
0.85
0.87
0.85
0.76
0.82
0.72
0.80
0.78
3.57
CMPip
CMAzep
CMAzoc
CMMorph
9
24

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M. Lukac et al. / European Journal of Medicinal Chemistry 44 (2009) 4970–4977
4976
species of Acanthamoeba: A. castellanii, A. polyphaga, A. lenticulata
M). The
low values of the MTC were also obtained by McBride et al. [42].
APCs and the QUATs against A. lugdunensis. Higher trophocidal
activity appertained to the QUATs. The APCs exhibited very low
trophocidal activity, only HPAzep and HPAzoc achieved activity
comparable with the activity of the QUATs. Haemolytic activity of
the tested compounds was similar. Haemolysis of erythrocytes
caused by the APCs and the QUATs only slightly depended on the
structure of the polar head group. The APCs were a little bit more
toxic than the QUATs.
and they observed lower values of the MTC (MTC ¼ 20–40
m
They studied the influence of HPC on two strains of Acanthamoeba:
A. polyphaga (MTC ¼ 31.25
m
M), A. castellanii (MTC ¼ 31.25
mM). In
our case, HPC was about 10-time less active against A. lugdunensis
[43] in comparison with its activity against A. castellanii, A. poly-
phaga and A. lenticulata which was observed by other authors. This
significant discrepancy can be most likely explained with testing
the compound on different species of the genus Acanthamoeba.
Dialkylamino analogues of APCs were low active compounds
similar to HPC. The same activity was shown by the heterocyclic
analogues HPPyr, HPPip and HPMorph. The expansion of hetero-
cyclic ring to 7–8 atoms caused an increase of the trophocidal
activity. HPAzep and HPAzoc were about 4-times more active than
other heterocyclic analogues and they were about 8-times more
effective than HPC.
Acknowledgements
This work was supported by grants No. UK/110/2008, VEGA
1/4300/07, VEGA 1/3416/06, VEGA1/4290/07, VEGA 1/0164/08 and
VEGA 1/4341/07.
References
[1] R. Zeisig, D. Arndt, H. Brachwitz, Pharmazie 45 (1990) 809–818.
[2] M. Lohmeyer, R. Bittman, Drugs Fut. 19 (1994) 1021–1037.
[3] H. Brachwitz, C. Vollgraf, Pharmacol. Ther. 66 (1995) 39–82.
[4] H. Eibl, C. Unger, Cancer Treat. Rev. 17 (1990) 233–242.
[5] R. Leonard, J. Hardy, G. van Tienhoven, S. Houston, S. Simmononds, M. David,
J. Mansi, Clin. Oncol. 19 (2001) 4150–4159.
[6] M. Koufaki, V. Polychroniou, T. Calogeropoulou, A. Tsotinis, M. Drees,
H.H. Fiebig, S. LeClerc, H.R. Hendriks, A. Makriyannis, J. Med. Chem. 39 (1996)
2609–2614.
[7] P. Hilgard, T. Klenner, J. Stekar, G. No¨ssner, B. Kutscher, J. Engel, Eur. J. Cancer 33
(1997) 442–446.
[8] D. Obando, F. Widmer, L.C. Wright, T.C. Sorrell, K.A. Jolliffe, Biorg. Med. Chem.
15 (2007) 5158–5165.
QUATs exhibited excellent activity against A. lugdunensis. We
have not found any investigation of antiprotozoal activity of CTAB
against Acanthamoeba recently, but the MTC of these QUATs is
comparable with the MTC of benzalkonium chloride, a commer-
cially available disinfectant [27]. CTAB and dialkylamino analogues
had the same values of MTC. In the series of the investigated
compounds, the activity did not depend on the alkyl chains length
of the head group. Heterocyclic compounds showed a 2-times
better activity than CTAB and dialkylamino analogues, only the
CMMorph exhibited the least activity of all.
ˇˇ
´
[9] J. Lorenzo-Morales, J. Kliescikova, E. Martinez-Carretero, L.M. De Pablos,
ꢁ
´
´
B. Profotova, E. Nohynkova, A. Osuna, B. Valladares, Eukaryot. Cell 7 (2008)
3.5. Haemolytic activity of APCs and QUATs
509–517.
ˆ
[10] J. Walochnik, M. Duchene, K. Seifert, A. Obwaller, T. Hottkowitz,
G. Wiedremann, H. Eibl, H. Aspo¨ck, Antimicrob. Agents Chemother. 46 (2002)
695–701.
The investigated APCs and QUATs are relatively haemolytic toxic
compounds (Table 1) because their toxicity depended on alkyl
chain length [8,44]. The haemolytic activity of the APCs did not
depend on head groups. Very low changes of this activity were
observed in dependence on structural modifications.
[11] P. Escobar, S. Matu, C. Marques, S.L. Croft, Acta Trop. 81 (2002) 151–157.
[12] V. Choubey, P. Maity, M. Guha, S. Kumar, K. Srivastava, S.K. Puri,
U. Bandyopadhyay, Antimicrob. Agents Chemother. 51 (2007) 696–706.
[13] F.L. Schuster, B.J. Guglielmo, G.S. Visvesvara, J. Eukaryot. Microbiol. 53 (2006)
121–126.
A different situation occurred in the case of QUATs. CTAB and
CMMorph exhibited the lowest haemolytic activity. The alkyl
chains length of dialkylamino analogues played an important role
in the haemolytic activity. The elongation of alkyl chains led to
a gradual increase of haemolytic activity. The haemolytic activity of
heterocyclic QUATs did not depend on ring size but they had
a higher activity than CTAB.
[14] C. Blaha, M. Ducheˆne, H. Aspo¨ck, J. Walochnik, J. Antimicrob. Chemother. 57
(2006) 273–278.
ˆ
[15] K. Seifert, M. Duchene, W.H. Wernsdorfer, H. Kollaritsch, O. Scheiner,
G. Wiedermann, T. Hottkowitz, H. Eibl, Antimicrob. Agents Chemother. 45
(2001) 1505–1510.
[16] S.L. Croft, W. Snowdon, V.Yardley, J.Antimicrob.Chemother. 38(1996)1041–1047.
[17] K. Seifert, A. Lemke, S.L. Croft, O. Kayser, Antimicrob. Agents Chemother. 51
(2007) 4525–4528.
[18] C.R. Birnie, D. Malamud, R.L. Schnaare, Antimicrob. Agents Chemother. 44
(2000) 2514–2517.
[19] D. Demberelnyamba, K.S. Kim, S. Choi, S.Y. Park, H. Lee, C.J. Kim, I.D. Yoo, Biorg.
Med. Chem. 12 (2004) 853–857.
[20] I. Lacko, F. Devı´nsky, L. Krasnec, Z. Naturforsch. 34c (1979) 485–486.
4. Conclusion
ˇ
ˇ´
[21] M. Pavlı´kova´, I. Lacko, F. Devı´nsky, D. Mlynarcık, Collect. Czech. Chem. Com-
16 compounds were prepared; 8 APCs and 8 QUATs. CTAB was
obtained as a commercially available substance. We investigated
the influence of a polar head group of the APCs and the QUATs on
cytotoxic, fungicidal, trophocidal and haemolytic activities. In this
study, we focussed on modifying the dimethylamino group of HPC
and CTAB as standard compounds for APCs and QUATs. We
prepared two series of the APCs and the QUATs: dialkylamino and
nitrogen heterocyclic analogues.
mun. 60 (1995) 1213–1228.
[22] E. Ob1ak, T.M. Lachowicz, J. quczyn´ ski, S. Witek, Cell. Mol. Biol. Lett. 7 (2002)
1121–1129.
[23] H. Kourai, T. Yabuhara, A. Shirai, T. Maeda, H. Nagamune, Eur. J. Med. Chem. 41
(2006) 437–444.
[24] M. Miko, F. Devınsky, Drug Metabol. Drug Interact. 10 (1992) 237–263.
[25] K.W. Yip, X. Mao, P.Y. Billie Au, D.W. Hedley, S. Chow, S. Dalili, J.D. Mocanu,
C. Bastianutto, A. Schimmer, F.F. Liu, Clin. Cancer Res. 12 (2006) 5557–5569.
[26] C.J. Marasco, C. Piantadosi, K.L. Meyer, S. Morris-Natschke, K.S. Ishaq,
G.W. Small, L.W. Daniel, J. Med. Chem. 33 (1990) 985–992.
´
The cytotoxic activity of APCs and QUATS were examined on six
human cancer cells lines – Jurkat, CCRF-CEM, HeLa, MDA-MB-231,
MCF-7 and A-549. The QUATs exhibited better cytotoxic activity in
comparison with the APCs. The most active compound was dibu-
tylamino analogue of CTAB. CMDBu exhibited the best activity on
tree cell lines, one leukemia (CCRF-CEM) and two carcinoma (HeLa,
MDA-MB-231). The APCs were about 10-times less active than the
QUATs. Generally, HPMorph exhibited the best cytotoxic activity on
all studied cancer cell lines. Generally, the best fungicidal activity
against C. albicans was exhibited by the APCs. The most active
[27] S. Zanetti, P.L. Fiori, A. Pinna, S. Usai, F. Carta, G. Fadda, Antimicrob. Agents
Chemother. 39 (1995) 1596–1598.
ˇ
ꢁ
´
E. Nohynkova´, Ann.
´
ˇ
[28] F. Ondriska, M. Mrva, M. Lichvar, P. Ziak, Z. Murgasova,
Agric. Environ. Med. 11 (2004) 335–341.
[29] D. Campbell, L.R. Dix, P. Rostron, Dyes Pigm. 29 (1995) 77–83.
[30] H.T. Clarke, H.B. Gillespie, S.Z. Weisshaus, J. Am. Chem. Soc. 55 (1933) 4571–4584.
[31] T. Mosmann, J. Immunol. Methods 65 (1983) 55–63.
[32] A. Fogt, H. Ha¨gerstrand, B. Isomaa, Chem. Biol. Interact. 94 (1995) 147–155.
[33] K. Ukawa, E. Imamiya, H. Yamamoto, K. Mizuno, A. Tasaka, Z. Terashita,
T. Okutani, H. Nomura, T. Kasukabe, M. Hozumi, I. Kudo, K. Inoue, Chem.
Pharm. Bull. 37 (1989) 1249–1255.
ˇ
´
[34] M. Luka´c, I. Lacko, F. Devınsky, Acta Facult. Pharm. Univ. Comenianae 55
(2008) 142–151.
compounds were HPC and HPDEt with MFC ¼ 4
mM. A different
ˇ
ˇ´
´
[35] F. Devı´nsky, I. Lacko, D. Mlynarcık, E. Svajdlenka, V. Borovska, Chem. Pap. 44
situation was observed in the case of antiprotozoal action of the
(1990) 159–170.

´ˇ
M. Lukac et al. / European Journal of Medicinal Chemistry 44 (2009) 4970–4977
4977
[36] R. Enomoto, C. Suzuki, M. Ohno, T. Ohasi, R. Futagami, K. Ishikawa, M. Komae,
T. Nishino, Y. Konishi, E. Lee, Ann. N.Y. Acad. Sci. 1095 (2007) 1–6.
[37] S.R. Vink, W.J. van Blitterswijk, J.H.M. Schellens, M. Verheij, Cancer Treat. Rev.
33 (2007) 191–202.
[38] Y. Martinova, M. Topashka-Ancheva, S. Konstantinov, S. Petkova,
M. Karaivanova, M. Berger, Arch. Toxicol. 80 (2006) 27–33.
[40] B. Ahlstro¨m, M. Chelminska-Bertilsson, R.A. Thompson, L. Edebo, Antimicrob.
Agents Chemother. 41 (1997) 544–550.
[41] P. Gilbert, L.E. Moore, J. Appl. Microbiol. 99 (2005) 703–715.
[42] J. McBride, P.R. Ingram, F.L. Henriquez, C. Roberts, J. Clin. Microbiol. 43 (2005)
629–634.
ˇ
´
[43] M. Luka´c, M. Mrva, F. Ondriska, I. Lacko, F. Devınsky, Chem. Listy 102 (2008) s199.
ˇ
´
¨
¨
¨
ˇ
[39] F. Widmer, L.C. Wright, D. Obando, R. Handke, R. Ganandren, D.H. Ellis,
T.C. Sorrell, Antimicrob. Agents Chemother. 50 (2006) 414–421.
[44] M. Dubnickova, M. Borowska-Hagerstrand, T. Soderstrom, A. Iglic,
H. Ha¨gerstrand, Acta. Biochim. Pol. 47 (2000) 651–660.