Antimicrobial activity of some thiourea derivatives and their nickel and copper complexes

Article abstract of DOI:10.3390/molecules14010519

Five thiourea derivative ligands and their Ni²⁺ and Cu ²⁺ complexes have been synthesized. The compounds were screened for their in vitro anti-bacterial activity using Gram-positive bacteria (two different standard strains of Staphyl

Full text of DOI:10.3390/molecules14010519

Molecules 2009, 14, 519-527; doi:10.3390/molecules14010519
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Antimicrobial Activity of Some Thiourea Derivatives and Their
Nickel and Copper Complexes
Hakan Arslan 1,2,*, Nizami Duran 3, Gulay Borekci 4, Cemal Koray Ozer 2 and Cevdet Akbay 1
1
Department of Natural Sciences, Fayetteville State University, Fayetteville, NC 28301, USA;
E-mail: cakbay@uncfsu.edu (C. A.)
2
Department of Chemistry, Faculty of Pharmacy, Mersin University, Mersin, TR 33169, Turkey;
3
Department of Microbiology, Faculty of Medicine, Mustafa Kemal University, Hatay, TR 31040,
Turkey; E-mail: nizamduran@hotmail.com (N. D.)
4
Health School, Mersin University, Mersin, TR 33169, Turkey; E-mail: gulay_borekci@yahoo.com
(G. B.)
* Author to whom correspondence should be addressed: E-mails: hakan.arslan.acad@gmail.com;
arslanh@mersin.edu.tr; harslan@uncfsu.edu.
Received: 2 December 2008; in revised form: 24 December 2008 / Accepted: 5 January 2009/
Published: 22 January 2009
Abstract: Five thiourea derivative ligands and their Ni2+ and Cu2+ complexes have been
synthesized. The compounds were screened for their in vitro anti-bacterial activity using
Gram-positive bacteria (two different standard strains of Staphylococcus aureus,
Staphylococcus epidermidis, Enterococcus faecalis, Streptococcus pyogenes, Bacillus
cereus) and Gram-negative bacteria (Esherichia coli, Pseudomonas aeruginosa,
Enterobacter cloacae, Proteus vulgaris, Enterobacter aerogenes) and in vitro anti-yeast
activity (Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis,
Candida parapsilosis). The minimum inhibitory concentration was determined for all
ligands and their complexes. In vitro anti-yeast activity of both ligands and their metal
complexes is greater than their in vitro anti-bacterial activity. The effect of the structure of
the investigated compounds on the antimicrobial activity is discussed.
Keywords: Thiourea derivatives; Complexes; Antibacterial activity; Antifungal activity;
In vitro studies; Lipophilicity.

Molecules 2009, 14
520
Introduction
Industrial production and the use of Fe, Co, Cu, Ni, Zn, Cd, and Pb elements can cause
environmental pollution. On the other hand, some of these metals are present in trace amounts as
essential elements for biological systems and these metal ions also play an important role in
bioinorganic chemistry. In order to understand the role of these metal ions in biological systems,
structural studies of the biological compounds and their metal complexes are extremely important.
Compounds containing carbonyl and thiocarbonyl groups occupy an important position among
organic reagents as potential donor ligands for transition metal ions [1-7]. Among these thiourea
derivatives are potentially very versatile ligands, able to coordinate to a range of metal centres as
neutral ligands, monoanions or dianions [1-12]. The oxygen, nitrogen and sulfur donor atoms of
thiourea derivatives provide a multitude of bonding possibilities. Both the ligands and their metal
complexes display a wide range of biological activity including antibacterial, antifungal,
antitubercular, antithroid, antihelmintic, rodenticidal, insecticidal, herbicidal, and plant-growth
regulator properties [13-17].
In view of this, our team focused on the synthesis, characterization, crystal structure, thermal
behavior and antimicrobial activity of new thiourea derivatives [1-7, 18-25]. Following our
examination of the antimicrobial activity of thiourea derivative ligands and their metal complexes, we
now report on the anti-bacterial and anti-yeast activity of five thiourea derivative ligands: (N-
(diethylcarbamothioyl)cyclohexanecarboxamide
[L1],
N-(di-n-propylcarbamothioyl)cyclohexane
carboxamide [L2] di-n-butylcarbamothioyl)cyclohexanecarboxamide [L3], N-(diphenylcarbamothioyl)
cyclohexanecarboxamide [L4], N-(morpholine-4-carbonothioyl)cyclohexanecarboxamide [L5]) and
their Ni2+ and Cu2+ metal complexes against standard bacterial (two different standard strains of
Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Streptococcus pyogenes,
Bacillus cereus, Esherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Proteus vulgaris,
Enterobacter aerogenes) and yeast strains (Candida albicans, Candida krusei, Candida glabrata,
Candida tropicalis, Candida parapsilosis).
Results and Discussion
The syntheses (Scheme 1) involve the reaction of a cyclohexanecarbonyl chloride with potassium
thiocyanate in acetone, followed by condensation of the resulting cyclohexanecarbonyl isothiocyanate
with an appropriate secondary amine (diethylamine, di-n-propylamine, di-n-butylamine,
diphenylamine ormorpholine). The ligands were purified by re-crystallization from an ethanol-
dichloromethane mixture (1:2). The reaction of the ligands with metallic salts at room temperature
with ethanol as solvent yielded the related metal complexes.
In the light of interesting antimicrobial activities of the coordination complexes, the thiourea
derivative ligands and their Ni2+ and Cu2+ metal complexes were screened for antibacterial and
antifungal activity against S. aureus, S. epidermidis, E. faecalis, S. pyogenes, B. cereus, B. cereus, E.
coli, P. aeruginosa, E. cloacae, P. vulgaris, E. aerogenes, C. albicans, C. krusei, C. glabrata, C.
tropicalis, C. parapsilosis by the broth microdilution procedure. The Gram positive anti-bacterial
agent, amikacin, the Gram negative anti-bacterial agent, gentamycin, and the anti-fungal agent,
nystatin, were used as controls. The in vitro antimicrobial properties against a number of Gram

Molecules 2009, 14
521
positive and Gram negative bacteria, and yeasts of both ligands and their metal complexes are
presented in Tables 1-3, respectively.
Scheme 1. Synthesis of the compounds.
O
S
O
O
KSCN
HNR2
N
H
NR2
Cl
N
C
S
M
M : Ni2+
Cu2+
NR2
HNR2 : HN(C2H5)2 L1
HN(C3H7)2 L2
O
S
S
HN(C4H9)2 L3
M
N
N
HN(C6H5)2 L4
HN(C4H8)O L5
O
R2N
Table 1. MIC values (μg/cm3) of the compounds against the tested Gram positive bacteria.
S. aureus
(ATCC
25923)
S. aureus
(ATCC
29213)
S. epidermidis E. faecalis
S. pyogenes
(Clinical
isolate)
B. cereus
(Clinical
isolate)
(ATCC
12228)
(ATCC
29212)
Compound
L1
L2
L3
L4
L5
200
400
200
100
400
200
200
200
400
200
100
100
200
200
50
200
200
400
100
200
200
200
400
400
200
200
200
400
200
100
2
100
200
200
100
100
200
200
100
100
200
100
200
200
200
100
0.5
400
200
200
100
100
200
200
200
200
400
200
200
200
400
200
4
200
200
400
200
200
200
100
100
100
200
400
400
400
400
200
1
200
100
200
100
200
200
200
200
100
100
100
100
200
200
100
2
NiL1
2
NiL2
2
NiL3
2
NiL4
2
NiL5
2
CuL1
2
CuL2
2
CuL3
2
CuL4
2
CuL5
2
Amikacin
2

Molecules 2009, 14
522
Table 2. MIC values (μg/cm3) of the compounds against the tested Gram negative bacteria.
E. coli
P. aeruginosa
E. cloacae
P. vulgaris
E. aerogenes
Compound
(ATCC 25922) (ATCC 27853) (ATCC 13047) (ATCC 13315) (Clinical isolate)
L1
L2
L3
L4
L5
400
200
200
200
200
200
200
400
400
200
200
400
400
400
200
0.5
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
1
400
200
200
200
400
200
200
400
400
400
400
200
200
400
400
2
200
200
200
200
200
200
200
200
400
400
400
200
100
200
400
2
200
400
200
400
400
400
200
400
200
400
200
200
400
400
200
0.5
NiL1
2
NiL2
2
NiL3
2
NiL4
2
NiL5
2
CuL1
2
CuL2
2
CuL3
2
CuL4
2
CuL5
2
Gentamycin
Table 3. MIC values (μg/cm3) of the compounds against the tested fungi.
C. albicans
C. krusei
C. glabrata
C. tropicalis
C. parapsilosis
Compound
(ATCC 90028) (ATCC 6258) (ATCC 32554) (ATCC 20336) (Clinical isolate)
L1
L2
L3
L4
L5
50
50
100
25
50
25
25
100
50
50
25
25
25
50
25
1
50
50
100
50
50
50
50
100
25
50
50
50
50
25
25
0.5
50
25
25
50
50
50
25
50
25
50
25
25
50
25
25
2
50
25
25
50
50
100
50
100
50
50
50
25
50
25
25
4
50
100
50
50
50
25
50
50
100
50
50
25
50
50
25
4
NiL1
2
NiL2
2
NiL3
2
NiL4
2
NiL5
2
CuL1
2
CuL2
2
CuL3
CuL4
CuL5
2
2
2
Nystatin

Molecules 2009, 14
523
All compounds inhibited the growth of bacteria with MIC values ranging between 50 and 400
μg/cm3 and showed anti-yeast activity with MICs between 25 and 100 μg/cm3. According to the
antimicrobial studies, all compounds showed such activity, albeit lower than their anti-yeast efficacy.
This difference may be due to the differences between the cell structures of bacteria and yeast. While
the cell walls of fungi contain chitin, the cell walls of bacteria contain murein [17]. In addition, fungi
contain ergosterol in their cell membranes instead of the cholesterol found in the cell membranes of
animals [4, 26].
CuL5 show good activity against C. albicans, C. krusei, C. glabrata, and C. tropicalis. NiL3
2
2
showed low activity against C. albicans, C. krusei, and C. tropicalis. When all the anti-yeast MIC
values are compared, twelve of fifteen compounds show good activity against C. glabrata and ten of
fifteen compounds show low activity against C. parapsilosis. According to the anti-bacterial studies,
the efficacy against Gram positive bacteria is higher than against Gram negative bacteria. Eleven of
fifteen compounds show good activity against S. epidermidis and eight of fifteen compounds show low
activity against S. pyogenes. In addition, L4 showed high activity against all Gram positive bacteria.
The investigated compound antimicrobial activity values in this research were lower than that
reported for other thiourea derivatives [4, 6, 17, 19]. The main difference in the thiourea derivatives
reported in this paper is the presence of the cyclohexyl moiety. The other derivatives included
substituted benzyl groups. Lipophilicity, which correlates well with the bioactivity of chemicals, is a
very important molecular descriptor and different lipophilic behaviour of compounds plays an
important role in their biological activity mechanisms. The n-octanol/water partition coefficient (log
Pow) is widely used as a general measure of lipophilicity. Compounds with benzyl groups have
relatively higher log Pow values and hence show more lipophilic character as compared to the
compounds with cyclohexyl groups [27].
It is interesting to note that the investigated compounds with cyclohexane group show lower anti-
microbial activity. This behavior can be attributed to the fact that due to their low lipophilicity, these
compounds do not penetrate into the microorganisms as easily as the thiourea derivatives with benzyl
group do. Similar behavior is observed in the anti-yeast activity MIC values for CuL5 and NiL5 . The
2
2
compound with a morpholine ring, which has the log Pow value of 2.55, show higher anti-microbial
activity than the other investigated compounds due probability to their higher lipophilic character.
The results show that the copper complexes are more active against the tested yeast as compared to
the nickel complexes. The increase in antifungal activity of the copper complexes can be ascribed to
the effect of the copper metal ion on the normal cell process. The complexation reaction reduces the
polarity of the metal ion by the partial sharing of metal ion’s positive charge with donor groups and
electron delocalization over the chelate ring. Thus, the lipophilic character of the central metal atom is
enhanced which results in a higher capability to penetrate the microorganisms through the lipid layer
of the cell membrane. Although MIC values for some compounds are good, unfortunately, the anti-
yeast and anti-bacterial activity values of all tested compounds are lower than the reference
compounds, thus these compounds cannot be suggested for clinical use.

Molecules 2009, 14
Experimental
Synthesis
524
The ligands were prepared by a procedure similar to that reported in the literature (Scheme 1) [3]. A
solution of cyclohexanecarbonyl chloride (0.005 mole) in acetone (30 mL) was added dropwise to a
suspension of potassium thiocyanate (0.005 mole) in acetone (30 mL). The reaction mixture was
heated (50 °C) under reflux for 30 min, and then cooled to room temperature. A solution of secondary
amine (0.005 mole) in acetone (30 mL) was added and the resulting mixture was stirred for 2 h.
Hydrochloric acid (0.1 N, 100 mL) was added and the solution filtered. The solid product was washed
with water and purified by recrystallization from an ethanol-dichloromethane mixture (1:2). Metallic
complexes were prepared according to the method described in the literature [3]. A solution of the
metallic acetate (0.01mole) in ethanol (30 mL) was added dropwise to a solution of the ligand in a 1:2
ratio for all metal with a small excess of ligand in ethanol (30 mL) at room temperature and the
resulting mixture was stirred for 30 min. The solid complexes were filtered and re-crystallized from a
1:2 ethanol-dichloromethane mixture.
Antimicrobial activity
The compounds were screened for their in vitro anti-bacterial and anti-yeast activities.
Antimicrobial activities of both ligands and complexes were determined by the broth microdilution
procedures and principles of the Clinical and Laboratory Standards Institute (CLSI) [28, 29]. Minimal
inhibitory concentrations for each compound were investigated against standard bacterial strains; S.
aureus (ATCC 25923), S. aureus (ATCC 29213), S. epidermidis (ATCC 12228), E. faecalis (ATCC
29212), S. pyogenes (clinical isolate), B. cereus (clinical isolate), E. coli (ATCC 25922), P.
aeruginosa (ATCC 27853), E. cloacae (ATCC 13047), P. vulgaris (ATCC 13315), E. aerogenes
(clinical isolate), and yeast-like fungi; C. albicans (ATCC 90028), C. krusei (ATCC 6258), C.
glabrata (ATCC 32554), C. tropicalis (ATCC 20336), C. parapsilosis (clinical isolate) obtained from
the Refik Saydam Hıfzıssıhha Institute, Ankara, Turkey, Microbiology Culture Collection of Inonu
University, Malatya, Turkey, and Department of Microbiology, Faculty of Medicine, Ege University,
İzmir, Turkey. Bacterial and fungal colonies of the test organisms were suspended directly into a small
volume of 0.9% saline and further diluted until turbidity matched the Mc Farland standard no: 0.5 Petri
dishes containing Mueller-Hinton agar for bacteria and Sabouraud Dextrose agar for fungi were
impregnated with these microbial suspensions. The stock solutions were prepared in dimethyl
sulfoxide (DMSO), which had no effect on the microorganisms in the concentrations studied. All of
the dilutions were done with distillated water. The concentrations of tested compounds were 400, 200,
100, 50, 25, 12.5, 6.25, 3.125 μg/cm3. DMSO was used as negative control. Amikacin, gentamycin,
and nystatin were used as reference drugs for Gram positive anti-bacterial activity, Gram negative
anti-bacterial activity and antifungal activity, respectively. All the inoculated plates were incubated at
35 °C and results were evaluated after 16-20 h for bacteria and 48 h for fungi. The lowest
concentration of the compounds that prevented visible growth was considered to be minimal inhibitor
concentrations (MICs).

Molecules 2009, 14
525
Acknowledgements
Support for this research was provided by grant P20 MD001089 and S06 GM078246-01 from the
USA National Institution of Health, NCMHD, and Department of Health and Human Services.
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Sample Availability: Samples of the compounds are available from the authors.
© 2009 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.
This article is an open-access article distributed under the terms and conditions of the Creative
Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).