Heteroleptic transition metal complexes of Schiff-base-derived ligands exert their antifungal activity by disrupting membrane integrity

Article abstract of DOI:10.1002/aoc.5128

Development of new treatment strategies and chemotherapeutic agents is urgently needed to combat the growing multidrug resistant species of Candida. In this direction, a new series of Cu (II), Co (II), Ni (II) and Zn (II) heteroleptic complexes were synth

Full text of DOI:10.1002/aoc.5128

Received: 15 April 2019
DOI: 10.1002/aoc.5128
Revised: 23 June 2019
Accepted: 28 June 2019
F U L L P A P E R
Heteroleptic transition metal complexes of Schiff‐base‐
derived ligands exert their antifungal activity by disrupting
membrane integrity
1
2
1
Ovas Ahmad Dar | Shabir Ahmad Lone
| Manzoor Ahmad Malik |
3
1
3
Mohmmad Younus Wani
| Md Ikbal Ahmed Talukdar | Abdullah Saad Al‐Bogami |
1
2,4
Athar Adil Hashmi | Aijaz Ahmad
1
Department of Chemistry, Jamia Millia
Development of new treatment strategies and chemotherapeutic agents is
Islamia, New Delhi 110025, India
2
Clinical Microbiology and Infectious
urgently needed to combat the growing multidrug resistant species of Candida.
In this direction, a new series of Cu (II), Co (II), Ni (II) and Zn (II) heteroleptic
complexes were synthesized, characterized and evaluated for antifungal
activity. Based on spectral characterization and physical measurements, an
Diseases, School of Pathology, Faculty of
Health Sciences, University of the
Witwatersrand, Johannesburg 2193, South
Africa
3
Chemistry Department, Faculty of
octahedral geometry was assigned to [Co(L1)(L2)ClH O] (C2), [Ni(L1)(L2)
2
Science, University of Jeddah, Jeddah,
Kingdom of Saudi Arabia
ClH O] (C3), [Zn(L1)(L2)ClH O] (C4) complexes, while a distorted octahedral
2
2
4
geometry was assigned to [Cu(L1)(L2)ClH O] (C1) complex. All the
2
Infection Control, Charlotte Maxeke
Johannesburg Academic Hospital,
National Health Laboratory Service,
Johannesburg 2193, South Africa
synthesized compounds were tested for antifungal activity against 11 Candida
albicans isolates, including fluconazole (FLC)‐resistant isolates, by determining
minimum inhibitory concentrations (MIC) and minimum fungicidal
concentrations (MFC), following CLSI guidelines. The mechanism of their
antifungal activity was assessed by studying their effect on the plasma
membrane using flow cytometry and quantifying the ergosterol contents. All
the test compounds showed varying levels of antifungal activity. Both the
ligands showed moderate antifungal activity with a median MIC value of
Correspondence
Mohmmad Younus Wani, Chemistry
Department, Faculty of Science,
University of Jeddah, P.O. Box 80327,
Jeddah 21589, Kingdom of Saudi Arabia.
Email: mwani@uj.edu.sa
Athar Adil Hashmi, Professor,
100 μg/mL with no fungicidal activity. Compound C3 was the most potent
Department of Chemistry, Jamia Millia
Islamia (Central University). New Delhi‐
compound with median MIC and MFC values of 0.10 and 1.60 μg/mL,
respectively. Flow cytometry analysis revealed that these compounds at MFC
values disrupt the cell membrane, resulting in propidium iodide entering the
cells. These compounds also reduced a considerable amount of ergosterol
content after treating the cells with MIC and sub‐MIC values. This study
indicates that these compounds have high antifungal activity against C.
albicans, and have the potential to be developed as novel antifungal drugs.
110025, India.
Email: aah_ch@yahoo.co.in
Aijaz Ahmad, Senior Medical Scientist,
Clinical Microbiology & Infectious
Diseases, University of the Witwatersrand.
Johannesburg, 2193, South Africa.
Email: aijaz.ahmad@wits.ac.za
Funding information
University Research Committee Grant for
KEYWORDS
2019 ‐ Friedel Sellschop Award, Grant/
Candida albicans, ergosterol, heteroleptic complexes, membrane disruption
Award Number: AZMD019
Appl Organometal Chem. 2019;e5128.
wileyonlinelibrary.com/journal/aoc
© 2019 John Wiley & Sons, Ltd.
1 of 14
https://doi.org/10.1002/aoc.5128

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DAR ET AL.
1
| INTRODUCTION
biologically important scaffolds in the form of heteroleptic
complexes, would deliver a synergistic effect and thus act
as a hopeful stratagem for development of effective
chemotherapeutic agents. Literature reveals that the Schiff
base ligands derived from 1,4‐benzodioxane‐6‐amine have
not been much studied, and the Schiff bases derived from
4‐(dimethyl‐amino)benzaldehyde show great pharmaco-
The growing incidences of life‐threatening systematic fun-
gal infections due to a rise in immunocompromised patient
populations and the limited availability of antifungal med-
ications necessitates the development of new antifungal
[1,2]
drugs or treatment strategies.
Fungal diseases, which
[
18–21]
include pulmonary aspergillosis, cryptococcal meningitis,
pneumocystis pneumonia, invasive candidiasis, invasive
aspergillosis, disseminated histoplasmosis, fungal asthma
and fungal keratitis, kill more than 1.5 million and affect
logical effects.
Therefore, in the present study some
new heteroleptic complexes derived from two different
bidentate Schiff base ligands have been synthesized, and
their promising antifungal potential was evaluated against
different fluconazole (FLC)‐susceptible and ‐resistant
Candida albicans isolates in a quest to find a better
antifungal agent.
[
3,4]
over a billion people worldwide.
Different antifungal
drugs belonging to either azoles or polyenes are currently
being used to treat fungal infections, but emergence
of drug resistance and treatment failures is a big
[5–7]
challenge.
Among the various strategies to tackle this
challenge and curb growing drug resistance and fungal
infections, development of new drugs based on a different
mechanism of action or drugs that target multiple path-
ways to evade drug resistance are currently being pur-
2 | EXPERIMENTAL
2.1 | Materials and physical methods
[8–10]
sued.
Complexation of potentially bioactive organic
All the chemicals used were of reagent grade,
and were used without further purification. 1,4‐
Benzodioxane‐6‐amine and 2‐hydroxy‐1‐naphthaldehyde
were purchased from Sigma‐Aldrich Chemie (Germany).
Ethylenediamine, metal chlorides and ethanol were
purchased from Merck. 4‐(dimethyl‐amino)Benzaldehyde
was purchased from Sd‐fine.
The prepared Schiff base ligands and metal complexes
were subjected to microanalyses (C, H and N) that were
carried out with Elementar (Thermo Scientific). The molar
conductivities of freshly prepared solutions at room
temperature were measured on a Syntronics type 302
molecules with metals results in a sharp increase in their
biological activity. A metal complex usually results in
enhanced efficacy compared with the ligand or the metal
ion alone, by sometimes the ligands protecting or
stabilizing the otherwise reactive metal ion or the metal
undergoing redox events that enhance the overall efficacy
of the complex, and sometimes the metal and the ligand
are mutually responsible for the biological activity of the
[11]
complex. Coordination compounds offer many binding
modes to polynucleotides, including outer‐sphere
non‐covalent binding, metal coordination to nucleobase
and phosphate backbone sites, as well as strand cleavage
conductivity bridge equilibrated at 25
±
0.01°C.
[
12]
induced by oxidation using redox‐active metal centers.
For conductivity measurements, 1mM solutions in
dimethylsulfoxide (DMSO) were prepared. Electrothermal
melting point apparatus was used to determine the melting
point of the synthesized compounds. UV–Vis spectra were
recorded between 200 and 800 nm using UV–Vis
spectrophotometer (UV‐260 Shimadzu, with 1‐cm quartz
cuvettes). Fourier transform‐infrared (FT‐IR) spectra of
the Schiff base ligands and metal complexes were
investigated using KBr pellets on a Perkin‐Elmer 283
The accessibility of different oxidation states of metals such
as Fe, Cu, Co, Ru, Mn, etc. may allow for redox chemistry
resulting in strand breakage and other alterations, which
result in the selective inhibition of the target cell or
[13]
pathogen.
A diverse range of ligands with different coordination
sites have been synthesized and used as chelators for
various metal ions. An important class of molecules is
the Schiff base ligands; they show great chelating proper-
ties due to their azomethine and other coordinating sites
or groups. They are stabile under various oxidative and
reductive conditions, which make them ideal candidates
for the development of new metal‐based antimicrobial
1
13
spectrophotometer. H‐NMR and C‐NMR measure-
ments were recorded using Brucker WH 300 (200 MHz)
and Brucker WH 270 (67.93 MHz) using DMSO‐d as a
6
solvent and tetramethylsilane as an internal standard.
ESI‐MS (AB‐Sciex 2000, Applied Biosystem) was used to
record mass spectra of the synthesized ligands and
complexes. The magnetic susceptibility of the investigated
compounds was measured using the Faraday balance.
Powder X‐ray diffraction (XRD) studies of the synthesized
compounds were carried out at room temperature using
Rigaku, Mini Flex II powder diffractometer using CuKα
[14–16]
and anticancer agents.
Keeping in view the imperative influence of Schiff bases
[17]
in medicinal chemistry, and the impact of metallodrugs
as potential chemotherapeutic agents, our main rationale
of this study was to explore whether the incorporation of
a transition metal ion, a Schiff base derived from

DAR ET AL.
3 of 14
monochromatic radiation (λ = 1.5406 Å) in the range of
8.65 (s, 2H, CH=N), 3.05 (s, 12H, N‐(CH ) ), 3.73 (s, 4H,
3
4
13
1
0° ≤ 2θ ≤ 80°. The thermogravimetric analysis (TGA/
DTG) was carried out in the temperature range of
0–1000°C in a dynamic nitrogen atmosphere with a
N‐CH ), 6.69–7.52 (m, 8H, ArH). C‐NMR (DMSO‐d6,
2
δ, ppm): 161.70 (‐HC=N‐), 40.60 (‐CH ), 61.86 (‐CH ‐),
3
2
2
111.94, 124.50, 129.54, 152.23 (‐C H ‐). Mass spectrum
6
4
o
+
heating rate of 10 C/min using NETZSCH STA 449F3
thermal analyzer.
(ESI) [M + H] = 323.2. UV–Vis (DMSO): λ/nm 286, 328.
2
.2.2 | Synthesis of Schiff base ligand (L2)
2
2
.2 | Synthesis
(E)‐1‐(((2,3‐dihydrobenzo[1,4]dioxin‐6‐yl)imino)methyl)
.2.1 | Synthesis of Schiff base ligand (L1)
Naphthalen‐2‐ol (L2) was synthesized from the conden-
sation of 2‐hydroxy‐1‐naphthaldehyde (0.86 g, 5 mmol)
and 1,4‐benzodioxane‐6‐amine (0.75 g, 5 mmol) dissolved
in ethanol in the molar ratio of 1:1, respectively. The
reaction mixture was stirred at room temperature for
4 hr. The yellow precipitate formed was filtered, and
washed thoroughly with the least amount of ethanol
and diethyl ether. Finally, the residue was collected and
dried in a desiccator over anhydrous calcium chloride
for further use.
The Schiff base ligand N,N′‐bis(4‐dimethylamino)
benzylidene‐ethane‐1,2‐diamine (L1) was synthesized by
the condensation reaction of 4‐(dimethyl‐amino)benzalde-
hyde (0.60 g, 4 mmol) and ethylenediamine (0.13 ml,
2
mmol) dissolved in ethanol in the stoichiometric ratio
of 2:1, respectively, and the reaction mixture was stirred
in a round‐bottom flask for 4 hr at room temperature
(approximately 25°C). The reaction was monitored by
taking thin‐layer chromatography at regular intervals.
The white precipitate obtained was separated by filtration,
washed with water and diethyl ether, and dried in vacuum
Color: yellow; yield: 78%. Elemental anal. calcd
forC H NO : C, 74.72; H, 4.95; N; 4.59; found: C,
74.80; H, 5.05; N, 4.62. IR (KBr pellet, cm ): 1610
19
15
3
−
1
over CaCl for further use. Synthetic outline of the Schiff
base ligand is shown in Scheme 1.
υ(‐CH=N‐), 3251 υ(OH), 1286 υ(C‐O), 1257 υ(C‐O‐C).
2
1
H‐NMR (DMSO‐d , δ, ppm): 9.59 (s, 1H, CH=N),
6
Color: off white; yield: 82%. Elemental anal. calcd for
C H N : C, 74.50; H, 8.13; N, 17.38; found: C, 73.77;
4.29(s, 4H, ‐OCH ), 13.15 (s, 1H, OH), 6.94–8.51 (m, 9H,
2
13
ArH). C‐NMR (DMSO‐d , δ, ppm): 170.05 (C‐OH),
20
26
4
6
−
1
H, 8.10; N, 17.40; IR (KBr pellet, cm ): 1590
υ(‐HC=N‐), 1344 υ(C‐N) , 1162 υ(C‐N)aliphatic, 1480
155.80 (CH=N), 144.96 (C‐N),143.38 (C‐O), 65.05–65.09
(‐OCH ), 109.46–138.73 (Ar Cs). Mass spectrum (ESI)
aromatic
1
2
+
υ(C=C), 2915υ(‐CH ‐). H‐NMR (DMSO‐d , δ, ppm):
[M + Na] = 328.09. UV–Vis (DMSO): λ/nm 272, 330.
2
6
SCHEME 1 Synthesis of Schiff base
ligands (L1 and L2) and their heteroleptic
complexes (C1–C4)

4
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DAR ET AL.
[
Zn(L1)(L2)Cl·H O] (C4)
2
2.2.3 | Synthesis of heteroleptic metal
Color: orange yellow; yield: 70%. Elemental anal. calcd
for [C H ClN O Zn]: C, 62.82; H, 5.68; N, 9.39; found:
C, 63.12; H, 5.72; N, 9.60. IR (KBr pellet, cm ): 1590
complexes (C1–C4)
39
42
5 4
−
1
Ethanolic solution (15 ml) of metal salts, [M(Cl )·nH O]
2
2
(2 mmol) was added to hot ethanolic solution (20 ml) of
υ(CH=N), 1302 υ(C‐Ophenol), 1353 υ(C‐N)aromatic, 1175
Schiff base ligand (L1) with constant stirring, and then
after a few minutes an ethanolic solution of Schiff base
ligand (L2) was added. Triethylamine was added to
maintain the pH of solution. The reaction mixture was
then refluxed for about 5–6 hr at 80°C. The reaction
mixture was then cooled to room temperature. The
precipitate formed was filtered, washed thoroughly with
ethanol followed by diethyl ether to remove the
unreacted compounds, and finally dried in a vacuum over
fused calcium chloride in a desiccator.
υ(C‐N)aliphatic
,
1264 υ(C‐O‐C), 3497 υ(OH), 570
1
υ(Zn‐O), 475 υ(Zn‐N). H‐NMR (DMSO‐d
8.72, (s, 2H, CH=N), 9.60 (s, 1H, CH=N), 3.14
(s, 12H, N‐(CH ), 3.80 (s, 4H, N‐CH ),4.37 (s, 4H,
‐OCH ), 6.69–7.89 (m, ArH). C‐NMR (DMSO‐d , δ,
ppm): 162.30, 156.85 (‐HC=N‐), 42.65 (‐CH ), 61.19
(‐CH ‐), 170.15 (C‐O), 145.51 (C‐N), 143.44 (‐O‐C),
65.10 (‐OCH ),+ 109.47–138.57 (Ar Cs). Mass spectrum
(ESI) [M + H] = 743.7.
, δ, ppm):
6
)
3
4
2
13
2
6
3
2
2
[
Cu(L1)(L2)Cl·H O] (C1)
2
Color: pale green; yield: 68%. Elemental anal. calcd
for [C H ClCuN O ]: C, 62.98; H, 5.69; N, 9.42; found:
C, 63.72; H, 5.72; N, 9.47. IR (KBr pellet, cm ): 1585
3
| BIOLOGICAL INVESTIGATIONS
.1 | Strains, media and chemicals
39
42
5 4
−
1
3
υ(CH=N), 1298 υ(C‐Ophenol), 1359 υ(C‐N)aromatic, 1178
υ(C‐N)aliphatic
,
1263 υ(C‐O‐C), 3490
υ
OH), 562
All the strains used in this study are detailed in Table 3,
which consists of eight FLC‐susceptible (including one
laboratory strain C. albicans SC5314 and seven clinical
isolates) and three FLC‐resistant clinical isolates. All
the clinical C. albicans strains were collected from
Charlotte Maxeke Johannesburg Academic Hospital,
Johannesburg, South Africa, and were stored in glycerol
stocks at−80°C in the Department of Clinical
Microbiology and Infectious Diseases. All the test strains
were isolated from patients under the ethical clearance
number M10102 obtained from the Human Research
Ethics Committee, University of the Witwatersrand.
Prior to experiments, Candida isolates were revived by
plating on Sabouraud Dextrose (SD) Agar (Sigma
Aldrich, USA), followed by growing single colonies in
fresh SD broth. FLC and amphotericin were purchased
from Sigma Fluke (USA). All other chemicals
and media were purchased from Sigma Aldrich and
Merck.
υ(Cu‐O), 448 υ(Cu‐N). Mass spectrum (ESI) [M + H]
+
=
742.3.
[
Co(L1)(L2)Cl·H O] (C2)
2
Color: black; yield: 65%. Elemental anal. calcd for
[
C H ClCoN O ]: C, 63.37; H, 5.73; N, 9.47; found: C,
39 42 5 4
−
1
6
3.40; H, 5.79; N, 9.52. IR (KBr pellet, cm ): 1592
υ(CH=N), 1350 υ(C‐N)aromatic, 1290 υ(C‐Ophenol), 1263
υ(C‐O‐C), 1166 υ(C‐N)aliphatic, 3497 υ(OH), 562 υ(Co‐O),
+
4
57 υ(Co‐N). Mass spectrum (ESI) [M + H] = 739.3.
[
Ni(L1)(L2)Cl·H O] (C3)
2
Color: brown; yield: 69%. Elemental anal. calcd for
[
C H ClN NiO ]: C, 63.39; H, 5.73; N, 9.48; found: C,
39 42 5 4
−
1
6
3.09; H, 5.76; N, 9.57. IR (KBr pellet, cm ): 1582
υ(CH=N), 1290 υ(C‐Ophenol), 1347 υ(C‐N)aromatic, 1165
υ(C‐N)aliphatic, 1262 υ(C‐O‐C), 3495 υ(OH), 556 υ(Ni‐O),
+
4
60 υ(Ni‐N). Mass spectrum (ESI) [M + H] = 738.8.
TABLE 1 Physico‐chemical properties of Schiff base ligands (L1 and L2) and their heteroleptic complexes (C1–C4)
(Ω ‐ cm mol
1
2
−1
)
m.p. (°C)
μeff (B.M.)
Compound Color
Mol. formula
Mol. wt. (m/z) ratio Yield (%)
Λ
m
L1
L2
C1
C2
C3
C4
Off white
C
C
20
19
H
26
N
4
322.4
305.3
742.2
738.2
737.2
323.2
328.0
742.3
739.3
738.8
743.7
82
78
68
65
69
70
–
190
192
252
285
310
290
–
–
Yellow
Pale green
Black
H15NO
3
–
26
20
17
22
[C39
[C39
[C39
H
H
H
H
42ClCuN
42ClCoN
5
O
4
]
1.90
5
O
4
]
4.90
Brown
42ClN
42ClN
5
5
NiO
4
]
3.70
Orange Yellow [C39
O
4
Zn] 743.2
Diamag.

DAR ET AL.
5 of 14
TABLE 2 Thermoanalytical results (TG and DTG) of heteroleptic complexes (C1–C4)
Found (calcd %)
TG
range
(°C)
DTGmax
(°C)
Weight
n* loss
Total weight
loss
Metallic
residue
Complex
Assignment
[C
39
[C
39
[C
39
[C
39
H
H
H
H
42ClCu N
5
O
4
]
100–230 148,222
2
6 (7.18)
1
O and Cl
2
Cu
Loss of one coordinated H
2
2
2
2
2
2
2
2
molecule
molecule
molecule
molecule
2
4
30–490
90–800
347
660
1
1
40 (41.07)
42 (43.37)
88 (91.62) Loss of C19
Loss of C20
H
H
15NO
3
26 4
N
42ClCoN
5
O
4
]
100–220 142,220
2
6.50 (7.24)
1
O and Cl
2
Co
Ni
Zn
Loss of one coordinated H
2
4
20–480
80–880
315
674
1
1
40.50 (41.33) 89.40 (92.21) Loss of C19
42.40 (43.64)
H
H
15NO
3
Loss of C20
26
N
4
42ClN
42ClN
5
5
NiO
4
]
100–240 130,229
2
6 (7.22)
1
O and Cl
2
Loss of one coordinated H
2
5
40–500
00–920
376
754
1
1
40 (41.37)
43 (43.8)
89 (92.39) Loss of C19
Loss of C20
H
H
15NO
3
26 4
N
O
4
Zn] 100–250 120,220
2
6 (7.18)
1
O and Cl
2
Loss of one coordinated H
2
5
50–540
40–790
287
710
1
1
41 (41.03)
43 (43.35)
90 (91.56) Loss of C19
Loss of C20
H
H
15NO
3
26 4
N
n* = number of decomposition.
TABLE 3 MICs (μg/ml) of ligands (L1, L2) and the complexes (C1–C4) against FLC‐susceptible and ‐resistant Candida albicans isolates
MICs (μg/mL)
L1
L2
C1
C2
C3
C4
FLC
Strains
MIC MFC MIC MFC MIC MFC MIC MFC MIC MFC MIC MFC MIC
Candida albicans SC5314 100
–
100
–
6.25 12.5 0.40 3.12 0.10 1.60 12.5 25
6.25 12.5 0.40 3.12 0.10 1.60 12.5 25
0.25
FLC‐susceptible
4175 100
–
–
–
–
–
–
–
100
50
100
100
50
–
–
–
–
–
–
–
0.12
0.25
0.25
0.25
0.25
0.25
0.25
4179 100
4180 100
4251 100
4554 100
4563 100
4576 100
12.5 12.5 3.12 6.25 0.20 3.12 50
50
6.25 12.5 0.40 3.12 0.10 1.60 12.5 25
6.25 12.5 0.40 3.12 0.10 1.60 12.5 50
6.25 12.5 0.40 3.12 0.10 1.60 12.5 50
6.25 12.5 1.60 3.12 0.20 1.60 12.5 25
6.25 12.5 0.40 3.12 0.10 1.60 12.5 25
100
100
FLC‐resistant
strains
4085 100
4122 100
–
–
–
100
100
100
–
–
–
12.5 50
12.5 50
12.5 50
1.60 6.25 0.20 3.12 25
3.12 6.25 0.40 3.12 50
1.60 6.25 0.20 3.12 25
50
50
50
16
32
32
4135 100
FLC, fluconazole; MIC, minimum inhibitory concentration.
[
22]
3
3
.2 | Antifungal susceptibility tests
.2.1 | Determination of minimum inhib-
Institute (CLSI)‐recommended guidelines M27‐A3,
with modifications. The initial concentrations of all the
newly synthesized compounds were 400 μg/mL, in order
to achieve the concentrations of 100, 50, 25, 12.5, 6.25,
3.12, 1.60, 0.8, 0.4, 0.2, 0.1 and 0.05 μg/mL in a 96‐well plate
after serial dilution. The positive control FLC (1000 μg/
mL) and the negative vehicle control (1% DMSO) were also
included in every set of experiments. Media and culture
controls were included to confirm the sterility and
viability, respectively. Further, minimum fungicidal
concentration (MFC) was determined by subculturing the
test dilutions from each well without color change on SD
itory concentrations and minimum fungi-
cidal concentrations
To determine the antifungal activity of newly synthesized
Schiff base ligands (L1 and L2) and their four complexes
(
C1–C4), minimum inhibitory concentrations (MICs)
were determined against tested FLC‐susceptible and
FLC‐resistant C. albicans isolates by serial micro‐broth
dilution assay following Clinical and Laboratory Standards

6
of 14
DAR ET AL.
agar plates and incubated for 24 hr. The lowest concentra-
tion that showed no fungal growth on the agar plate was
defined as the MFC value. All the experiments were done
in triplicate, and all the results were expressed in μg/mL.
cell analyzer (Becton Dickinson, USA). The results were
analyzed using FlowJo software version 10.0. For data
analysis, quadrants were adjusted, analyzed and
[
24]
interpreted as reported earlier.
3.2.2 | Sterol quantification
4 | RESULTS AND DISCUSSION
To study the effect of newly synthesized compounds on the
ergosterol biosynthesis pathway, total intracellular sterols
were extracted and quantified using a spectrophotometer,
The heteroleptic complexes (C1–C4) were successfully
prepared by reacting equimolar amounts of Schiff base
ligands (L1 and L2) with the corresponding metal salts
in the presence of triethylamine. The metal complexes
were neutral, colored, air‐stable, and insoluble in water
and common organic solvents but remarkably soluble in
DMF and DMSO. Synthesis and the final structures of
the ligands and their complexes were verified by different
physical and spectroscopic techniques. The different
physico‐chemical properties of Schiff base ligands (L1
and L2) and metal complexes (C1–C4) are shown in
Table 1. The molar conductivity (Λm) of the complexes
[23]
as reported previously.
One FLC‐susceptible and one
‐resistant isolate of C. albicans cells were treated with
MIC and ½ MIC values of both the ligands and their
four derivatives. In all the tests, there is a negative
control (without test compound) and positive control
(2 μg/mL FLC). All the sterol samples were scanned
spectrophotometrically between 240 and 300 nm, using a
spectrophotometer. The presence of ergosterol and the late
sterol intermediate 24(28) DHE in the extracted sample
resulted in a characteristic four‐peaked curve. The absence
of detectable ergosterol in extracts was indicated by a flat
line. Ergosterol content was calculated as a percentage of
the wet weight of the cell, using the following equations:
−
3
was carried out using DMSO (10 M) at 25°C. The results
−
1
2
−1
obtained were in the range of 17–26 Ω cm m , which
showed their non‐electrolytic nature.
4
.1 | Magnetic susceptibility and
%
%
%
ergosterol þ %24ð28Þ DHE ¼ ½ðA281:5=290Þ × Fꢀ=pellet weight
24ð28Þ DHE ¼ ½ðA230=518Þ × Fꢀ=pellet weight
electronic spectral studies
ergosterol ¼ ½%ergosterol þ %24ð28Þ DHEꢀ − %24ð28Þ DHE
The geometry of the synthesized heteroleptic complexes
was obtained from the electronic spectral data and
magnetic susceptibility measurements. The electronic
absorption spectra of the ligands (L1 and L2) and their
complexes (C1–C4) were recorded in 10
where F is the factor for dilution in ethanol, and 290 and
18 are the E values (in percentages per centimeter) deter-
5
mined for crystalline ergosterol and 24(28) DHE,
respectively.
−3
M DMSO
solution in the range of 200–800 nm using the same
solvent as blank. The UV–Vis spectrum of L1 displayed
two absorption bands at 286 and 328 nm, and L2 also
showed two absorption bands at 272 and 330 nm
corresponding to π‐π* and n‐π* transitions, respectively.
In complexes, these bands were shifted towards lower or
higher wavelengths signifying the coordination of ligands
to metal ions, as shown in Figure 1. The addition of metal
ion to ligands produced characteristic changes in the
visible absorption spectrum of the ligands, signifying
the complex formation. In complexes, a band appeared
in the region 352–424 nm corresponding to charge trans-
fer from ligand to metal. In complex C1 the absorption
band appearing at 398 nm can be attributed to charge
transfer from ligand to metal. The broad asymmetric
band in C1 appearing at 681 nm corresponded to a com-
3
.2.3 | Effect on membrane permeability
Based on the MIC results, all the active compounds (C1,
C2, C3 and C4) except the ligands were tested to study
the effect on membrane integrity, by doing flow cytome-
try using propidium iodide (PI). PI is a fluorescent dye
with a characteristic to enter into necrotic cells with
disintegrated membranes. Mid‐exponentially grown C.
albicans cells were exposed to MFC values of the test enti-
ties for 2 hr. After the exposure time, cells were harvested
and resuspended in phosphate‐buffered saline. To this
solution, 1 mL of PI was added with a final concentration
of 1 mg/mL of PI. All the samples were incubated at 35°C
for 15 min in the dark at room temperature. In every set
of experiments, negative (untreated cells) and positive
2
2
2
2
bination of three bands B → A (υ ), B → B (υ2)
1
g
1g
1
1g
2g
2
2
(2 μg/mL amphotericin B‐treated cells) controls were
and B1g → E (υ ), which are similar in energy. The
g 3
included. Unstained cells were always included as
autofluorescence controls. Cell‐associated fluorescence
was measured using a flow cytometry BD LSRFortessa
broadness of the band showed its distortion from
the octahedral geometry and provided the indication for
[
25,26]
Jahn Teller distortion.
The magnetic moment of this

DAR ET AL.
7 of 14
FIGURE 1 UV–Vis spectra of: (a) Schiff
base (L1); (b) L2; (c) Zn (II); (d) Cu (II);
(
e) Ni (II); and (f) Co (II) complexes in
−
3
1
0
M dimethylsulfoxide (DMSO)
solution
complex was found to be 1.90 B.M., which lies within the
normal range for octahedral Cu (II) complexes. The
electronic absorption spectrum of C2 shows bands at
In complexes, the position or intensity of the peaks
obtained for ligands are expected to change upon chela-
tion. The characteristic peaks are given in Section 2.2.
4
4
−1
4
60, 550 and 630 nm that are assigned to T (F) → T (P),
The sharp band observed at 1602 cm in the spectrum
1g
1g
4
4
4
4
T (F) → A (F) and T (F) → T (F), respectively.
of free Schiff base ligand (L1) was assigned to υ
(CH=N) stretching vibrations. In Schiff base ligand
(L2), the υ(CH=N) stretching vibration was found at
1g
2g
1g
2g
The band at 382 nm corresponds to ligand to metal
[27]
charge transfer band.
The magnetic moment of
−
1
complex (C2) was found to be 4.90 B.M. The absorption
bands and the magnetic moment suggested an octahedral
geometry around the Co (II) ion. The absorption
spectrum of C3 displayed three absorption bands at 521,
1610 cm . In the spectra of metal complexes, the
υ(CH=N) stretching vibrations of azomethine were
shifted to lower wavenumbers, which revealed the
participation of the azomethine nitrogen in coordination.
The participation of deprotonated phenolic OH in
complexation was verified by the blue shift of υ(C‐O)
6
34 and 705 nm, which may be attributed to
3
3
3
3
3
3
A2g → T (P), A → T (F) and A → T transi-
tions, respectively.
1g
2g
28]
1g
2g
2g
[
−1
The spectrum also displayed a band
stretching band, present at 1286 cm in the free ligand
−
1
at 398 nm, which may be assigned to ligand to metal
charge transfer. The magnetic moment observed for
complex C3 was found to be 3.7 B.M. The absorption
spectra and magnetic moment reveal that there is an
octahedral geometry around the Ni (II) ion. In C4 the
absorption band found at 424 nm may be attributed to
the ligand to metal charge transfer with zero magnetic
moment. Due to the diamagnetic nature of complex C4,
no d‐d bands were observed. An octahedral geometry
has been proposed for the complex C4.
L2, which shifted to higher wavenumbers by 4–16 cm
in complexes. The IR spectrum of the L2 ligand displayed
−
1
a broad band at 3152 cm , which could be assigned to
the phenolic OH group.
In complexes (C1–C4), the broad bands present at
−
1
3490–3497 cm might be attributed to the stretching
frequencies of the υ(OH) of coordinated water molecules
present in these complexes. In Schiff base ligand L2 and
−
1
complexes, the bands present at 1256–1263 cm were
assigned to C‐O‐C stretching vibrations. In complexes
C1–C4, new bands are found in the regions 556–570 cm
−
1
,
which were attributed to υ(M‐O) stretching vibrations,
−
1
4.2 | FT‐IR spectra
and the bands present at 448–475 cm in complexes
have been ascribed to υ(M‐N) of the azomethine mode.
Therefore, from the IR spectra data it was revealed that
the ligand (L1) behaves as a bidentate ligand with NN
donor sites coordinating to the metal ions through
The IR spectra of the complexes were compared with
those of the free Schiff base ligands (L1 and L2) to verify
the coordination sites that may be involved in chelation.

8
of 14
DAR ET AL.
azomethine N atoms. The ligand (L2) also behaves as a
bidentate ligand with NO donor sites coordinating to
metal ions through azomethine N and phenolic O atoms.
The IR spectra of the Schiff base ligands (L1 and L2) and
complex C3 as an example is shown in Figure 2, and the
spectra of complexes (C1, C2 and C4) are given in
the supplementary data.
of L2 exhibited a sharp singlet at 4.29 ppm attributed to
methylene protons (‐OCH ). The Ph‐OH proton of L2
2
[
29]
appeared as a singlet at 13.15 ppm.
In C4 complex,
the Ph‐OH proton signal was found absent confirming
the complexation through phenolic oxygen. In C4
complex, a new signal has been found at 3.48 ppm due
to the presence of coordinated H O protons. The aromatic
2
protons in C4 complex appeared as a set of multiplets in
the region of 6.69–7.89 ppm. The positions of the main
1
13
1
13
4
.3 | H‐NMR and C‐NMR spectra
signals in the H‐NMR and C‐NMR are stated in the
13
Experimental section. The attained C values of C4
complex were compared with the corresponding ligands
and were in good agreement with the proposed structure
of the C4 complex. In Schiff base ligands (L1 and L2), the
azomethine carbon atoms were observed at 161.70
and 155.80 ppm, which were shifted to 162.30 and
156.85 ppm in C4 complex, respectively. The paramag-
netic C1, C2 and C3 complexes did not show fine NMR
spectra and were therefore not included in this study.
1
The H‐NMR spectral data of the Schiff base ligands (L1,
L2) and C4 complex were recorded in DMSO‐d for the
interpretation of number and nature of protons relative
to TMS with chemical shifts given in ppm. The H‐NMR
spectra of the Schiff bases (L1 and L2) displayed singlet
signals at 8.65 and 9.59 ppm, respectively, attributed to
the protons of azomethine nitrogen (CH=N), which
confirmed the formation of Schiff base ligands. In C4
complex, a shift of electron density from ligands to metal
ion has been detected. The signal for the azomethine
protons is slightly deshielded and appeared at 8.72 and
6
1
1
13
The H‐NMR of Schiff base ligands and C‐NMR of L1,
L2 and C4 are shown in the supplementary data.
9
.60 ppm, and this downfield shift confirms the coordina-
tion of azomethine nitrogen with the Zn (II) metal ion
Figure 3). In L1, the two sharp singlets at 3.73 and
.05 ppm are ascribed to active methylene protons
4.4 | Mass spectra
(
3
The mass spectra of the Schiff base ligands (L1, L2) and
complexes (C1–C4) are characterized by moderate to high
1
(
‐CH and ‐N‐(CH ) ), respectively. The H‐NMR spectra
2
3 4
FIGURE 2 Fourier transform‐infrared (FT‐IR) spectra of: (a) Schiff base (L1); (b) Schiff base (L2); and (c) Ni (II) complex (C3)

DAR ET AL.
9 of 14
1
FIGURE 3 H‐NMR spectrum of C4 complex in dimethylsulfoxide (DMSO)‐d
6
relative intensity molecular ion peaks. It is noticeable that
the molecular ion peaks are in good agreement with the
suggested empirical formula of the compounds. The mass
spectra [M + H] of Schiff base ligand (L1) showed the
molecular ion peak at m/z 323.2, and the mass spectra
Figure 4. In complex C3, the trend of curves decreases
from maximum to minimum intensity indicating its
amorphous nature. Some new extra peaks were found
in complexes as compared with ligand, signifying the
coordination of ligand to metal ions. The XRD of ligand
(L1) displayed reflecting peaks at 2θ scattering angles of
11.50, 14.30, 17.97, 23.92, 25.73 and 27.10. The L2
exhibited reflecting peaks at 2θ scattering angles of
15.94, 19.62, 20.64, 23.78, 24.98 and 28.86, while
complex C1 exhibited reflecting peaks at 2θ scattering
angles of 15.87, 17.10, 20.10, 20.42, 22.50, 25.10, 26.94
and 29.50 assigned to (100), (110), (111), (021), (111),
+
+
[
3
(
7
M + Na] of L2 showed the molecular ion peak at m/z
+
28.0. The mass spectra [M + H] of metal complexes
C1, C2, C3 and C4) showed molecular ion peaks at
42.3, 739.3, 738.8 and 743.7, respectively. The mass spec-
tra of the synthesized compounds are given in the supple-
mentary data.
(
121), (102) and (022) crystal planes, respectively, char-
4
.5 | Powder X‐ray diffraction studies
acteristic of well‐ordered monoclinic arrangement of
Cu atoms (space group: P21/c, JCPDS: 72–0438). The
complex C2 displayed fine reflecting peaks at 2θ scatter-
ing angles of 15.03, 15.70, 17.38, 18.70, 23.15, 23.40,
25.70, 31.30 and 33.28 ascribed to (100), (001), (110),
(111), (120), (021), (101), (131) and (202) crystal planes,
respectively, characteristic of well‐ordered monoclinic
arrangement of Co atoms (space group: P21/m, JCPDS:
75–2160), whereas complex C4 showed reflecting peaks
at 2θ scattering angles of 11.35, 13.54, 15.12, 19.63,
20.43, 22.12 and 29.85 assigned to (010), (110), (111),
(210), (211), (113) and (123) crystal planes, respectively,
All the efforts failed to grow single crystals of synthe-
sized compounds suitable for single X‐ray crystallogra-
phy. So, X‐ray powder diffraction studies of the
compounds were carried out to get more details about
their structure. The powder XRD of the compounds
o
were carried out in the range 10‐80 (θ) at a wavelength
of 1.54 Å. The XRD pattern indicates that the ligands
(
L1, L2) and complexes (C1, C2, C4) have well‐defined
crystalline patterns with various degrees of crystallinity.
The XRD pattern of L1, L2 and complex C1 is shown in

10 of 14
DAR ET AL.
FIGURE 4 Powder X‐ray diffraction
(
(
XRD) pattern of: (a) Schiff base ligand
L1); (b) L2; and (c) C1 complex
characteristic of well‐organized orthorhombic arrange-
temperature range of 148–222°C with DTG peaks
observed at 148 and 222°C. This weight loss is due to
the simultaneous loss of coordinated water molecule
and chlorine atom. The second stage continued with
one degradation step with the weight loss of 40% (calcd
41.07%) in the temperature range of 230–490°C with
DTG peak observed at 347°C. This weight loss is due to
the loss of C H NO fragment. The third stage occurred
ment of Zn atoms (space group: Pca2 , JCPDS:
1
7
6–0778). The detailed XRD parameters of the com-
plexes are listed in Table S1.
The average crystallite size of the ligands (L1 and L2)
and the complexes (C1, C2 and C4) was estimated using
[30]
the Debye Scherrer's formula.
19
15
3
at the temperature range of 490–800°C with the weight
D ¼ 0:9λ= βcos θ
loss of 42% (calcd 43.37%) due to the loss of C H N
2
0
26 4
fragment with DTG peak observed at 660°C. The overall
weight loss observed in C1 was found to be 88% (calcd
where constant 0.9 is the shape factor, D is the particle
size, λ is the X‐ray wavelength of CuKα radiation
9
1.62%). The metallic Cu left as the residue has the
observed weight of 10% against the calculated value of
1%.
Complex C2 underwent degradation in three stages.
The first stage occurred at the temperature range of
00–220°C with a weight loss of 6.50% (calcd 7.24%).
(
1.5406 Å), θ is the Bragg diffraction angle and β is the
[30]
full‐width at half‐maximum value of θ.
The average
1
crystallite sizes of the ligands (L1 and L2) and the
complexes (C1, C2 and C4) were found to be 30.79,
3
0.18, 39.87, 26.68 and 34.28 nm, respectively.
1
4
.6 | Thermal analysis (TG and DTG)
The DTG peaks were observed at 142 and 220°C. This
weight loss is due to the simultaneous loss of one
coordinated water molecule and chlorine atom. The com-
plex underwent further degradation at the temperature
range of 220–480 C, with the loss of 40.50% (calcd
41.33) and the DTG peak was observed at 315°C. This
The thermal analysis of the complexes (C1–C4) was
studied by using thermogravimetric techniques (Thermal
Gravimetric Analysis and Derivative Thermogravimetry)
within a temperature range from room temperature to
o
1
000°C at a heating rate of 10°C/min. As a representative
weight loss is due to the loss of C H NO fragment.
19
15
3
case, the TG/DTG curve of C1 is depicted in Figure 5. The
TGA/DTG data of complexes are summarized in Table 2.
The thermal decomposition of C1 occurs in three
stages. The first stage proceeded with two degradation
steps with the weight loss of 6% (calcd 7.18%) in the
The third stage of degradation occurred at the
temperature range of 480–880°C, with the loss of 42.40%
(calcd 43.64%) and the DTG peak was observed at
674°C. This weight loss is due to the loss of C H N
26 4
2
0
fragment. The overall weight loss was found to be

DAR ET AL.
11 of 14
FIGURE 5 Thermogravimetric analysis
(
TGA) and DTG curves of complex C1
8
9.40% (calcd 92.21%). The metallic Co left as a residue
has the observed weight of 10% against the calculated
value of 8.44%.
has the observed weight of 10.60% against the calculated
value of 7.79%.
The thermogram of C3 showed degradation in three
stages. The first degradation occurred at the temperature
range of 100–240°C with the loss of 6% (calcd 7.22%), and
the DTG peaks were observed at 130 and 229°C. This
weight loss is due to the loss of one coordinated water
molecule and chlorine atom. The second stage of degra-
dation occurred at the temperature range of 240–500°C
with the loss of 40% (calcd 41.37%) with the DTG peak
observed at 376°C. This weight loss is due to the loss of
C H NO fragment. The third stage of degradation
4.7 | Minimum inhibitory concentration
and minimum fungicidal concentrations
The antifungal activity of all the newly synthesized ligands
and their complexes was evaluated by determining their
MIC and MFC values against 11 different C. albicans
strains, including one laboratory standard strain, seven
FLC‐susceptible clinical strains and three FLC‐resistant
clinical strains. All the MIC results are summarized in
Table 3. Evaluation of MIC depicts that among the two
ligands and their four complexes, C3 showed the highest
inhibitory activity, while both ligands (L1 and L2) showed
the least inhibitory activity against all the tested strains.
Based on the MIC values, the order of potency of the test
compounds was C3 > C2 > C1 > C4 > L1 ≥ L2. The
median MIC values for C3, C2, C1, C4, L1 and L2 are
0.10, 0.40, 6.25, 12.5, 100 and 100, respectively. FLC was
also tested, and the isolates with MIC values < 8 μg/mL
for FLC were considered FLC‐susceptible while the
isolates with MIC ≥ 8 μg/mL were categorized as
FLC‐resistant, following the CLSI Interpretive Guidelines
19
15
3
occurred at the temperature range of 500–920°C with
the loss of 43% (calcd 43.8%), and the DTG peak was
observed at 754°C. This weight loss is due to the loss of
C H N fragment. The total weight loss was found to
20
26 4
be 89% (calcd 92.39%). The metallic Ni left as a residue
has the observed weight of 11% against the calculated
value of 7.61%.
The thermogram of C4 showed the first stage of degra-
dation due to the simultaneous loss of one coordinated
water molecule and one chlorine atom at the temperature
range of 100–250°C with the loss of 6% (calcd 7.18%). The
DTG peaks were observed at 120 and 220°C. The complex
underwent further degradation due to the loss of
C H NO fragment at the temperature range of
[31]
for In vitro Susceptibility Testing of Candida species.
Furthermore, all the test compounds were prepared to
different concentrations using 1% DMSO, and therefore
DMSO was used as negative vehicle control and was
observed to have no inhibitory activity against any of the
tested isolates. The MIC data revealed that the structural
changes from ligand to its metal complexes produced
the marked enhancement in their potency as
antifungal agents.
19
15
3
2
50–540°C with the loss of 41% (calcd 41.03%). The
DTG peak was observed at 287°C. The third stage of
degradation occurred due to the loss of C H N
26 4
20
fragment at the temperature range of 540–790°C with
the loss of 43% (calcd 43.35%). The DTG peak was
observed at 710°C. The overall weight loss was found to
be 90% (calcd 91.56%). The metallic Zn left as a residue

12 of 14
DAR ET AL.
The MFC is the lowest concentration of the test
isolates, the decrease in total cellular ergosterol content
ranged from 28% to 63% when treated with ½ MIC, and
71% to 97% when treated with MIC values of the test com-
pounds, respectively. As expected, the decrease in total cel-
lular ergosterol content of susceptible cells treated with
FLC was measured to be 96%. In contrast to this,
resistant isolates only showed decline in ergosterol
content by 37%.
Ergosterol and its biosynthesis pathway is an
established drug target for different classes of antifungal
drugs including that of azoles and polyenes. There are
several studies reporting that the manipulation of this
drug target is responsible for drug resistance in different
fungal species including C. albicans. Our results clearly
indicate that the resistant isolates used in this study are
resistant due to the overexpression of ergosterol. The
survival of these cells at ½ MIC values of the test
compounds, where more than 50% ergosterol biosynthe-
sis was inhibited, indicate that these C. albicans isolates
belong to ergosterol‐tolerant class. Due to the structural
variability of these compounds, it is possible that they
interact with different enzymes of the ergosterol pathway
and thereby inhibit its biosynthesis. However, further
in‐depth studies are required to elucidate the actual bind-
ing of these compounds with the specific enzymes.
compounds that causes death of cells that can be
observed on clear agar plates. Table 3 summarizes the
MFC values of all the test compounds against 11 tested
C. albicans isolates. Except two ligands (L1, L2), all the
derivatives showed MFC values, depicting the fungicidal
activity of newly synthesized compounds. The MFC
values are generally two–fourfold higher than
MIC values.
4
.8 | Sterol quantitation
The antifungal effect of the tested compounds was rapid
and lethal. The rapid irreversible action of the active
complexes suggests that there may be a cellular target
accessible to the compound externally. Therefore, we
studied the effect of these active complexes on sterol
biosynthesis. Figure 6 and Table S2 summarize the effect
of these compounds on ergosterol biosynthesis in
FLC‐susceptible and ‐resistant C. albicans isolates at MIC
and ½ MIC values of all the newly synthesized
compounds. Sterol patterns were altered in a similar
manner in FLC‐susceptible and ‐resistant isolates when
cells were treated with subinhibitory concentrations of
the test compounds. These compounds completely blocked
ergosterol synthesis at MIC values (Figure 6). As shown in
Table S2, the decrease in total cellular ergosterol content
for susceptible isolates ranged from 21% to 57% for cells
grown in ½ MIC values to 62% to 93% for cells grown with
MIC values of the test compounds. Similarly, for resistant
4.9 | Effect on membrane integrity
The effect of active compounds (C1–C4) on disruption of
membrane integrity in C. albicans was studied using flow
FIGURE 6 UV spectrophotometric sterol profiles of representative fluconazole(FLC)‐susceptible (a) and FLC‐resistant (b) Candida
albicans isolates. Isolates were grown for 16 hr in Sabouraud Dextrose (SD) broth containing ½ minimum inhibitory concentration (MIC)
and MIC values of two ligands and four derivatives. Sterols were extracted from cells, and spectral profiles in the range 240–300 nm were
determined. Control represents untreated cells, while positive control represents cells treated with 2 μg/mL of FLC

DAR ET AL.
13 of 14
FIGURE 7 Sequence of density plots showing Candida albicans SC5314 cells analyzed by flow cytometry for the samples treated with
minimum fungicidal concentrations (MFCs) of compounds C1, C2, C3 and C4. Negative and positive controls are untreated and 2 μg/ml
amphotericin B‐treated cells, respectively. Density plots show relative fluorescence intensities of Candida cells marked with propidium iodide
(
PI) on the y‐axis. The lower left quadrant (Q4) marked the morphologically intact live cells observed with higher relative fluorescence
intensity, while the upper left grid (Q1) marked dead cells
cytometer. PI is the most commonly used fluorescent dye
to study the effect of drugs on cell membranes. Live cells
with integrated membranes will not allow PI to internal-
ize the cells; however, dead cells with disintegrated
membranes internalize PI, resulting in an increase in
products have been reported to disrupt fungal membrane
integrity causing cell death.
[
24,32]
5 | CONCLUSIONS
[24]
red fluorescence.
The PI penetration in C. albicans
cells treated with MFC values of test compounds along
with negative and positive controls are summarized in
Figure 7. All the four tested compounds along with
amphotericin B at MFC values showed >90% PI
internalization, indicating that these compounds have a
potential to disintegrate cell membranes at the higher
concentrations similarly to that of amphotericin B.
The membrane damage in collaboration with the
inhibition of ergosterol biosynthesis by these derivatives
can contribute to membrane malfunctioning resulting in
membrane depolarization, intracellular content leakage,
disturbance in membrane permeability and ultimately
cell death. The results in this study are in congruent with
the previous findings where derivatives from natural
In this study, some heteroleptic complexes derived from
two Schiff base ligands, obtained from biologically
important scaffolds were synthesized in a quest to find
better antifungal therapeutic agents. With the help of
spectral and other physical measurements, an octahedral
geometry was assigned to the complexes C2, C3 and C4,
while a distorted octahedral geometry was assigned to
the complex C1. The antifungal activity evaluation
against susceptible and resistant C. albicans revealed that
both the ligands have moderate activity, while derivative
complexes have strong antifungal activity. Insight
mechanisms revealed that these complexes disrupt cell
membranes and also deplete ergosterol content. To
conclude, this work suggests that these compounds could

14 of 14
DAR ET AL.
be promising antifungal drugs, and also advocates the
determination of optimal concentrations for clinical
applications for the treatment and prevention of candidi-
asis. The excellence of these compounds demands more
insight studies into all other possible mechanisms.
[15] X. Liu, C. Manzur, N. Novoa, S. Celedón, D. Carrillo, J.‐R.
Hamon, Coord. Chem. Rev. 2018, 357, 144.
[16] K. Mancha Madha, P. Gurumoorthy, S. Arul Antony, N.
Ramalakshmi, J. Mol. Struct. 2017, 1143, 478.
[17] M. A. Malik, O. A. Dar, P. Gull, M. Y. Wani, A. A. Hashmi,
Medchemcomm. 2018, 9, 409.
ACKNOWLEDGEMENTS
[18] A. M. Asiri, S. A. Khan, H. M. Marwani, K. Sharma,
J. Photochem. Photobiol. B Biol. 2013, 120, 82.
The author OAD is thankful to University Grants
Commission (UGC), Government of India for financial
assistance through Central University Ph.D. Students
Fellowship. AA acknowledges financial support from
University Research Committee Grant for 2019 ‐ Friedel
Sellschop Award (Grant No: AZMD019). The funders
had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
[
[
[
[
[
[
[
19] M. Y. Nassar, H. M. Aly, M. E. Moustafa, E. A. Abdelrahman,
J. Inorg. Organomet. Polym. Mater. 2017, 27, 1220.
20] M. Y. Nassar, H. M. Aly, E. A. Abdelrahman, M. E. Moustafa,
J. Mol. Struct. 2017, 1143, 462.
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Organomet. Chem. 2018, 32, e4051.
22] CLSI‐Clinical and Laboratory Standards Institute Standards,
Wayne, PA, USA, 2008, 40.
23] A. Ahmad, M. Y. Wani, A. Khan, N. Manzoor, J. Molepo, PLoS
ONE 2015, 10, e0145053.
CONFLICT OF INTEREST
24] A. Khan, A. Ahmad, F. Akhtar, S. Yousuf, I. Xess, L. A. Khan,
The authors declare that they have no competing interests.
N. Manzoor, Res. Microbiol. 2010, 161, 816.
25] G. Ahumada, T. Roisnel, S. Kahlal, D. Carrillo, R. Córdova, J.‐
Y. Saillard, J.‐R. Hamon, C. Manzur, Inorgan. Chim. Acta 2018,
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ORCID
Shabir Ahmad Lone
625
Mohmmad Younus Wani
838-1337
https://orcid.org/0000-0003-1850-
9
[26] J. Devi, N. Batra, Spectrochim. Acta Part A Mol. Biomol.
Spectrosc. 2015, 135, 710.
https://orcid.org/0000-0002-
1
[27] M. M. Abo‐Aly, A. M. Salem, M. A. Sayed, A. A. Abdel Aziz,
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How to cite this article: Dar OA, Lone SA, Malik
MA, et al. Heteroleptic transition metal complexes
of Schiff‐base‐derived ligands exert their antifungal
activity by disrupting membrane integrity. Appl
Organometal Chem. 2019;e5128. https://doi.org/
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