Spectral, thermal and in vitro antimicrobial studies of cyclohexylamine-N-dithiocarbamate transition metal complexes

Article abstract of DOI:10.1016/j.saa.2010.06.002

Transition metal complexes of the type [M(L)₂] and those containing monodentate phosphines of the type [M(L)₂(PPh ₃)] {M = Ni, Co, Cu and Zn; L = cyclohexylamine-N-dithiocarbamate; PPh₃ = triphenylphosphine} have been synthesized. The complexes were characterized using IR, UV-vis, NMR spectroscopy, and thermal analysis (TGA). The ¹H NMR, ¹³C NMR and ³¹P NMR showed the expected signals for the dithiocarbamate and triphenylphosphine moieties. The spectral studies in all compounds revealed that the coordination of metals occurs via the sulphur atom of the dithiocarbamate ligand in a bidentate fashion. Thermal behavior of the complexes showed that the complexes were more stable than their parent ligands. The ligand moiety is lost in the first step and the rest of the organic moiety decomposes in the subsequent steps. Furthermore, the ligand and their metal complexes were screened in vitro for their antibacterial activity against Escherichia coli, Staphylococcus aureus, Salmonella typhi, Enterococcus faecalis, Pseudomonas aeruginosa and Bacillus cereus and antifungal activities against Aspergillus flavus, Aspergillus carbonarius, Aspergillus niger and Aspergillus fumigatus. The metal complexes exhibited higher antimicrobial activity than the parent ligands. Generally, the zinc complexes were effective against the growth of bacteria with Zn(L) ₂ displaying broad spectrum bacteriocidal activity at concentrations of 50 μg/mL; and Ni(L)₂ was more effective against the growth of fungi at concentrations of 100-400 μg/mL under laboratory conditions.

Full text of DOI:10.1016/j.saa.2010.06.002

Spectrochimica Acta Part A 77 (2010) 579–587
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Spectral, thermal and in vitro antimicrobial studies of
cyclohexylamine-N-dithiocarbamate transition metal complexes
Saul M. Mambaa, Ajay K. Mishraa,∗, Bhekie B. Mambaa, Patrik B. Njobehb,
Mike F. Duttonb, Elvis Fosso-Kankeuc
a Department of Chemical Technology, University of Johannesburg, PO Box 17011, Johannesburg, Gauteng, Doornfontein 2028, South Africa
b Food, Environment and Health Research Group, Faculty of Health Sciences, University of Johannesburg, PO Box 17011, Doornfontein 2028, South Africa
c Water and Health Research Unit, Faculty of Health Sciences, University of Johannesburg, PO Box 17011, Doornfontein 2028, Johannesburg, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
Transition metal complexes of the type [M(L)2] and those containing monodentate phos-
phines of the type [M(L)2(PPh3)] {M = Ni, Co, Cu and Zn; L = cyclohexylamine-N-dithiocarbamate;
PPh3 = triphenylphosphine} have been synthesized. The complexes were characterized using IR, UV–vis,
NMR spectroscopy, and thermal analysis (TGA). The 1H NMR, 13C NMR and 31P NMR showed the expected
signals for the dithiocarbamate and triphenylphosphine moieties. The spectral studies in all compounds
revealed that the coordination of metals occurs via the sulphur atom of the dithiocarbamate ligand in
a bidentate fashion. Thermal behavior of the complexes showed that the complexes were more stable
than their parent ligands. The ligand moiety is lost in the first step and the rest of the organic moiety
decomposes in the subsequent steps. Furthermore, the ligand and their metal complexes were screened
in vitro for their antibacterial activity against Escherichia coli, Staphylococcus aureus, Salmonella typhi, Ente-
rococcus faecalis, Pseudomonas aeruginosa and Bacillus cereus and antifungal activities against Aspergillus
flavus, Aspergillus carbonarius, Aspergillus niger and Aspergillus fumigatus. The metal complexes exhibited
higher antimicrobial activity than the parent ligands. Generally, the zinc complexes were effective against
the growth of bacteria with Zn(L)2 displaying broad spectrum bacteriocidal activity at concentrations of
50 ␮g/mL; and Ni(L)2 was more effective against the growth of fungi at concentrations of 100–400 ␮g/mL
under laboratory conditions.
Received 8 January 2010
Received in revised form 6 May 2010
Accepted 4 June 2010
Keywords:
Spectral
Thermal
Antimicrobial
DTC
Transition metal
Triphenylphosphine
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
while dimeric complexes have a five-coordinate geometry around
the copper ion [13]. Zinc(II) complexes generally show dimeric
Dithiocarbamate-containing coordination compounds are well
known and have been widely studied because of their wide biolog-
ical, industrial, agricultural and chemical applications [1–9]. The
dithiocarbamates can be used as nitrogen–oxygen trapping agents
[1], chelating agents of heavy metals [2–4], vulcanizers, fungicides,
lubricants and catalysts. They have also been used in medicine
since the dithiocarbamate moiety has been found in a variety of
biologically active molecules [5–9].
Transition metal DTC complexes exhibit both mono- and binu-
clear types of molecular structure depending on the preparation
procedures [10–12]. Nickel(II) complexes of the type Ni(DTC)2
have a mononuclear square planar structure and similar cobalt(II)
complexes also exhibit square planar coordination. Copper(II)
bis(dithiocarbamate) complexes can be dimeric or monomeric with
the monomeric structures adopting the square planar arrangement
non-planar molecular configurations which may be described as
distorted tetrahedral, trigonal-bipyramidal or square-pyramidal
of the type Zn2(DTC)4 [14]. Furthermore, zinc(II) complexes
can also be monomeric adopting the square planar geometry,
which usually depends on the size of the coordinated groups
[12–15].
Transition metal derivatives of cyclohexylamine dithiocarba-
mates have been reported to be promising fungicides [16]. Recently
it has been reported that platinum(II) and palladium(II) complexes
of substituted cyclohexylamine-N-dithiocarbamates displayed low
cytotoxicity and antitumor activity [17,18]. However, to the best of
our knowledge the biological profiles of the 3-d metal complexes
of cyclohexylamine dithiocarbamates have not been studied and
reported.
In this paper, we report on the synthesis and characteri-
zation of cyclohexylamine-N-dithiocarbamate, and mixed-ligand
complexes containing triphenylphosphine adducts of nickel(II),
cobalt(II), copper(II) and zinc(II). All the complexes were character-
ized by spectroscopic techniques and thermogravimetric analysis.
∗
Corresponding author. Tel.: +27 11 559 6180; fax: +27 11 559 6425.
E-mail address: amishra@uj.ac.za (A.K. Mishra).
1386-1425/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2010.06.002

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S.M. Mamba et al. / Spectrochimica Acta Part A 77 (2010) 579–587
Fig. 1. 1H NMR spectrum of Ni(L)2.
We also report on the antifungal and antibacterial activity of the
ligand and selected metal complexes.
mode. Themogravimetric analysis was performed on a PERKIN-
ELMER Pyris 1 TG Analyzer under nitrogen atmosphere using
alumina pan as a reference. The weight of the sample was between
4 mg and 8 mg and the heating rate was maintained at 10 ◦C/min
from 20 to 900 ◦C.
2. Experimental
2.1. Materials and reagents
All reagents and solvents used were of the analytical
reagents (AR) grade and were used as supplied. The cop-
per(II) chloride dehydrate, cobalt(II) acetate tetrahydrate, nickel(II)
acetate tetrahydrate, zinc chloride, potassium hydroxide, carbon
disulphide and triphenylphosphine were obtained either from
Sigma–Aldrich or from Merck.
2.3. Preparation of cyclohexylamine-N-dithiocarbamate (L)
The ligand was prepared by following a standard method [13].
The potassium salt of the DTC ligand was prepared by nucle-
ophilic addition of carbon disulphide (CS2) to cyclohexylamine.
A methanolic solution of cyclohexylamine (5.7 mL, 50 mmol,) in
methanol (15 mL) was added to CS2 (3.0 mL, 50 mmol) in methanol
(10 mL) in the presence of KOH (2.8 g, 0.05 mol) in methanol (20 mL)
in a 2 neck round bottom flask. The reaction was maintained at
0–5 ◦C using a water-ice bath. The resultant yellowish solution
was subjected to continuous stirring for 5 h and allowed to evapo-
rate. The yellowish-white solid obtained was then recrystallized in
methanol. The product was dried in a vacuum over calcium chloride
(CaCl2) overnight (yield: 84%, M.pt.: 72 ◦C).
2.2. Instruments
IR spectra were recorded on a MIDAC FTIR spectrophotome-
ter in the range 4000–400 cm−1 using KBr pellets. The UV–vis
spectra were recorded in DMSO on a SHIMADZU 2450 UV-Visible
spectrometer. NMR spectra were recorded on a GEMINI-300 MHz
spectrophotometer at room temperature using d6-DMSO as a sol-
vent. 13C NMR spectra were recorded in the proton decoupled
Scheme 1. Preparation of metal complex.

S.M. Mamba et al. / Spectrochimica Acta Part A 77 (2010) 579–587
581
2.4. Preparation of the complexes
where r = r2 − r1; r2: radius of zone of inhibition of the control
(AmB), r1: radius of zone of inhibition of the test compound and
R: radius of petri dish.
Minimum inhibition dose (MID) was defined as the lowest con-
centration of the tested complex, which showed the least visible
fungal growth inhibition.
2.4.1. Preparation of bis(dithiocarbamato) transition metal
complexes
Warm solutions of metal salts (2.5 mmol) {CuCl2·2H2O (0.43 g),
Co(CH3COO)2·4H2O (0.62 g), Ni(CH3COO)2·4H2O (0.62 g) and ZnCl2
(0.34 g)} in methanol (10 mL) were added drop wise to a solution
of L (1.06 g, 5 mmol) in methanol (15 mL). The mixture was then
refluxed for 3 h. The precipitates formed were filtered off, washed
with water, methanol and petroleum ether. The solids were then
dried overnight over CaCl2 under vacuum. Scheme 1 shows a gen-
eral scheme for accessing the metal complexes.
2.5.1.2. Statistical analysis. A one-way analysis of variance
(ANOVA) was performed to derive mean values, which were then
compared by least significant difference (LSD) using all pairwise
multiple comparison procedures (Holm-Sidak method) (Systat
Inc., 2006). Mean values among treatment groups were deemed to
have significant differences if the level of probability was ≤0.05.
Ni(L)2: Colour: green, yield: 50%, M.pt.: 160 ◦C; Co(L)2: colour:
dark, yield: 43%, M.pt.: 154 ◦C; Cu(L)2: Colour: yellow, yield: 66%,
M.pt.: charring; Zn(L)2: Colour: white, yield: 54%, M.pt.: 151 ◦C.
2.5.2. Antibacterial screening
Commercial reference strains of Gram-negative {Escherichia coli
(E. coli), Pseudomonas aeruginosa (P. aeruginosa) and Salmonella
typhi (S. typhi)} and Gram-positive {Staphylococcus aureus (S.
aureus), Enterococcus faecalis (E. faecalis) and Bacillus cereus (B.
cereus)} bacteria were selected to assess the potential antibacte-
rial effects of selected ligands and corresponding transition metal
complexes.
2.4.2. Preparation of phosphine based complexes
A mixture of Ni(L)2 (0.102 g, 0.25 mmol) and PPh3 (0.066 g,
0.25 mmol) or Co(L)2 (0.204 g, 0.5 mmol) or Cu(L)2 (0.206 g,
0.5 mmol) and PPh3 (0.13 g, 0.5 mmol) was refluxed in THF (50 mL)
for 3 h. The coloured solutions were filtered off and allowed to
evaporate without heating. The solids obtained were washed with
petroleum ether and dried under vacuum over CaCl2.
Typically, a solution of PPh3 (0.066 g, 0.25 mmol) in THF (10 mL)
was added drop wise to a solution of Zn(L)2 (0.207 g, 0.25 mmol)
in THF (50 mL) with stirring. The mixture was refluxed for 3 h and
the solvent was evaporated. The white solid obtained was washed
with petroleum ether and dried under vacuum over CaCl2.
[Ni(L)2PPh3]: Colour: green, yield: 69%, M.pt.: 68 ◦C;
[Co(L)2PPh3]: Colour: dark, yield: 51%, M.pt.: 72 ◦C; [Cu(L)2PPh3]:
Colour: orange (rusty), yield: 71%, M.pt.: 78 ◦C; [Zn(L)2PPh3]:
Colour: white, yield: 38%, M.pt.: 75 ◦C.
2.5.2.1. Disc diffusion method. Sterile blank discs (6 mm from
Davies Diagnostics (Pty) Ltd., SA) were impregnated with 15 ␮L of
known concentration of stock solution of tested compounds as to
obtain discs containing 15, 30, 100, 200 and 400 ␮g of each com-
pound. Impregnated discs were air-dried and cautiously placed
onto the surface of Mueller-Hinton agar (MHA) plates freshly inoc-
ulated with microorganisms. After 10 min at room temperature the
plated cultures were incubated in an inverted position for 20–24 h
at 37 ◦C.
All microorganisms’ culture solutions were adjusted to the
turbidity of the 0.5 Mc Farland prior to inoculation onto Mueller-
Hinton agar. Experiments were conducted in quadruplicate (four
discs with identical concentration ofthe samecompound) and com-
mercial antibiotic (Vancomycin 30 ␮g and Carbenicillin 100 ␮g)
impregnated discs were used as positive controls. Susceptibility
diameter zone was reported as the average value of replicates mea-
surements.
2.5. Antimicrobial screening
The antimicrobial activity of the ligand and selected synthesized
complexes were tested against some Gram (+) and Gram (−) bac-
teria and fungi. The activity of the compounds was compared with
standard reference drugs.
2.5.1. Antifungal screening
2.5.2.2. Broth dilution method. Stock solution of tested compounds
were twofold serially diluted in nutrient broth (“Lab-Lemco” pow-
der: 1.0 g/L; yeast extract 2.0 g/L; peptone 5.0 g/L; sodium chloride
5.0 g/L; pH 7.4 0.2 at 25 ◦C; Merck Chemicals, SA) contained in
sterile test tube to obtain experimental concentrations of 1.53, 3.06,
6.25, 12.5, 25, 50, 100 and 200 ␮g/mL. Overnight culture (2 mL) of
each microorganism was added to the tube, constituting a final
concentration of approximately 105 CFU/mL in the final volume
(4 mL). The mixture was incubated for 18 h at 37 ◦C. The growth
monitored as absorbance at 600 nm using a UV spectrophotometer
(Helios Epsilon-USA) and the minimum inhibitory concentration
(MIC) determined as the lowest concentration of compound that
maintain the turbidity of the solution unchanged through inhibi-
tion of growth. The solution in each tube was then inoculated onto
nutrient agar plate, and incubated at 37 ◦C for 24 h to determine the
Minimum Bactericidal Concentration (MBC). The highest dilution of
a compound that killed >99.9% of the bacteria was considered as the
MBC.
2.5.1.1. Disc diffusion method. A disc application technique was
employed in vitro to evaluate the antifungal activities of bio-
chemical complexes [19]. Fungi of the Aspergillus spp. (A. flavus,
A. carbonarius, A. niger and A. fumigatus) were used in the
study. Mature conidia of fungal isolates were harvested from
potato dextrose agar (PDA) plates and suspended in ringer solu-
tion and spore suspensions standardized with a haemocytometer
(104 conidia/mL). Conidial suspension (1 mL) representing each
fungal isolate was then spread on a 90-mm petri dish containing
PDA (20 mL) with the excess of the conidial suspension decanted
and allowed to dry. The compounds were dissolved in dimethyl
sulphoxide (DMSO). Sterile 6-mm diameter test discs (AB BioDisk,
Solna) were impregnated with 15 ␮L of the solution of each test
compound to contain 100, 200 and 400 ␮g/disc in triplicates.
Amphotericin B (AmB) (20 ␮g/disc) was used as a reference drug
for fungal inhibition, while DMSO was used as a negative control.
Plates were incubated at room temperature (22–25 ◦C) for 7 days.
The radius of the inhibition zone of fungal growth on two axes at
right angle to each other was measured after 3, 5 and 7 days and
expressed as the percentage minimum inhibition zone calculated
as follows:
3. Results and discussion
The TLC and elemental analysis results (Table 1) revealed
that the synthesized ligand and metal complexes were obtained
in high purity. The ligand was soluble in most solvents while
ꢀr2
ꢀR2
% minumun inhibition zone (MIZ) =
× 100

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S.M. Mamba et al. / Spectrochimica Acta Part A 77 (2010) 579–587
Table 1
Elemental analysis.
Compound
Formula
Found (calcd) (%)
C
H
N
S
L
C7H12KNS2
C14H24N2NiS4
C14H24CoN2S4
39.05(39.40)
41.03(41.28)
41.36(41.26)
40.56(40.80)
40.46(40.61)
57.02(57.40)
57.45(57.38)
56.57(56.98)
56.23(56.83)
5.80(5.67)
5.88(5.94)
5.82(5.94)
5.96(5.87)
5.70(5.84)
5.77(5.87)
5.83(5.87)
5.78(5.83)
5.82(5.81)
6.89(6.56)
6.82(6.88)
6.82(6.87)
6.10(6.80)
6.81(6.77)
4.25(4.18)
4.11(4.18)
4.10(4.15)
4.67(4.14)
29.95(30.05)
31.16(31.94)
31.95(31.47)
30.98(31.12)
30.15(30.98)
18.99(19.15)
19.06(19.16)
18.83(19.02)
19.02(18.97)
Ni(L)2
Co(L)2
Cu(L)2
Zn(L)2
[Ni(L)2PPh3]
[Co(L)2PPh3]
[Cu(L)2PPh3]
[Zn(L)2PPh3]
C
C
14H24CuN2S4
14H24N2S4Zn
C32H39N2NiPS4
C32H39N2CoPS4
C32H39N2CuPS4
C32H39N2ZnPS4
the metal complexes were insoluble in most organic sol-
vents.
[23]. The absence of bands in the region above 900 nm ruled out the
possibility of a tetrahedral or pseudo-tetrahedral geometry around
the Cu(II) complex [11].
Zinc complexes showed three bands at 260, 278 and 324 nm.
The band at 324 nm was assigned to the charge transfer transitions
and the other two bands were assigned to n → ␲* and ␲ → ␲* tran-
sitions. The absorption spectra also showed no d–d bands. There
was no chance of transition because there was no empty d-orbital
[14,24].
3.1. Electronic spectra
The electronic spectral data of the ligand and metal com-
plexes is summarized in Table 2. Dithiocarbamates generally show
three bands in the UV region. These bands are ascribed to the
intra-molecular intra-ligand transitions corresponding to ␲ → ␲*
transitions of the N–C S system, ␲ → ␲* transitions of the S–C
S
group and a n → ␲* transition located on the sulphur atom [20–22].
3.2. Infrared (IR) spectroscopy
The electronic spectra of the ligand showed three electronic
bands which were assigned to the ␲ → ␲* transitions of the N–C
S
Table 3 summarizes the significant IR bands, along with their
proposed assignments of the ligand and its complexes. The sim-
ilarities in the IR spectra of the complexes indicated that they
have similar structures. On complexation, the peak at 1482 cm−1
attributed to the ꢂ (C N) in the ligand was shifted to higher
frequency (1500–1522 cm−1) in the complexes. This confirmed an
increase in the C–N double bond character due to the mesomeric
drift of an electron cloud of the –NCS2 moiety towards the metal
ion suggesting a strong contribution of the thioureide resonance
form to the structure of dithiocarbamates [11,23]. The ꢂ (C S)
stretching vibrations are observed at ca. 1000 cm−1 without any
splitting in the complexes suggesting the bidentate coordination
of the dithiocarbamate moiety [25]. The presence of only one band
in the region 1000 70 cm−1 was characteristic of the bidentate
nature of the dithiocarbamate ligand. The appearance of two bands
within a difference of 20 cm−1 in the same region was characteristic
of the monodentate binding nature of the ligand [25]. This band was
shifted slightly to a higher frequency in the complexes suggesting
coordination via the C–S group [26].
system, ␲ → ␲* transitions of the S–C S group and a n → ␲* tran-
sition located on the sulphur atom. The electronic spectra of Ni(II)
complexes showed two weak d–d bands at 482 and 632 nm. These
1
2
1
1
bands can be assigned to the A1g → B2g and A1g → A2g tran-
sitions [11,12]. This suggests the octahedral geometry for the
Ni(II) complex. A band at 382 nm was assigned to metal → ligand
charge transfer transitions. The spectrum of the mixed-ligand
complex showed an additional band at 412 nm as a result of triph-
enylphosphine ligand → metal charge transfer transition. Bands
below 300 nm were ascribed to ␲ → ␲* transitions of the N–C
S
and the S–C S systems.
Two d–d bands were observed in the spectra of cobalt(II) com-
4
plexes at 642 and 498 nm which were due to the 4
A
2g(F) → T2g(F)
and 4A2g(F) → T1g(F) transitions eventhoughtherewerethreespin
allowed transitions [11]. The presence of these bands suggesting
the octahedral complexes. Metal → ligand charge transfer (MLCT)
transitions were observed at 400 nm and intra-ligand transitions at
about 260 and 324 nm.
4
The copper complexes, [Cu(L)2] and [Cu(L)2PPh3] showed
two bands between 250 and 320 nm as a result of intra-ligand
and charge transfer transitions, and an unresolved d–d band at
nearly 400 nm. These complexes are anticipated as octahedral.
For square planar Cu(II) complexes two d–d bands corresponding
3.3. NMR spectral studies
Chemical shifts of all the synthesized compounds are summa-
rized below.
2
2
2
2
to B1g → Eg and B1g → A1g were anticipated while octahedral
Cu(II) complexes typically exhibit three unresolved d–d bands
2
2
2
assigned to the 2B1g → B2g
,
2B1g → Eg and 2B1g → A1g transitions
Table 3
IR spectral data of L and its complexes.
Table 2
Wavenumbers (cm−1
)
Electronic data of the ligand and its metal complexes.
Compound
Compound
Wavelength, ꢁ (nm)
ꢂ (N–H)
ꢂ (C
N)
ꢂ (C
S)
L
257, 308
L
3318
3235
3245
3230
3310, 3249
3234
3267
3231
3306, 3250
1482
1513
1500
1511
1513
1522
1501
1511
1511
974
977
979
977
980
979
983
971
984
Ni(L)2
Co(L)2
Cu(L)2
Zn(L)2
[Ni(L)2PPh3]
[Co(L)2PPh3]
[Cu(L)2PPh3]
[Zn(L)2PPh3]
252, 327, 381, 482, 632
269, 323, 400, 550, 658
263, 300, 398
Ni(L)2
Co(L)2
Cu(L)2
Zn(L)2
[Ni(L)2PPh3]
[Co(L)2PPh3]
[Cu(L)2PPh3]
[Zn(L)2PPh3]
263, 278, 334
256, 327, 381, 418, 486, 632
256, 324, 400, 498, 642
256, 302, 318
260, 278, 324

S.M. Mamba et al. / Spectrochimica Acta Part A 77 (2010) 579–587
583
3.3.1. 1H NMR spectra
The –CH2 (methylene) protons of all the complexes appeared
between 1.00 and 1.95 ppm. The CH–N signal was observed around
4.00 ppm in the ligand and shifted on complexation due to coordi-
nation of the ligand to the metal. Fig. 1 shows the NMR spectra of
the of Ni(L)2 complex. The deshielding of the protons was attributed
to the release of electrons of nitrogen forcing a high electron den-
sity towards the sulphur (or metal) via the thioureide ␲-system
[27]. Signals between 9 and 11 ppm were assigned to the N–H of
the thioureide system [18] and the aromatic protons for the PPh3
moiety were observed at 7.0–7.8 ppm.
•
•
•
•
•
L, 1H NMR, ı (ppm): 1.00–1.79 (m, 10H, –CH2), 3.72 (s, 1H,
–CHN), 9.61 (s, 1H, –NH)
13C NMR, ı (ppm): 22.6 (2C), 25.9 (1C), 31.9 (2C), 57.7 (1C,
–CHN), 212.6 (1C, –NCS2)
Ni(L)2, 1H NMR, ı (ppm): 1.1–1.9 (m, 20H, –CH2), 3.8 (brs,
1H, –CHN), 10.4 (brs, 1H, –NH)
13C NMR, ı (ppm): 24.2 (4C), 24.6(2C), 31.2(4C), 52.8 (2C),
203.7 (2C)
Co(L)2, 1H NMR, ı (ppm): 1.11–1.81 (m, 20H, –CH2), 3.74
(brs, 1H, –CHN), 9.99 (brs, 1H, –NH)
Fig. 2. TG curves of the ligand and selected complexes.
13C NMR, ı (ppm): 24.1 (4C), 24.6(2C), 31.2(4C), 52.8 (2C),
203.7 (2C)
Cu(L)2, 1H NMR, ı (ppm): 1.11–1.97 (m, 20H, –CH2), 3.90
(brs, 1H, –CHN), 10.3 (brs, 1H, –NH)
3.4. Thermogravimetric analysis (TGA)
13C NMR, ı (ppm): 24.8 (4C), 25.6(2C), 31.2(4C), 52.8 (2C),
203.7 (2C)
The thermal decomposition of L and its metal complexes is pre-
sented in Fig. 2. The ligand, L, decomposed in two steps and it
proceeded via a potassium thiocyanate intermediate [30]. The first
step took place in the range 60–180 ◦C. Aqueous methanol was lost
first at 60–104 ◦C and this was followed by a sharp decomposi-
tion after 104 ◦C which was assigned to the loss of cyclohexane
(found: 53.68, calcd: 54.5%). The decomposition of the intermedi-
ate took place in the second step leading to the formation of the final
product. Ni(L)2 also decomposed via a Ni(SCN)2 intermediate. The
first step of the decomposition began at 137 ◦C and was completed
at 208 ◦C with the loss of 64.0% (calcd: 63.4%). Two cyclohexane
molecules and C2S2 were eliminated. The decomposition of the
intermediate was slow at 211–796 ◦C. The final product was NiS
(found: 20.9%, calcd: 22.3%). The first step in the decomposition
of Zn(L)2, from 131 to 346 ◦C was assigned to the loss of one DTC
and one cyclohexane group (found: 61.74%, calcd: 62.3%). The final
product was ZnS (found: 26.0%, calcd: 23.2%).
[Ni(L)2PPh3] decomposed in three steps. The first step was
assigned to the loss of CS2 (found: 11.2%, calcd: 11.4%). The second
step was rapid and total mass loss at this stage was 42.5% (calcd:
42.5%) which matched the loss of two cyclohexane molecules, 2N
and a C atom. PPh3 was lost in the last and final step (found:
79.9%, calcd: 81.6%). The end product of the decomposition was
NiS (found: 11.6%, calcd: 13.6%). [Zn(L)2PPh3] decomposed in two
steps with the first step assigned to the elimination of CS2 (found:
11.2%, calcd: 11.4%). The elimination of the PPh3 and the organic
moiety occurred in the second and last step. The final product was
Zn (found: 7.6%, calcd: 9.6%).
Zn(L)2, 1H NMR, ı (ppm): 1.06–1.83 (m, 20H, –CH2), 3.67
(brs, 1H, –CHN), 9.80 (brs, 1H, –NH)
13C NMR, ı (ppm): 24.7 (4C), 25.0(2C), 31.2(4C), 57.5 (2C),
203.7 (2C)
•
•
NiL2PPh3, 1H NMR, ı (ppm): 1.02–1.56 (m, 20H, –CH2), 3.4
(brs, 1H, –CHN), 7.0–7.6(m, 15H, Ar), 10.25 (brs, 1H, –NH)
31P NMR, ı (ppm): 42.3
CoL2PPh3, 1H NMR, ı (ppm): 1.23–1.85 (m, 20H, –CH2),
3.76 (brs, 1H, –CHN), 7.2–7.7 (m, 15H, Ar), 9.9 (brs, 1H,
–NH)
31P NMR, ı (ppm): 41.0
•
•
CuL2PPh3, 1H NMR, ı (ppm): 1.1–2.1 (m, 20H, –CH2), 3.4
(brs, 1H, –CHN), 7.2–7.8 (m, 15H, Ar), 10.0 (brs, 1H, –NH)
31P NMR, ı (ppm): 42.6
ZnL2PPh3, 1H NMR, ␦ (ppm): 1.22–1.81 (m, 20H, –CH2), 3.4
(brs, 1H, –CHN), 7.12–7.7 (m, 15H, Ar), 9.8 (brs, 1H, –NH)
31P NMR, ␦ (ppm): 43.0
3.3.2. 13C NMR spectroscopy
The ı(N13CS2) appeared in the expected region (above 202 ppm)
for normal oxidation state transition metal dithiocarbamates
[28]. In all metal complexes these signals were observed around
204 ppm. An upfield shift was observed compared to the chem-
ical shift of the parent ligand (at 212 ppm). This upfield shift
was caused by the mesomeric shift of the electron density
from the dithiocarbamate moiety towards the metal centre
[27–29]. The –C–NH carbons of the complexes were observed
around 55 ppm and were not greatly affected during complex
formation.
3.5. Antifungal activity
The antifungal activities of the ligand and its corresponding
complexes using Aspergillus spp. were studied in vitro and results
are summarized in Tables 4–6. The antifungal activity of the com-
pounds as compared with that of reference drug, AmB showed
significant activity. In this case, all compounds tested were active
against fungi on Days 3, 6 and 7, with maximum activity observed
on Ni(L)2 having an average % MIZ of 4.78 recorded on Day 3.
Ni(II) complexes have been found to exhibit maximum biological
activity against pathogenic fungi when compared with their par-
ent ligands and similar metal complexes [11,31]. The activity of the
metal complexes against the tested fungi was worth noting since
3.3.3. 31P NMR spectroscopy
31P NMR was recorded in d6-DMSO with H3PO4 being an inter-
nal reference. 31P chemical shifts are observed 42.3, 41.0, 42.6 and
43.0 ppm at for the Ni, Co, Cu and Zn complexes, respectively. The
signals are observed in the deshielded region taking into account
the free PPh3 signal at −5.5 ppm. This deshielding effect confirmed
the coordination of the phosphorus to the metal and was attributed
to the movement of electron density from the phosphorus to the
metal atom [29].

584
S.M. Mamba et al. / Spectrochimica Acta Part A 77 (2010) 579–587
Table 4
Effects of ligand complexes on % MIZ of fungi on Day 3.
Complexes
L
Conc.
% MIZ
A. flavus
A. carbonarius
A. niger
A. fumigatus
100
200
400
Probability
1.53 0.22a
2.66 0.08b
4.30 0.52c
***
1.81 0.34a
2.53 0.31a
5.28 0.17b
***
1.71 0.38a
2.85 0.12b
5.11 0.17c
***
1.36 0.06a
1.49 0.00ab
1.64 0.06b
*
Ni(L)2
100
200
400
Probability
5.31 0.41a
5.40 0.47a
6.09 1.09b
*
2.41 0.28a
2.84 0.50a
4.89 1.51b
**
3.92 0.61a
3.96 0.88a
7.06 0.56b
**
3.05 0.61a
5.28 0.61ab
7.16 0.94b
**
Zn(L)2
100
200
400
Probability
1.01 0.08a
2.54 0.08b
3.01 0.30b
***
1.47 0.30a
1.66 0.19a
2.27 0.16b
*
2.44 0.21a
2.56 0.88a
4.13 0.65b
*
1.82 0.24a
3.70 0.44b
4.47 0.21b
***
[Zn(L)2PPh3]
[Ni(L)2PPh3]
100
200
400
Probability
1.12 0.05
1.12 0.08
1.29 0.14
NS
0.86 0.04a
1.00 0.00ab
1.17 0.10b
*
1.34 0.25
1.46 0.14
1.59 0.06
NS
1.99 0.14a
4.87 0.27b
5.43 0.54c
***
100
200
400
Probability
2.04 0.10
2.59 0.34
2.39 0.40
NS
1.08 0.05
1.28 0.08
1.33 0.08
NS
2.54 0.08b
2.68 0.26a
3.47 0.35b
*
2.80 0.52a
3.58 0.19ab
4.35 0.38b
*
a,b,cMean values in the same column for each complex not sharing the same letters are significantly different *p < 0.05, **p < 0.01, ***p < 0.001. Values within columns are
means (left) and standard error of the mean (SEM) (right). NS: not significant; MIZ: minimum inhibitory zone.
Table 5
Effects of ligand complexes on % MIZ of fungi on Day 5.
Complexes
L
Conc.
% MIZ
A. flavus
A. carbonarius
A. niger
A. fumigatus
100
200
400
Probability
0.76 0.06a
0.86 0.04a
1.14 0.37b
*
0.67 0.06
0.83 0.06
1.01 0.10
*
0.68 0.07
0.88 0.12
1.06 0.21
NS
0.88 0.13a
1.24 0.00b
1.41 0.05b
***
Ni(L)2
100
200
400
Probability
1.83 0.04
1.91 0.00
1.98 0.00
NS
1.50 0.06a
1.59 0.00a
1.83 0.04b
**
1.26 0.17
1.46 0.08
1.57 0.09
NS
1.02 0.11a
1.17 0.07a
1.24 0.06b
*
Zn(L)2
100
200
400
Probability
0.61 0.00a
0.61 0.00a
0.79 0.00b
*
0.64 0.07a
0.70 0.07b
0.90 0.05b
*
0.79 0.00a
0.86 0.04a
1.17 0.10b
**
0.44 0.00a
0.79 0.00b
0.90 0.05c
**
[Zn(L)2PPh3]
[Ni(L)2PPh3]
100
200
400
Probability
0.68 0.07
0.68 0.07
0.79 0.00
NS
0.68 0.07
0.68 0.07
0.79 0.00
NS
0.82 0.15
0.88 0.13
1.25 0.11
NS
0.79 0.00
0.79 0.00
0.79 0.00
NS
100
200
400
Probability
0.79 0.00
0.94 0.09
0.94 0.11
NS
0.88 0.12
0.88 0.12
1.03 0.05
NS
0.79 0.00a
1.03 0.17ab
1.41 0.05b
*
0.55 0.03a
0.67 0.04b
0.79 0.00c
***
a,b,cMean values in the same column for each complex not sharing the same letters are significantly different *p < 0.05, **p < 0.01, ***p < 0.001, NS: not significant. Values within
columns are means (left) and standard error of the mean (SEM) (right). MIZ: minimum inhibitory zone.
such activity may either be associated with cell wall destruction
[31], DNA damage [32], protein synthesis inhibition or chelation
with metal ions in fungal cells thus depriving them of the needed
ions which would lead to cell mortality [11,33]. Data obtained also
indicate that with increasing dosage level of Ni(L)2 from 100 to
400 ␮g/disc, % MIZ significantly (p < 0.01) increased by approxi-
mately twofolds, except for A. flavus (5.31–6.09%) (Table 4) while %
MIZ slightly increased from 5.31 to 6.09 (p < 0.05). Although L2 was
less active than Ni(L)2, its activity increased threefolds with dose
increment (p < 0.001), except in the case of A. fumigatus whose MIZ
also marginally increased from 1.36 to 1.64%.
activities on the growth of A. flavus, A. niger and A. fumigates. L
showed maximum activity against A. carbonarius with low activity
exhibited against the growth of A. fumigatus.
The compounds reported herein exhibited antifungal activity
against fungi after Day 5 (Tables 5 and 6). Such inhibitory data
provided by the tested compounds should be considered they are
effective, especially when such activity is comparable with that
of AmB. More importantly in this context, was the broad spec-
trum activity exhibited by the tested compounds in inhibiting the
growth of more than one fungus. In most situations, biological
activities of compounds are often evaluated after 2 days [31] or
4 days [11,34], but in this case, the long lasting efficacy of these
compounds was determined up till the seventh day, which still
Generally, the results obtained suggested that while some com-
plexes tested were less active against A. carbonarius, have higher

S.M. Mamba et al. / Spectrochimica Acta Part A 77 (2010) 579–587
585
Table 6
Effects of ligand complexes on % MIZ of fungi on Day 7.
Complexes
L
Conc.
% MIZ
A. flavus
A. carbonarius
A. niger
A. fumigatus
100
200
400
Probability
0.86 0.04a
0.93 0.04a
1.08 0.05b
**
0.64 0.03a
1.21 0.49a
1.45 0.53b
NS
0.44 0.00a
0.64 0.07ab
0.73 0.04b
**
0.73 0.06
0.75 0.12
0.80 0.09
NS
Ni(L)2
100
200
400
Probability
0.96 0.15
1.12 0.08
1.08 0.05
NS
0.94 0.09a
1.24 0.09b
1.28 0.04b
*
1.09 0.09
1.09 0.09
1.24 0.00
NS
0.98 0.11a
1.24 0.00ab
1.42 0.12b
**
Zn(L)2
100
200
400
Probability
0.44 0.00a
0.44 0.00a
0.79 0.00b
**
0.61 0.06a
0.67 0.04a
0.86 0.04b
**
0.87 0.08
0.79 0.05
0.80 0.09
NS
0.44 0.00a
0.61 0.00b
0.68 0.07b
*
[Zn(L)2PPh3]
[Ni(L)2PPh3]
100
200
400
Probability
0.44 0.00a
0.73 0.04b
0.79 0.00b
**
0.61 0.10
0.68 0.03
0.70 0.04
NS
0.88 0.12
0.94 0.18
1.08 0.05
NS
0.85 0.13a
1.04 0.07a
1.33 0.10b
*
100
200
400
Probability
0.68 0.07a
0.94 0.09ab
1.17 0.13b
*
0.86 0.04a
0.89 0.00a
1.00 0.00b
***
0.67 0.04a
1.09 0.09a
2.65 0.70b
*
0.75 0.12
0.82 0.15
0.94 0.09
NS
a,b,cMean values in the same column for each complex not sharing the same letters are significantly different *p < 0.05, **p < 0.01, ***p < 0.001, NS: not significant. Values within
columns are means (left) and standard error of the mean (SEM) (right). MIZ: minimum inhibitory zone.
Table 7
Determination of antibacterial activity by disc diffusion.
Selected bacteria
Carbenicillin 100 ␮g
Vancomycin 30 ␮g
L
Ni(L)2
Co(L)2
Zn(L)2
[Cu(L)2PPh3]
[Zn(L)2PPh3]
Disc content (␮g/disc)
Inhibitory activity
+
+
Escherichia coli
−
−
+
+
+
−
−
−
−
−
−
−
−
−
−
+
+
+
+
+
−
−
−
−
−
+
+
+
+
+
15
30
100
200
400
−
−
+
+
+
+
+
+
Pseudomonas aeruginosa
Salmonella typhi
−
−
−
−
−
+
+
+
+
+
−
−
+
+
+
−
−
−
−
−
+
+
+
+
+
−
−
−
−
−
15
30
100
200
400
−
−
−
+
−
−
−
−
−
−
−
−
−
−
−
−
+
+
+
−
−
−
−
−
−
−
+
+
+
15
30
100
200
400
+
Staphylococcus aureus
Enterococcus faecalis
Bacillus cereus
−
−
+
+
+
+
+
+
+
+
−
−
−
−
−
+
+
+
+
+
−
−
−
−
−
−
−
+
+
+
15
30
100
200
400
+
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
+
+
+
−
−
−
−
−
−
−
−
−
−
15
30
100
200
400
+
−
−
+
+
+
−
−
−
−
−
−
−
−
−
−
−
+
+
+
+
−
−
−
−
−
−
+
+
+
+
15
30
100
200
400
(−): resistant; (+): susceptible.
showed some activity. The decrease in biological activity of these
compounds over time might be attributed to the reduced stability
of such compounds, especially under neutral and acidic conditions
[35]. Alternatively, the reduced activity of compounds with time
could be that, there is likely a tendency for some fungi to reduce
sensitivity and develop resistance over time by adapting to such
applications [36].
When making a choice between various therapeutic applica-
tions, the chosen antifungal drug among other factors, must have
a long lasting potential. In this study, the prolonged effect of

586
S.M. Mamba et al. / Spectrochimica Acta Part A 77 (2010) 579–587
Table 8
MIC and MBC values of tested compounds.
Selected bacteria
L
Ni(L)2
Co(L)2
Zn(L)2
[Cu(L)2PPh3]
[Zn(L)2PPh3]
MIC(MBC) (␮g/mL)
Escherichia coli
Pseudomonas aeruginosa
Salmonella typhi
Staphylococcus aureus
Enterococcus faecalis
Bacillus cereus
100–100
100–200
100–200
100–200
100–nf
200–nf
nf–200
nf–nf
nf–nf
200–nf
200–200
nf–nf
100–50
100–100
50–50
50–50
100–200
100–50
nf–100
nf–100
nf–200
nf–200
nf–200
nf–50
nf–nf
nf–200
200–nf
200–nf
200–nf
nf–nf
100–100
nf–100
200–200
nf–nf
100–100
100–100
compounds against fungal growth typically in the case of nickel
complexes as earlier indicated as well as their broad spectrum
activity, was noted. It is, therefore, necessary to evaluate the
cytotoxic effects of these compounds as their applications in the
formulation of novel antifungal therapeutic drugs seem promising.
also show broad antifungal activity and maximum activity being
exhibited by the nickel(II) complex. Because of the broad spectrum
activity displayed by some of the tested compounds, it is would be
necessary to evaluate the cytotoxicity of these compounds as their
applications in the formulation of novel antimicrobial therapeutic
drugs seem promising.
3.6. Antibacterial studies
Acknowledgements
3.6.1. Bacterial susceptibility to tested compounds
The authors are thankful to the National Research Foundation
(NRF) and Nanotech Innovation Centre (NIC) for financially sup-
porting this work.
The disc diffusion assay was conducted to determine if the
tested compounds could inhibit the growth of bacteria. The results
obtained revealed inhibitory activity of all tested compounds
against at least one microorganism (Table 7) and the inhibition
concentration ranged from 15 to 400 ␮g/disc. Zn(L)2 generally
exhibited strong inhibitory activity than any other compound. The
compounds inhibitory activity was greater against S. aureus, while
E. faecalis was resistant to most of the complexes and compounds
tested.
References
[1] K.S. Siqqidi, S.A.A. Nami, L. Chebude, Y. Chebude, Journal of Brazilian Chemical
Society 17 (1) (2006) 107–112.
[2] R. Say, E. Birlik, Z. Erdemgil, A. Denizli, A. Ersoz, Journal of Hazardous Materials
150 (2008) 560–564.
[3] N. Unlu, M. Ersoz, Separation and Purification Technology 52 (2007) 461–469.
[4] F. Fu, H. Zeng, Q. Cai, R. Qiu, J. Yu, Y. Xiong, Chemosphere 69 (2007) 1783–1789.
[5] Y. Yoshikawa, Y. Adachi, H. Sukurai, Life Sciences 80 (2007) 759–766.
[6] S. Ozkirimli, T.I. Apak, M. Kiraz, Yegenoglu, Archives of Pharmacal Research 28
(11) (2005) 1213–1218.
3.6.2. Minimum inhibitory and Minimum Bactericidal
Concentration
Microorganisms are developing resistance against some of the
antibacterial compounds available on the market. Candidate com-
pounds for substitution of existing one, should exhibit strong
inhibitory activity at very low concentration. The effectiveness of
tested compounds in this study was then assessed through deter-
mination of MIC and MBC. Results recorded revealed that among
all the compounds Zn(L)2 was effective at lower concentration
(50 ␮g/mL) and also exhibit bactericidal activity against all the bac-
teria (Table 8).
On the other hand Co(L)2 was the compound with the weakest
activity against bacteria and mostly static except with P. aerugi-
nosa. Although [Cu(L)2PPh3] exhibited bactericidal activity against
all bacteria, it was not possible to determine the MIC from its activ-
ity up to a concentration of 200 ␮g/mL. This implies that inhibitory
activity of [Cu(L)2PPh3] against bacteria was very slow and did
not prevent the growth from the first hours of incubation, hence
the increase of turbidity observed in the tube; copper is a bio-
essential element which acts as cofactor for many enzymes [37].
The inhibitory effect of copper results from DNA damage due to the
ease of reduction of Cu(II) to Cu(I) [38,39]. However the inhibitory
activity of complexes is the summation of the effects of the lig-
and and the metal which affect both the bacterial cell wall and
metabolism [39,40].
[7] F. Shaheen, A. Badshah, M. Gielen, C. Gieck, M. Jamil, D. de Vos, Journal of
Organometallic Chemistry 693 (2008) 1117–1126.
[8] V. Alverdi, L. Giovagnini, C. Marzamo, R. Seraglia, F. Bettio, S. Sitran, R. Graziani,
D. Fregona, Journal of Inorganic Biochemistry 98 (2004) 1117–1128.
[9] R. Singh, N.K. Kaushik, Spectrochimica Acta Part A 71 (2) (2008) 669–675.
[10] H.D. Yin, J. Zhai, Y.Y. Sun, D.Q. Wang, Polyhedron 27 (2008) 663–670.
[11] A. Mohammad, C. Varshney, S.A.A. Nami, Spectrochimica Acta Part A 73 (2009)
20–24.
[12] K.D. Karlin, Progress in Inorganic Chemistry, vol. 53, John Wiley and Sons, Inc.,
New Jersey, 2005, p. 71.
[13] D. Chen, L. Powers, Journal of Inorganic Biochemistry 58 (1995) 245–253.
[14] A.V. Ivanov, T. Rodyna, O.N. Antzutkin, Polyhedron 17 (18) (1998) 3101–3109.
[15] Y. Yang, B. Zuo, J. Li, G. Chen, Spectrochimica Acta Part A 52 (1996) 1915–1919.
[16] T.S. Carswell, H.L. Morill, Industrial and Engineering Chemistry (1937)
1247–1251.
[17] N. Manav, A.K. Mishra, N.K. Kaushik, Spectrochimica Acta Part A 65 (2006)
32–35.
[18] N. Manav, A.K. Mishra, N.K. Kaushik, Spectrochimica Acta Part A 60 (2004)
3087–3092.
[19] A.P. Rauter, Lucas, T. Almeida, D. Sacoto, V. Ribeiro, J. Justino, A. Neves, F.V.M.
Silva, M.C. Oliveira, M.J. Ferreira, M.S. Santosa, E. Barbosaa, Carbohydrate
Research 340 (2005) 191–201.
[20] M.R.L. Oliviera, V.M. de Bellis, Transition Metal Chemistry 24 (1999) 127–130.
[21] A. Vandebeek, S.J. Joris, K.I. Aspila, C.L. Chakrabarti, Canadian Journal of Chem-
istry 48 (1970) 2204–2209.
[22] N. Geetha, S. Thirumaran, Journal of the Serbian Chemical Society 73 (2) (2008)
169–177.
[23] K.S. Siddiqi, S. Khan, S.A.A. Nami, M.M. El-ajaily, Spectrochimica Acta Part A. 67
(2007) 995–1002.
[24] R.M. Desai, M.K. Shah, V.H. Shah, E-Journal of Chemistry 3 (12) (2006) 137–141.
[25] S.A.A. Nami, K.S. Siddiqi, Synthesis and Reactivity in Inorganic, Metal-Organic
and Nanometal Chemistry 34 (9) (2005) 1581–1590.
[26] G.G. Mohamed, A.N. Ibrahim, A.E.H. Attia, Spectrochimica Acta Part A 72 (2009)
610–615.
4. Conclusions
[27] A.B. Prakasam, K. Ramalingam, G. Boceli, A. Cantoni, Polyhedron 26 (2007)
4489–4493.
[28] H.L.M. Van Gaal, J.W. Diesveld, F.W. Pijpers, J.G.M. Van der Linden, Inorganic
Chemistry 18 (11) (1979) 3251–3260.
[29] A.B. Prakasam, K. Ramalingam, G. Boceli, A. Cantoni, Polyhedron 26 (2007)
1133–1138.
[30] V.T. Yilmaz, T.K. Yazicilar, H. Cesur, R. Ozkanca, F.Z. Maras, Synthesis and Reac-
tivity in Inorganic, Metal-Organic and Nanometal Chemistry 33 (4) (2003)
589–605.
The characterization techniques confirm that both dithiocar-
bamate ligands are bidentately coordinated to the metal atom
via the sulphur atoms. The metal complexes exhibit antibacterial
activity against the chosen bacterial species. The zinc dithiocarba-
mate complex exhibited antibacterial activity against five bacterial
species. Broad spectrum bactericidal activity is displayed by the
triphenylphosphine adduct of the copper complex. The complexes

S.M. Mamba et al. / Spectrochimica Acta Part A 77 (2010) 579–587
587
[31] A.K. Mishra, N.K. Kaushik, Spectrochimica Acta Part A 69 (2008) 842–848.
[32] C. Pellerito, L. Nagy, L. Pellerito, A. Szorcsik, Journal of Organometallic Chemistry
691 (2006) 1733–1747.
[36] M.B. Klix, J.A. Verreet, M. Beyer, Crop Protection 26 (2007) 683–690.
[37] L.Z. Li, C.T. Zhao, T. Xu, H.W. Ji, Y.H. Yu, G.Q. Guo, H. Chao, Journal of Inorganic
Biochemistry 99 (2005) 1076–1082.
[33] H. van den Bossche, F. Dromer, L. Improvissi, M. Lozane-Chiu, J.H. Hex, D.
Sanglard, Medical Mycology 36 (1998) 119–128.
[38] K.R. Rupesh, S. Deepalatha, M. Krishnaveni, Venkatesan, S. Jayachandran, Euro-
pean Journal of Medicinal Chemistry 41 (2006) 1494–1503.
[34] N.V. Loginova, T.V. Koval’chuk, G.I. Polozov, N.P. Osipovich, P.G. Rytik, I.I.
Kucherov, A.A. Chernyavskaya, V.L. Sorokin, O.I. Shadyro, European Journal of
Medicinal Chemistry 43 (2008) 1536–1542.
[35] I. Rogachev, V. Gusis, A. Gusis, J.L. Cortina, J. Gressel, A. Warshawsky, Reactive
and Functional Polymers 42 (1999) 243–254.
[39] S. Khan, S.A.A. Nami, K.S. Siddiqi, E. Husain, I. Naseem, Spectrochimica Acta Part
A: Molecular and Biomolecular Spectroscopy 72 (2009) 421–428.
[40] J.G. Cappuccino, N. Sherman, Microbiology—A Laboratory Manual, 6th ed., Pear-
son Education, San Francisco, USA, 2002, p. 263.