X-ray crystallographic, FT-IR and NMR studies as well as anticancer and antibacterial activity of the salt formed between ionophore antibiotic Lasalocid acid and amines

Article abstract of DOI:10.1016/j.molstruc.2012.07.036

Two new complexes of the ionophore antibiotic Lasalocid acid (LAS) with phenylamine (PhA) and butylamine (BuA) were synthesized and their molecular structures were studied using single crystal X-ray diffraction and spectroscopic methods. In the solid state both amines are protonated and all NH3+ protons are hydrogen bonded to etheric, hydroxyl and carboxylic oxygen atoms of the LAS anion. In chloroform solutions the structure observed in the crystal of LAS-BuA complex is preserved and an equilibrium between the LAS-PhA complex and dissociated Lasalocid acid and phenylamine is observed. In vitro antimicrobial tests of the complexes showed a significant activity towards some strains of Gram-positive bacteria. For the first time Lasalocid acid and its complexes with amines were tested in vitro for cytotoxic activity against human cancer cell lines: A-549 (lung), MCF-7 (breast), HT-29 (colon) and mouse cancer cell line P-388 (leukemia). We found that LAS and its complexes are strong cytotoxic agents towards all tested cell lines. The cytostatic activity of the compounds studied is greater than that of cisplatin, indicating that Lasalocid and its complexes are promising candidates for new anticancer drugs.

Full text of DOI:10.1016/j.molstruc.2012.07.036

Journal of Molecular Structure 1032 (2013) 69–77
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
X-ray crystallographic, FT-IR and NMR studies as well as anticancer
and antibacterial activity of the salt formed between ionophore
antibiotic Lasalocid acid and amines
a,
a
b
c
b
⇑
´
´
Adam Huczynski , Jacek Rutkowski , Joanna Wietrzyk , Joanna Stefanska , Ewa Maj ,
Małgorzata Ratajczak-Sitarz a, Andrzej Katrusiak a, Bogumil Brzezinski a, Franz Bartl d
a Faculty of Chemistry, Adam Mickiewicz University, Grunwaldzka 6, 60-780 Poznan, Poland
b Ludwik Hi-rszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114 Wrocław, Poland
c Medical University of Warsaw, Department of Pharmaceutical Microbiology, Oczki 3, 02-007 Warsaw, Poland
d Institute of Medical Physics and Biophysics, Charité Universitätsmedizin Berlin, Campus Charité Mitte, Ziegelstr. 5/9, 10117 Berlin, Germany
h i g h l i g h t s
"
"
"
"
Ionophore antibiotic complexes with aromatic and aliphatic amines were obtained.
Antimicrobial tests demonstrated activity of all compounds studied against Gram-positive bacteria.
Lasalocid acid and its complexes can be recognized as potential anticancer drug candidates.
A pseudo-cyclic structure was found to be strictly requested for cytotoxic activities.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 June 2012
Received in revised form 23 July 2012
Accepted 23 July 2012
Available online 1 August 2012
Two new complexes of the ionophore antibiotic Lasalocid acid (LAS) with phenylamine (PhA) and butyl-
amine (BuA) were synthesized and their molecular structures were studied using single crystal X-ray dif-
À
Á
fraction and spectroscopic methods. In the solid state both amines are protonated and all NHþ3 protons
are hydrogen bonded to etheric, hydroxyl and carboxylic oxygen atoms of the LAS anion. In chloroform
solutions the structure observed in the crystal of LAS–BuA complex is preserved and an equilibrium
between the LAS–PhA complex and dissociated Lasalocid acid and phenylamine is observed. In vitro anti-
microbial tests of the complexes showed a significant activity towards some strains of Gram-positive bac-
teria. For the first time Lasalocid acid and its complexes with amines were tested in vitro for cytotoxic
activity against human cancer cell lines: A-549 (lung), MCF-7 (breast), HT-29 (colon) and mouse cancer
cell line P-388 (leukemia). We found that LAS and its complexes are strong cytotoxic agents towards all
tested cell lines. The cytostatic activity of the compounds studied is greater than that of cisplatin, indi-
cating that Lasalocid and its complexes are promising candidates for new anticancer drugs.
Ó 2012 Elsevier B.V. All rights reserved.
Keywords:
Ionophores
Polyether antibiotics
Spectroscopy
Complexes
Structural studies
1. Introduction
agents are other than synthetic, with almost 50% actually being
either natural products or directly derived from them [4].
Natural products have proven to be the most reliable single
source of new and effective anticancer agents. Nearly 60% of anti-
cancer and anti-infective agents that are commercially available or
in the late stages of clinical trials originate from natural products
[1]. Available literature data suggest that the utility of natural
products as sources of novel structures, but not necessarily the fi-
nal drug entity, is still topical [2,3]. Thus, in the field of anticancer
drugs, in the time range from the 1940s to date, over 70% of the
Lasalocid acid is a representative of naturally occurring poly-
ether ionophore antibiotics [5,6]. Ionophores are lipophilic chelat-
ing agents that transport cations across lipid bilayer membranes,
such as the plasma and subcellular membranes of cells. This unreg-
ulated membrane transports of various ions leads to mitochondrial
injury and cell swelling, vacuolisation, and finally death [5–7].
To date, over 120 polyether ionophore structures have been char-
acterized [8]. Ionophore antibiotics show a broad spectrum of
bioactivity ranging from antibacterial, antifungal, antiparasitic,
antiviral, and recently discovered anti-tumour cell cytotoxicity
[9]. The discovery of strong anticancer properties of one of them
⇑
Corresponding author. Tel.: +48 618291287.
E-mail address: adhucz@amu.edu.pl (A. Huczyn´ ski).
0022-2860/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2012.07.036

´
A. Huczynski et al. / Journal of Molecular Structure 1032 (2013) 69–77
70
solution using a 1:1 molar ratio of LAS (50 mg) and respective
amine. Mp = 208–210 °C (LAS–PhA) and 180–182 °C (LAS–BuA).
O(2)
OH
O(1)
O
31
32
33
CH3
H
CH3 CH3
1
2
30
CH3
2.3. X-ray measurements
O(3)
O
11
13
3
8
15
18
28
CH3
The crystals of both complexes selected for single-crystal X-ray
diffraction measurements formed colourless parallelepipeds with
well-developed faces. They were stable under normal conditions
and their X-ray diffraction measurements were carried out on an
Oxford Diffraction Super Nova diffractometer (Oxford Diffraction,
OH
O(4)
O
O
H33C4
O(5)
O(6)
27
19
O
O(7)
26
CH3
23
22
H32C4
Abingdon, UK) using CuK
a radiation at room temperature. Their
OH25
structures were solved by direct methods with SHELXS-97 and re-
fined by full-matrix least-squares with SHELXL-97 [25]. Nine H-
atoms at N(1) in LAS–PhA and LAS–BuA complexes and at O(3),
O(4) and O(8) in LAS–BuA complex were located from the differ-
ence Fourier maps and refined with isotropic temperature factors.
All other H-atoms were located from the molecular geometry (CAH
0.93–0.98 and OAH 0.82 Å) and their Uiso’s were related to the
thermal vibrations of their carriers. Selected details about the crys-
tal structure, experiment, structure solution and refinement are gi-
ven in Table 1.
O(8)
Scheme 1. Structure of Lasalocid acid (LAS).
– salinomycin has received much attention in recent years [10–15].
It has been shown that salinomycin is 100 times more effective
against breast cancer stem-like cells than Taxol (paclitaxel), a com-
monly chemo-therapeutic drug used against breast cancer [15].
After discovery of the anticancer properties of salinomycin, which
has been so far the top candidate for a chemotherapeutic agent
among the ionophore antibiotics, other ionophore antibiotics have
been more thoroughly explored [14]. It has been also demon-
strated that several candidates for chemotherapeutic drugs can
be found among other naturally occurring ionophore antibiotics
such as monensin A, inostamycin and nigericin [9,16–20].
Lasalocid acid (Scheme 1), isolated from Streptomyces lasaliensis,
is able to form pseudomacrocyclic complexes with monovalent
and divalent cations and to transport these cations across cell
membranes [21]. It is commercially used as a coccidiostat for poul-
try and as growth promoter for ruminants [22].
In previous publications, we have shown that Lasalocid acid
forms complexes with allylamine [23] and tetramethylguanidine
[24]. The first complex shows higher antibacterial activity than
pure Lasalocid and the second complex has antibacterial activity
comparable to that of Lasalocid. As an extension of these studies
we synthesized and investigated two new hydrogen-bonded com-
plexes of Lasalocid acid (LAS) with aromatic amine (phenylamine,
PhA) and aliphatic amine (butylamine, BuA) by X-ray and FT-IR,
and NMR spectroscopy. We determined the antimicrobial and
cytotoxic activity of the Lasalocid complexes for the first time
and compared them to the respective activity of uncomplexed
Lasalocid.
2.4. Spectroscopic measurements
The 1H and 13C NMR spectra of LASA, LAS–PhA and LAS–BuA
(0.1 mol dmꢀ3) were recorded in CDCl3 solutions using Bruker
Avance 600 MHz spectrometer. All spectra were locked to deute-
rium resonance of CDCl3. The 1H NMR measurements were car-
ried out at the operating frequency 600.0018 MHz and the 13C
NMR spectra at the operating frequency 150.885 MHz. The tem-
perature 298.0 K and TMS as the internal standard were used in
both cases. No window function or zero filling was used. The er-
rors of the 1H and 13C NMR chemical shift values were 0.01 ppm
and 0.1 ppm, respectively. The 1H and 13C NMR signals were as-
signed using 2-D (COSY, HETCOR, NOESY and HMBC) whose
examples are shown in the Supplementary Materials. 2-D spectra
were recorded using standard pulse sequences from Bruker pulse-
sequence libraries.
The mid infrared region the FT-IR spectra of LAS, LAS–PhA com-
plex and LAS–BuA complex were recorded in chloroform solution
(0.1 mol dmꢀ3) and the FT-IR spectra of LAS–PhA and LAS–BuA
crystals were recorded in nujol and fluorolube mulls. For solutions,
a cell with Si windows and wedge-shaped layers was used to avoid
interferences (mean layer thickness 170 lm). The spectra were ta-
ken with an IFS 113v FT-IR spectrophotometer (Bruker, Karlsruhe)
equipped with a DTGS detector; resolution 2 cmꢀ1, NSS = 64. The
Happ–Genzel apodization function was used.
2. Experimental
The ESI-MS spectra were obtained on a Waters/Micromass
(Manchester, UK) ZQ2000 mass spectrometer (single quadrupole
type instrument, Z-spray, software MassLynx V3.5, Manchester,
UK). The sample solutions were prepared in chloroform (concen-
tration 5 ꢁ 10ꢀ5 mol/dm3). The solutions of the sample were in-
fused into the ESI source using a Harvard pump, the flow rate of
80 dm3/min. The ESI source potentials were capillary 3 kV, lens
0.5 kV, extractor 4 V and cone voltage 10 V. The source tempera-
ture was 120 °C and the dessolvation temperature was 150 °C.
Nitrogen was used as the nebulizing and dessolvating gas at the
flow-rates of 100 and 300 dm3 hꢀ1, respectively.
2.1. General
Lasalocid sodium salt was extracted from Avatec. Phenylamine
(PhA) and n-butylamine (BuA) and solvents were obtained from
Sigma–Aldrich or Fluka and used without any further purification.
2.2. Preparation of Lasalocid–Phenylamine (LAS–PhA) and Lasalocid–
Butylamine (LAS–BuA) complexes
Lasalocid sodium salt (1.0 g) was dissolved in dichloromethane
(150 ml) and stirred vigorously with a layer of diluted aqueous sul-
phuric acid (pH = 1.5) (100 ml). The organic layer containing Lasa-
locid acid (LAS) was washed three times with distilled water.
Subsequently dichloromethane was evaporated under reduced
pressure to dryness giving LAS (0.75 g).
2.5. Antimicrobial activity
Micro-organisms used in this study were as follows: Gram-po-
sitive cocci: Staphylococcus aureus NCTC 4163, Staphylococcus aur-
eus ATCC 25923, Staphylococcus aureus ATCC 6538, Staphylococcus
aureus ATCC 29213, Staphylococcus epidermidis ATCC 12228,
Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 11778, Enterococcus
The crystals of 1:1 complex of LAS–PhA and 1:1 complex of
LAS–BuA were obtained by crystallization from dried acetonitrile

´
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A. Huczynski et al. / Journal of Molecular Structure 1032 (2013) 69–77
Table 1
Crystal data and structure refinement for LAS–PhA and LAS–BuA.
LAS–PhA
LAS–BuA
Empirical formula
Formula weight
C
40H61NO8
C38H65NO8
663.91
683.90
Temperature (K)
293(2)
293(2)
Wavelength
1.54178 Å
1.54178 Å
Crystal system, space group
Unit cell dimensions
Orthorhombic, P212121,
a = 10.6585(1) Å
b = 18.9220(2) Å
c = 19.3361(2) Å
3899.71(7) Å3
Orthorhombic, P212121
a = 10.1671(1) Å
b = 18.7745(1) Å
c = 20.4549(1) Å
3904.48(3) Å3
Volume
Z,
4
4
Calculated density
Absorption coefficient
F(000)
1.165 g cmꢀ3
1.129 g cmꢀ3
0.640 mmꢀ1
1488
0.621 mmꢀ1
1456
Crystal size
h Range for data collection
Limiting indices
Reflections collected/unique
Completeness to h = 26.63
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
0.25 ꢁ 0.20 ꢁ 0.10 mm
0.25 ꢁ 0.20 ꢁ 0.15 mm
3.27–76.49°
3.19–73.82°
ꢀ13 6 h 6 13, ꢀ23 6 k 6 22, ꢀ24 6 l 6 24
61008/8122 Rint = 0.0178
99.5%
ꢀ12 6 h 6 9, ꢀ23 6 k 6 23, ꢀ25 6 l 6 25
30662/7816 Rint = 0.0121
99.3%
Full-matrix least-squares on F2
8122/0/454
Full-matrix least-squares on F2
7816/0/447
1.092
1.005
Final R indices [I > 2
R indices (all data)
Absolute structure parameter
Largest diff. peak and hole
r
(I)]
R1 = 0.0344, wR2 = 0.1045
R1 = 0.0365, wR2 = 0.1062
ꢀ2.2(6)
R1 = 0.0421, wR2 = 0.1375
R1 = 0.0433, wR2 = 0.1406
0.09(15)
0.173 and ꢀ0.120 eÅꢀ3
0.431 and ꢀ0.213 eÅꢀ3
Table 2
Dimensions of the hydrogen bond (Å and °).
ium ranged from 3.125 to 400 lg/ml. The final inoculum of all
studied organisms was 104 CFU mlꢀ1 (colony forming units per
ml). Minimal inhibitory concentrations were read after 18 h of
incubation at 35 °C. The data on the antimicrobial activity of the
compounds are summarized in Table 4.
DAHꢂ ꢂ ꢂA
d(DAH)
d(Hꢂ ꢂ ꢂA)
1.72
1.88
1.96
1.88(2)
1.94(2)
2.13(2)
2.56(2)
2.50(2)
1.39(4)
2.03(3)
1.78(3)
1.96(2)
1.86(4)
2.08(3)
d(Dꢂ ꢂ ꢂA)
<(DHA)
LAS–PhA
O(3)AH(3)ꢂ ꢂ ꢂO(1)
O(8)AH(8)ꢂ ꢂ ꢂO(1)
O(4)AH(4)ꢂ ꢂ ꢂO(2)
N(1)AH(2 N)ꢂ ꢂ ꢂO(8)
N(1)AH(1 N)ꢂ ꢂ ꢂO(2)
N(1)AH(3 N)ꢂ ꢂ ꢂO(6)
N(1)AH(3 N)ꢂ ꢂ ꢂO(5)
N(1)AH(2 N)ꢂ ꢂ ꢂO(7)
O(3)AH(3)ꢂ ꢂ ꢂO(1)
O(8)AH(8)ꢂ ꢂ ꢂO(1)
O(4)AH(4)ꢂ ꢂ ꢂO(2)
N(1)AH(1 N)ꢂ ꢂ ꢂO(8)
N(1)AH(3 N)ꢂ ꢂ ꢂO(2)
N(1)AH(2 N)ꢂ ꢂ ꢂO(6)
0.82
0.82
0.82
2.448(2)
2.674(2)
2.740(2)
2.745(2)
2.820(2)
2.935(2)
3.043(2)
3.089(1)
2.460(2)
2.717(2)
2.707(2)
2.784(2)
2.813(2)
2.954(2)
148
163
159
160(2)
168(2)
159(2)
118(2)
123(1)
165
132
153
144
168
0.91(2)
0.90(2)
0.85(2)
0.85(2)
0.91(2)
1.08(4)
0.90(3)
1.00(3)
0.95(3)
0.96(4)
0.97(3)
2.6. Cytotoxic activity
2.6.1. Cell lines
LAS–BuA
To evaluate antiproliferative activity of Lasalocid acid and its
complexes, six different cell lines were used: A-549 (human lung
adenocarcinoma), MCF-7 (human breast adenocarcinoma), HT-29
(human colon adenocarcinoma), P-388 (murine leukemia), HLMEC
(human lung microvascular endothelial cells) and BALB/3T3 (mur-
ine embryonic fibroblast cell line). The cell lines were obtained
from the American Type Culture Collection (ATCC) and are/were
maintained in the Institute of Immunology and Experimental Ther-
apy, Wroclaw, Poland.
Twenty four hours before adding the compounds tested, the
cells were seeded in 96-well plates (Sarstedt, Germany) at a den-
sity of 104 cells per well (for HLMEC 103 cells per well) and cul-
tured in RPMI 1640 medium (HLMEC, P388) (IIET, Wroclaw),
OptiMEM + RPMI 1640 (1:1) medium (A-549, HT-29) (OptiMEM
from Gibco), Dulbecco medium (BALB/3T3) or Eagle medium
(MCF-7) (IIET, Wroclaw). RPMI medium was supplemented with
150
hirae ATCC 10541, Micrococcus luteus ATCC 9341, Micrococcus luteus
ATCC 10240; Gram-negative rods: Escherichia coli ATCC 10538,
Escherichia coli ATCC 25922, Escherichia coli NCTC 8196, Proteus vul-
garis NCTC 4635, Pseudomonas aeruginosa ATCC 15442, Pseudomo-
nas aeruginosa NCTC 6749, Pseudomonas aeruginosa ATCC 27863,
Bordetella bronchiseptica ATCC 4617 and yeasts: Candida albicans
ATCC 10231, Candida albicans ATCC 90028, Candida parapsilosis
ATCC 22019.
Antimicrobial activity was examined by the disc-diffusion
method under standard conditions using Mueller-Hinton II agar
medium (Becton Dickinson) for bacteria and RPMI agar with 2%
glucose (Sigma) according to CLSI (previously NCCLS) guidelines
[26].
10% foetal bovine serum (HyClone, UK), 2 mM
L-glutamine
(HLMEC, P388) and 1 mM sodium puryvate (P388) (Sigma–Aldrich,
Germany). OptiMEM + RPMI medium was supplemented with 5%
foetal bovine serum, 2 mM
tria) and 1 mM sodium puryvate (HT-29). Eagle medium for
MCF-7 was supplemented with 10% fetal bovine serum, 2 mM
L-glutamine (A549, HT-29) (PAA, Aus-
Sterile filter paper discs (9 mm diameter, Whatman No. 3 chro-
matography paper) were dripped with tested compound solutions
L
-
(in MeOH or MeOH/DMSO 1:1) to load 400
l
g of a given compound
glutamine, aminoacids and insulin (Sigma–Aldrich, Germany). Dul-
becco medium was supplemented with 10% foetal bovine serum
(PAA, Austria), 4 mM L-glutamine and glucose 4,5 g/l (Sigma–Al-
per disc. Dry discs were placed on the surface of appropriate agar
medium. The results (diameter of the growth inhibition zone) were
read after 18 h of incubation at 35 °C. Compounds with recognized
activity in disc-diffusion tests were examined by the agar dilution
method to determine their MIC – Minimal Inhibitory Concentra-
tion (CLSI) [27]. Concentrations of the agents tested in solid med-
drich, Germany). All media contained antibiotics: 100 lg/ml strep-
tomycin and 100 U/ml penicillin (Polfa–Tarchomin). During entire
experiment all cell lines were incubated at 37 °C in a humid atmo-
sphere saturated with 5% CO2.

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A. Huczynski et al. / Journal of Molecular Structure 1032 (2013) 69–77
72
Table 3
The most important 1H and 13C NMR chemical shifts d (ppm) of LAS–PhA, and LAS–BuA complexes in CDCl3 and differences
D
(ppm) between shifts and respective chemical shifts
for LAS in CDCl3.
LAS
LAS–BuA
LAS–PhA
No. atoms
d
1H
d
13C
d
1H
d
13C
d
1H
d
13C
D1 1H
D1 13C
D2 1H
D2 13C
1
2
3
4
5
6
7
–
–
–
–
173.2
111.0
161.6
124.2
134.5
121.4
144.1
73.0
48.8
214.4
55.0
83.8
86.2
70.7
72.6
76.0
–
–
–
–
–
7.05
6.51
–
4.30
2.76
–
2.48
4.05
–
3.43
–
3.82
3.02
–
14.90
4.87br
4.07
7.35
175.4
115.5
161.6
123.6
132.0
120.7
144.2
71.7
48.7
217.8
55.9
83.1
87.2
70.2
71.1
76.8
39.3
–
–
–
–
–
7.15
6.61
–
4.26
2.79
–
2.53
3.88
–
174.4
113.1
161.6
123.8
132.1
119.7
144.2
71.9
48.8
216.3
55.4
83.4
86.9
70.3
71.4
76.4
133.6
–
–
–
–
–
2.2
4.5
0.0
ꢀ0.6
ꢀ2.5
ꢀ0.7
0.1
–
–
–
–
1.2
2.1
0.0
ꢀ0.4
ꢀ2.4
ꢀ1.7
0.1
7.17
6.63
–
4.08
2.84
–
2.62
3.88
–
3.50
–
ꢀ0.12
ꢀ0.12
–
0.22
ꢀ0.08
–
ꢀ0.14
0.17
–
ꢀ0.02
ꢀ0.02
–
0.18
ꢀ0.05
–
ꢀ0.09
0.00
–
11
12
13
14
15
18
19
22
23
35
O(1)H
O(3)H
O(4)H
O(8)H
ꢀ1.3
ꢀ1.1
0.1
0.1
3.4
0.9
1.9
0.4
ꢀ0.7
ꢀ0.4
1
0.7
3.45
–
3.95
–
ꢀ0.07
ꢀ0.5
ꢀ1.5
0.8
–
–
ꢀ0.05
ꢀ0.4
ꢀ1.2
0.4
–
–
–
–
3.93
–
ꢀ0.11
–
–
3.06
ꢀ1.27
ꢀ2.07
–
0.02
–
–
6.14br
11.84
6.14br
6.14br
–
–
–
–
–
–
–
–
–
–
11.95
3.80vbr
3.80vbr
9.34
–
–
–
–
0.11
ꢀ2.34
ꢀ2.34
ꢀ0.02
–
–
–
–
–
–
À
Á
NHþ3
NH2
–
–
–
–
–
3.80vbr
–
–
–
–
–
D1 = dLAS–BuA – dLAS; D2 = dLAS–PhA – dLAS.
br – broad signal; vbr – very broad signal.
Table 4
Each compound at every concentration was tested in triplicates
in a single experiment, and each experiment was repeated at least
three times.
Antimicrobial activity of Lasalocid complexes: LAS–PhA, LAS–BuA and Lasalocid acid
(LAS), diameter of the growth inhibition zone (Giz, mm) and Minimal Inhibitory
Concentration (MIC,
lg/ml).
LAS
Giz
LAS–PhA
LAS–BuA
2.6.3. MTT assay
MIC
Giz
MIC
Giz
MIC
After 72 h of incubation of the agents tested with leukaemia
S. aureus NCTC 4163
S. aureus ATCC 25923
S. aureus ATCC 6538
S. aureus ATCC 29213
S. epidermidis ATCC 12228
B. subtilis ATCC 6633
B. cereus ATCC 11778
E. hirae ATCC 10541
E. faecalis ATCC 29212
M. luteus ATCC 9341
M. luteus ATCC 10240
30
29
24
24
28
31
29
20
25
28
31
4
4
4
4
4
2
2
8
4
4
4
28
29
23
23
26
28
30
21
25
27
31
4
2
2
2
4
2
2
4
2
2
2
25
27
21
22
26
26
30
20
25
26
29
4
4
4
4
8
2
4
8
4
4
4
cells, 20 ll of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazo-
lium bromide solution (Sigma–Aldrich, Germany) were added to
each well and cells were incubated for (another) 4 h at 37 °C as de-
scribed above. Viable cells metabolize yellow MTT to navy blue for-
mazan, so that the more viable cells in the well the more formazan
is produced. Then, 80 ll of the lysing mixture, consisting of 225 ml
dimethylformamide, 67.5 g sodium dodecyl sulphate (both from
Sigma–Aldrich, Germany) and 275 ml of distilled water, were
added to each well. After 24 h, when the formazan crystals had
been dissolved, optical densities of all solutions were read on the
Multiskan RC photometer (Labsystems) at 570 nm wavelength.
2.6.2. The antiproliferative assays in vitro
The antiproliferative activity of all compounds tested was eval-
uated after 72 h exposure of cultured cells to four different concen-
trations (in the range 100 lg/ml–0.1 lg/ml) of each agent and to
reference drug cisplatin at similar concentrations in a positive con-
trol of the test. Additionally, the cells were exposed to DMSO (Sig-
ma–Aldrich, Germany), solvent used to dissolve the compounds
tested, at the same concentrations as it was present in examined
agents’ concentrations.
2.6.4. SRB assay
Adherent cells cultured in 96-well plates were fixed by gently
layering of 50% cold trichloroacetic acid TCA (POCH, Poland) on
the top of culture medium and incubated at 4 °C for 1 h. All wells
were washed then four times with water. Then, fixed cells were
stained with 0.4% sulforodamine B solution (Sigma–Aldrich, Ger-
many) in 1% acetic acid (POCH, Poland) for 0.5 h and then each well
was washed with 1% acetic acid and protein bound dye was
Table 5
Cytotoxicity of Lasalocid acid (LAS) and its complexes with phenylamine (LAS–PhA) and n-butylamine (LAS–BuA) against various cell lines.
Compound
IC50
(l
g/ml)
A-549
MCF-7
HT-29
HLMEC
BALB/3T3
P388
LAS
1.35 0.25
1.32 0.10
1.36 0.12
3.35 0.30
4.51 1.83
4.17 0.61
4.08 0.64
3.71 0.62
2.99 1.07
3.06 0.55
3.12 0.54
11.18 1.98
0.45 0.20
0.37 0.02
0.39 0.06
1.95 0.13
5,01 1,88
4,57 1,17
5,12 1,02
1,81 0,67
2.13 0.90
2.14 0.86
2.72 0.46
2.98 0.08
LAS–BuA
LAS–PhA
Cisplatin
Values are mean values SD measured using SRB or MTT assay from at least three independent experiments.

´
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A. Huczynski et al. / Journal of Molecular Structure 1032 (2013) 69–77
extracted with 10 mM TRIS base (Sigma–Aldrich, Germany) for
optical density reading of each well in Multiskan RC photometer
(Labsystems, Helsinki, Finland) at 540 nm wavelength.
The results are presented as the IC50 values: the concentration
of the agent tested which inhibits proliferation of 50% of cells pop-
ulation. IC50 values were calculated in Cheburator 0.4, Dmitry Nev-
ozhay software for each experiment and the results are presented
as a mean IC50 standard deviation calculated from each experi-
ments performed. The results are summarized in Table 5.
In the spectrum of LAS–BuA complex, the bands with maxima at
3379 cmꢀ1 and 3166 cmꢀ1 can be assigned to O(8)AHꢂ ꢂ ꢂO(1)ꢀ and
O(4)AHꢂ ꢂ ꢂO(2)ꢀ intramolecular hydrogen bonds on the basis of the
hydrogen bonds parameters collected in Table 2.
The most important information provided by the FT-IR spectra
concerning the structures of LAS–PhA and LAS–BuA complexes is
included in the region 1750–1500 cmꢀ1 (Fig. 3c). In the spectrum
of Lasalocid acid (dashed line) the band assigned to the
m(C@O)
vibrations of the carboxylic group is observed at 1652 cmꢀ1. In
the spectra of both complexes (solid and dashed-dotted lines,
respectively) this band is no longer observed and instead a new
complex band arises in the region below 1600 cmꢀ1 with maxima
at 1587 cmꢀ1 and 1571 cmꢀ1. This new composite band is a super-
3. Results and discussion
À
þÁ
position of the
m
as(COOꢀ), d NH3 and
m
(C@C) vibrations and its
3.1. Crystal structure of the Lasalocid–Phenylamine (LAS–PhA) and
Lasalocid–Butylamine (LAS–BuA) complexes
presence indicates a proton transfer from the carboxylic group of
LAS to the NH2 group of the respective amines within the complex.
The band assigned to the m(C@O) vibrations of the ketone group
The crystals of Lasalocid–Phenylamine (LAS–PhA) and Lasalo-
cid–Butylamine (LAS–BuA) complexes are isostructural. Both com-
plexes crystallise in the same space group and the crystals have
very similar unit-cell dimensions (Table 1). The unit-cell dimen-
sion a is longer by about 0.5 Å in LAS–PhA, but parameter c is more
than 1 Å longer in LAS–BuA, and the unit-cell volumes of both com-
pounds differ in only 5 Å3. It is characteristic that LAS anions as-
sume very similar pseudo-cyclic conformations, with the same
system of hydrogen bonds OAHꢂ ꢂ ꢂO, as illustrated in Fig. 1 and in
Table 2. The shortest of OAHꢂ ꢂ ꢂO bonds links hydroxyl group
O(3)AH and carboxylate oxygen O(1) (Table 2). The other two
O(8)AHꢂ ꢂ ꢂO(1) and O(4)AHꢂ ꢂ ꢂO(2) intramolecular hydrogen bonds,
are similar in length in LAS–PhA and in LAS–BuA complexes, too.
Likewise, the complexes host the PhA and BuA cations in a similar
manner (see Table S1, Supplementary material). They are included
in the void inside the pseudo-cyclic structure, with practically
identical position of the nitrogen atom. The location of the N-atoms
is fixed by intermolecular NAHꢂ ꢂ ꢂO hydrogen bonds to LAS oxygen
atoms O(2), O(6) and O(8) (Fig. 1 and Table 2). These host-guest
NAHꢂ ꢂ ꢂO hydrogen bonds additionally stabilize the LAS conforma-
tion. The crystal packing of LAS–PhA and LAS–BuA is similar
(Fig. 2), and dominated by van der Waals interactions.
in Lasalocid and its both complexes is observed at ca. 1712 cmꢀ1
indicating that this group is not involved in any hydrogen bond.
Formation of protonated 1:1 LAS–BuA and LAS–PhA complexes
in chloroform solution is indicated by their ESI-MS spectra (Fig. S1,
supplementary data) in which at cv = 10 V only one signal at m/
z = 664 and 684 respectively, characteristic of such complexes, is
observed.
Comparison of the FT-IR spectra of LAS–BuA complex in chloro-
form and in the solid state (Fig. 4) shows small changes associated
only with changes in hydrogen bond strength, indicating that in
the solution the crystal structure is practically conserved. Protonic
vibrations within the O(8)AHꢂ ꢂ ꢂO(1)ꢀ and O(4)–Hꢂ ꢂ ꢂO(2)ꢀ hydro-
gen bonds are indicated by a broad band in the same region in
which the respective vibrations in the spectrum of the crystalline
LAS–BuA complex are found.
In Fig. 5 the spectra of LAS–PhA complex in chloroform and in
the solid state and the spectrum of LAS in solution are compared
showing very strong changes. The spectral features of the complex
in solution compared with those in the spectrum of LAS indicate
clearly a partial dissociation of the complex with formation of
Lasalocid acid. This statement is proved by the appearance of a
band at 1653 cmꢀ1, 3597 cmꢀ1 and a shoulder at 3430 cmꢀ1 as well
as by the 1H and 13C NMR data of LAS and its complexes given in
Table 3. Most informative are the signals of the C(1) atom of the
carboxylic group and its C(2) neighbouring atom. In the 13C NMR
spectrum of LAS the signal of C(1) atom is found at 173.2 ppm
and in the spectrum of LAS–BuA at 175.4 ppm due to the proton
transfer from the carboxylic group to the amine group. In the spec-
trum of LAS–PhA the respective signal is observed at 174.4 ppm
indicating that the proton transfer process is incomplete. The same
conclusion follows from a comparison of the chemical shifts of C(2)
carbon atom.
3.2. Spectroscopic studies
In Fig. 3 the FT-IR spectra of crystalline Lasalocid acid (dashed
line) and its crystalline complexes with phenylamine (LAS–PhA)
(solid line) and butylamine (LAS–BuA) (dashed-dotted line) are
compared. We observe clear differences between these structures,
especially regarding the formation of hydrogen bonds (Fig. 3b).
According to the X-ray data, in the spectrum of the LAS–PhA
and LAS–BuA complexes, the bands labelled in Fig. 3b should be as-
signed to the inter- and intramolecular hydrogen bonds of different
strength that exist within the structure of these complexes. The
hydrogen bonds and their parameters are given in Table 2. The
strongest hydrogen bond observed in both complexes is formed
between the O(3)AH group and the O(1) atom of the carboxylic
group, in LAS–BuA it is more symmetrical than in the LAS–PhA
structure. These intramolecular hydrogen bonds belong to the so
called quasi-aromatic hydrogen bonds [28] characterized by very
low intensity and limited shape of the band assigned to the
stretching protonic vibrations occurring usually in the region ca.
2200–3000 cmꢀ1 [29,30]. Thus, the broad band with maxima at
The analysis of the shift of the 13C NMR signal assigned to the
carbon atom of ketone group from 214.4 ppm (LAS) to 217.8 ppm
(LAS–BuA complex) or 216.3 ppm (LAS–BuA complex) can indicate
that oxygen atom of carbonyl group interacts with the hydrogen
atoms of protonated amines by weak hydrogen bond. On the other
hand, the band assigned to the C@O stretching vibrations in the FT-
IR spectra of both complexes shifts very slightly toward lower
wave numbers in comparison with the position of this band in
the FT-IR spectrum of LAS. Thus, it seems that carbonyl group is
À
Á
not engaged in the hydrogen bond formation with NHþ3 group
and the shifts of the 13C NMR signal carbonyl carbon atom is con-
nected with conformational changes of LAS molecule after com-
plexation of the respective amines.
À
Á
2595 cmꢀ1 and 2310 cmꢀ1 should be assigned to the
m
NHþ3 vibra-
tions of the protonated and hydrogen bonded amine group.
According to the crystal data (Table 2) in the spectrum of the
LAS–PhA complex, the bands with maxima at 3394 cmꢀ1 and
3062 cmꢀ1 are assigned to O(4)AHꢂ ꢂ ꢂO(2)ꢀ and O(8)AHꢂ ꢂ ꢂO(1)ꢀ
intramolecular hydrogen bonds, respectively.
The dissociation of LAS–PhA complex in solution is also con-
À
Á
firmed by the proton signals assigned to NHþ3 in the spectrum
of the complex at 9.34 ppm and of the free, non-hydrogen-bonded
NH2 protons of the aniline molecule at 3.80 ppm. The dissociation

´
74
A. Huczynski et al. / Journal of Molecular Structure 1032 (2013) 69–77
Fig. 1. A perspective view of: (a) LAS–PhA and (b) LAS–BuA complexes in the crystal structure. The hydrogen bonds have been indicated by dashed lines.
of the LAS–PhA complex is not surprising because phenylamine
(aniline) belongs to very weak N-bases (pKa = 4.6).
complexes with phenylamine and butylamine against typical
Gram-positive cocci, Gram-negative rods and yeast-like
organisms.
3.3. Antimicrobial activity evaluation of the compounds
The data concerning the antimicrobial activity of the com-
pounds studied are summarized in Table 4. Lasalocid acid (LAS),
and its complexes LAS–PhA and LAS–BuA are very active against
Gram-positive bacteria. LAS–PhA shows slightly higher antibacte-
rial activity in comparison to that of Lasalocid (Table 4). The anti-
bacterial activity of LAS and LAS–BuA is similar. Lasalocid acid,
pure amines and their complexes are inactive against strains of
Candida and Gram-negative bacteria.
Preliminary tests of the antimicrobial activity of Lasalocid
acid have shown that it is active against Gram-positive standard
bacterial strains [23]. Recently, we have proved that the com-
plex of Lasalocid acid with allylamine exhibited higher antibac-
terial activity than pure Lasalocid acid [23]. This result inspired
us to verify in vitro the antibacterial activity of other Lasalocid

´
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A. Huczynski et al. / Journal of Molecular Structure 1032 (2013) 69–77
100
50
(a)
LAS-PhA
LAS
LAS-BuA
0
4000
3500
3000
2500
2000
1500
1000
500
100
3394
50
2310
3166
2595
3379
(b)
3062
0
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800
100
50
0
(c)
1652
1571
1587
1600
Wavenumber [cm-1]
1712
1700
1500
1400
Fig. 3. The FT-IR spectra of crystals of: (---) LAS, (––) 1:1 LAS–PhA and (-ꢂ-) 1:1
LAS–BuA complexes in nujol/fluorolube mulls in the ranges: (a) 4000 ꢀ 400 cmꢀ1
;
(b) 3800 ꢀ 1800 cmꢀ1 and (c) 1750 ꢀ 1400 cmꢀ1
.
(human lung microvascular endothelial cells), P-388 (murine leu-
kemia), Balb3T3 (mouse embryonic fibroblasts). In view of the po-
tential clinical application of Lasalocid in combination with
different chemotherapeutic drugs, this study was undertaken to
examine the antitumor activity of Lasalocid in combination with
various amines such as aromatic amine – phenylamine (PhA) and
aliphatic amine – butylamine (BuA), which form relatively stable
and structurally well defined complexes discussed above.
As shown in Table 5, all compounds tested showed moderate to
potent anticancer activities against all the cells selected. All the
compounds are very promising because they are outranged of
the positive control cisplatin. Lasalocid acid and its complexes
showed almost equipotent activity with cisplatin only against
P388 and MCF-8 cell lines. All compounds tested revealed stronger
cytotoxicity against A-549, HT-29, HLMEC cell lines, about three-
fold stronger than that of the positive control cisplatin.
4. Conclusions
Fig. 2. Autostereographic projections [32] of complexes: (a) LAS–PhA and (b) LAS–
BuA structures, viewed along the 001 crystal direction.
Two new complexes of ionophore antibiotic Lasalocid acid (LAS)
and amines such as phenylamine (PhA) and butylamine (BuA) have
been synthesised and their molecular structures have been fully
characterized using crystallographic and spectroscopic methods.
In the solid state both complexes of Lasalocid acid are com-
pletely deprotonated and the amine groups are protonated. The
structures of the complexes are stabilized by the intra- and inter-
molecular hydrogen bonds formed between the Lasalocid anion
3.4. Cytotoxic activity evaluation of the compounds
The cytotoxic activity of Lasalocid acid (LAS) and its complexes
(LAS–PhA and LAS–BuA) was tested against 6 different cancer cell
lines: A-549 (human lung adenocarcinoma), MCF-7 (human breast
adenocarcinoma), HT-29 (human colon adenocarcinoma), HLMEC
À
Á
and the respective protonated amine. The NHþ3 protons of the

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76
A. Huczynski et al. / Journal of Molecular Structure 1032 (2013) 69–77
100
50
0
100
50
LAS-PhA
LASA in CHCl
3
LAS-BuA crystal
LAS-BuA in CHCl
(a)
(a)
(b)
LAS-PhA in CHCl
3
3
0
4000
3500
3000
2500
2000
1500
1000
500
4000
100
3500
3000
3394
2500
2000
1500
1000
500
100
50
0
3298
3168
50
2310
2595
2310
3597
3373
3430
2595
3062
3178
3230
3379
(b)
3166
0
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800
100
100
1653
50
0
50
0
1617
1595
1614
1653
(c)
1571
1592
1600
1572
(c)1709
1587
1600
Wavenumber [cm-1]
1700
1500
1400
1750
1700
1650
1550
1500
1450
1400
Wavenumber [cm-1]
Fig. 5. The FT-IR spectra of the crystals of (—) 1:1 LAS–PhA complex in nujol/
fluorolube mulls, (-ꢂ-) LAS–PhA complex in chloroform solution and (---) LAS in
Fig. 4. FT-IR spectra of the crystals of (-ꢂ-) 1:1 LAS–BuA complex in nujol/fluorolube
mulls and (—) LAS–BuA complex in chloroform solution: (a) 4000 ꢀ 400 cmꢀ1; (b)
chloroform solution: (a) 4000 ꢀ 400 cmꢀ1
;
(b) 3800 ꢀ 1800 cmꢀ1 and (c)
1750 ꢀ 1400 cmꢀ1
.
3800 ꢀ 1800 cmꢀ1 and (c) 1750 ꢀ 1400 cmꢀ1
.
HLMEC cell line may suggest potential antiangiogenic activity. On
the other hand, their moderate activity against normal fibroblast
cell line may predict its lower toxicity in further studies in vivo.
The structure activity relationship study revealed that the com-
pounds holding the aromatic amine species showed cytotoxicity
similar to that of aliphatic amine species and slightly lower than
that of pure Lasalocid acid.
The compounds reported in this paper could serve as potential
leads for cytotoxic activity. Further investigation is needed to rec-
ognize the exact mechanism of action of these molecules which is
clearer after their structural characterizations preformed in this
paper.
protonated amine molecules are hydrogen bonded with the etheric
oxygen atom O(6), the hydroxyl oxygen atom O(8), and one car-
boxylate oxygen atom O(2) of the LAS anion. In both complexes
the O(1) oxygen atom of the carboxylate group is involved in a rel-
atively strong intramolecular quasi-aromatic O(1)AHꢂ ꢂ ꢂO(3)
hydrogen bond. The pseudo-cyclic structure of Lasalocid anion is
stabilized by two O(4)AHꢂ ꢂ ꢂO(2) and O(8)AHꢂ ꢂ ꢂO(1) intramolecu-
lar hydrogen bonds binding the ends of the LAS anion.
In chloroform solutions the crystal structure of the LAS–BuA
complex is preserved and the equilibrium between LAS–PhA com-
plex and its dissociated mixture of Lasalocid acid and phenylamine
is observed. Thus, in the solution the proton transfer process from
Lasalocid acid to phenylamine is incomplete.
All compounds were examined for their in vitro antimicrobial
activities against Gram-positive and Gram-negative bacteria and
Candida fungi. Lasalocid complexes with amines (LAS–PhA and
LAS–BuA) holding the aromatic amine moiety and the aliphatic
amine moiety showed significant antibacterial activity against
Gram-positive bacteria compared to that of Lasalocid. All com-
pounds showed negative antifungal activity and antibacterial
activity against Gram-positive bacteria. For the first time, cytotox-
icity studies of Lasalocid and its new complexes with amines were
carried out against A-549 (lung), MCF-7 (breast), HT-29 (colon) and
P-388 (leukemia) cancer cell lines as well as normal BALB/3T3
(murine embryonic fibroblasts) and HLMEC (human lung micro-
vascular endothelial) cell lines.
Acknowledgments
Financial support from budget funds for science in years 2012-
2013 – Grant ’’Iuventus Plus’’ of the Polish Ministry of Science and
Higher Education-No. 0179/IP3/2011/71, is gratefully acknowl-
´
edged by Adam Huczynski.
Appendix A. Supplementary material
Supplementary data (exemplary NMR, ESI-MS spectra and Ta-
bles) associated with this article can be found, in the online
version.
Lasalocid and their complexes show good cytotoxic activity and
outperform the reference drug cisplatin against all cancer cell lines
examined. Moreover, their high antiproliferative activity against
CCDC 867826 and 867827 contains the supplementary crystal-
lographic data for complex LAS-PhA and complex LAS-BuA. These
data can be obtained free of charge via http://www.ccdc.cam.a-

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A. Huczynski et al. / Journal of Molecular Structure 1032 (2013) 69–77
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