S-benzyldithiocarbazate imine coordinated metal complexes kill Candida albicans by causing cellular apoptosis and necrosis

Article abstract of DOI:10.1016/j.bioorg.2020.103771

Development of new chemotherapeutic agents and strategies are urgently needed to curb and halt the growing menace caused by hard-to-treat microbes. Coordination of metals to bioactive organic ligands is now considered to be an efficient strategy for delivering bioactive compounds inside the microbial cell membranes. Metal complexes have been effectively used to treat many dreadful diseases were other treatment modalities had failed. Use of metal complexes to treat microbial infections is now conceived to be an alternative and efficient strategy. Towards this, some new homoleptic transition metal complexes, obtained by coordination of metal ions to bioactive S-benzyldithiocarbazate Schiff-base ligands were evaluated for their anti-Candida activity and their potential to disrupt the membrane architecture. The complexes displayed remarkable antifungal activity against a wide spectrum of fluconazole susceptible and resistant Candida albicans isolates, with Ni complex (dtc3) being highly active with minimum inhibitory concentration (MIC) values ranging from 1 to 32 μg/mL. Cell viability assay confirmed the fungicidal activity of these metal complexes, especially the complex dtc3. These metal complexes kill Candida albicans by inducing cellular apoptosis and necrosis thereby causing phosphatidylserine externalization as revealed by Annexin V-FITC and propidium iodide staining assays.

Full text of DOI:10.1016/j.bioorg.2020.103771

Journal Pre-proofs
S-benzyldithiocarbazate imine coordinated metal complexes kill Candida al-
bicans by causing cellular apoptosis and necrosis
Manzoor Ahmad Malik, Shabir Ahmad Lone, Mohmmad Younus Wani, Ikbal
Ahmed Talukdar, Ovas Ahmad Dar, Aijaz Ahmad, Athar Adil Hashmi
PII:
S0045-2068(19)32008-5
DOI:
https://doi.org/10.1016/j.bioorg.2020.103771
YBIOO 103771
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
22 November 2019
24 January 2020
16 March 2020
Please cite this article as: M. Ahmad Malik, S. Ahmad Lone, M. Younus Wani, I. Ahmed Talukdar, O. Ahmad
Dar, A. Ahmad, A. Adil Hashmi, S-benzyldithiocarbazate imine coordinated metal complexes kill Candida
albicans by causing cellular apoptosis and necrosis, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/
j.bioorg.2020.103771
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
©
2020 Published by Elsevier Inc.

S-benzyldithiocarbazate imine coordinated metal complexes kill
Candida albicans by causing cellular apoptosis and necrosis
1
2
3
Manzoor Ahmad Malik , Shabir Ahmad Lone , Mohmmad Younus Wani , Ikbal
1
1
2,4
1
Ahmed Talukdar , Ovas Ahmad Dar , Aijaz Ahmad *, and Athar Adil Hashmi *
1Bioinorganic Chemistry Lab. Department of Chemistry, Jamia Millia Islamia, New Delhi-
1
10025, India
2Clinical Microbiology and Infectious Diseases, School of Pathology, Faculty of Health
Sciences, University of the Witwatersrand, Johannesburg, South Africa
3Chemistry Department, Faculty of Science, University of Jeddah, P.O. Box 80327, Jeddah
2
1589,Kingdom of Saudi Arabia.
4Division of Infection Control, Charlotte Maxeke Johannesburg Academic Hospital, National
Health Laboratory Service, Johannesburg, South Africa
ORCID:
https://orcid.org/0000-0003-2845-0727 (Aijaz Ahmad)
https://orcid.org/0000-0002-1838-1337 (Mohmmad Y. Wani)
https://orcid.org/0000-0003-1850-9625 (Shabir Lone)
*
Corresponding authors
Athar Adil Hashmi (ahashmi@jmi.ac.in)
Aijaz Ahmad (aijaz.ahmad@wits.ac.za)

Abstract
Development of new chemotherapeutic agents and strategies are urgently needed to curb and
halt the growing menace caused by hard-to-treat microbes. Coordination of metals to
bioactive organic ligands is now considered to be an efficient strategy for delivering bioactive
compounds inside the microbial cell membranes. Metal complexes have been effectively used
to treat many dreadful diseases were other treatment modalities had failed. Use of metal
complexes to treat microbial infections is now conceived to be an alternative and efficient
strategy. Towards this, some new homoleptic transition metal complexes, obtained by
coordination of metal ions to bioactive S-benzyldithiocarbazate Schiff-base ligands were
evaluated for their anti-Candida activity and their potential to disrupt the membrane
architecture. The complexes displayed remarkable antifungal activity against a wide spectrum
of fluconazole susceptible and resistant Candida albicans isolates, with Ni complex(dtc3)
being highly active with minimum inhibitory concentration (MIC) values ranging from 1 – 32
µg/mL. Cell viability assay confirmed the fungicidal activity of these metal complexes,
especially the complex dtc3. These metal complexes kill Candida albicans by inducing
cellular apoptosis and necrosis thereby causing phosphatidylserine externalization as revealed
by Annexin V-FITC and propidium iodide staining assays.
Keywords: Dithiocarbazate; metal complexes; apoptosis; necrosis; Candida albicans

1
. Introduction
Microbial infections have crumbled our society without any boundaries of rich and poor. The
unrelenting rise of microbial infections has emerged as a serious global concern intimidating
the effective prevention and treatment of an ever-increasing range of infections [1-3].
Enormous effort is being invested in the development of effective antimicrobial agents to
eliminate this rising clinical concern. In addition, the development of resistance to current
antimicrobial agents has resulted in high rates of morbidity and mortality [4-6]. Among the
various therapeutic approaches to wipe out microbial menace, development of metal
containing chemotherapeutic agents is now conceived to be an efficient strategy to disrupt the
microbial membrane architecture and the microbial biofilms which are considered as defence
armour of the microbes against any antibiotics. Because of the range of coordination
geometries, metal complexes provide more stereochemical variability than is possible for
organic molecules and the coordination of ligands to metal ions often introduces new
elements of chirality which may be important for biological molecule recognition and
interaction [7]. The metal complexes can also be highly positively charged that could aid
their interaction with negatively charged biological molecules such as DNA, RNA, several
types of phospholipids, and some regions of membrane proteins. It has been reported that
silver, gold, platinum, copper and other transition metal complexes interact with different
cellular targets and display efficient antimicrobial properties [8-15]. Another crucial
determinant of the antimicrobial property appears to be the cellular uptake. It is generally
accepted that most hydrophobic antibiotics cross the membrane by energy independent
passive diffusion, but some lipophilic antibiotics cross the cell membrane by active transport.
Since metal complexes contain hydrophobic as well as lipophilic regions, these allow the
complexes to interact with the membrane components differently, thus causing disruption of
the membrane architecture [16]. Also, because of the different microbial microenvironment,

metal complexes could undergo redox reactions and ligand exchange outside or inside the
microbial membranes therefore targeting the solvent exposed [4Fe-4S] clusters of proteins,
undergoing thiol mediated reductions, and inducing ROS-mediated cellular damage [17].
S-benzyldithiocarbazate structural moiety is considered to be of great relevance and
importance for designing flexible ligands and their metal complexes with diverse range of
biological activities [18, 19]. In addition, due to the limited number of currently available
antifungal drugs and the increasing incidences of invasive fungal infections in
immunocompromised patients such as HIV, cancer, tuberculosis etc. drug resistance become
a major challenge for clinicians to provide an appropriate antifungal therapy. Based on these
facts, there is an urgent need for novel antifungal molecules that could be employed to
overcome antifungal drug resistance. In this work, we report the synthesis of new transition
metal complexes, derived from an s-benzyldithiocarbazate Schiff base ligand with interesting
antifungal activities. The antifungal potential of these complexes was studied against a panel
of different susceptible and resistant Candida albicans. An attempt was also made to
delineate the mechanism of action of these active complexes by studying the mode of cell
death by Annexin V and PI staining methods.
2
2
2
1
. Experimental
.1 Chemistry
.1.1 Materials and physical measurements
,3-Benzodioxole-5-carboxaldehyde, Cu(OAc) .H O, Co(OAc) .4H O and Ni(OAc) .4H O
2
2
2
2
2
2
−
1
were purchased from Merck. Ethanol was distilled prior to use. IR spectra (4000–400 cm )
1
were obtained as KBr pellets using an FT-IR 8400-Shimadzu spectrophotometer. H NMR
1
3
(
400 MHz) and C NMR (200 MHz) spectra were recorded on a JEOL JNM-A400

spectrometer in DMSO with TMS as internal standard. Mass spectra were obtained on a
JEOL-JMS-D300 mass spectrometer. Molar conductance measurements were obtained with a
heavy-duty conductivity meter (Extech Instruments, USA, model No. 407303) and magnetic
susceptibilities were measured at room temperature (20°C). The UV–Vis spectra were
recorded on a Perkin Elmer Lambda 40 UV–Vis spectrophotometer between 200 and 800 nm
−
5
with 10 M solutions in DMSO. Melting points were measured on a melting point apparatus,
Gallenkamp, England. XRD studies were carried out using XPERT-PRO, X-ray
diffractometer. Morphological evaluation of the synthesized compounds was done by SEM
analysis. Thermal stability of the complexes was performed in a nitrogen environment on a
Thermogravimetric analyzer Exstar 6300.
2
.1.2 Preparation of the Schiff baseligand (sbl)
A previously reported procedure was followed to synthesize dithicarbazate[18], according to
which hydrazine hydrate (2.50 g, 0.05 mol, 99%) was added to an absolute ethanol solution
of KOH (2.81 g, 0.05 mol) and the mixture was stirred at 0 °C. Carbon disulphide (3.81 g,
0
0
.05 mol) was added dropwise with constant stirring over 1 h. Then benzyl chloride (8.25 g,
.05 mol) was added dropwise with vigorous stirring at 0 °C, over an additional hour. A
white precipitate of S-benzyldithiocarbazate was formed and collected. Finally, a solution of
,3-Benzodioxole-5-carboxaldehyde (6.81 g, 0.05 mol) in methanol was added to the
1
obtained S-benzyldithiocarbazate and refluxed for an hour. The mixture was filtered while
hot and the filtrate was cooled, giving a light-yellow precipitate of the desired Schiff base
product which was dried in a vacuum desiccator over anhydrous CaCl . The physical and
2
spectroscopic data of sbl are as follows:
Color: Light yellow, yield: 79%; m. p. (185–190 °C). Anal. Calcd forC H N O S : C,
1
6
14
2
2 2
5
8.16%; H, 4.27%; N, 8.48%; S, 19.41. Found: C,58.72%; H, 4.32%; N, 8.61%; S, 19.26.IR

−
1
data (KBr pellets, cm ): ν(N–H) 3124 m, ν(C–H) 2918 m, ν(C=N) 1600 s, ν (C=C) 1492 s,
1
ν(C=S) 1091 m, ν(N–N) 1033 s, ν(CSS) 921 m (Figure S3). H NMR (500 MHz, DMSO-d6,
ppm) δ: 13.26(s, 1H, NH), 8.15(s, 1H, CH=N), 7.68(d, 2H, C-4,6), 6.93(d, 2H, C-3,7),
1
3
4
1
3
.49(2H, –SCH ), 6.08(2H, -OCH O-). CNMR(100 MHz, CDCl , ppm) δ: 196.31(C=S),
2 2 3
46.90(CH=N),137.41(C-g), 124.67(C-h), 109.13(C-f), 102.21(C-d), 105.71(C-e),
7.94(C-c). UV–Vis spectrum [DMSO, λmax nm]: 256, 275, 328, 376.
2
.1.3 Synthesis of the metal complexes: dtc1-dtc3
To a solution of sbl(0.078 g, 0.25 mmol) in (1:1) methanol/dichloromethane (20 mL) was
added a solution of Cu(OAc) .H O (0.031 g, 0.125 mmol) in methanol (10 mL). The resulting
2
2
mixture was stirred at room temperature for 4 h. The dark-brown precipitate that formed was
filtered off, washed with ethanol, methanol, diethylether and dried in a desiccator over
anhydrous calcium chloride.
The corresponding Co(II) and Ni(II) complexes were prepared by a similar procedure as
described for Co(II) complex, using Co(OAc) ·4H O and Ni(OAc) .4H O, respectively.
2
2
2
2
Brick-red and light greenish precipitate of Co(sbl) and Ni(sbl) was obtained. Physical and
2
2
spectroscopic data for the complexes are given below.
[
Cu(sbl) ].dtc1
2
−
1
Dark brown, yield: 74%; m. p. (210–220 °C). Selected IR data (KBr pellets, cm ): ν(C=N)
1
2
572 m, ν(N–N) 1027 s, ν(CSS) 834 m (Figure S4). UV–Vis spectrum [DMSO, λ max nm]:
79, 365, 439, 568. ESI MS Calc. for C H N O S Cu: 721.01; found (M + 1): 721.57.
3
2
26
4
4
4
−
5
−1
2
−1
Molar conductivity (1.0 × 10 M; DMSO, Ω cm mol ): 13.8.
[
Co(sbl) ].dtc2
2

−
1
Brick red, yield: 66%; m. p. (227–230 °C). Selected IR data (KBr pellets, cm ): ν(C=N)
1
3
574 s, ν(N–N) 1032 s, ν(CSS) 827 m(Figure S5). UV–Vis spectrum [DMSO, λ max nm]: 282,
21, 383, 703. ESI MS Calc. for C H N O S Co: 717.01; found (M + 1): 717.04. Molar
3
2
26
4
4
4
−
5
−1
2
−1
conductivity (1.0 × 10 M; DMSO, Ω cm mol ): 10.3.
[
Ni(sbl) ].dtc3
2
−
1
Light green, yield: 71%; m. p. (215–220 °C). Selected IR data (KBr pellets, cm ): ν(C=N)
1
3
569 s, ν(N–N) 1032 s, ν(CSS) 827 m(Figure S6). UV–Vis spectrum [DMSO, λ max nm]: 281,
54, 413, 515. ESI MS Calc. for C H N O S Ni: 716.01; found (M + 1): 716.01. Molar
3
2
26
4
4 4
−
5
−1
2
−1
conductivity (1.0 × 10 M; DMSO, Ω cm mol ): 14.6.
2
.2 Microbiology
2
.2.1. Candida strains and culture conditions
Candida albicans strains including fluconazole susceptible (n =9), fluconazole resistant (n
5) and a control laboratory strain C. albicans SC5314 (ATCC MYA- 2876) were used.
=
These clinical isolates were collected previously from HIV and cancer patients visiting
different clinics at Charlotte Maxeke Johannesburg Academic Hospital and were stored at -80
°
C in the Department of Clinical Microbiology and Infectious Diseases, University of the
Witwatersrand, Johannesburg. The ethical clearance number M000402 obtained from Human
Research Ethics Committee, University of the Witwatersrand was used to collect these
isolates. As the isolates were stored in glycerol stocks at -80 ºC for long time, identification
and drug susceptibility was done prior to the start of this study. Rapid tests for presumptive
identification of C. albicans from clinical isolates was carried out using the germ tube assay
®
and the API 20 C AUX test kit (Biomerieux, France). All the isolates were also screened for
flucoazole (FLC) susceptibility by determining minimum inhibitory concentrations,

following CLSI guidelines. FLC was tested and the isolates having an MIC values ≤2 µg/mL
for FLC were considered FLC susceptible, MIC values 4 µg/mL for FLC were considered
FLC susceptible dose dependent, while as the isolates having MIC 8 µg/mL were
categorized as FLC resistant, following the CLSI Interpretive Guidelines for In vitro
Susceptibility Testing of Candida species.
2
.2.2. Antifungal Susceptibility testing
Minimum Inhibitory Concentrations (MIC) and Minimum Fungicidal Concentrations (MFC)
of the test compounds sbl and dtc1-dtc3against C. albicans isolates were determined by
following Clinical and Laboratory Standards Institute (CLSI) guidelines M27-A3 [20].
Briefly, two-fold dilutions of test compounds (100 µL) were prepared in 96-well flat-bottom
6
microtiter plates, followed addition of 100 µL culture inoculum (1  10 cells/mL)and
incubated at 37 ºC for 24 hours. In every set of experiment fluconazole and 1% DMSO were
used as positive and negative controls respectively. The MIC’s were determined as the lowest
concentration of the test compound that resulted in complete inhibition of growth. To
determine MFC, each well without visible growth was sub-cultured onto agar plates and
incubated at 37 ºC for 24 hours. Concentration of test compound in the first well with no
growth on agar plate was taken as the MFC value.
2
.2.3.Cell viability assay
Muse Count and Viability kit (EMD Millipore, USA) was used for cell viability assay
following the manufacturer’s instructions. Cells were differentiated in to viable and non-
viable cells based on their permeability to the dyes present in the reagent. Briefly, C. albicans
cells were treated with MIC values of test entities followed incubation at 37°C for 3 h. Cell
7
suspension (1  10 cells/mL) were prepared in saline, then 380 µL of viability reagent was

added to 20 µL of prepared cell suspension and incubated at room temperature for 5 minutes.
After incubation sample analysis was done by using MUSE cell analyser (EMD Millipore,
Germany). Heat killed cells and untreated cells were used positive and negative controls
respectively.
2
.2.4.Annexin V and PI staining
To determine the mode of cell death by the test compounds, apoptosis was studied. FITC
Annexin V Apoptosis Detection Kit I (BD Biosciences, CA, USA) was used following the
manufacturer’s instructions. C. albicans cells were exposed to MIC value of test compounds
sbl, dtc1-dtc3for 3h at 37 ºC. After incubation protoplasts of C. albicans cells were prepared
as described in our previous study [21], then resuspended in annexin V binding buffer at a
6
concentration of 1  10 cells/mL. This prepared solution (100 µL) was taken into a new tube
and then FITC annexin V (5 µL) and Propidium iodide (5 µL) were added followed by
incubation in the dark at room temperature for 15 min. After incubation annexin V binding
buffer (400 µL) was added to each tube and analysed by flow cytometry BD LSRFortessa
cell analyser (Becton Dickinson, USA). Untreated cells and cells exposed to 2.5 mM H O
2
2
were used as negative and positive controls respectively. The cells in four different quadrants
were analysed and interpreted as Q1: necrosis (Annexin V-/PI+); Q2: late apoptosis (Annexin
V+/PI+); Q3: early apoptosis (Annexin V+/PI-) and Q4: viable cells (Annexin V-/PI-).
FlowJo_V10 software was used to analyse the final data.
2
.2.5.Haemolytic assay
Haemolytic assay was used to determine the cytotoxic effect of the test compounds following
method described by Ahmad et al; 2009 with modifications. Briefly, 10 mL of Horse blood
(NHLS, Johannesburg, South Africa) was centrifuged at 2000 rpm for 10 min. The obtained

cell pellet was washed twice with chilled PBS, followed by preparation of 10% RBC
suspension in the same solution. The 10% suspension was further diluted to 1:10 in PBS.
From this suspension aliquot of 100 µL was taken and added to varying concentrations (1/2
MIC, MIC and 2 MIC) of test entities. Tubes were incubated at 37°C for 1 h, followed by
centrifugation at 2000 rpm for 10 min. After centrifugation supernatant (200 µL)was
transferred to a flat-bottomed 96-well plate and absorbance was read spectrophotometrically
at 450 nm using iMark microplate reader (Bio-Rad laboratories, CA, USA). Triton X-100 and
PBS were used as positive and negative controls respectively. The percentage of haemolysis
was calculated by the following equation:
퐴450 표푓 푡푒푠푡 푐표푚푝표푢푛푑 푡푟푒푎푡푒푑 푠푎푚푝푙푒 ― 퐴450 표푓 푏푢푓푓푒푟 푡푟푒푎푡푒푑 푠푎푚푝푙푒
%
퐻푎푒푚표푙푦푠푖푠 =
푋100%
[
]
퐴450 표푓 1% 푡푟푖푡표푛 푋 ― 퐴450 표푓 푏푢푓푓푒푟 푡푟푒푎푡푒푑 푠푎푚푝푙푒
2
.2.6.Statistical analysis
The statistical analysis was performed using Dunnett’s multiple comparisons of a Two-way
ANOVA test by GraphPad Prism 8.1 software. All the experiments were performed
independently in triplicates (n=3), and data were presented as mean ± standard deviation
(SD). P value ****P < 0.0001 was considered as statistically significant.
3
. Results and discussion
Metal complexes (dtc1-dtc3) with a bidentate NS ligand obtained via the condensation
reaction between the respective S-benzyl dithiocarbazate and 1,3-Benzodioxole-5-
carboxaldehyde in equimolar amounts was prepared as described previously [18]. (Scheme
1
). The physico-analytical data of sbl and complexes (dtc1-dtc3) are provided in the
experimental section.
<
Scheme 1>

Reaction of S-benzyldithiocarbazate with 1,3-Benzodioxole-5-carboxaldehyde was carried
out in methanolic solution at 60 °C to recover a light-yellow precipitate of sbl. Based on the
ligand structure, different reaction conditions were chosen to optimize the yield and purity of
the complexes. The structures of the complexes dtc1-dtc3 were proposed on the basis of the
bidentate nature of ligand (sbl),established by infrared spectroscopy, mass spectrometry and
elemental analysis. The mass spectra ofthe complexes (Figure S7-S9) reported in the
experimental section agreed with the molecular mass of the complexes thereby confirming
2
:1 ligand to metal stoichiometry in the complexes. The purity of the ligand (sbl)was also
evaluated using Reversed Phase-High Performance Liquid Chromatography (RP-HPLC)on a
C8 column. A mixture of ACN and H O (20:80) was used as a mobile phase to elute the
2
compound(sbl). The chromatogram obtained after evaluation using RP-HPLC with a single
1
peak shown in Figure S10 confirms the purity of sbl. Furthermore, the H NMR spectrum of
sbl (Figure 1) displayed peaks at ca. 8.15 ppm(-C=N-), 13.26 ppm(-NH-), 4.49 ppm(-CH -),
2
6
.08 ppm(-CH -) and 7.21-7.43 ppm(aromatic protons).To confirm the formation of the
2
ligand and its complexes and to better characterize them, few other techniques like FT-IR,
UV-Visible, SEM, and XRD were employed. The FT-IR spectra of the sbl exhibited
−
1
-1
-1
-
characteristic bands at ca. 3124cm (νNH),2918cm (νCH ),1600cm (νC=N), 1091cm
2
1(
νC=S) and 1033cm (νN-N), respectively. Upon complex formation, the band due to the
-1
-1
azomethine ν(C=N) vibration experienced a downward shift of ca. 26−31cm validating its
coordination to the metal ions. The disappearance of ν(C=S) at ca. 1091 cm−1 and the
−
1
splitting of asymmetric ν(CSS) of the free ligand at ca. 990−923 cm in the spectra of the
complexes strongly backed the evidence of coordination via the thiolate sulphur atoms.
-1
Furthermore, in the complexes dtc1-dtc3new bands were found in the regions 544-574 cm ,
attributed to υ(M-O) stretching vibrations and the bands present at 440-475 cm-1 in
complexes have been ascribed to υ(M-N) stretching vibrations.

<
Figure 1>
The electronic spectra of the ligand (sbl) and complexes dtc1-dtc3 (Fig.S12) were recorded
in DMSO in the region 200–800 nm. The spectra of the ligand (sbl) exhibited two bands at
ca. 256nm and 275nm, attributed to the π-π* transitions in aromatic rings andtheiminic(-
C=N-) group, respectively. Other bands at 328nm and 376nm are allotted to π-π* and n-π*
transitions of thedithiocarbazate group. For all the complexes, the spectrum displayed strong
bands at ca. 280 and 345 nm (sh) corresponding to the intra-ligand π- π* and n- π * electronic
transitions. The spectrum of the dtc1 complex exhibited a d–d transition band at 568 nm due
2
2
to B → E corresponding to a square planar geometry with a magnetic moment value of
1
g
g
1
.82 BM due to a single unpaired electron [22]. The electronic absorption spectra of the
7
Co(II), d , complex(dtc2)displayed a d–d band in the visible region at ca. 703 nm due to
1
1
B → A indicating a distorted square planar geometry for the complex also supported by
1
g
1g
the observed effective magnetic moment of2.61BM[23]. The UV-Vis spectrum of
dtc3complex displays absorption peaks at 281 nm, 354 nm and 515 nm. The bands at 281 nm
and 354 nm corresponds to π- π* and n- π* electronic transitions, while the absorption band
1
1
at 515 nm is assigned a d–d transition due to B → A characteristic of low-spin square
1
g
1g
planar diamagnetic dtc3 complexes[24, 25].
The room temperature molar conductance values of the complexes (dtc1-dtc3)were obtained
in 10-5 M DMSO solutions. The low molar conductivity values 13.8 Ω cm mol ,
−1
2
−1
−
1
2
−1
−1
2
−1
1
0.3Ω cm mol and 14.6 Ω cm mol for complexes dtc1, dtc2 anddtc3respectively, are
indicative of the non-electrolytic nature of all the complexes [26].
Thermo grams of dtc1-dtc3complexes were recorded in the temperature range of 40- 800 °C.
The results obtained are in close agreement with the theoretical formula of the compounds as
suggested by the analytical data. The obtained thermo grams revealed that all the complexes

were stable up to a temperature of about 210-230 °C exhibiting a two-step decomposition in
each complex. All the complexes did not show any loss of hydrated or coordinated water
molecules, which is in accordance with the predicted structures of the complexes. In the
dtc1complex the first decomposition occurred in the temperature range of 210-430 °C may
be ascribed to the loss of C H N O S group and the second decomposition observed in the
1
6
14
2
2 2
range of 430-760 °C may be assigned to the removal of other C H N O S group, thereby
1
6
14
2
2 2
confirming the loss of one part of the ligand in the first step and the remaining ligand
molecule in the second step with Cu metal as the residue. The dtc2 anddtc3complexes
showed similar decomposition patterns like that of dtc1complex with slight deviations in the
recorded thermo grams. Residues of (Co, Ni) metal were recovered in all the cases. A
characteristic TGA diagram of the complexes is given in Fig. S13.
Single crystal X-Ray diffraction is a more precise technique that furnishes complete
information about the structure of the compounds. But despite of our several efforts of
recrystallization, none of the compounds was obtained in the crystalline form. Therefore,
powder XRD of the ligand and the complexes was performed to describe the structure of the
synthesized compounds. The powder XRD pattern of the ligand (sbl), dtc1, dtc2
anddtc3complexes recorded in the range of 2θ = 10-80° are shown in Figure2. From the
XRD data, the ligand(sbl) and complexes, display sharp crystalline peaks confirming their
crystalline nature. The average crystallite size of the synthesized compounds was calculated
using Scherrer equation [27]. The ligand and complexes have an average crystallite size of
3
1.23, 13.25, 25.70 and 17.21nm, respectively, suggesting that the compounds are
polycrystalline.
<
Figure 2>

SEM analysis was performed to determine the surface morphology of the ligand (sbl) and its
complexesdtc1-dtc3. The SEM images given in Figure3clearly indicate that the compounds
exhibited different morphological structures. The ligand sbl shows irregularly shaped
particles while as the complexes exhibit rod like, grass like and fibre/needle like
morphologies. The different surface morphologies of metal complexes from that of the ligand
further confirm the interaction of the metal ions with the ligand molecule. The average grain
size (17-36nm) obtained from SEM analysis confirm that the complexes are polycrystalline in
nature which is well in accordance with XRD analysis.
<
Figure 3>
4
. Biological activity
4
.1 Antifungal activity
The in vitro susceptibility of C. albicans isolates (n=15) against the test compounds (sbl and
complexes dtc1-dtc3), were evaluated by determining MIC and MFC values and the results
are summarized in Table 1. From the results, it is evident that all the three metal complexes
dtc1-dtc3 exhibited enhanced antifungal activity than the ligand (sbl). The mean range of
MIC and MFC values for all test compounds against different FLC susceptible C. albicans
strains were 1 – ≥ 512 µg/mL and 4 – 1024 µg/mL respectively. These figures for different
FLC resistant C. albicans strains were in the range of 16 – 512 µg/mL and 32 – 1024 µg/mL
respectively. The highest antifungal activity was observed for the nickel (II) metal complex
dtc3 with MIC and MFC range of 0.5 – 32 µg/mL and 1 – 64 µg/mL respectively, followed
by dtc2 (MIC 4 – 128 µg/mL and MFC 16 – 256 µg/mL), and dtc1 (MIC 64 – 1024 µg/mL
and MFC 128 – 2048 µg/mL ). The ligand showed the least antifungal effect with MIC and
MFC values of 256 – 2048 µg/mL and 512 – > 2048 µg/mL respectively. These results

indicated that metalation of dithiocarbazate derived Schiff base compounds significantly
improved their antifungal activity.
<
Table 1>
4
.2. Cell viability
The cell viability of C. albicans SC5314 at MIC values of different test entities are presented
in Fig.4. The results demonstrated these test compounds considerably decreased the cell
viability when compared to untreated control cells. After treatment, the ligandsbl showed
only 23% ± 2.5% cell death, while there was a drastic increase in percentage of cell death
after treatment with metal complexes dtc1-dtc3. Ni complex (dtc3)exhibited highest
percentage of cell death 80% ± 8.5% followed by Co(dtc2) 70% ± 7.5% and Cu (dtc1)with
4
1
7% ± 7.5%. The negative (untreated) and positive (heat killed) controls exhibited 2% ±
.5% and 95% ± 2.5% cell death respectively.These results revealed that after metalation test
compounds showed improved antifungal activity with fungicidal nature. The viability results
reflect the same pattern as observed in the MIC determination, which also advocates the
reliability of this technique to be used in antifungal studies. To further differentiate the results
between the cells and debris and/or non-nucleated cells, cell size index was also done in the
same assay and it was confirmed that the results are due to live and dead cells (Figure 4).
<
Figure4>
4
.3 Annexin V and PI staining
Annexin V-FITC and Propidium iodide double staining was performed to observe the
apoptotic effect of the test compounds against C. albicans SC5314. The annexin V detects
phosphatidylserine (PS) externalization and PI stains necrotic and dead cells. C. albicans
cells after exposed to MIC values of test compounds showed varying degree of apoptosis

represented in Figure 5 and summarized in Figure 6. Among the test compounds, the Ni
metal complex dtc3was the most active which induced early apoptosis (17% ± 4.2%), late
apoptosis (38% ± 2.6%) and necrosis (20% ± 2.5%). Co and Cu metal complexes dtc2 and
dtc1induced early apoptosis (6% ± 3.5%), late apoptosis (34% ± 3.6%), necrosis (23% ±
1
2
.5%) and early apoptosis (11% ± 1.2%), late apoptosis (27% ± 2.5%) and necrosis (13% ±
.0%), respectively. In contrary, the ligand sbl showed only 2% ± 1.0%, 9% ± 3.6% and 5%
±
2.3% of early apoptosis, late apoptosis and necrosis respectively. Negative control
untreated sample) exhibited early apoptosis (2% ± 1.0%), late apoptosis (1% ± 1.5%) and
necrosis (1% ±1.0%) and positive control (H O exposed sample) showed early apoptosis
(
2
2
(32% ± 2.5%), late apoptosis (25% ± 3.0%) and necrosis (2% ± 1.5%). From the results it
was observed that all these test compounds can induce apoptosis in C. albicans cells mainly
through late apoptosis and necrotic pathways.
<
<
Figure5>
Figure6>
4
.5. Haemolytic activity
To determine the cytotoxicity of these test compounds, haemolytic assay using horse red
blood cells was performed. When compared to positive control (Triton X-100), which causes
1
00% lysis these test compounds exhibited significantly less haemolysis. PBS was used as a
negative control which showed no lysis of RBCs. Metal complexes (dtc1-dtc3) showed cell
haemolysis in the range of 4% - 6%, 8% - 12% and 12 % - 23 % at 1/2 MIC, MIC and 2 MIC
values respectively while as the ligand sbl exhibited 7% ± 3%, 12% ± 3.5% and 27% ± 5.5%
cell haemolysis at 1/2 MIC, MIC and 2 MIC values, respectively. Further, results also

indicated that the haemolytic activity of these compounds is concentration dependent. Red
blood cells provide a handy tool for toxicity studies of new compounds, because they are
readily available, their membrane properties are well known, and their lysis is easily
monitored by measuring the release of haemoglobin [28]. The comparative study of sbl and
its complexes(dtc1-dtc3)with Triton-X, which is well known to cause haemolysis in RBCs,
indicated that these compounds within their MIC ranges and above showed significantly less
cytotoxicity (Figure 7).
<
Figure7>
C. albicans is the most widespread opportunistic fungal pathogen in the human body, causing
mucosal and systemic infections. Fungal-selective targets are insufficient due to the fact that
most eukaryotes share similar metabolic pathways and essential cellular machinery with
humans. Therefore, new compounds are needed to be identified or synthesized that can cause
fungal programmed cell death also called as apoptosis to allow specific manipulation of cell
death without costing human counterparts.Apoptosis in C. albicans can be induced by several
external and internal factors and has been regulated at biochemical and molecular levels [29].
Flow cytometry analysis revealed that the C. albicans cells exposed to sub-MIC
concentrations of the test compounds showed the movement of phosphatidylserine to the
outer leaflet of the plasma membrane. The externalisation of phospholipids results with the
binding of annexin V, which was detected by flow cytometer. It has been extensively
observed that fungicidal compounds at sub-MIC values show programmed cell death [29,30].
Previous studies have shown that several stimuli including hydrogen peroxide, metal
complexes, diallyl disulfide, farnesol, lactoferrin, defensins and the antifungal agents
amphotericin B and caspofungin cause Candida albicans cells to undergo apoptosis [31-36].
Cell wall stress stemming from inhibition of (1,3)-β-D-glucan, which is essential for

ergosterol biosynthesis is also a trigger for apoptosis. In our previous studies we found that
metal complexes have the potential to inhibit ergosterol biosynthesis, resulting in apoptotic
death of C. albicans [37-39]. It has also been established that metal complexes affect
mitochondrial function, retard the synthesis of cytochrome b and c and uncouple respiration.
Treatment of fungal cells with Cu(II) and Ag(I) complexes results in a reduced amount of
ergosterol in the cell membrane and subsequent increase in its permeability [34]. Also cells
exposed to Cu(II) and Mn(II) complexes demonstrate an elevation in oxygen uptake, which
goes to show that the metal complexes damage mitochondria and uncouple respiration. Ag(I)
complexes have been shown to cause extensive, non-specific DNA cleavage to C. albicans,
disrupt cell division and induce gross distortions in fungal cell morphology [34]. Since metal
complexes are known to affect mitochondrial function and it is the site for both the origin and
target of ROS, mitochondria and ROS play an essential role in apoptotic processes [40,41].
During apoptosis, oxidative stress induces the mitochondrial permeability transition pore
(
PTP) to open, which then triggers the mitochondrial outer membrane permeabilization
MOMP) that is involved in the release of cytochrome c and other proapoptotic factors,
(
leading to subsequent metacaspase activation [42]. ROS can also directly induce apoptosis in
a metacaspase-dependent manner [43].
Besides ROS, Ca2+ is also closely related to apoptosis [44]. Under oxidative stress, Ca2+
signal can lead to PTP opening and release of cytochrome c and other proapoptotic factors,
which ultimately results in the formation of apoptotic bodies and cell death. Although there is
no MCU complex (mitochondrial calcium uniporter) in yeast mitochondria, apoptotic stimuli
could induce enough mitochondrial Ca2+ to undergo the permeability transition, which is
2
+
2+
accompanied by increased ROS and cytosolic Ca levels. Once PTP opens, Ca levels in the
cytosol and matrix equilibrate and stabilize PTP in the open conformation, leading to matrix
swelling, outer mitochondrial membrane damage, and release of proapoptotic proteins,

thereby causing cell death. Several studies reported that antioxidant or mitochondrial Ca2+
channel inhibitors could rescue cells from natural product-induced apoptosis [45-47],
2
+
indicating that oxidative stress or elevated mitochondrial Ca might be the reasons leading to
apoptosis. High antifungal activity of the complexes, especially the Ni complex dtc3 demands
further in vivo studies against C. albicans and other non-albicans Candida species. Low
toxicity and potential to cause apoptosis and necrosis magnifies its potential to be developed
as a novel antifungal drug.
5
. Conclusion
Metal-based drugs have several promising advantages in comparison to organic ligands and
have gained trust from the researches after the worldwide approval of the drug cisplatin. The
distinct mechanism of action from conventional drugs makes them perfect alternatives to the
conventional drugs where resistance has already appeared. In this direction, a series of new
transition metal complexes were synthesized, characterized and evaluated for their antifungal
activities. The biological profiles revealed that the complexes (dtc1-dtc3) were more active
than the free ligand (sbl). Complex dtc3 exhibited a minimum inhibitory concentration (MIC)
value ranging from 1 – 32 µg/mL. The mechanism of action appears to be due to apoptosis
in C. albicans, causing phosphatidylserine externalization as revealed by Annexin V-FITC
and propidium iodide staining assays. Furthermore, low toxicity and potential to cause
apoptosis and necrosis further amplifies its potential to be developed as a novel antifungal
drug.
Funding
We gratefully acknowledge financial support from Council for Scientific and Industrial
Research (Grant No: 09/466(0215)2K19EMR-1).
Ethics approval

This study was approved by the Human Research Ethics Committee of University of the
Witwatersrand (Johannesburg, South Africa).Existing stock cultures of Candida
albicans used in this study were stored in the department of Clinical Microbiology and
Infectious Diseases, University of the Witwatersrand, Johannesburg, South Africa.
Competing interests
We have no competing interests to declare
6
. References
[
1]
2]
www.wto.org/english/news_e/news16_e/heal_29aug16_e.pdf.
[
N. S. Ng, P. Leverett, D. E. Hibbs, Q. Yang, J. C. Bulanadi, M. J. Wua and J. R.
Aldrich-Wright, The antimicrobial properties of some copper(II) and platinum(II)
1
,10-phenanthroline complexes, Dalton Transactions, (2013) 3196–3209.
[3]
A.S. Abd-el-aziz, C. Agatemor, N. Etkin, P. David, M. Lanteigne, K. Mcquillan, R.G.
Kerr, Antimicrobial Organometallic Dendrimers with tunable activity against
multidrug-resistant bacteria, (2015). doi: 10.1021/acs.biomac.5b01207.
[
4]
5]
F. Li, J.G. Collins, F.R. Keene, Chem Soc Rev Ruthenium complexes as antimicrobial
agents, Chem. Soc. Rev. 44 (2015) 2529–2542.
[
J. Duan, H. Liu, P. Jeyakkumar, L. Gopala, S. Li, R. Geng, C. Zhou, Design, synthesis
and biological evaluation of novel Schiff base-bridged tetrahydroprotoberberine
triazoles as a new type of potential antimicrobial agents, Medchemcomm. 8 (2017)
9
07–916.
[6]
M. A. Malik, O. A. Dar, P. Gull, M. Y. Wani, A. A. Hashmi, Heterocyclic schiff base
transition metal complexes in antimicrobial and anticancer chemotherapy.
Medchemcomm 2018, 9, 409–436.

[
7]
8]
9]
F. Li, J. G. Collins and F. R. Keene, Ruthenium complexes as antimicrobial agents,
Chem. Soc. Rev., 44 (2015) 2529-2542.
[
S. Rafique, M. Idrees, A. Nasim, H. Akbar and A. Athar, Transition metal complexes
as potential therapeutic agents, Biotechnol. Mol. Biol. Rev., 5 (2010) 38-45.
V. Bobbarala, A Search for Antibacterial Agents, InTech Press, Rijeka, Croatia, 2012.
[
[
10] M. D. Rhodes, P. J. Sadler, M. D. Scawen and S. Silver, Effects of gold(I) antiarthritic
drugs and related compounds on Pseudomonas putida, J. Inorg. Biochem., 46 (1992)
1
29-142.
[
11] A. D. Richards, A. Rodger, M. J. Hannon and A. Bolhuis, Antimicrobial activity of an
iron triple helicate, Int. J. Antimicrob. Agents, 33 (2009) 469-472.
[
12] N. S. Ng, P. Leverett, D. E. Hibbs, Q. Yang, J. C. Bulanadi, M. J. Wu and J. R.
Aldrich-Wright, The antimicrobial properties of some copper(ii) and platinum(ii)
1
,10-phenanthroline complexes, Dalton Trans., 42 (2013) 3196-3209.
[
13] M. A. Neelakantan, M. Esakkiammal, S. S. Mariappan, J. Dharmaraja and T.
Jeyakumar, Synthesis, characterization and biocidal activities of some Schiff base
metal complexes, Indian J. Pharm. Sci., 72 (2010) 216-222.
[
[
[
[
14] B. S. Sekhon, Matrix metalloproteinases – an overview, J. Pharm. Educ. Res., 1
(
2010) 1-20.
15] L. J. Ming, Structure and function of metalloantibiotics, Med. Res. Rev., 23 (2003)
97-762.
6
16] S. K. Nandanwar and H. J. Kim, Anticancer and Antibacterial Activity of Transition
Metal Complexes, Chemistry Select, 4 (2019) 1706 –1721.
17] J. A. Lemire, J. J. Harrison and R. J. Turner, Antimicrobial activity of metals:
mechanisms, molecular targets and applications, Nature reviews Microbiology, 11
(2013) 371.

[
18] M. L. Low, L. Maigre, P. Dorlet, R. Guillot, M. Pagès, K. A. Crouse, C. Policar, N.
Delsuc, M. L. Low, L. Maigre, P. Dorlet, R. Guillot, J. Pagès, K. A. Crouse,
Conjugation of a New Series of Dithiocarbazate Schiff Base Copper (II) Complexes
with Vectors Selected to Enhance Antibacterial Activity Conjugation of a New Series
of Dithiocarbazate Schiff Base Copper (II) Complexes with Vectors Selected to
Enhance
Anti-
bacterial
Activity,
Bioconjugate
Chem.,
(2014).
doi:10.1021/bc5004907.
[
19] M. A. Ali, A. H. Mirza, R. J. Butcher, M. T. H. Tarafder, T. Boon, A. M. Ali,
Biological activity of palladium ( II ) and platinum (II) complexes of the acetone
Schiff bases of S -methyl- and S -benzyldithiocarbazate and the X-ray crystal
structure of the [ Pd ( asme ) 2 ] ( asme 5 anionic form of the acetone Schiff base of S
-methyldithiocarbazate ) complex, J. Biol. Inorg. Chem., 92 (2002) 141–148.
[
[
[
20] CLSI. Performance Standards for Antifungal Susceptibility Testing of Yeasts. 1st ed.
CLSI supplement M60. Wayne, PA: Clinical and Laboratory Standards Institute
(2017).
21] A. Khan, A. Ahmad, L. A. Khan, & N. Manzoor, Ocimum sanctum (L.) essential oil
and its lead molecules induce apoptosis in Candida albicans. Res. Microbiol. 165
(2014) 411-419.
22] S. Tejaswi, M.P. Kumar, A. Rambabu, Synthesis, structural, DNA binding and
cleavage studies of Cu (II) complexes containing benzothiazole cored schiff bases, J.
Fluoresc. (2016).
[
23] A. B. P. Lever, 1984. Inorganic Electronic Spectroscopy. Elsevier, Amsterdam.
24] R. Takjoo, R. Centore, Synthesis, X-ray structure, spectroscopic properties and DFT
studies of some dithiocarbazate complexes of nickel (II), J. Mol. Struct. 1031 (2013)
[
1
80–185.

[
25] C. F. Wise, D. Liu, K. J. Mayer, P. M. Crossland, Carolyn L. Harley, and William R.
McNamara, A nickel complex of a conjugated bisdithiocarbazate schiff base for the
photocatalytic production of hydrogen, Dalton Transactions, 00 (2013) 1-3.
26] M.S. Nair, D. Arish, R.S. Joseyphus, and DNA cleavage studies of some heterocyclic
Schiff base metal complexes, J. Saudi Chem. Soc. 16 (2012) 83–88.
[
[
27] C.J. Dhanaraj, I.U. Hassan, J. Johnson, J. Joseph, R.S. Joseyphus, Synthesis, spectral
characterization, DNA interaction, anticancer and molecular docking studies on some
transition metal complexes with bidentate ligand, J. of Photochem. PhotoBio. (2016).
doi: 10.1016/j.jphotobiol.2016.06.032.
[
28] A. Ahmad, A. Khan, N. Manzoor, L. A. Khan, Evolution of ergosterol biosynthesis
inhibitors as fungicidal against Candida, 48 (2010) 35-41.
[
29] D.Carmona-Gutierrez, T. Eisenberg, S. Buttner, C. Meisinger, G. Kroemer, F. Madeo,
Apoptosis in yeast: triggers, pathways, subroutines. Cell Death Differ, 17 (2010) 763-
7
3.
[
30] B. Almeida, A. Silva, A. Mesquita, B. Sampaio-Marques, F. Rodrigues, P. Ludovico,
Drug-induced apoptosis in yeast. Biochim. Biophys Acta1783 (2008) 1436-1448.
31] M. E. Shirtliff, B. P. Krom, R. A. Meijering, B. M. Peters, J. Zhu, M. A. Scheper, M.
L. Harris, M. A. Jabra-Rizk, Farnesol-induced apoptosis in Candida albicans.
Antimicrob. Agents Chemother. 53 (2009) 2392–2401.
[
[
32] H. Lu,Z. Zhu, L. Dong, X. Jia, X. Sun, L. Yan, Y. Chai, Y. Jiang, Y. Cao, Lack of
trehalose accelerates H O -induced Candida albicans apoptosis through regulating
2
2
2
+
Ca signaling pathway and caspase activity. PLoS One 6 (2011) 15808.
33] I. S. Hwang, J. Lee, J. H. Hwang, K. J. Kim, D. G. Lee, Silver nanoparticles induce
apoptotic cell death in Candida albicans through the increase of hydroxyl radicals.
FEBS J. 279 (2012) 1327–1338.
[

[
[
[
[
34] A. Eshwika, B. Coyle, M. Devereux, M. McCann, K. Kavanagh, Metal complexes of
1
,10-phenanthroline-5,6-dione alter the susceptibility of the yeast Candida albicans to
amphotericin B and miconazole. Biometals 17 (2004) 415–422.
35] K. M. Lemar, M. A. Aon, S. Cortassa, B. O’Rourke, C. T. Muller, D. Lloyd, Diallyl
disulphide depletes glutathione in Candida albicans: oxidative stress-mediated cell
death studied by two-photon microscopy. Yeast 24 (2007) 695–706.
36] H. Nikawa, L. P. Samaranayake, J. Tenovuo, K. M. Pang, T. Hamada, The fungicidal
effect of human lactoferrin on Candida albicans and Candida krusei. Arch. Oral Biol.
3
8 (1993) 1057–1063.
37] O. A. Dar, S. A. Lone, M. A. Malik, M. Y. Wani, Md. I. A. Talukdar, A. S.
Al‐Bogami, A. A. Hashmi, A. Ahmad, Heteroleptic transition metal complexes of
Schiff‐base‐derived ligands exert their antifungal activity by disrupting membrane
integrity, Applied Organometall. Chem. 33 (2019) 5128.
[
38] O. A. Dar, S. A. Lone, M. A. Malik, M. Y. Wani, A. Ahmad and A. A. Hashmi, New
transition metal complexes with a pendent indole ring: insights into the antifungal
activity and mode of action, RSC Adv., 9 (2019) 15151-15157.
[
39] O. A. Dar, S. A. Lone, M. A. Malik, F. M. Aqlan, M. Y. Wani, A. A. Hashmi, A.
Ahmad, Synthesis and synergistic studies of isatin based mixed ligand complexes as
potential antifungal therapeutic agents, Heliyon, 5, (2019) 02055.
[
40] A. Terman, B. Gustafsson, and U. T. Brunk, The lysosomal-mitochondrial axis theory
of postmitotic aging and cell death. Chem. Biol. Interact. 163 (2006) 29–37.
41] C. Pereira, R. Silva, L. Saraiva, B. Johansson, M. Sousa, and M. Côrte-Real,
Mitochondria-dependent apoptosis in yeast. Biochim. Biophys. Acta. 1783 (2008)
[
1
286–1302.
[42] H. U. Simon, A. Hajyehia, and F. Levischaffer, Role of reactive oxygen species
(ROS) in apoptosis induction. Apoptosis 5 (2000) 415–418.
[
43] X. Z. Wu, W. Q. Chang, A. X. Cheng, L. M. Sun, and H. X. Lou, Plagiochin E, an
antifungal active macrocyclic bis(bibenzyl), induced apoptosis in Candida albicans

through a metacaspase-dependent apoptotic pathway. Biochim. Biophys. Acta 1800
2010) 439–447.
(
[
44] M. Carraro, and P. Bernardi, Calcium and reactive oxygen species in regulation of the
mitochondrial permeability transition and of programmed cell death in yeast. Cell
Calcium 60 (2016) 102–107.
[
45] R. Zhang, I. Humphreys, R. P. Sahu, Y. Shi, and S. K. Srivastava, In vitro and in vivo
induction of apoptosis by capsaicin in pancreatic cancer cells is mediated through
ROS generation and mitochondrial death pathway. Apoptosis 13 (2008) 1465–1478.
46] D. G. Yun, and D. G. Lee, Silibinin triggers yeast apoptosis related to mitochondrial
[
2
+
Ca influx in Candida albicans. Int. J. Biochem. Cell Biol. 80 (2016) 1–9.
47] W. Lee, and D. G. Lee, Reactive oxygen species modulate itraconazole induced
apoptosis via mitochondrial disruption in Candida albicans. Free Radic Res. 52
[
(2017) 39–50.

O
O
S
N
O
O
N
M
N
N
S
(a)
N
S
O
O
S
S
H
S
N
sbl
M= Cu (dtc1), Co (dtc2), Ni (dtc3)
Scheme 1: Synthesis of dithiocarbazate Schiff base ligand-based metal complexes.
Reaction conditions: (a) MeOH/CH CN, 1:1, r.t., 4-8 h.
3

1
Figure 1: H NMR spectrum of the ligand sbl.

Figure 2: Powder XRD patterns of ligand sbl and its complexesdtc1, dtc2 and dtc3.

Figure3: SEM images of ligand (sbl) and complexes dtc1, dtc2 anddtc3.