Mononuclear transition metal complexes containing iodo-imidazole ring endowed with potential anti-Candida activity

Article abstract of DOI:10.1007/s00044-016-1653-4

Special attention is directed to design and synthesize antimicrobial drug candidates by the complexation of bioactive ligands with transition metals. In this pursuit a ligand with imidazole ring was synthesized and treated with Cu^II, Co^II and ,Ni^II salts to afford the mononuclear metallic complexes IHC1, IHC2 and IHC3, respectively, being assigned the general formula [M(L)₂]. Physical and spectral characterization supported octahedral geometry for the complexes. Both the ligand and the synthesized metal complexes were evaluated for their antifungal activity against three different strains of Candida, by determining the minimum inhibitory concentrations and minimum fungicidal concentrations. The antifungal activity results showed that the target compounds display remarkable antifungal activity, with metal complex IHC1 showing the most potent antifungal activity. Mechanism of action of the ligand and metal complexes appears to originate from membrane disruption as revealed by confocal scanning laser microscopy.

Full text of DOI:10.1007/s00044-016-1653-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1653-4
ORIGINAL RESEARCH
Mononuclear transition metal complexes containing iodo-imidazole ring
endowed with potential anti-Candida activity
1 ●
2 ●
3 ●
Mohmmad Younus Wani Aijaz Ahmad Manzoor Ahmad Malik
Abilio J.F.N. Sobral
1
Received: 25 April 2015 / Accepted: 4 July 2016
©
Springer Science+Business Media New York 2016
Abstract Special attention is directed to design and syn-
thesize antimicrobial drug candidates by the complexation
of bioactive ligands with transition metals. In this pursuit a
ligand with imidazole ring was synthesized and treated with
ligand and metal complexes appears to originate from
membrane disruption as revealed by confocal scanning laser
microscopy.
II
II
II
●
●
●
Cu , Co and ,Ni salts to afford the mononuclear metallic
Keywords Hydrazone Imidazole Candida Metal
●
complexes IHC1, IHC2 and IHC3, respectively, being
complexes Confocal microscopy
assigned the general formula [M(L) ]. Physical and spectral
2
characterization supported octahedral geometry for the
complexes. Both the ligand and the synthesized metal
complexes were evaluated for their antifungal activity
against three different strains of Candida, by determining
the minimum inhibitory concentrations and minimum fun-
gicidal concentrations. The antifungal activity results
showed that the target compounds display remarkable
antifungal activity, with metal complex IHC1 showing the
most potent antifungal activity. Mechanism of action of the
Introduction
In antifungal drug designing, the lack of selectivity of
conventional chemotherapeutic agents and the acquisition
of multiple-drug resistance are two major challenging pro-
blems. With the frequent use of antifungal drugs, resistance
has become a global concern (Chi et al., 2011; Kathiravan
et al., 2012). The clinical efficacy of many existing anti-
fungal drugs is being threatened by the emergence of
multidrug resistant pathogens (Vandeputte et al., 2012).
Despite the currently applied antifungal therapies, both
mortality and morbidity caused by human pathogens are
still unacceptably high (Poulain, 2013). Besides increasing
the morbidity and mortality rates, resistance to conventional
drugs have resulted in treatment failures and increased
health care costs. Therefore, new prophylactic and ther-
apeutic strategies are urgently needed to prevent fungal
infection.
Electronic supplementary material The online version of this article
(
doi:10.1007/s00044-016-1653-4) contains supplementary material,
which is available to authorized users.
*
Abilio J.F.N. Sobral
asobral@ci.uc.pt
Manzoor Ahmad Malik
wani@uc.pt
Various attempts have been undertaken in this pursuit
and an effective drug designing strategy is the complexation
of bioactive ligands with transition metal complexes.
Among the important pharmacophores responsible for
antifungal activity, the azole scaffold is still considered a
viable lead structure for the synthesis of more efficacious
and broad-spectrum antimicrobial agents. Modifying the
structure of the so far effective azole drugs in order to
1
2
Departmento de Quimica, FCTUC, Universidade de Coimbra, Rua
Larga, 3004-535 Coimbra, Portugal
Department of Clinical Microbiology and Infectious Diseases,
School of Pathology of NHLS and University of the Witwatersrand,
Faculty of Health Sciences, 2193 Johannesburg, South Africa
3
Department of Chemistry, Jamia Millia Islamia (Central Uni-
versity), New Delhi 110025, India

Med Chem Res
improve their antimicrobial potency and selectivity is of
scientific focus (Liu et al., 2008; Giraud et al., 2008).
Imidazole ring is a part of many existing drugs and the
interesting and versatile biological activities of imidazoles
established them as important pharmacophores (Sadek,
complexes was performed on a TG 51 thermogravimetric
analyzer under nitrogen atmosphere with the heating rate of
10 °C min . Reactions were monitored using thin-layer
chromatography using commercially available precoated
plates (Merck Kieselgel 60 F254 silica). Visualization was
−1
2
011; Zhang et al., 2014). Additionally, acyl-hydrazones
achieved with UV light at 254 nm or I vapour staining.
2
exhibit keto-enoltautomerism and –CO–NH–N< moiety of
these molecules is suggested to be responsible for their
important biological activities (Rollas and Kucukguzel,
General procedures for the synthesis of ligand (L)
2
007). A significant number of transition metal complexes
Synthesis of ethyl 2-(5-iodo-2-methyl-1H-imidazole-1-yl)
of acyl-hydrazones are known for wide spectrum of biolo-
gical activities, therefore a successful drug development
strategy is the complexation of transition metals with such
ligands, as it is possible to enhance the pharmacological and
chemical properties (such as potency, selectivity, chemical
stability and lipophilicity) of the target compounds (Stadler
and Harrowfield, 2009; Nandy et al., 2015; Babahan et al.,
acetate (2)
A mixture of 5-iodo-2-methyl-1H-imidazole (1) (1 mmol),
ethylchloroacetate (1 mmol) and potassium carbonate (1.5
mmol) in dry acetone (5–10 mL) was refluxed for 50 h. The
reaction mixture was filtered hot and the solvent was dis-
tilled off from the filtrate. The crude ester thus obtained was
purified by recrystallization from ethanol.
2
015). Another important fact is that the metals can coor-
dinate with O- or N-terminals from proteins in a variety of
models and play a crucial role in the conformation and
function of biological macromolecules (Bruijnincx and
Sadler, 2008).
Yield: 62.5 %; anal. calc. For C H N O : C 32.67,
8
11 2 2
H 3.77, N 9.53 %; found: C 32.48, H 3.92, N 9.23 %; IR
−1
νmax cm : 3028 (C–H, Ar), 1722 (C=O), 1635 (C=N),
1
1584 (C=C, Ar), 758 (C–I); H NMR (DMSO ) δ(ppm):
d
Considering our previous work on azoles (Wani et al.,
7.91 (s, 1H, imidazole ring), 4.80 (s, 2H, CH ), 4.10 (q, 2H,
2
2
012; Wani et al., 2012; Wani et al., 2013) and taking
CH ); 2.20 (s, 3H, CH , imidazole ring), 1.35 (t, 3H, CH ,
2
3
3
1
3
inspiration from the above facts, in this study we synthe-
sized imidazole ring bearing ligand with a hydrazone pen-
dent and its metal complexes as antifungal leads and made
an attempt to unravel the mechanism of action of the syn-
thesized compounds by confocal microscopy.
J = 6.8, Hz); C-NMR (DMSO ) δ (ppm): 164.21 (C=O)
d
148.51 (C=N), 128.50, 126.08 (C=C, imidazole ring),
64.51 (–O–CH ), 48.24 (CH ), 16.15 (CH ), 14.11 (CH ,
2
2
3
3
+
imidazole ring); FAB MS m/z: 294.06 [M + H] , 317.18
+
[M + Na] .
Synthesis of 2-(5-iodo-2-methyl-1H-imidazol-1-yl)
Materials and methods
Chemistry
acetohydrazide (3)
2-(5-iodo-2-methyl-1H-imidazol-1-yl)acetohydrazide
(3)
was synthesized as by a reported procedure (Wani et al.,
2013).
Solvents and organic reagents were purchased from Sigma
Aldrich and Merck (Germany) and were used without fur-
ther purification. Melting points (mps) were performed
using a Mel-temp instrument, and the results are uncor-
rected. Elemental analyses were performed on Her-
aeusVario EL III analyzer. The results were within ±0.4 %
of the theoretical values. The absorption spectra were
recorded using an OCEAN OPTICS USB 4000 UV spec-
trometer. IR spectra were recorded on Perkin-Elmer model
Yield 70 %; anal. calc. for C H N O: C 25.73, H 3.24,
6
9 4
−1
N 20.00 %; found: C 25.45, H 3.42, N 20.26 %;IR νmax cm :
3280 (NH), 3022 (C–H, Ar), 1711 (C=O), 1628 (C=N),
1
1595 (C=C, Ar), 786 (C–I); H NMR (DMSO ) δ (ppm):
d
8.32 (bs, 1H, NH), 7.68 (s, 1H, imidazole ring), 4.74 (s, 2H,
1
3
CH ), 2.34 (s, 3H, CH ) 2.18 (d, 2H, NH ); C-NMR
2
3
2
(DMSO ) δ (ppm): 162.11 (C=O) 148.48 (C=N), 128.30,
d
126.10 (C=C, imidazole ring), 26.15 (CH ), 12.15 (CH ,
2
3
+
1
600 FT IR RX1 spectrophotometer as KBr discs or ATR
imidazole ring); FAB MS m/z: 281.05 [M + H] .
1
13
mode. H and C-NMR spectra were recorded at ambient
temperature on a Bruker AVANCE 400 NMR spectrometer
using standard parameters. All chemical shifts are reported
in δ units with reference to TMS. The FAB mass spectra of
all the complexes were recorded on a JEOL SX 102/DA-
Synthesis of the ligand
(2-(5-iodo-2-methyl-1H-imidazol-1-yl)-N′-((pyridin-2-yl)
methylene)acetohydrazide) L
6
000 Mass Spectrometer/Data System using argon/xenon
(
(
6 kV, 10 mA) as the FAB gas and m-nitro benzyl alcohol
NBA) as the matrix. Thermogravimetric analysis of the
2-Pyridinecarbaldehyde (10 mmol) was added over a solu-
tion consisting of 2-(5-iodo-2-methyl-1H-imidazol-1-yl)

Med Chem Res
acetohydrazide (3) (10 mmol) in 20 ml ethanol and the
reaction mixture was kept under reflux for about 3 h.
The solvent was removed under reduced pressure and the
microcrystalline mass obtained was recrystallized from
methanol.
also included in every set of experiments. To determine
MFC values, after reading the corresponding MIC values,
20 µL samples from all optically clear tubes (complete
growth inhibition) plus the last tube showing growth were
sub-cultured on YPD agar plates. The plates were incubated
at 37 °C for a minimum of 3 days, until growth was clearly
visible in the control samples, and MFC values were
determined as the lowest concentration of the test com-
pounds for which there was no visible growth on plates.
−1
Yield: 89.5 %; IR νmaxcm : 3570 (NH), 3022 (C–H,
1
Ar), 1675 (C=O), 1628 (C=N), 1595 (C=C, Ar); H NMR
(
DMSO ) δ (ppm): 8.69 (bs, 1H, NH), 8.38–7.54 (m, 4H,
d
Ar), 7.38 (s, 1H, imidazole ring), 7.30 (s, 1H, CH), 4.72 (s,
1
3
2
H, CH ), 2.36 (s, 3H, CH ); C-NMR (DMSO ) δ (ppm):
2 3 d
1
68.12 (C=O) 152.64 (C=N, pyridine ring), 148.48 (C=N,
Disc diffusion halo assays
imidazole ring), 148.11 (C=C, pyridine ring), 138.63
C=N, hydrazone),136.12, 129.10 (C=C, pyridine ring),
28.30, 126.10 (C=C, imidazole ring), 26.45 (CH ), 12.28
(
1
Strains were inoculated into liquid YPD medium and grown
overnight at 37 °C. The cells were then pelleted and washed
three times with distilled water. Approximately, 10 cells/
2
+
+
5
(
3
CH , imidazole ring); FAB MS m/z: 369.03 [M + H] ,
92.08 [M + Na] .
3
+
+
mL were inoculated in molten agar media at 40 °C and
poured into 100 mm diameter petriplates. Filter discs were
kept on solid agar and test compounds were spotted on the
disc. Test compounds (four fold more than MIC) were
pipetted onto 4 mm diameter filter discs and the plates were
incubated for 48 hours at 37 °C. After incubation period, the
diameter of zone of inhibition was recorded in millimetres
and was compared with that of control. Experiments were
performed thrice in replicate on separate days and values
were calculated in terms of meanof all three respective
categories.
Synthesis of the metal complexes
To a hot solution of the ligand (L), (2 mmol) in methanol
(
10 mL) was added a solution of respective metal chloride
salt (Cu, Co, Ni) (1 mmol) dissolved in minimum quantity
of ethanol and the reaction mixture was heated under reflux
for 3–4 h. After keeping the solution at 0 °C overnight, the
coloured solid separated out. This was filtered off and
washed with hot water followed by small quantity of
ethanol and dried to give amorphous solids in 65–75 %
isolated yield. (For spectral data see supporting
information).
Growth curve studies
To further check the effect of the ligand and its metal
complexes on the growth of the Candida cells, growth
curve study was performed as described previously (Khan
et al., 2010). Candida cells from the overnight grown pri-
mary culture were inoculated into 100 mL of fresh YPD
broth and incubated at 37 °C for 24 h. Both ligand and its
metal complexes were added at MIC and half MIC values to
the cultures along with the positive and negative controls.
After every 2 h aliquots were taken and growth was recor-
ded turbidometrically at 595 nm using a LaboMed Inc.
Spectrophotometer (USA). All the experiments were done
in triplicate and the results were shown as mean ± standard
deviation.
Biology
Strains and media
Stock cultures of the C. albicans, C. glabrata and C. tro-
picalis were maintained on nutrient agar slants and stored at
4
°C. Prior to experiment, cells were grown and sub-cul-
tured in yeast extract, peptone and dextrose (YPD) media at
7 °C in orbital shaker at 200×rpm (REMI CIS 24 BL).
3
Prior to each experiment, overnight grown microorganism
suspensions in YPD broth were standardized to 10 CFU/
mL.
6
Determination of minimum inhibitory concentrations (MIC)
and minimum fungicidal concentrations (MFC)
Confocal scanning laser microscopy (CSLM)
Confocal scanning laser microscopy (CSLM) was used to
evaluate the effect of test compounds on the architecture of
yeast cells using propidium iodide (PI). PI is a fluorescent
probe used to study the effect of drugs on membranes. It
only penetrates cells with severe membrane lesions, show-
ing increased red fluorescence (Ahmad et al., 2011; Naskar
Minimum inhibitory concentration was defined as the
lowest concentration of the test compound that causes
inhibition of visible growth (turbidity). MIC was deter-
mined in vitro in liquid medium by the macrobroth dilution
method as per the guidelines of CLSI reference document
M27-A3 (CLSI, 2008). Positive control fluconazole, nega-
tive vehicle control (1 % DMSO) and culture control were
6
et al., 2005). Candida albicanscells (10 /mL) were incu-
bated at 37 °C up to mid-exponential phase and were then

Med Chem Res
treated with four times MIC of test compounds along with
negative (no compound) and positive (5 mg/L amphotericin
B) controls. The samples were then inoculated at 37 °C for
percentage of stained dead cells were shown as mean of
these counts.
3
0 min, in dark. Unstained cells were always included as
auto-fluorescence controls. The suspensions were cen-
trifuged, washed and resuspended in phosphate-buffered
saline. Five microlitres of PI were added to the cell sus-
pensions in order to obtain a final concentration of 1 mg/L
to determine the changes in cellular morphology. The cells
were examined with Olympus Laser Confocal Scanning
Microscope equipped with green helium neon laser (543
nm) for PI. The objective used was an oil immersion
C-Apochromat lens (20×). Image acquisition was done by
FV10-ASW1.6 Software.Four different slots per slide were
selected independently and stained and unstained cells were
counted. All the experiments were done in triplicate and
Results and discussion
Chemistry
The starting material 5-iodo-2-methyl-1H-imidazole (1)
employed in the preparation of the ligand was obtained
from Sigma and used after purification by recrystallization.
2-(5-iodo-2-methyl-1H-imidazol-1-yl)acetohydrazide
(3)
was obtained by refluxing the starting material 1 with
ethylchloroacetate under basic conditions and subsequent
treatment with hydrazine hydrate in ethanol. Final step
involved the treatment of acetohydrazide (3) with equimolar
ratio of 2-pyridine carboxaldehyde in ethanol (Scheme 1).
The ligand (L) was obtained in 75 % yield and recrys-
tallized from ethanol and found to be highly soluble in
methanol, ethanol, DMSO, DMF, acetonitrile, dichlor-
omathane and chloroform. Structure was established by
N
I
O
N
N
Cl
H C
3
H C
3
O
N
H
O
I
1
13
K CO , dry acetone
FTIR, H NMR, C-NMR and FAB MS spectral analysis.
The complexes were prepared by mixing 2:1 ratio of the
appropriate ligand (L) and metal chloride salts (Cu, Co, Ni)
in ethanol under reflux for 3–4 h (Scheme 2). After keeping
the solution at 0 °C overnight the coloured solid separated
out. This was filtered off and washed with hot water fol-
lowed by small quantity of methanol, and dried in vacuo
over silica gel to give amorphous solids with >75 % yield.
Our attempt to get single crystals of the complexes was a
failure. The obtained complexes are stable in air, are soluble
in DMF, DMSO, sparingly soluble in MeOH, EtOH and
2
3
O
(
1)
(
2)
NH NH .H O
2
2
2
C H OH
2
5
N
H C
3
I
N
O
N
O
H
N
I
HN
H C
3
N
N
O
HN
DCM, but insoluble in water and having mps higher than
N
C H OH, AcOH
2 5
−3
H
300 °C. The molar conductance values in DMSO (10 M)
reflux
NH2
are too low to account for any dissociation, therefore the
complexes are considered to be non-electrolytes (Chandra
et al., 2009). IHC1, IHC2 and IHC3 showed value of 28,
L
(3)
−
1
2
−1
Scheme 1 Synthesis of ligand (L)
36 and 29 Ω cm mol , respectively. The elemental
Scheme 2 Synthesis of metal
complexes (IHC1–IHC3)
H C
3
N
N
O
N
N
O
I
NH
H
dry Methanol
reflux
2
L
+
MCl2
Complex
N
HN
M
N
I
H
M
N
Cu IHC1
Co IHC2
N
CH3
Ni IHC3

Med Chem Res
analyses data along with some physical properties of the
ligand (L) and its complexes (IHC1, IHC2 and IHC3) are
reported in Table 1. The chemical structures of all the
compounds were confirmed by means of elemental analysis
and electronic, IR, H NMR and C-NMR spectral studies.
The structures of complexes were further established by
thermogravimetric analysis and FAB MS. The analytical
data of these compounds are in good agreement with their
composition.
The UV-vis spectroscopy is a versatile technique, which
enables establishing structure of metal complexes with
utmost certainty. Electronic spectra of the ligand, shows two
bands at 255 nm and 335 nm due to the benzene ring and
the π→π* transition of the chromophores (–C=N–NH–
CO–), respectively. These bands get red shifted by 5–10 nm
and 10–40 nm respectively, in the electronic spectra of the
complexes. In addition to ligand-originated bands, addi-
tional absorption bands due to metal-originated d-d transi-
tions and charge transfer spectra were observed in the
spectra of the complexes, characteristic to their geometries.
The electronic spectra of the copper (II) complex (IHC1)
1
13
Selected significant IR bands revealed the structural
characteristics of the compounds. The Ligand (L) may
exhibit keto-enol tautomerism since it contains a keto group
2
2
(
C=O) and a proton adjacent to carbonyl group (Fig. 1).
display bands at 770 nm and 368 assigned to B → E
1g g
−1
II
The absence of υ (OH) stretch at 3550–3200 cm and the
presence of υ (N–H) stretch at 3570 cm in the spectra of
transitions and, charge transfer bands (L→Cu CT),
respectively, indicating its tetragonally distorted octahedral
geometry (Yan-Hui et al., 2007; Lever, 1968). This com-
plex has magnetic moment 1.96 BM, which is higher than
the spin-only value (1.70 BM) expected for one unpaired
electron and offers possibility of an octahedral geometry
−1
the ligand suggest that the ligand remains in the keto form
−1
in the solid state. The amide C=O stretch at 1675 cm of
−1
ligand is shifted to lower wave number by ca. 15–30 cm ,
indicating that it participates as a coordinating site. This
coordination behaviour of the ligand is also proved by the
appearance of IR bands due to υ M–O and υ M–N vibra-
II
(Singh et al., 2007). Cu ion has one unpaired electron in
the 3d shell, therefore, its compounds have magnetic
moment close to spin-only value of 1.73 BM. Due to spin
orbital coupling, higher values are observed. Cobalt(II)
complex (IHC2) gave rise to three absorption bands in the
−
1
−1
tions in the range 478–498 cm
and 428–450 cm ,
respectively (Abu-Melha and El-Metwally, 2007; Chandra
and Sharma, 2009). The pyridine-ring-stretching, in-plane-
ring-bending and out of-plane-ring-bending vibrations are
−1
found at 1502, 628 and 502 cm , respectively. These
absorptions are highly affected when nitrogen atom of
pyridine ring takes part in coordination. The position of
these absorption bands is shifted to higher region, indicating
that the nitrogen of pyridine ring is involved in coordina-
N
N
I
I
N
N
O
H
HO
−
1
tion. The negative shift of 25–50 cm of υ (C=N) stretch in
the complexes indicates the involvement of azomethine
nitrogen in complexation (Lever, 1968). The broad band
N
N
N
N
N
N
−1
observed in region 3570 cm due to υ (N–H) stretch is
slightly shifted in complex probably due to the adjustment
of current arising due to coordination of amide C=O, thus,
confirming the fact that ligand behaves as a neutral tri-
dentate NNO donor in these complexes.
amido-imino form
imido-imino form
A
B
Fig. 1 Possible Keto-Enol tautomerism of the Ligand (L) (A = Keto;
B = Enol)
−
1
2
−1
Table 1 Elemental analyses
data along with some physical
properties of the ligand (L) and
its complexes (IHC1, IHC2 and
IHC3)
Compound Molecular formula MW calculated Elemental analysis
Λ Ω cm mol
µ (BM)
C
H
N
M
L
C
12
H
12
N
5
OI
369.03
Cu) 802.84
Co) 797.92
Ni) 797.95
39.04 3.28 18.97
9.28 3.40 18.78
35.95 3.02 17.47 7.48 28
6.15 3.12 17.22 7.60
36.15 3.03 17.57 10.98 36
6.35 3.18 17.76 10.72
36.17 3.04 17.57 7.36 29
6.10 3.22 17.85 7.58
—
—
—
3
IHC1
IHC2
IHC3
(C24
(C24
(C24
H
24
N
24
N
24
N
10
10
10
O
I
2 2
I
2 2
I
2 2
1.96
4.85
2.75
3
H
H
O
O
3
5
−
1
2
−1
MW Molecular Weight, Molar conductance Λ Ω cm mol , Magnetic moment µ (BM)

Med Chem Res
visible region, under the influence of octahedral field, by the
signal is shifted downfield by 2–5 ppm in their respective
complexes, which indicates coordination of nitrogen lone
pair to metal.
4
excitation of the electron from the ground state T (F) to
1
g
4
4
4
the excited states T (F), A (F) and T (P). 940 nm
assigned to T (F)→ T (F) ν , 710 nm assigned to
2
g
2g
1g
4
4
Thermal behaviour of the complexes (IHC1, IHC2 and
IHC3) was studied in the temperature range 25–900 °C. All
the dehydrated complexes are stable upto ca.150 °C and
then decompose in three major steps. The temperature range
for the first endothermic step being 150–290 °C followed by
second exothermic step in the temperature range of 290–
380 °C. Differential Thermal Analysis plots for complexes
suggest their exothermic nature of decomposition in the
subsequent stages. Third stage of decomposition starts
immediately after second one and continues until complete
decomposition of the ligand and formation of MO (M=Cu,
Co, Ni) as the end product. The final temperature at which
MO obtained varied from complex to complex, but in any
case the formation of final stable product does not go
beyond 900 °C. Although decomposed fragments of the
ligand could not be approximated due to continuous weight
loss, the total percentage of weight loss of the complexes
corresponds to the loss of the ligand after considering the
transfer of one oxygen atom to the metal ion and residue
corresponds to the metal oxide. Additionally, a general
splitting pathway with the characteristic peaks was observed
by the metal complexes. The positive ion FAB mass spec-
trum of the complexes was recorded using n-NBA as the
matrix. The spectra of the complexes (IHC1, IHC2 and
IHC3) showed a number of informative fragment ions of
different intensities confirming their molecular weights. The
mass spectrum of the metal complexes (IHC1, IHC2 and
IHC3) obeyed a similar pattern of fragmentation with the
1
g
2g
1
4
4
4
4
T (F)→ A (F) ν , and 440 nm assigned to T (F)→
1
g
2g
2
1g
7
T (P) ν transitions in a high-spin d octahedral environ-
1g
3
ment (Rakha et al., 2014). The room temperature magnetic
moment value of 4.85 BM demonstrates that IHC2 is
paramagnetic and has a high-spin octahedral configuration
4
with T (F) ground state (Subramanya et al., 2003). The
1
g
electronic spectra of Nickel (II) complex (IHC3) display
three absorption bands at 915, 660 and 395 nm, assigned to
3
3
3
3
3
A (F)→ T (F) υ , A (F)→ T (F) υ and A (F)→
2
g
2g
1
2g
1g
2
2g
3
T (P) υ , respectively (Ibrahim et al., 2014). The magnetic
1g
3
moment of the Ni(II)complex at room temperature lies in
the range of 2.75 BM corresponding to two unpaired elec-
trons. Thus, on the basis of spectral studies complexes
IHC1, IHC2 and IHC3 were assigned to have a six coor-
dinated distorted octahedral geometry as shown in
Scheme 2.
Further evidence for the formation of compounds was
1
obtained from the H NMR spectra. Assignments of the
signals are based on the chemical shifts and intensity pat-
terns. The ligand (L) did not show any resonance at ca. 0.5–
5
.0 ppm, attributed to –OH proton resonance, while the
appearance of a broad peak at 8.63 ppm due to the –NH
proton of amide group indicates that even in a polar solvent
such as DMSO they remain in the keto form. The –NH
proton signal of the ligand usually shifts 1.50–2.50 ppm
upfield in the complexes (IHC1, IHC2 and IHC3). How-
ever, in complexes, we could not calculate the coupling
constant values for aromatic region, possibly due to the
merging of peaks upon coordination. This information
suggests the adjustment of electronic current upon coordi-
nation of >C=O group to the metal ion. The aromatic ring
protons were observed with the expected chemical shift and
integral values in the same region as those of free ligand.
+
+
+
molecular ion peaks observed as [M + Na +H] , [M + K +
+
+
+
H] , [M + Na ] (Metal adduct ions) and [M + H] and the
major fragmentation pathway involved the cleavage of
amide group.
1
3
Moreover, the C-NMR spectra of the ligand taken in
DMSO gave the spectral signals in good agreement with the
probable structures. The ligand showed two signals at 173.5
and 164.5 ppm assigned to the amide (C=O) and azo-
methine carbon (C=N), respectively. The signals from
Antifungal Studies
MICs and MFCs
MICs and MFCs of the ligand and its metal complexes have
been determined against three different laboratory Candida
species. Table 2 summarizes the in vitro susceptibilities
MICs and MFCs of all the test compounds against these
fungal isolates. Evaluation of MIC and MFC values showed
that both the ligand and its metal complexes were active in
vitro against all the tested isolates, however, the MIC/MFC
values of the ligand against all the three types of Candida
strains were higher when compared to the metal complexes
(Table 2, Fig S1), but are within the reference range. The
lowest MIC/MFC results obtained with metal complex
IHC1 suggest an increase in the antimicrobial activity after
1
26.8 to 148.5 ppm were due to the imidazole, pyridine and
1
3
phenyl ring carbons. C-NMR spectra were also used for
the elucidation of the coordination mode of the ligand in
complexes. Assignments of the signals are based on the
chemical shifts and intensity patterns and coordination
induced shift (CIS), Δδ [Δδ = δ (complex)−δ (free ligand)],
of the signals for carbon atom in the vicinity of the coor-
dinating functions. Thus the C=O carbon in the ligand
experiences CIS value of 5–8 ppm in complexes, indicating
the coordination of carbonyl oxygen. As a result of varia-
tion of electron density on coordination, azomethine carbon

Med Chem Res
Table 2 Minimum inhibitory concentrations of L, IHC1, IHC2 and
IHC3
Compound
Mean MIC/MFC (mg/L)
C. albicans
C. glabrata
C. tropicalis
ATCC 10261
ATCC 90030
ATCC 750
L
50/100
6. 25/12.5
12.5/25
12.5/100
2
50/100
6. 25/12. 5
6. 25/25
25/200
4
50/100
6. 25/12. 5
6. 25/12.5
25/200
8
IHC1
IHC2
IHC3
Fluconazole
Amphotericin B
0.125
2
2
II
the interaction of the ligand with Cu ions. These results are
in congruent with the previous findings where metal com-
plexes were observed to easily disrupt the cell walls leading
to cell death (Goulart, 2011; Antonio et al., 2014).
Disc diffusion assay
Fig. 2 Representative plate of disc diffusion assay of C. albicans
0261 treated with ligand (b), IHC1 (c), IHC2 (d) and IHC3 (e).
Negative solvent control (1 % DMSO) is represented by (a) in the
centre. Zone of inhibitions were observed after plates were incubated
for 48 h at 37 °C
1
Index of sensitivity (SI) was defined as the diameter of
inhibition zone(mm)/concentration (mg/mL) = clearing
(
mm/mg). The sequence of the SI for all the test compounds
decreases in the order given as IHC1>IHC2>IHC3>L.
The SI values for IHC1, IHC2, IHC3 and L are 3.4, 3.2,
depicts the higher effect of the complexes than the ligand.
A known drug (2 mg/Lamphotericin B) was included as a
positive control, depicting flat lines with complete inhibi-
tion of growth in all the three isolates. Flat lines at MIC
values of the ligand and its metal complexes as well as that
of known drug against all the three types of Candida iso-
lates showed potent antifungal efficacy of the legend and its
metal complexes. These results also highlight that the
antifungal effects of ligand and its metal complexes are
primarily concentration dependent.
2
.5 and 1.2, respectively. While variability may occur
among the antifungal activity of the test compounds, it is
exciting that the complexes except IHC3 showed fungicidal
potential, as is evident from the clear zones of inhibition in
disc diffusion assay; whereas in contrast IHC3 showed a
visible turbid halo, an indication of its fungistatic nature
(
Fig. 2). There was a complete growth observed around the
control disc impregnated with the solvent (1 % DMSO),
which indicates that all the inhibition of the Candida growth
is because of the test compounds and not of the solvent.
Growth curve studies
Confocal laser microscopy
Effect of different concentrations of the ligand and its metal
complexes on the growth pattern of C. albicans, C. tropi-
calis and C. glabrata are shown in Fig. 3 (a–c), respec-
tively. The control Candida cells showed a normal pattern
of growth, with a lag phase of 2–4 h and an active expo-
nential phase of 14–16 h before attaining the stationary
phase. The test compounds suppressed growth and delayed
exponential phases of all three tested isolates of Candida as
shown in Fig. 3. Both the ligand and the complexes affect
the growth pattern of Candida species almost in a similar
manner. At MIC values of the test compounds flat lines
were observed indicating the complete inhibition of growth,
whereas, ½ MIC values showed reduced growth pattern in
comparison with the control. The growth pattern clearly
Candida albicans cells were grown and then stained with PI
as described in experimental section. PI penetrates only
those cells which have severe membrane lesions; the entire
yeast cell appears red. The laser confocal images of stained
Candida cells exposed to test compounds are shown in
Fig. 4. The dead stained cells were counted from four dif-
ferent slots per slide in three different experiments and
mean percentage was expressed. Our results showed that
maximum of PI penetrates (dead cells) 75 % of the yeast
cells when treated with 4×MIC of IHC1, while as 65 %
cells showed PI uptake when treated with 4×MIC of IHC2
and 65 % with 4×MIC of IHC3. 30 % cells showed PI
uptake when treated with 4×MIC of the ligand (L).
This indicates that the structure of the cell membrane was

Med Chem Res
Fig. 3 Growth curve study of representative standard—Candida
albicans (a), Candida tropicalis (b) and Candida glabrata (c) isolates
grown with half MIC of ligand (triangles) and MIC of ligand (plus),
half MIC of IHC1 (cyrillic) and MIC of IHC1 (minus), half MIC of
IHC2 (stars) and MIC of IHC2 (hyphen), half MIC of IHC3 (circles),
and MIC of IHC3 (black lozenge). Squares and diamonds represent
negative and positive controls, respectively. Values shown in figure
are mean ± SD of three independent recordings
disrupted by these test compounds to a large extent indi-
cating that these compounds display a membrane disruption
mechanism of action. Under these conditions, 5 mg/L
amphotericin B-induced PI staining in more than 90 % of
Candida cells (Fig. 4).
metals and drug action is increased due to effective per-
meability of the drug into the site of action. In fact, metal
complex provide better opportunities to be used as ther-
apeutic agents. The enhanced antifungal activity of the
complexes over the ligand can also be attributed to the fact
that the acyl-hydrazone ligand contains a polar head; the
hydrazide part and an aromatic hydrophobic part. The
This study supports the fact that the lipophilicity of the
drug is increased through the formation of chelates with

Med Chem Res
Fig. 4 Laser confocal images of
Candida albicans cells. Cells
with membrane damage are seen
stained with PI (red signals)
Control -ive (0%)
Control +ive Amp B
L (30%)
IHC1 (75%)
IHC2 (65%)
IHC3 (50%)
coordination allows the ligand to hide the acyl-hydrazone
polar part around the metal and the metal complex thus
exposes the hydrophobic moiety to the solvent endowing it
of the features necessary to cross the cell membrane.
Another assumption is that the combination of two ligands
with a divalent metal ion results in a charge neutral complex
Additional in vitro and in vivo tests are required to be
performed on new fungal strains including the resistant
species and other pathogens to evaluate antimicrobial
potentials of these novel complexes.
(
Bernhardt et al., 2009). This charge neutrality means that
Conclusions
the complexes themselves may also be able to cross the cell
membrane, and this provides a novel method of delivery of
the compounds to the cell in the form of a labile divalent
complex which may then dissociate within the cell
II
II
In the present paper, we discussed the synthesis of Cu , Co
II
and Ni metal complexes of a ligand bearing an iodo-imi-
dazole ring and hydrazone pendent with a pyridine ring. In
all complexes, the ligand L acted as a tridentate NNO donor
favouring an octahedral geometry. Further antifungal activity
evaluation by determining MIC and MFC, disc diffusion
assay and growth curve studies revealed that the metal
complexes show enhanced activity compared to the ligand
that can be attributed to their increased lipophilicity com-
pared to the ligand alone. Laser confocal images showed
remarkable membrane disruption due to the target com-
pounds. Therefore, these preliminary results indicate that
iodo-imidazole and pyridine ring bearing transition metal
complexes can be promising therapeutic agents against fun-
gal infections, which, however, needs further optimization.
(
Bernhardt et al., 2009). The reason for high antifungal
activity of copper complex (IHC1) can be explained in
terms of the effect of 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 positive
charge with donor groups and electron delocalization over
the chelate ring (Özer et al., 2009). Thus, the lipophilic
character of the central metal atom is enhanced that results
in a higher capability to penetrate the microorganisms
through the lipid layer of the cell membrane.
Rapidity of action and low MIC values of these novel
complexes offered an opportunity to develop new anti-
fungals in the continued quest to control important infec-
tious pathogens. However, the need for the second step
testing, which involves the trialling of these compounds for
toxicity and side effects at cellular levels is highly required.
Acknowledgments MAM is thankful to UGC India for financial
support. Fundação para a Ciência e a Tecnologia (FCT-Portugal) grant
SFRH/BPD/86581/2012 (M.Y. Wani) is highly acknowledged.

Med Chem Res
Compliance with ethical standards
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