Biological activity and molecular structures of bis(benzimidazole) and trithiocyanurate complexes

Article abstract of DOI:10.3390/molecules200610360

1-(1H-Benzimidazol-2-yl)-N-(1H-benzimidazol-2-ylmethyl)methanamine (abb) and 2-(1H-benzimidazol-2-ylmethylsulfanylmethyl)-1H-benzimidazole (tbb) have been prepared and characterized by elemental analysis. These bis(benzimidazoles) have been further used in combination with trithiocyanuric acid for the preparation of complexes. The crystal and molecular structures of two of them have been solved. Each nickel atom in the structure of trinuclear complex [Ni₃(abb)₃(H₂O)₃(μ-ttc)](ClO₄)₃?3H₂O?EtOH (1), where ttcH3 = trithiocyanuric acid, is coordinated with three N atoms of abb, the N,S donor set of ttc anion and an oxygen of a water molecule. The crystal of [(tbbH₂)(ttcH₂)₂(ttcH₃)(H₂O)] (2) is composed of a protonated bis(benzimidazole), two ttcH₂ anions, ttcH3 and water. The structure is stabilized by a network of hydrogen bonds. These compounds were primarily synthesized for their potential antimicrobial activity and hence their possible use in the treatment of infections caused by bacteria or yeasts (fungi). The antimicrobial and antifungal activity of the prepared compounds have been evaluated on a wide spectrum of bacterial and yeast strains and clinical specimens isolated from patients with infectious wounds and the best antimicrobial properties were observed in strains after the use of ligand abb and complex 1, when at least 80% growth inhibition was achieved.

Full text of DOI:10.3390/molecules200610360

Molecules 2015, 20, 10360-10376; doi:10.3390/molecules200610360
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Biological Activity and Molecular Structures of
Bis(benzimidazole) and Trithiocyanurate Complexes
Pavel Kopel 1,2,*, Dorota Wawrzak 3, Vratislav Langer 4, Kristyna Cihalova 1,2
Dagmar Chudobova 1,2, Radek Vesely 5, Vojtech Adam 1,2 and Rene Kizek 1,2
,
1
Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno,
Zemedelska 1, Brno CZ-613 00, Czech Republic; E-Mails: kriki.cihalova@seznam.cz (K.C.);
dagmar.chudobova@centrum.cz (D.C.); vojtech.adam@mendelu.cz (V.A.);
kizek@sci.muni.cz (R.K.)
2
3
Central European Institute of Technology, Brno University of Technology, Technicka 3058/10,
Brno CZ-616 00, Czech Republic
Institute of Chemistry, Environmental Protection and Biotechnology,
Jan Dlugosz University of Czestochowa, Armii Krajowej 13/15, Czestochowa PL-42-201, Poland;
E-Mail: d.wawrzak@ajd.czest.pl
4
5
Environmental Inorganic Chemistry, Department of Chemical and Biological Engineering,
Chalmers University of Technology, Göteborg SE-412 96, Sweden; E-Mail: langer@chalmers.se
Department of Traumatology at the Medical Faculty, Masaryk University and Trauma Hospital of Brno,
Ponavka 6, Brno CZ-662 50, Czech Republic; E-Mail: ves.radek@seznam.cz
* Author to whom correspondence should be addressed; E-Mail: paulko@centrum.cz;
Tel.: +420-5-4513-3350; Fax: +420-5-4521-2044.
Academic Editor: Derek J. McPhee
Received: 31 March 2015 / Accepted: 29 May 2015 / Published: 4 June 2015
Abstract: 1-(1H-Benzimidazol-2-yl)-N-(1H-benzimidazol-2-ylmethyl)methanamine (abb)
and 2-(1H-benzimidazol-2-ylmethylsulfanylmethyl)-1H-benzimidazole (tbb) have been
prepared and characterized by elemental analysis. These bis(benzimidazoles) have been
further used in combination with trithiocyanuric acid for the preparation of complexes. The
crystal and molecular structures of two of them have been solved. Each nickel atom in the
structure of trinuclear complex [Ni3(abb)3(H2O)3(μ-ttc)](ClO4)3·3H2O·EtOH (1), where
ttcH3 = trithiocyanuric acid, is coordinated with three N atoms of abb, the N,S donor set of
ttc anion and an oxygen of a water molecule. The crystal of [(tbbH2)(ttcH2)2(ttcH3)(H2O)]
(2) is composed of a protonated bis(benzimidazole), two ttcH2 anions, ttcH3 and water. The

Molecules 2015, 20
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structure is stabilized by a network of hydrogen bonds. These compounds were primarily
synthesized for their potential antimicrobial activity and hence their possible use in the
treatment of infections caused by bacteria or yeasts (fungi). The antimicrobial and antifungal
activity of the prepared compounds have been evaluated on a wide spectrum of bacterial and
yeast strains and clinical specimens isolated from patients with infectious wounds and the
best antimicrobial properties were observed in strains after the use of ligand abb and
complex 1, when at least 80% growth inhibition was achieved.
Keywords: coordination compounds; biological activity; trithiocyanuric acid;
trimercaptotriazine; single crystal X-ray diffraction; benzimidazole
1. Introduction
Heterocyclic compounds are very often used in the development of novel drugs with new mechanisms
of action. Among heterocyclic compounds, arylaziridines [1] and benzimidazole derivatives have very
important role because of their wide spectrum of biological activities. Benzimidazole and its derivatives are
reported to be physiologically and pharmacologically active and some applications are found in the
treatment of several diseases including epilepsy, diabetes and infertility. These compounds show a wide
range of biological activities like antibacterial, antifungal, antitubercular [2], antimalarial [3], anti-
inflammatory, analgesic, antiamoebic [4] antihistamine [5], antiulcerative, antioxidant [6,7],
antiproliferative [8], antihypertensive [9], antiallergic [10], antitumor [11–13], antikinase and anti-HIV-1
properties [14]. Some benzimidazoles have also been evaluated as cholinesterase inhibitors [15,16] and
drugs against parasites [17,18].
Trithiocyanuric acid (ttcH3), also referred as trimercaptotriazine, has a symmetric structure with three
nitrogen atoms and three sulphur atoms in a ring (see Figure 1). These donor atoms can be used for
coordination to central atoms and can also form bridges among central atoms. Trithiocyanuric acid is
used as remediation agent for the removal of heavy metals from industrial wastewaters [19,20].
A very important application of ttcNa3 is the removal of residual palladium from reaction mixtures,
especially in drug production [21,22]. It was also applied in plating processes, in the production of
composite materials containing metals and rubbers and as an anticorrosion agent [23,24].
The biological activity of trithiocyanuric compound was also evaluated as it can serve as a ligand of
Toxoplasma gondii orotate phosphoribosyltransferase [25]. Trinuclear ruthenium complexes including
[(Ru(L)2)3(μ-ttc)](ClO4)3 (L = phenylazopyridine) with proved tris-S,N chelating mode were characterized
by Kar et al. [26,27]. The interactions of the trinuclear complexes with the circular and linear forms
of
p-Bluescript DNA show reduced ethidium bromide fluorescence on gel electrophoresis. Some
polynuclear zinc complexes with macrocyclic ligands and ttc3− were studied by
very interesting
Aoki et al. [28–30]. The (Zn4(L4)3(ttc)4)12+ complex (L = cyclen = 1,4,7,10-tetraazacyclododecane), with
a trigonal prism configuration is very stable in aqueous solution at neutral pH. The complex does not
dissociate into the starting building blocks in the presence of Zn2+ binding anions such as phosphates
and double-stranded DNA. The results of the competitive binding assays with ethidium bromide and
calf-thymus DNA, thermal melting experiments, and gel mobility shift assays altogether indicated that

Molecules 2015, 20
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the complex induced the aggregation of double-stranded DNA. The antitumor and antimicrobial
activities were evaluated for Ni(II), Fe(II) and Mn(II) trithiocyanurate complexes [31]. The antitumor
activity of the complexes was assayed in vitro against G-361 (human malignant melanoma), HOS
(human osteogenic sarcoma), K-562 (human chronic myelogenous leukaemia) and MCF-7 (human
breast adenocarcinoma) tumour cell lines. The IC50 values of the Fe(II) and Mn(II) compounds turned
out to be lower than those of cisplatin and oxaliplatin. The complexes of Fe(II), Mn(II) and Ni(II) with
a combination of a Schiff base, nitrogen-donor ligand or macrocyclic ligand and trithiocyanuric acid
were prepared and their anticholinesterase activity was studied [32]. It was shown that the tested
compounds inhibit cholinesterases and can be considered as potential drugs for Alzheimer’s disease or
as prophylactics in the case of nerve agent exposure or poisoning by pesticides.
abb
ttcH3 thione – thiol
Complex cation 3
tbb
Figure 1. Structural formulas of the ligands used and the complex cation
[Ni3(pmdien)3(μ-ttc)](ClO4)3 (3). abb = 1-(1H-benzimidazol-2-yl)-N-(1H-benzimidazol-2-
ylmethyl)methanamine, tbb
=
2-(1H-benzimidazol-2-ylmethylsulfanyl-methyl)-1H-
benzimidazole, ttcH3 = trithiocyanuric acid in its thione and thiol forms.
In this paper, we present the possible application of some bis(benzimidazole) (their formulas are given in
Figure 1) and trithiocyanuric complexes in the treatment of severe and difficult to treat bacterial or yeast
infections. The major aim of this study was to test the biological activity of these compounds
on the commercially supplied microorganisms (Staphylococcus aureus, Escherichia coli and
Saccharomyces cerevisiae) and three bacterial strains isolated from patients from non-healing wounds
infected by bacterial strains (Streptococcus agalactiae, Corynebacterium striatum and Serratia marcescens).
For the microbiological testing, an already known and well characterized coordination compound,
[Ni3(pmdien)3(μ-ttc)](ClO4)3 (3), where pmdien = N,N,N′,N′′,N′′-pentamethyldiethylenetriamine, was
also prepared according to published method [33]. The complex 3 has a similar structure (see Figure 1) to
1 and it was of interest to compare their biological activities. The successful preparation of the ligands was
unambiguously proved by single crystal X-ray analysis of two compounds.

Molecules 2015, 20
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2. Results and Discussion
2.1. Synthesis and Characterization
Bis(benzimidazole) ligands abb and tbb were prepared by well-known methods. This method is time
consuming but it gave good yields, especially in the case of tbb. The composition of both compounds was
confirmed by elemental analyses and moreover by single crystal X-ray analysis of reaction product with
nickel salt and ttcH3, respectively. The trinuclear complex [Ni3(abb)3(H2O)3(μ-ttc)](ClO4)3·3H2O·EtOH (1)
was prepared by the reaction of nickel perchlorate, abb ligand and very slow addition of ttcNa3 in an
ethanol‒water mixture. Previously, we used the same strategy for the preparation of similar trinuclear
complexes just like in case of [Ni3(pmdien)3(μ-ttc)](ClO4)3 (3). Without the presence of terdentate ligand
abb a polymeric poorly soluble precipitate should be formed. The perchlorate salt was shown to be a
good counterion in preparation of bi- or trinuclear complexes. The composition of 1 was proposed on
the basis of the elemental analysis and confirmed by single crystal X-ray analysis. The formation of
co-crystals between organic compounds containing N, S or O atoms is well known. For example,
Ranganathan characterized channel structures of melamine with ttcH3 [34] and recently co-crystals of
N-donor ligands (4,4′-bipyridine, 1,2-bis-(4-pyridyl)ethylene, 1,7-phenanthroline) and ttcH3 were reported
by Nagarajan [35]. In our work, we succeeded in preparation of [(tbbH2)(ttcH2)2(ttcH3)(H2O)] (2)
co-crystals by reaction of tbb with ttcH3 in EtOH solution. The formation of 2 was also confirmed by
X-ray structural analysis. The solubility of all compounds was checked. All the compounds are soluble
in methanol, ethanol, DMSO and DMF and insoluble in water. For the biological testing and cyclic
voltammetry measurements, the samples were dissolved in a small amount of DMSO and after that
diluted with water. The higher amount of DMSO was used to dissolve abb, tbb and molecular crystals of
2. The crystals of 2 are more soluble in comparison with abb and tbb. The stability of coordination
compounds 1 and 3 was proved by cyclic voltammetry measurements after 1, 2 and 3 days.
2.2. X-ray Structures of the Complexes [Ni3(abb)3(H2O)3(μ-ttc)](ClO4)3·3H2O·EtOH (1) and
[(tbbH2)(ttcH2)2(ttcH3)(H2O)] (2)
The molecular structure of 1 is depicted in Figure 2, while selected bond lengths and angles are listed
in Table 1. The crystal structure (see Figure 3) is stabilized by hydrogen bonds but hydrogen atoms
belonging to water molecules were not resolved. The molecular structure of 1 consists of trinuclear
−
nickel(II) complex, ClO4 anions, three crystal water and EtOH molecules. Three central nickel atoms
are coordinated by three N atoms of abb, a water molecule O atom and the S,N-donor set of a ttc trianion
in a deformed octahedral arrangement. The ttc trianion forms a bridge connecting the nickel centers.
The bond lengths of the abb ring nitrogens to the central atoms are in the range of 2.041(3)–2.062(4) Å,
abb chain Ns are in the range of 2.101(4)–2.121(3) Å and ttc N are in the range of 2.030(3)–2.047(3) Å,
that is significantly longer bond lengths are observed for the Ni-N(abb chain). The Ni-S bond lengths
are quite similar (2.5549(12)–2.5622(11) Å) and these lengths are much longer than Ni-S bonds found
in pentacoordinated complexes [Ni(pmdien)(ttcH)] (2.3392(13) Å) [36] and [Ni3(pmdien)3(μ-
ttc)](ClO4)3 (2.3821(9) Å) [33], respectively. A very complicated situation occurs in the heptanuclear
complex [Ni7(pmdien)6(H2O)2(μ-ttc)3](ClO4)5·3H2O [37]. In this complex the central nickel atom is
coordinated by three ttc anions only with Ni-S bond lengths in the 2.4663–2.4740 Å range. Two nickel

Molecules 2015, 20
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atoms are hexacoordinated (three N atoms of pmdien, a water molecule O atom and the S,N set of ttc)
with Ni-S lengths 2.4094 and 2.5150 Å, whereas the remaining four Ni atoms are pentacoordinated with
shorter Ni-S bond lengths in the 2.3643–2.4032 Å range. A longer Ni-S bond (2.573(2) Å) was found in
octahedral [Ni(dpta)(H2O)(ttcH)]·H2O, with terdentate dpta = dipropylenetriamine [38] and there is only
an interaction with a Ni-S distance 2.665 Å in [Ni(taa)(ttcH)], where taa = tris (2-aminoethyl)amine [39].
Table 1. Selected bond lengths (Å) and angles (°) for 1.
Ni1-N1B
Ni1-N1
2.041(3)
2.047(3)
2.057(3)
2.102(3)
2.121(3)
Ni2-N5
2.030(3)
2.056(4)
2.062(4)
2.101(4)
2.109(3)
Ni3-N1F
Ni3-N3
2.044(3)
2.043(3)
2.057(3)
2.100(3)
2.107(3)
100.24(13)
Ni2-N1C
Ni2-N1D
Ni2-N22
Ni2-O2
Ni1-N1A
Ni1-O1
Ni3-N1E
Ni3-O3
Ni1-N11
N1B-Ni1-N1
Ni3-N33
101.50(14) N5-Ni2-N1C
99.59(14) N1F-Ni3-N3
N1B-Ni1-N1A 159.97(15) N5-Ni2-N1D
98.94(13) N1F-Ni3-N1E 160.83(14)
N1-Ni1-N1A
N1B-Ni1-O1
N1-Ni1-O1
97.76(13) N1C-Ni2-N1D 161.06(14) N3-Ni3-N1E 97.92(13)
92.65(13) N5-Ni2-N22
170.79(14) N1F-Ni3-O3
90.79(14)
98.86(13)
97.07(12) N1C-Ni2-N22 80.76(15) N3-Ni3-O3
90.41(13) N1D-Ni2-N22 80.33(14) N1E-Ni3-O3
N1A-Ni1-O1
92.42(14)
N1B-Ni1-N11 80.27(14) N5-Ni2-O2
N1-Ni1-N11 165.69(14) N1C-Ni2-O2
N1A-Ni1-N11 79.71(14) N1D-Ni2-O2
96.53(12) N1F-Ni3-N33 79.92(14)
90.31(13) N3-Ni3-N33
163.52(14)
91.45(13) N1E-Ni3-N33 80.92(14)
O1-Ni1-N11
N1B-Ni1-S6
N1-Ni1-S6
N1A-Ni1-S6
O1-Ni1-S6
N11-Ni1-S6
97.03(13) N22-Ni2-O2
89.81(10) N5-Ni2-S4
92.67(14) O3-Ni3-N33
67.54(9) N1F-Ni3-S2
97.62(13)
88.42(11)
67.60(9)
67.36(9)
N1C-Ni2-S4
89.22(10) N3-Ni3-S2
94.26(10) N1E-Ni3-S2
103.30(11) O3-Ni3-S2
163.73(9) N33-Ni3-S2
92.54(10) N1D-Ni2-S4
164.40(9) N22-Ni2-S4
98.56(10) O2-Ni2-S4
92.90(10)
166.03(10)
95.98(10)
Figure 2. Numbering scheme of 1 with atomic displacement ellipsoids drawn at 30%
probability level. Hydrogen atoms are omitted for clarity.

Molecules 2015, 20
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Figure 3. Projection of the contents of the unit cell along a-axis for 1.
The molecular structure of [(tbbH2)(ttcH2)2(ttcH3)(H2O)] (2) is depicted in Figure 4, while selected
bond lengths and angles are listed in Table 2. The structure consists from protonated tbb, two ttcH2
anions, ttcH3 and a water molecule. The two benzimidazole rings of tbb form a sandwich-like structure
probably due to π-π interactions. The crystal structure is stabilized by weak hydrogen bonds
(see Supplementary Tables S1 and S2, and Figure 5). The tbb behaves as a strong base with hydrogen
atoms on all nitrogens but from the molecular structure, it is seen that in the structure there are tbb and
ttc planes connected through hydrogen bonds. The S-C bond lengths (1.810 and 1.828 Å) of tbb are in
the range typical of a single bond. The C-N bond lengths in five membered rings close to the bridge are
significantly shorter (in the 1.324–1.335 Å range) than the rest of bond lengths (range of 1.389–1.405 Å).
−
The S-C bond lengths of ttcH3 are in the 1.635–1.666 Å range, whereas those of ttcH2 are in the
1.647–1.681 Å and 1.645–1.692 Å range, respectively. Similar S-C bond lengths were found in the structure
of [Na(phen)3][(ttcH2)] [40] and [Fe(phen)3] (ttcH2)(ClO4) [41], where phen = 1,10-phenanthroline.
Figure 4. Numbering scheme of 2 with atomic displacement ellipsoids drawn at 30%
probability level.

Molecules 2015, 20
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Figure 5. Projection of the contents of the unit cell along a-axis for 2.
Table 2. Selected bond lengths (Å) and angles (°) for 2.
S1-C1A
S1-C1B
1.810(4) S1C-C1C
1.828(4) S2C-C2C
1.666(4)
1.635(4)
1.648(4)
1.656(4)
1.647(4)
1.681(4)
1.675(4)
1.692(4)
1.645(4)
C8A-C3A-N1A 106.9(3)
N2A-C8A-C3A 105.9(3)
C1A-C2A 1.490(5) S3C-C3C
N1A-C2A 1.332(5) S1D-C1D
N1A-C3A 1.405(5) S2D-C2D
N2A-C2A 1.335(5) S3D-C3D
N2A-C8A 1.389(5) S1E-C1E
C3A-C8A 1.404(5) S2E-C2E
C1B-C2B 1.485(5) S3E-C3E
N1B-C2B 1.331(5) C1A-S1-C1B
N1B-C3B 1.397(5) C2A-C1A-S1
C2B-C1B-S1
111.4(3)
109.4(3)
109.8(3)
C2B-N1B-C3B
C2B-N2B-C8B
N2B-C2B-N1B 109.0(3)
N1B-C3B-C8B 105.9(3)
N1C-C1C-N3C 115.0(3)
N2C-C2C-N1C 113.5(3)
101.20(18) N2C-C3C-N3C 114.1(3)
115.2(3) N1D-C2D-N2D 113.5(3)
N2E-C3E-N3E 113.4(3)
N2B-C2B 1.324(5) C2A-N1A-C3A 107.7(3)
N2B-C8B 1.392(5) C2A-N2A-C8A 109.1(3)
C3B-C8B 1.397(5) N1A-C2A-N2A 110.4(3)
2.3. Biological Activity
The mechanism of the action of ligands or complexes on bacteria is very similar. When these
compounds enter bacterial cells, they condense DNA to prevent DNA from replicating and cells from
reproducing [42]. The basic mechanism of the action is therefore among others based on membrane
protein inactivation, reduced transcription ability of mRNA into the vital cell proteins, inactivation of
cytochrome b and consequent bactericidal activity [43,44].
The biological activity of nickel(II) perchlorate hexahydrate, nickel(II) chloride hexahydrate, the
ligands abb, tbb, ttcNa3 and complexes 1–3 was studied. The results of the study are presented in Figure 6.
The antimicrobial effect of ligands and complexes was tested on commercial Staphylococcus aureus
(S. aureus), Escherichia coli (E. coli), Saccharomyces cerevisiae (S. cerevisiae) and on bacteria isolated
from the infected wounds Streptococcus agalactiae (S. agalactiae), Corynebacterium striatum
(C. striatum) and Serratia marcescens (S. marcescens).

Molecules 2015, 20
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S. aureus
250 µg/mL tbb
250 µg/mL complex (1)
250 µg/mL abb
S. agalactiae
250 µg/mL tbb
250 µg/mL complex (1)
250 µg/mL abb
A
D
250 µg/mL complex (2)
250 µg/mL complex (3)
250 µg/mL ttc Na3
250 µg/mL complex (2)
250 µg/mL complex (3)
250 µg/mL ttc Na3
0.3
0.2
0.1
0
0.3
0.2
0.1
0
0
6
12
Time (h)
18
24
0
6
12
Time (h)
18
24
C. striatum
250 µg/mL tbb
250 µg/mL complex (1)
250 µg/mL abb
E. coli
250 µg/mL tbb
250 µg/mL complex (1)
250 µg/mL abb
E
B
250 µg/mL complex (2)
250 µg/mL complex (3)
250 µg/mL ttc Na3
250 µg/mL complex (2)
250 µg/mL complex (3)
250 µg/mL ttc Na3
0.5
0.4
0.3
0.2
0.1
0
0.5
0.4
0.3
0.2
0.1
0.0
0
6
12
Time (h)
18
24
0
6
12
Time (h)
18
24
S. marcescens
250 µg/mL tbb
250 µg/mL complex (1)
250 µg/mL abb
S. cerevisiae
250 µg/mL tbb
250 µg/mL complex (1)
250 µg/mL abb
C
F
250 µg/mL complex (2)
250 µg/mL complex (3)
250 µg/mL ttc Na3
250 µg/mL complex (2)
250 µg/mL complex (3)
250 µg/mL ttc Na3
0.3
0.2
0.1
0
0.3
0.2
0.1
0
0
6
12
18
24
0
6
12
Time (h)
18
24
Time (h)
Figure 6. Testing of antimicrobial activity of 250 μg/mL concentration of ligands and
complexes after 24 h of treatment on: (A) S. aureus; (B) E. coli; (C) S. cerevisiae; (D)
S. agalactiae; (E) C. striatum; (F) S. marcescens.
The salts, ligands and complexes were applied in concentrations of 0; 3.9; 7.8; 15.6; 31.3; 62.5; 125
and 250 μg/mL on the three model organisms (representatives of Gram-negative and Gram-positive
bacteria and yeasts) and the three bacterial strains isolated from the patient’s wounds and the effect was
subsequently evaluated spectrophotometrically by decreasing the absorbance values in comparison with
the control strains without the treatment. The highest inhibition of all tested organisms growth was
observed after the treatment of ligand abb (S. aureus—97.8%, E. coli—94.4% and S. cerevisiae—
86.0%, S. agalactiae—80.8%, C. striatum—93.8% and S. marcescens—95.5%) and complex 1
(S. aureus—99.2%, E. coli—99.8% and S. cerevisiae—94.0%, S. agalactiae—94.4%, C. striatum—99.6%
and S. marcescens—88.7%), which has also in its structure the ligand abb (Figure 6A–F).
The eight increasing concentrations were also used to observe antimicrobial effect of each tested
compounds for the statistical analysis of the viability of bacterial cells. The IC50 values were calculated
from the absorbance measurements after the application of the concentration range in 24 h of measurements
(Table 3). By these values, the previous results of antimicrobial activity were confirmed, the lowest IC50
values were observed in case of ligand abb and complex 1. The other low values of IC50 were caused by
uneven inhibition after the increasing concentrations of applied components. The nickel salts did not
inhibit the growth of the test organisms except for C. striatum. The growth of this bacterium was inhibited
and the IC50 values 16.5 and 160.3 μg/mL were calculated for nickel perchlorate and nickel
chloride, respectively.

Molecules 2015, 20
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Table 3. Statistical calculation of IC50 by the STATISTICA software version 10.0 from
absorbance values after the application of 0; 7.8; 15.6; 31.3; 62.5; 125 and 250 μg/mL
concentrations of tested compounds.
IC50 (μg/mL) Measurement after 24 h
Compounds
S. aureus E. coli S. cerevisiae S. agalactiae C. striatum S. marcescens
Ligand abb
Ligand tbb
Ligand ttcNa3
Complex 1
Complex 2
Complex 3
5.6
485.2
625
19
21.4
204.7
86.4
15.4
64.3
18.2
22.4
126.1
119.5
21.4
30.8
112.5
115.8
24.3
26.7
122.6
123.9
27.2
182.3
636.8
23.7
4.8
562.3
1036.7
257.6
12.6
102.1
113.4
119.5
124.9
61.5
110.4
The highest antimicrobial activity on all studied microorganisms was observed for abb and complex 1.
The IC50 values are in these cases comparable although the synergistic effect of nickel and ligands in 1
must play an important role when we take in to account the amount of abb in the tested samples. When
we compare the activities of abb and tbb, which only differ in the chain atoms in the middle (N or S),
the activity of tbb is very low. The combination of tbb and ttc in complex 2 leads to a better activity
against S. cerevisiae only, but the sodium salt of trithiocyanuric acid shows similar activity. The
coordination compound 3 was prepared to compare the effect of ligands pmdien and abb, which are
coordinated to central nickel atoms. The trinuclear complexes 1 and 3 are structurally very similar
although nickel atoms in 1 are octahedral due to the presence of coordinated water molecules whereas
the bulky pmdien ligand in 3 does not allow coordination of water and the nickel atoms are
pentacoordinated. These changes in coordination modes and ligands also lead to different biological
properties of the complexes. Complex 1 is active against all the treated microorganisms whereas complex
3 shows no activity against S. aureus, but it shows better activity against E. coli and similar activity against
S. cerevisiae in comparison with 1.
3. Experimental Section
3.1. General Information
Safety note: Caution! Perchlorate salts of metal complexes with organic ligands are potentially
explosive and should be handled with great care.
The chemicals and solvents were supplied by Aldrich (St. Louis, MO, USA) and used without further
purification. The C, H, N, and S analyses were carried out on an EA 1108 instrument (Fisons
Instruments, Rodano, Italy). IR spectra (400–4000 cm−1) were recorded with the help of a Tensor 27
FTIR spectrometer (Bruker, Billerica, MA, USA) using KBr pellets. The absorption spectra in MetOH
(230–1100 nm) were obtained on a Specord 210 UV/VIS spectrometer (Analytik Jena AG, Jena,
Germany). The crystallographic data for the structures 1 and 2 has been deposited with the Cambridge
Crystallographic Data Centre as supplementary publications No. 1033059 and 1033060. Copies of the data
can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK, (Fax:
+44-0-1223-336033 or E-Mail: deposit@ccdc.cam.ac.uk).

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3.2. Preparation of the Compounds
3.2.1. 1-(1H-Benzimidazol-2-yl)-N-(1H-benzimidazol-2-ylmethyl)methanamine (abb)
The ligand was prepared according to Matthews et al. [45]. Iminodiacetic acid (6.65 g, 50 mmol) and
o-phenylenediamine (10.82 g, 100 mmol) were mixed in a solution of 18% HCl (100 mL).
The solution was heated under reflux for 3 days. A green precipitate formed after cooling was dissolved
after the addition of water (300 mL). The solution was stirred and ammonia (28%) was added until the
pH of the solution became 9. During the addition of ammonia, a light brown precipitate was obtained.
The precipitate was collected on a frit, washed several times with water and dried at 40 °C. Yield: 32.3%.
Anal. Calcd. for abb·4H2O: C, 55.0; H, 6.6; N, 20.0; S, 0.0. Found: C, 55.0; H, 6.6; N, 19.5; S, 0.0%.
IR (cm−1): 405w, 447w, 480w, 533w, 742vs, 767m, 876m, 1015m, 1110m, 1222m, 1273vs, 1310w,
1331m, 1438vs, 1454s, 1532w, 1623m, 2851m, 3055s, 3226s, 3368m. UV-Vis (MeOH/nm): 324,
378, 428.
3.2.2. 2-(1H-Benzimidazol-2-ylmethylsulfanylmethyl)-1H-benzimidazole (tbb)
The ligand was prepared according to Matthews et al. [45]. Thiodiacetic acid (15.02 g, 100 mmol)
and o-phenylenediamine (21.63 g, 200 mmol) were mixed in a solution of 18% HCl (230 mL). The
solution was heated under reflux 3 d. A green precipitate formed after cooling was dissolved after
addition of water (500 mL). The solution was stirred and ammonia (28%) was added until the pH of the
solution became 9. During the addition of ammonia, a light brown precipitate was obtained. The precipitate
was collected on a frit, washed several times with water and dried at 40 °C. Yield: 32.3%. Anal. Calcd.
for tbb·3/2H2O: C, 59.8; H, 5.3; N, 17.4; S, 10.0. Found: C, 60.1; H, 5.2; N, 17.2; S, 9.8%. IR (cm−1):
437w, 736vs, 844w, 1027m, 1129w, 1159m, 1226m, 1273s, 1313m, 1440vs, 1454s, 1535m, 1625w,
2784m, 2939s, 3059s, 3377m. UV-Vis (MeOH/nm): 290, 322, 340.
3.2.3. [Ni3(abb)3(H2O)3(μ-ttc)](ClO4)3·3H2O·EtOH (1)
The ligand abb (0.35 g, 1 mmol) was dissolved in EtOH (100 mL) and added with stirring to a solution
of nickel(II) perchlorate hexahydrate (0.36 g, 1 mmol) in 10 mL of EtOH. This red solution was heated
to boiling and after cooling ttcNa3·9H2O (0.13 g, 0.33 mmol) in water: EtOH (1:1) mixture was added
with stirring. A light red solution was left for crystallization. After a week, grey green plate crystals
suitable for X-ray analysis were collected. Yield: 64%. Anal. Calcd.: C, 38.9; H, 3.9; N, 15.4; S, 5.9.
Found: C, 38.7; H, 3.5; N, 15.6; S, 5.7%. IR (cm−1): 430w, 627s, 764s, 913m, 1088vs, 1111vs, 1144s,
1239m, 1322w, 1338w, 1437s, 1454s, 1542w, 1624m, 2918m, 3064br, 3193br, 3381s. UV-Vis
(MeOH/nm): 371, 492, 541, 585, 801, 997.
3.2.4. [(tbbH2)(ttcH2)2(ttcH3)(H2O)] (2)
The crystals of 2 were prepared by mixing tbb and ttcH3 ethanol solutions in 1:3 molar ratio. Yellow
needle-like crystals suitable for X-ray analysis were obtained after a week. Yield: 75%.
Anal. Calcd.: C, 35.6; H, 3.0; N, 21.6; S, 38.0. Found: C, 35.4; H, 3.1; N, 21.3; S, 37.7%. IR (cm−1):

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457m, 665w, 746w, 782w, 1120vs, 1258w, 1296w, 1360s, 1541vs, 1574s, 1715w, 2522w, 2907m,
3039m, 3142s, 3444br. UV-Vis (MeOH/nm): 300, 341.
3.2.5. [Ni3(pmdien)3(μ-ttc)](ClO4)3 (3)
The complex [Ni3(pmdien)3(μ-ttc)](ClO4)3, where pmdien = N,N,N′,N′′,N′′-pentamethyldiethylene-
triamine, was prepared according to Kopel et al. [33], by reaction of pmdien (0.2 mL, 1 mmol) with
Ni(ClO4)2·6H2O (0.37 g, 1 mmol) in EtOH, followed by the addition of a solution of ttcNa3·9H2O
(0.14 g, 0.33 mmol). Yield: 72%. Anal. Calcd.: C, 30.8; H, 6.0; N, 14.4; S, 8.2. Found: C, 30.4; H, 5.9;
N, 14.1; S, 7.7%.
3.3. X-ray Crystallography
X-ray data of 1 and 2 were collected on a SMART CCD diffractometer (Siemens, Madison, WI, USA)
with Mo-Kα radiation (λ = 0.71073 Å, graphite monochromator). The crystal was cooled to 173(2) K by
a flow of nitrogen gas using the LT-2A device. A full sphere of reciprocal space was scanned by
0.3 steps in ω with a crystal-to-detector distance of 3.97 cm. Preliminary orientation matrices were
obtained from the first frames using SMART [46]. The collected frames were integrated using the
preliminary orientation matrix which was updated every 100 frames. Final cell parameters were obtained
by refinement of the positions of reflections with I > 10σ (I) after integration of all the frames using
SAINT software [46]. The data were empirically corrected for absorption and other effects using the
SADABS program [47]. The structures were solved by direct methods and refined by full-matrix least
squares on all |F2| data using SHELXTL software [48].
X-ray data of 1: Important crystallographic parameters are as follows: C53H48Cl3N18O19S3Ni3,
wavelength 0.71073 Å, triclinic, space group Pī, a = 14.7409(7), b = 16.2290(8), c = 16.3145(8) Å,
α = 103.367(1)°, β = 108.538(1)°, γ = 93.087(1)°, volume 3565.7(3) Å3, Z = 2, density (calc.) 1.509 Mg/m3,
absorption coefficient 1.063 mm−1, F(000) = 1654, crystal size 0.42 mm × 0.32 mm × 0.08 mm, index ranges
−19 ≤ h ≤ 19, −21 ≤ k ≤ 21, −22 ≤ l ≤ 22, reflections collected/independent 51144/18385 (Rint = 0.0648),
data/restraints/parameters 18385/0/894, goodness-of fit on F2 = 1.027, final R1 (I > 2σ(I) data) = 0.0713,
wR2 = 0.2059, final R1 (all data) = 0.0986, wR2 = 0.2259. The largest peak and hole on the final difference
map were 2.900 and −1.379 e.Å−3.
X-ray data of 2: Important crystallographic parameters are as follows: C25H25N13OS10, wavelength
0.71073 Å, triclinic, space group Pī, a = 11.2463(10), b = 13.0425(12), c = 13.6109(13) Å, α = 74.757(2)°,
β = 77.406(2)°, γ = 65.299(2)°, volume 1736.6(3) Å3, Z = 2, density (calc.) 1.614 Mg/m3, absorption
coefficient 0.681 mm−1, F(000) = 868, crystal size 0.44 mm × 0.08 mm × 0.02 mm, index ranges
−13 ≤ h ≤ 13, −15 ≤ k ≤ 15, −16 ≤ l ≤ 16, reflections collected/independent 18662/6149 (Rint = 0.0682),
data/restraints/parameters 6149/0/448, goodness-of fit on F2 = 1.018, final R1 (I > 2σ(I) data) = 0.0558,
wR2 = 0.1327, final R1 (all data) = 0.0832, wR2 = 0.1443. The largest peak and hole on the final difference
map were 0.382 and −0.571 e.Å−3.

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3.4. Biological Activity Testing
3.4.1. Cultivation of Commercially Supplied Bacteria and Yeasts
Staphylococcus aureus (NCTC 8511), Escherichia coli (NCTC 13216) and Saccharomyces cerevisiae
(ATCC 9763) were obtained from the Czech Collection of Microorganisms, Faculty of Science, Masaryk
University (Brno, Czech Republic). The strains were stored as a spore suspension in 20% (v/v) glycerol
at −20 °C. Prior to use in this study, the strains were thawed and the glycerol was removed by washing
with distilled water. The composition of cultivation medium was as follows: glucose 10 g/L, tryptone 10 g/L
and yeast extract 5 g/L, sterilized MilliQ water with 18 MΩ. pH of the cultivation medium was adjusted
at 7.4 before sterilization. The sterilization of the media was carried out at 121 °C for 30 min in sterilizer
(Tuttnauer 2450EL, Beit Shemesh, Israel). The prepared cultivation media were inoculated with
bacterial cultures or yeasts into 25 mL Erlenmeyer flasks. After the inoculation, bacteria and yeasts were
cultivated for 24 h on a shaker at 600 rpm and 37 °C. The strains, cultivated under these conditions, were
diluted by cultivation medium to OD600 = 0.1 and used in the following experiments.
3.4.2. Preparation of Hospital Samples and Their Cultivation
Cohort of Patients with Bacterial Infections
For the evaluation, patients with surface or deep infection severity were selected. The patients were
with the age between 19 and 93 years. Enrollment of the patients into the clinical study was approved
by the Ethics Committee of Trauma Hospital in Brno.
Collection of Wound Swabs from Patients with Bacterial Infections
Smears were collected from the infected wounds with the agreement of the patients. The smear was
sampled by a rolling motion at the site of skin puncture using a sterile swab sampler. Subsequently,
the swab was removed into a tube with a semi-solid transport medium (inorganic salts, sodium
thioglycolate, 1% agar, activated charcoal) and carefully sealed and labeled. The marked tubes were
immediately examined microbiologically.
Cultivation of Clinical Specimens
The isolation of bacterial strains was performed using selective blood agar. The swab samples were
cultivated on blood agar with 10% of NaCl, blood agar with no other compounds [49], Endo agar [50]
and blood agar with amikacin [51]. These Petri dishes were cultivated for 24–48 h at 37 °C. For the
cultivation of all bacterial specimens TGY medium (glucose 1 g/L, tryptone 5 g/L, yeast extracts 2.5 g/L)
was used.
3.4.3. Determination of Growth Curves
The procedure for the evaluation of the antimicrobial effect of tested compounds consisted in
measuring of the absorbance using the apparatus Multiskan EX (Thermo Fisher Scientific, Vantaa,
Finland) and subsequent analysis in the form of growth curves. Bacteria and yeasts were cultivated in

Molecules 2015, 20
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GTY medium for 24 h with shaking and were diluted with GTY medium to absorbance 0.1 before
analysis using a Specord spectrophotometer 210 at a wavelength of 600 nm. On the microplate, these
cultures were mixed with various concentrations of four types of antibiotics (ampicillin, streptomycin,
penicillin and tetracycline) or S. aureus alone as a control for the measurements. The concentrations of
antibiotics were 0, 7.8, 15.6, 31.3, 62.5, 125 and 250 μg/mL. The total volume in the microplate wells
was always 300 μL. The measurements were carried out at time 0, then each half-hourly for 24 h at 37 °C
and a wavelength of 600 nm. The obtained values were analysed in a graphical form as growth curves
for each variant individually.
3.4.4. Statistical Analyses
The software STATISTICA (data analysis software system), version 10.0 (Statsoft Inc. ed., Tulsa,
OK, USA) was used for the data processing. The half-maximal concentrations (IC50) were calculated
from logarithmic regression of sigmoidal dose-response curve. The general regression model was used
to analyse differences between the combinations of the compounds.
4. Conclusions
In this work we present our results for bis(benzimidazole) and trithiocyanurate complexes and
their use as antimicrobial agents in the treatment of bacterial infections. For these purposes
bis(benzimidazoles) were synthesized and then further used for the preparation of coordination
compound and molecular crystals. The X-ray single crystal analysis proved the structures of trinuclear
Ni(II) complex and bis(benzimidazole)‒trithiocyanurate co-crystals. It was shown that these compounds
are biologically active. Especially the ligand abb and the complex 1, where the ligand is coordinated,
showed very good activities against the wide spectrum of commercially available bacterial and yeast
strains (S. aureus, E. coli and S. cerevisiae) and clinical specimens from real patients with non-healing
infections and complicated courses of treatment.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/20/06/10360/s1.
Acknowledgments
The financial support from the project CEITEC CZ.1.05/1.1.00/02.0068 is highly acknowledged.
The authors thank Radek Chmela for his skilful technical assistance and Amitava Moulick for language
correction. The authors thank Richard Sevcik for FTIR measurements.
Author Contributions
Pavel Kopel synthesized the ligands, participated in the design and coordination of the study and
drafted the manuscript. Dorota Wawrzak prepared complexes and participated in paper preparation.
Vratislav Langer characterized complexes using X-ray crystallography. Kristyna Cihalova and
Dagmar Chudobova tested the biological activity of the compounds and participated in preparation of
the manuscript. Radek Vesely took samples (smears) from the patients with bacterial infections.

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Rene Kizek participated in design and coordination of the study. Vojtech Adam participated in design
of study and in drafting the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the prepared compounds are available from the authors.
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