Synthesis and evaluation of Zn(II) dithiocarbamate complexes as potential antibacterial, antibiofilm, and antitumor agents

Article abstract of DOI:10.1080/00958972.2019.1693041

Four complexes having the formula [Zn(L)₂] [L1 = (C₁₈H₂₀NS₂ ^–), N-(4-isopropyl-benzyl)-(benzyl)-dithiocarbamate], [L2 = (C₁₀H₁₂NS₂ ^–), N-(benzyl)-(ethyl)-dithiocarbamate], [L3 = (C₁₉H₂₂ONS₂ ^–), N-(4-isopropyl-benzyl)-(4-methoxy-benzyl)-dithiocarbamate], and [L4 = (C₁₆H₁₆NS₂ ^–), N-(benzyl)-(4-methyl-benzyl)-dithiocarbamate] have been contemplated, synthesized, and characterized by elemental analysis and IR, ¹H, ¹³C NMR and UV–visible absorption spectra. All Zn(II) complexes have similar geometry and coordination number. Complex A2 (with ligand L2) crystallizes in triclinic system with space group P-1 having distorted square pyramidal geometry which was stabilized by weak C–H···π and C–H···S intramolecular interactions. The antibacterial, antibiofilm, and antitumor activities of the complexes have been screened and A2 and A3 showed their prominence. Interestingly, both A2 and A3 showed more killing potential against multi-drug resistant gram-positive isolates with MIC indices of 16 μg mL^?1 and 16 μg mL^?1, respectively, against both MRSA and MSSA, while the antitumor agent A3 showed its prominence with GI₅₀ and LC₅₀ 41.15 and 133.73 μg mL^?1, respectively.

Full text of DOI:10.1080/00958972.2019.1693041

Journal of Coordination Chemistry
ISSN: 0095-8972 (Print) 1029-0389 (Online) Journal homepage: https://www.tandfonline.com/loi/gcoo20
Synthesis and evaluation of Zn(II) dithiocarbamate
complexes as potential antibacterial, antibiofilm,
and antitumor agents
Vinay Kumar Maurya, Ashish Kumar Singh, Ravi Pratap Singh, Shivangi
Yadav, Krishna Kumar, Pradyot Prakash & Lal Bahadur Prasad
To cite this article: Vinay Kumar Maurya, Ashish Kumar Singh, Ravi Pratap Singh, Shivangi
Yadav, Krishna Kumar, Pradyot Prakash & Lal Bahadur Prasad (2019): Synthesis and evaluation
of Zn(II) dithiocarbamate complexes as potential antibacterial, antibiofilm, and antitumor agents,
Journal of Coordination Chemistry, DOI: 10.1080/00958972.2019.1693041
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JOURNAL OF COORDINATION CHEMISTRY
https://doi.org/10.1080/00958972.2019.1693041
Synthesis and evaluation of Zn(II) dithiocarbamate
complexes as potential antibacterial, antibiofilm, and
antitumor agents
a
b
a
Vinay Kumar Maurya , Ashish Kumar Singh , Ravi Pratap Singh ,
b
a
b
a
Shivangi Yadav , Krishna Kumar , Pradyot Prakash and Lal Bahadur Prasad
a
b
Department of Chemistry, Institute of Science, Banaras Hindu University, Varanasi, India; Bacterial
Biofilm and Drug Resistance Research Laboratory, Department of Microbiology, Institute of Medical
Sciences, Banaras Hindu University, Varanasi, India
ABSTRACT
ARTICLE HISTORY
Received 27 July 2019
Accepted 1 November 2019
–
Four complexes having the formula [Zn(L)
2
] [L1 ¼ (C18
H
1
20NS
2
), N-
2
–
(
4-isopropyl-benzyl)-(benzyl)-dithiocarbamate], [L2 ¼ (C H NS ),
0
12
–
N-(benzyl)-(ethyl)-dithiocarbamate], [L3 ¼ (C19
H22ONS ), N-(4-
2
KEYWORDS
isopropyl-benzyl)-(4-methoxy-benzyl)-dithiocarbamate], and [L4 ¼
–
Dithiocarbamate; transition
metal complexes; bacterial
biofilm; minimum inhibitory
concentration; antitumor;
drug resistance
(
C H NS ), N-(benzyl)-(4-methyl-benzyl)-dithiocarbamate] have
16 16 2
been contemplated, synthesized, and characterized by elemental
1
13
analysis and IR, H, C NMR and UV–visible absorption spectra. All
Zn(II) complexes have similar geometry and coordination number.
Complex A2 (with ligand L2) crystallizes in triclinic system with
space group P-1 having distorted square pyramidal geometry which
was stabilized by weak C–Hꢀꢀꢀp and C–HꢀꢀꢀS intramolecular interac-
tions. The antibacterial, antibiofilm, and antitumor activities of the
complexes have been screened and A2 and A3 showed their prom-
inence. Interestingly, both A2 and A3 showed more killing potential
against multi-drug resistant gram-positive isolates with MIC indices
ꢁ
1
ꢁ1
of 16 lg mL and 16 lg mL , respectively, against both MRSA and
MSSA, while the antitumor agent A3 showed its prominence with
GI50 and LC50 41.15 and 133.73 mg mL , respectively.
ꢁ
1
CONTACT Lal Bahadur Prasad
lbprasad@bhu.ac.in
Department of Chemistry, Institute of Science, Banaras
Hindu University, Varanasi 221005, India
Supplemental data for this article can be accessed here.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
V. K. MAURYA ET AL.
1. Introduction
The upsurge of “bad bugs” in recent years has jolted our belief and effectively stated
that our win over drug-resistant bacteria was mere an illusion with resistance
spreading faster than the development of newer antimicrobials [1–4]. These bacterial
infections are more prevalent among immune-compromised individuals especially in
cases of organ transplantation or in the treatment of critical diseases, such as cancer
and AIDS [5–7]. Recent antibacterial therapy includes two main approaches. The
first approach targets the bacterial membrane and alters the permeability of the
membrane, causing loss of macromolecules and ions essential to the bacterial survival.
Another approach is rather old but fascinating which targets cell wall biosynthesis.
Faulty cell wall architecture ultimately influences the membrane permeability and
several other interactive cascades [8].
The study of Zn(II) complexes containing N- and S-donors has increased in recent
past with the aim to synthesize antibacterial and antitumor drugs that may show
higher activity, improved selectivity, and bioavailability, along with reduced toxicity
with respect to the current therapeutic alternatives. Reports suggest that Zn(II) com-
plexes have wide applications. Tanaka et al. reported the chemical and biological
properties of zinc complex of L-carnosine and its advantages as an anti-ulcer drug of
membrane protective type. They reported the slow dissociation rate of Zn from the
complex in gastric juice that lingered the stability of the complex for prolonged heal-
ing effects [9]. Reports suggest that about 10% of human proteins potentially bind
zinc, in addition to hundreds which are involved in zinc homeostasis [10].
Indispensability of zinc can be understood by the fact that it is the second most abun-
dant transition metal in organisms after iron and it is the only metal which appears in
all enzyme classes [11]. Zinc orchestrates a wide array of functions in living systems
such as: 1) maintenance of growth and metabolism of cells; 2) wound healing; 3)

JOURNAL OF COORDINATION CHEMISTRY
3
endocrine functionality maintenance like activation and secretion of hormones; 4)
neurotransmission maintenance; 5) memory processing; 6) maintenance of carbohy-
drate metabolism; 7) maintenance of lipid metabolism; 8) cell membrane stability; 9)
skin tone maintenance; 10) immuno-modulation; 11) maintenance of retina and eye
lens; and 12) reduction of hazardous effect of xenobiotics. Literature documents that
zinc ions are potential antimicrobial agents even at low concentrations. For instance,
gastroenteritis is strongly attenuated by ingestion of zinc [12]. Besides direct anti-
microbial effect, zinc has immunomodulatory functions as well. Cell-mediated immune
dysfunctions are noted in the case of Zn-deficiency which worsen the outcome in the
response towards bacterial infection and sepsis [10, 12]. Having small ionic radius the
zinc ion acts as a Lewis acid and, therefore, its stability with some cellular ligands sat-
isfactorily high making the zinc reactive in such complexation. This simply means high
probability of ligand-exchange reactions in zinc(II) complexes in living systems. Zn(II) is
stable even in highly oxidizing and reducing environments because of the fully occu-
pied 3d-orbital. In addition, it has high affinity for the main coordination atoms, such
as sulfur, nitrogen, and oxygen. These salient features bestow it with high versatility in
structure, coordination mode, and coordination number of the complexes produced
[12]. This establishes the utility of zinc in designing of less cytotoxic but effective com-
plexes. The dithiocarbamate ligand chemistry is expeditiously progressing owing to
the ease of functionalization on the N atom by different substituent, which facilitates
the making of more complex structures via secondary interactions and the tuning of
their physical properties [13–18]. Dithiocarbamates (DTCs) find application in diverse
therapeutic paradigm. Dithiocarbamate ligands have previously been used for the
treatment of bacterial and fungal infections and in the treatment of AIDS [19–24]. In
addition, dithiocarbamates are known to exert pro-oxidant and antioxidant effects [10,
12]. Recent report from Fregona et al. showed the activity of Cu(II)/Zn(II) dithiocarba-
mates to induce apoptosis in wide array of cancer cells [25]. Therefore, it became pru-
dent to take advantages of both the zinc(II) ion and DTCs, to explore the role of their
complexes in the growth and survival of bacteria, its biofilm, and even the cancer cells
with new cellular targets and mode of action if any, in comparison to the traditional
metal-based complexes, which may be potential drug candidates.
We, in the present work, successfully attached the functionalized DTC ligands to
Zn-cation in order to evaluate their antibacterial, antibiofilm, and antitumor potential.
The current study reports the synthesis and characterization of four new homoleptic
–
Zn-complexes with formula [Zn(L) ] [L1 ¼ (C H NS ), N-(4-isopropyl-benzyl)-(ben-
2
18 20
2
–
zyl)-dithiocarbamate], [L2 ¼ (C H NS ), N-(benzyl)-(ethyl)-dithiocarbamate], [L3 ¼
10
12
2
–
(
(
C H ONS ), N-(4-isopropyl-benzyl)-(4-methoxy-benzyl)-dithiocarbamate], and [L4 ¼
1
9
22
2
–
C H NS ), N-(benzyl)-(4-methyl-benzyl)-dithiocarbamate]. The complexes were iso-
1
6
16
2
lated in high yield by the metathetical reaction of an aqueous methanolic solution of
Zn(OOCCH ) ꢀ2H O and potassium salts of ligands L1, L2, L3, and L4 in a 1:2 molar
3
2
2
ratio. However, N-(benzyl)-(ethyl)-dithiocarbamate complex (A2) exhibited potential
antibacterial and antibiofilm properties, while potential antitumor activity was exhib-
ited by N-(4-isopropyl-benzyl)-(4-methoxy-benzyl)-dithiocarbamate complex (A3).
Although various analyses were done for all the synthesized complexes, but owing
to the better activities on bacterial systems, we primarily executed the finer structural

4
V. K. MAURYA ET AL.
details of [N-(benzyl)-(ethyl)-dithiocarbamate] complex (A2) utilizing its single-crystal
X-ray structure. Elemental analysis, FT-IR, UV–vis, and NMR spectroscopy of all Zn(II)
complexes were performed.
2
. Experimental
.1. Materials and instrumentation
All starting materials were procured from Sigma-Aldrich, Merck or SRL laboratories,
2
1
13
India and used as received. H and C NMR spectra were recorded in CDCl solution
3
on a JEOL 300 FT NMR spectrometer. Chemical shifts are quoted in parts per million
(
d, ppm) with (CH ) Si (TMS) as internal standard. The melting point was determined
3 4
in open capillaries on a Gallenkamp’s apparatus and is uncorrected. The infrared spec-
tra were recorded with samples pressed as KBr pellets on a Varian Excalibur 3100-FT
ꢁ
1
IR spectrophotometer in the range of 4000–400 cm . Carbon, hydrogen and nitrogen
analysis were performed on an Exeter CE-440 element analyzer CHN-analyzer using tin
sample-tubes. The UV–vis absorption spectra (200–800 nm) were recorded on
Shimadzu UV-1800 spectrophotometers. Intensity data for monocrystalline sample A2
were collected on an Oxford Diffraction X-caliber CCD diffractometer (genteel-focus,
sealed tube equipped radiation source) with a graphite monochromator. Data integra-
tion, scaling of the reflections for the said complex A2, and the analytical and semi-
empirical absorption corrections were performed with the CRYSALIS suite. Final unit
cell parameters were based on the fitting of all reflection positions. Program XPREP
was used in the space group identification. The structure of compound A2 was solved
by direct methods using the SHELXS-97 program.
2.2. Synthesis of the dithiocarbamate ligands (KL1–KL4)
In preparation of dithiocarbamate ligands, the secondary amines were synthesized
by the reaction of 4-isopropyl-benzaldehyde (0.148 g, 1 mmol) and benzylamine
(0.108 g, 1 mmol) for KL1; benzaldehyde (0.102 g, 1 mmol) and ethylamine (0.056 g,
1
mmol) for KL2; 4-isopropyl-benzaldehyde (0.148 g, 1 mmol) and 4-methoxybenzyl-
amine (0.137 g, 1 mmol) for KL3; 4-methyl-benzeldehyde (0.118 g, 1 mmol) and eth-
ylamine (0.056 g, 1 mmol) for KL4 dissolved in 15 mL ethanol with 8 h refluxing at
ꢂ
7
0–80 C [26]. The obtained reaction mixture(s) contain Schiff base, which was then
placed in ice-cold conditions. To reduce the obtained Schiff base solution, metha-
nolic solution of NaBH was added with constant stirring and the obtained product
4
was extracted with dichloromethane (45 mL), with subsequent washing with dis-
tilled water five times (25 mL). The oily yellow mixture was extracted on a rotary
evaporator. The potassium salts of KL1–KL4 dithiocarbamate ligands were made by
the reaction of KOH (0.056 g, 1 mmol) and carbon disulfide (0.076 g, 1 mmol) with
relevant secondary amine (25 mL THF solvent) prepared as given in Scheme 1
ꢂ
under ice-cold conditions and maintained temperature below 8 C. The final prod-
uct was further stirred for 5 h at room temperature and the solvent was removed
over a rotary evaporator. The obtained product was washed thrice with ether to
yield a pale yellow solid product.

JOURNAL OF COORDINATION CHEMISTRY
5
EtOH, Reflux
-8 h
R1
CHO + NH CH R
2
2
2
R
1
CH
N
CH
2
R
2
7
MeOH, NaBH4
Ice-cold cond.
R
1
CH
2
CH
2
R
2
KOH, CS2
Ice-cold cond.
R1
CH2
CH2 R2
N
NH
KS
S
Zn(OOCCH
3
)
2
2
.2H O
(KL )
MeOH, r.t.
H C
R
2
S
S
2
R1
CH
2
N
N
Zn
H C
R1
R
2
CH2
S
2
S
(
ML2 )
Where R
1
= C
4
H
4
CH(CH
3
)
2
, (L1), (A1); C
2
H
5
, (L2), (A2); C
4
H
4
3 2 4 4 3
CH(CH ) , (L3), (A3);C H CH , (L4),
(A4)
R = C H (L1), (A1); C H , (L2), (A2); C H OCH , (L3), (A3); C H , (L4), (A4).
2
7
8,
6
5
6
4
3
6
5
Scheme 1. Generalized synthesis of dithiocarbamate ligands and metal complexes.
2
.2.1. Characterization data of ligands
1
KL1, Yield: 0.27 g, 74%. H NMR (500.15 MHz, CDCl ), d (ppm): 7.08–6.99 (m, 5H,
3
–
–
C H ), 7.01–6.89 (m, 4H, –C H CH(CH ) ), 4.89 [s, 2H, –CH C H CH(CH ) ], 4.42 (s, 2H
6 5 6 4 3 2 2 6 4 3 2
13
1
CH C H ), 2.80–2.73 (m, 1H, –CH(CH3)2), 1.42–1.40 (2 –CH ). C f Hg NMR
2
6
5
3
(500.15 MHz, CDCl3),
[–C H CH(CH ) ], 140.41,
(–CH C H CH(CH ) –CH C H ), 33.67, 24.58 (2 –CH ).
d
(ppm): 217.11 (CS2), 147.58, 141.86,135.44, 128.92
128.51,
128.31,
127.28
(–C H ),
52.02,
50.84
6
4
3 2
6 5
2
6
4
3 2,
2
6
5
3
1
KL2, Yield: 0.18 g, 72%. H NMR (500.15 MHz, CDCl ), d (ppm): 7.34–7.29 (m, 5H,
3
13
1
–
C H ), 5.27 (s, 2H, –CH C H ), 3.91 (q, –CH CH ), 1.10 (t, 3H –CH ). C f Hg NMR
6
5
2
6
5
2
3
3
(500.15 MHz, CDCl ), d (ppm): 209.36 (CS ), 137.07, 128.82, 128.29, 127.29 (–C H ),
3 2 6 5
5
6.49, (–CH C H ), 49.15 (–CH CH ), 11.31 (–CH ).
KL3, Yield: 0.28 g, 73%. H NMR (500.15 MHz, CDCl ), d (ppm): 7.25–6.83 (m, 4H,
2 6 5 2 3 3
1
3
–
C H OCH ), 7.17–6.79 [m, 4H, –C H CH(CH ) ], 5.32 [s, 2H, –CH C H CH(CH ) ], 5.29 (s,
6
4
3
6
4
3 2
2
6
4
3 2
2
H –CH C H OCH ), 3.66 (s, 3H, –OCH ) 2.81 (m, 1H, –CH(CH ) ), 1.25–1.15 (2 –CH ).
2 6 4 3 3 3 2 3
1
3
1
C f Hg NMR (500.15 MHz, CDCl ), d (ppm): 213.38 (CS ), 158.70, 129.39, 128.66,
3
2
1
5
14.23 (–C H OCH ), 147.77, 134.76, 127.38, 126.83 [–C H CH(CH ) ], 55.50,
6 4 3 6 4 3 2
5.24, [–CH C H CH(CH ) –CH C H OCH ], 55.35 (–OCH ), 33.75 [–CH(CH ) ], 24.08
2
6
4
3 2,
2
6
4
3
3
3 2
(
2 –CH3).
1
KL4, Yield: 0.27 g, 84%. H NMR (500.15 MHz, CDCl ), d (ppm): 7.14–6.92 (m, 5H,
3
–
2
1
C H ), 6.85–6.83 (m, 4H, –C H CH ), 5.04 (s, 2H, –CH C H ), 5.02 (s, 2H, –CH C H CH ),
6
5
6
5
3
2
6
5
2
6
4
3
13
1
.05 (s, 3H, –CH3). C f Hg NMR (500.15 MHz, CDCl ), d (ppm): 212.37 (CS ), 133.39,
3
2
27.29, 127.00, 126.83 (–C H ), 137.25, 136.53, 129.33, 128.78 (–C H CH ), 55.91
6
5
6
5
3
(
–CH C H ), 55.95 (–CH C H CH ), 20.29 (–CH ).
2 6 5 2 6 4 3 3

6
V. K. MAURYA ET AL.
2
.3. Synthesis of the complexes [Zn(L) ] [L ¼ L1 (A1), L2 (A2), L3 (A3), L4 (A4)]
2
Homoleptic complexes A1–A4 were synthesized according to the method described earlier
with minor modifications [27, 28]. The stirred methanolic solution of the ligands (25 mL) KL1
(0.37 g, 1 mmol), KL2 (0.25 g, 1 mmol), KL3 (0.38 g, 1 mmol), and KL4 (0.32 g, 1 mmol) was
added gradually to a 15-mL aqueous methanolic solution of Zn(OOCCH ) ꢀ2H O (0.09 g,
3
2
2
0.5 mmol). The reaction mixture was stirred for 5 h in each case, resulting in the formation of
a white precipitate that was filtered off and washed with methanol followed subsequent
washing with diethyl ether. The compounds were recrystallized in dichloromethane.
2
.3.1. Characterization data of complexes
1
A1, Yield: 0.30 g, 83%. H NMR (500.15 MHz, CDCl ), d (ppm): 7.45–7.36 (m, 5H, –C H ),
3
6 5
7
.21–7.04 [m, 4H, –C H CH(CH ) ], 4.91 [s, 1H, –CH C H CH(CH ) ], 4.88 (s, 2H
6
4
3 2
2
6
4
3 2
13
1
–
CH C H ), 2.92–2.86 (m, 1H, –CH(CH ) ), 1.23–1.21 (2 –CH ).
C f Hg NMR
2
6
5
3 2
3
(500.15 MHz, CDCl3),
[–C H CH(CH ) ], 139.91,
(–CH C H CH(CH ) –CH C H ), 33.54, 24.42 (2 –CH ).
d
(ppm): 207.61 (CS2), 147.11, 134.94, 129.12, 128.77
128.41,
128.31,
127.07
(–C H ),
53.97,
52.75
6
4
3 2
6 5
2
6
4
3 2,
2
6
5
3
1
A2, Yield: 0.27 g, 84%. H NMR (500.15 MHz, CDCl ), d (ppm): 7.37–7.31 (m, 5H,
3
13
1
–
C H ), 5.15 (s, 2H, –CH C H ), 3.77 (q, –CH CH ), 1.14 (t, 3H –CH ). C f Hg NMR
6
5
2
6
5
2
3
3
(500.15 MHz, CDCl ), d (ppm): 202.00 (CS ), 137.34, 128.98, 128.04, 127.88 (–C H ),
3 2 6 5
5
7.97, (–CH C H ), 49.71 (–CH CH ), 12.26 (–CH ).
A3, Yield: 0.31 g, 82%. H NMR (500.15 MHz, CDCl ), d (ppm): 7.22–6.85 (m, 4H,
2 6 5 2 3 3
1
3
–
C H OCH ), 7.20–6.83 [m, 4H, –C H CH(CH ) ], 4.93 [s, 2H, –CH C H CH(CH ) ], 4.92 (s, 2H
6 4 3 6 4 3 2 2 6 4 3 2
13
1
–CH C H OCH ), 3.75(s, 3H, –OCH ) 2.88 [m, 1H, –CH(CH ) ], 1.20–1.17 (2 –CH ). C f Hg
2
6
4
3
3
3 2
3
NMR (500.15 MHz, CDCl ), d (ppm): 205.40 (CS ), 159.62, 129.70, 128.20, 114.35 (–C H OCH ),
3
2
6
4
3
1
49.04, 131.99, 127.04, 126.80 [–C H CH(CH ) ], 55.40, 55.28, [–CH C H CH(CH )
6 4 3 2 2 6 4 3 2,
–
CH C H OCH ], 58.56 (–OCH ), 33.94 [–CH(CH ) ], 24.03, 18.51 (2 –CH ).
2 6 4 3 3 3 2 3
1
A4, Yield: 0.26 g, 82%. H NMR (500.15 MHz, CDCl ), d (ppm): 7.39–7.34 (m, 5H,
3
–
2
1
C H ), 7.24–7.19 (m, 4H, –C H CH ), 5.04 (s, 2H, –CH C H ), 5.01 (s, 2H, –CH C H CH ),
6
5
6
5
3
2
6
5
2
6
4
3
13
1
.37 (s, 3H, –CH3). C f Hg NMR (500.15 MHz, CDCl ), d (ppm): 205.81 (CS ), 131.45,
3
2
28.17, 128.08, 128.05 (–C H ), 138.05, 134.57, 129.60, 128.92 (–C H CH ), 55.57
6
5
6
5
3
(
–CH C H ), 55.52 (–CH C H CH ), 21.17 (–CH ).
2 6 5 2 6 4 3 3
2.4. X-ray crystallography
The X-ray diffraction data were collected by mounting single crystals of C H N S Zn
20 24 2 4
3
(
A2) with dimensions 0.27 ꢃ 0.23 ꢃ 0.23 mm on glass fibers. Single-crystal X-ray data
for complex A2 were collected on an Oxford Diffraction X-caliber CCD diffractometer
genteel-focus, sealed tube equipped radiation source) with a graphite monochrom-
(
ator and Mo-Ka radiation wavelength 0.71073 Å. The intensity data and unit cell
dimensions were measured at 150 K. Using the CrysAlis program data reductions were
carried out [29]. By using SHELXS-97 direct methods structure was solved and refined
2
on F by a full matrix least squares technique [30]. All non-hydrogen atoms were
refined anisotropically with thermal parameters and hydrogen atoms were geometric-
ally fixed corresponding to 1.2 times the Ueq data of the appropriate carrier atom.

JOURNAL OF COORDINATION CHEMISTRY
7
2.5. Biological investigations
2.5.1. Bacterial growth condition
Followed by the synthesis and characterization of the complexes, we investigated their
effect(s) over various bacterial isolates both clinical and control isolates. We used
multi-drug resistant clinical isolates of Escherichia coli (Lab code: 2042/2018) and
Staphylococcus aureus (Lab code: 764/2018), representatives of gram-negative and
gram-positive classes. In addition, we also utilized control bacterial strains namely
Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922). We defined
multi-drug resistance as resistance against at least five different classes of drugs.
Screening was performed utilizing modified Kirby-Bauer method in accordance with
the Clinical and Laboratory Standards Institute guidelines 2018. Total 11 antibiotics
were used namely Ampicillin (10 mg), Amikacin (30 mg), Amoxicillin/clavulanate (20/
10 mg), Ciprofloxacin (5 mg), Co-trimoxazole (23.75/1.25 mg), Ertapenem (10 mg),
Gentamicin (120 mg), Imipenem (10 mg), Levofloxacin (5 mg), Meropenem (10 mg),
Piperacillin, and tazobactum (100/10 mg).
2.5.2. Preliminary screening of complexes using disc diffusion method
Initially the screenings of the synthesized complexes were done for the evaluation of
their antimicrobial activity using disc diffusion method against representative gram
positive (S. aureus) and gram-negative (E. coli) strains. The blank disc was loaded with
five microliters of the drug in the concentrations 10 (A1), 20 (A2), 30 (A3), 40 (A4) mg
ꢁ
1
disc (Supplementary Figure S26).
2.5.3. Minimum inhibitory concentration (MIC) determination
After the preliminary screening, minimum inhibitory concentrations of the complexes
were determined against the above listed bacterial strains. MIC of the complexes was
determined by the broth micro dilution method as described earlier with minor modi-
fications [6]. Briefly, overnight solid cultures of staphylococcal strains (Lab code: 764/
2
018 and ATCC 25923) were diluted 1:100 in brain heart infusion (BHI) broth (HiMedia
laboratories, India) supplemented with 4% glucose at pH 7, whereas E. coli (Lab code:
042/2018 and ATCC 25922) were grown in 5 mL Luria Bertani broth (Miller, HiMedia
laboratories, India) aerobically for 18 h. Both the bacterial cultures (500 mL) were then
2
ꢁ
1
diluted to 1.5 mL fresh broth. The stock solutions of the complexes (2.5 mg mL
)
were prepared in DMSO. The stock was subsequently diluted in two-fold dilutions in
respective broths (HiMedia laboratories, India) in microtiter wells. Each well of 96-well
6
microtiter plate was then inoculated with 160 mL of standardized cell suspension (10
ꢁ
1
ꢂ
CFU mL ), and incubated at 37 C for the next 18 h along with the 40 mL of complex
suspension. The MIC was defined as the lowest concentration of the compounds at
which no obvious growth was manifested. Positive controls did not contain any test
compound while the sterile broth was used as negative control, and all the experi-
ments were performed in triplicate.
2.5.4. Antibiofilm activity determination using tissue culture plate assay (TCP)
We performed the antibiofilm assay in 96-well tissue culture plates as described previ-
ously with minor modifications [1, 5]. Briefly, Staphylococcus epidermidis (ATCC 35984),

8
V. K. MAURYA ET AL.
Staphylococcus aureus (ATCC 25923), and Escherichia coli (ATCC 25922) were grown on
ꢂ
BHI agar overnight at 37 C. A few typical colonies from the BHI agar plates of these
ꢂ
isolates were inoculated in BHI broth and incubated for 12 h at 37 C to generate fresh
early exponential-phase cultures and later on diluted 1:100 in brain heart infusion
(BHI) broth (HiMedia laboratories, India) containing glucose (4%) at pH 7.
A volume of 170 mL of each diluted bacterial suspension was dispensed into flat-
bottom polystyrene 96-well tissue culture plates (Nest, Tarsons, India) and 30 ml of
ꢁ
1
complex suspensions (Stock 2.5 mg mL ) were added to each well. Wells without
ꢂ
compounds were set up as controls. Plates were incubated at 37 C to evaluate biofilm
inhibitory effects over 72 h old preformed biofilm. After respective incubations, biofilm
was quantitated by crystal violet (CV) assay (Merck, Germany) as described earlier. The
assays were performed in triplicate, and the results were expressed as mean OD570
±
the standard deviation of the mean (SD). The MBIC was defined as the lowest concen-
tration of compounds at which >75% reduction in biofilm absorbance was observed
(Supplementary Figure S25).
2.5.5. Bacterial growth rate analysis
The effect of complex A2 on growth rates of bacterial isolates were analyzed spectro-
photometrically using Synergy H1 Hybrid Multi-Mode Reader described earlier [30].
Briefly, the bacterial cells at early exponential phase were inoculated into TCP with OD
at kmax 600 nm adjusted to 0.01. The optical density was monitored periodically every
2
1
5 min for 4.5 h. The slope of the linear part of the growth curve (R ꢄ0.98) gave the
growth rate which was determined for at least 4 data points of the semi-logarithmic
plot of absorbance (ln [OD600]) versus incubation time (in hours).
2.5.6. Fluorescent microscopy
For finer details of the effects of compounds (here shown the activity of complex A2)
over clinical isolates namely S. aureus biofilm, fluorescent microscopy was done. The
viability of bacteria in the biofilm after treatment with respective compounds was
determined using Sytox green. Sytox green penetrates bacteria with damaged mem-
branes. Briefly, the overnight grown bacterial isolates were diluted 1:100 in fresh BHI
broth with absorbance at kmax 600 nm ꢅ0.2. Hundred-microliters of diluted bacteria
were dispensed into chambered slide containing 400 mL of BHI broth (Nunc, Denmark).
The biofilm was grown statically for 72 h followed by treatment with the sub inhibitory
concentration of compound A2. Prior to staining, residual broth was removed by gen-
tle tapping and washed thrice by phosphate buffer (pH 7.5) followed by fixation using
4% (v/v) paraformaldehyde for 30 min. The Sytox green (stock 5 mM) was reconstituted
with DMSO to get the working aliquot of strength 1 mM. For use, 50 mL aliquots were
prepared and used. Ten-microliter sample of Sytox green was applied directly to the
top of the growth. One can note the abundance of green signals coming from the
bacterial lawn, which is indicative of bacterial death.
We used the Nikon eclipse inverted fluorescent microscope to detect the green
fluorescence from the test isolates. Sytox green was excited with the HeNe2 530 nm
laser and emission fluorescence was collected with 620 nm filter. Images were
obtained via a Plan-Neofluar 20X objective.

JOURNAL OF COORDINATION CHEMISTRY
9
Figure 1. Structure of ligands used in this work.
2.6. Elucidation of antitumor activity
2.6.1. Cell culture
We cultured HCT 116 cells in Dulbecco’s modified Eagle medium supplemented with
ꢁ
1
ꢁ1
1
0% fetal bovine serum added with 100 U mL penicillin and 100 mg mL streptomycin
ꢂ
in a 5% CO atmosphere at 37 C in a CO incubator. The cells were exposed to the metal
complexes for 24 h. The 2 mg mL stock solution of said complex suspension was pre-
pared in DMSO and stored as small aliquots at 4 C and diluted two folds in a different
dose ranging from 2 to 1024 mg mL in Dulbecco’s modified Eagle’s medium. The com-
plex solution was added to the growth medium such that the final solvent concentration
remained less than 1% which had no obvious deleterious effects on cell viability.
2
2
ꢁ
1
ꢂ
ꢁ
1
2.6.2. Cell proliferation assay
Cell proliferation was determined using the sulforhodamine B (SRB) assay. Growing
3
HCT116 cells were seeded into 96-well plates at a density of 5 ꢃ 10 cells per well and
allowed to adhere overnight. Twenty-four hours later, cells were incubated for 48 h
ꢁ
1
with a range of concentrations of complexes A2 and A3 (2–256 mg mL ). Cells were
fixed with 50% (w/v) TCA and stained with 0.4% (w/v) sulforhodamine B (SRB) for
30 min before washing with 1% (v/v) acetic acid. SRB was solubilized with 10 mM Tris
pH ꢅ10.5, the absorbance read at 510 nm, and cell growth expressed as the percent-
age (%) of the growth of untreated cells. The concentration of QDs that resulted in
5
0% growth inhibition (GI50) was calculated by interpolation from the growth inhib-
ition versus concentration curves using GraphPad Prism version 5.01 for Windows
GraphPad Software Inc., San Diego, CA).
(
3. Results and discussion
Dithiocarbamate ligands (Figure 1) and their complexes were prepared according to
the synthetic procedure as shown in Scheme 1. All the complexes were isolated as air

1
0
V. K. MAURYA ET AL.
ꢁ1
Table 1. IR spectral data (cm ) of the ligand and its complexes.
Compound
m(C–N)/(C ¼ N)
m(C–S)
m(¼C–H)
m(-C–H)
m(Zn–S)
C
18
C
10
C
19
C
16
C
36
C
20
C
38
C
32
H
H
H
H
H
H
H
H
20NS
12NS
23ONS
2
2
K (KL1)
K (KL2)
1330
1354
1346
1354
1417
1429
1416
1453
1000
1028
1017
1028
963
978
996
997
2960
3026
2999
3025
2959
3026
2999
3025
2925
2920
2929
2916
2922
2918
2918
2918
–
–
–
2
K (KL3)
16NS K (KL4)
2
–
40
24
46
32
N
N
O
2
S
S
4
Zn (A1)
Zn (A2)
427
445
461
422
2
4
2
N
2
S
4
Zn (A3)
N
2
S
4
Zn (A4)
stable crystalline solids, in yields varying from 69% to 98%. However, the complexes
ꢂ
were found to decompose from 185 to 197 C. The purity of complexes A1–A4 had
been corroborated in terms of the satisfactory melting points and C, H, and N analysis
(Supplementary Table S1).
1
3
.1. H NMR spectra
1
H NMR spectra of the ligands and complexes were recorded in CDCl . The chemical
3
1
shifts of the different types of the protons observed in the H NMR spectra of the
ligands were compared with their Zn(II) complexes. H NMR spectra of the ligands
1
show proton signals at between d 7.34–6.79, 3.66, and 5.32–4.42 ppm due to benzene
protons, –OCH and methylene protons of aryl group, respectively [31, 32]. The methy-
3
lene protons (adjacent to nitrogen atom) of alkyl chain are observed at d 3.91 ppm
and alkyl chain of methyl proton are observed around d 2.05–1.05 ppm [33]. It is
noticed that signals due to benzene, –OCH , aromatic and aliphatic methylene protons
3
and methyl protons do not show significant shift upon complexation.
1
3
3
.2. C NMR spectroscopy
13
The expected regions for C NMR spectra in normal oxidation state of transition metal
13
dithiocarbamate appeared above 190 ppm for d (CS ). In C NMR spectra, signals
2
between d 217.11 and 209.36 ppm appeared due to the NCS carbon of the free ligands
2
while after complexation, d values were observed between 207.61 and 202.00 ppm for all
13
complexes [34]. In the C NMR spectra, a perceivable upfield shift of d 10–7 ppm for the
NCS carbon in the complexes was witnessed as compared to the potassium salts of the
2
13
ligands that indicate M–S bonding. C NMR signal of methoxy carbon was observed at d
5.35 ppm, methylene carbons (adjacent to nitrogen atom) of aryl group were observed
5
around d 56.49–50.84 ppm, methylene carbon (adjacent to nitrogen atom) of alkyl chain
was observed at d 49.15 ppm and alkyl chain of methyl carbon (adjacent to methylene
carbon) was observed at d 11.31 ppm. It was observed that signals due to benzene,
–
OCH , aromatic and aliphatic methylene carbon, including methyl carbon, exhibited
3
slight up and downfield shifts upon complexation.
3.3. Infrared spectra
The important infrared (IR) bands of ligands and their zinc complexes are listed in
Table 1. Infrared spectral bands of the complexes indicate that they are analogous in

JOURNAL OF COORDINATION CHEMISTRY
11
ꢁ
5
Figure 2. UV–vis absorption spectra of complexes A1–A4 in CH Cl solution (10 M).
2
2
structures. Strong m(C–N) bands of zinc dithiocarbamate complexes were found in the
ꢁ
1
range 1453–1416 cm . This high stretching vibration may be ascribed to the increase
in the carbon nitrogen double bond character due to delocalization of electrons
towards the metal atom in each zinc dithiocarbamate complex [35]. A shift of m(C–N)
values to higher wavenumbers as compared to the dithiocarbamate ligands is due to
the mesomeric effect of the electrons from dithiocarbamate moiety towards the metal
center. In the dithio-ligands, two types of m(C–S) bands namely, the m(CS )
and
2
asym
ꢁ
1
m(CS2)sym which appear at 1028 and 1000 cm , respectively, are replaced by a strong
ꢁ
1
single peak observed approximately at 996 cm
in all complexes signifying that
dithiocarbamate coordination moiety is symmetrical to the metal ion. The m(M–S)
ꢁ
1
peaks are observed in between 461–422 cm in all complexes [36, 37]. The m(¼C–H)
ꢁ
1
and m(–C–H) stretching vibrations are found in the region 3026–2960 cm
and
ꢁ
1
2929–2916 cm , respectively, in ligands as well as their complexes.
3.4. Electronic excitation and emission spectra
For deeper insights to the coordination properties of the metal ions, the electronic
spectra of all the synthesized complexes have been recorded in the range 200–800 nm
in CH Cl as shown in Figure 2. The electronic absorption spectra of complexes A1–A4
2
2
show very similar absorption features near 229–234 nm which depicts characteristic
ꢆ
metal perturbed p–p and intra-ligand charge-transfer (ILCT) transitions [38]. The band
at 260–282 nm is assigned to M!L charge transfer (MLCT) transition [22]. Upon excita-
tion with 230 nm for A1, 233 nm for A2, 232 nm for A3, and 229 nm for A4 we
observed the emission peaks at 306 nm, 312 nm, 311 nm, and 306 nm, respectively, for
complexes A1, A2, A3, and A4. The Stokes shift of 76 nm, 79 nm, 79 nm and 77 nm
was observed in the complexes (Figure 3). This emission band originates due to the

1
2
V. K. MAURYA ET AL.
ꢁ
5
Figure 3. Photoluminescence spectra of complexes A1–A4 in CH Cl solution (10 M).
2
2
metal perturbed metal-to-ligand charge-transfer (MLCT) states of the dithiocarbamate
metal complexes.
3
.5. Crystal structure of [C H N Zn S ] (A2)
40 48 4 2 8
A single-crystal of complex A2 was collected from a dichloromethane solution within
–4 weeks for the determination of its molecular structure. Complex A2 crystallizes in
3
triclinic system with space groups P-1 and the N-benzyl-ethyl-dithiocarbamate ligand
is asymmetrically bonded to zinc metal centers. Lattice parameters and refinement for
complex A2 are given in Table 2. The crystallographic details and selected bond
lengths and angles are listed in Table 3. Complex A2 is a dinuclear molecule where
2
four dithiocarbamate ligands are uniquely bonded to two zinc atoms in a l ,ΠS,S-
2
chelating-bridging, as well as S,S-chelating fashion as shown in Figure 4. The Zn(1)
atom occupies distorted square pyramidal geometry. The Zn(1) atoms are asymmetric-
ally S,S-chelated by two dithiocarbamate ligands with distances to the S(11) and S(13)
atoms of 2.846(13) and 2.334(12) Å and to the S(41) and S(43) atoms of 2.339(12) and
2.453(14) Å, respectively. The larger bond length of S(11) atom indicates a bridge to
the Zn(1) atom and the geometry is distorted square pyramid as evident from the
intermediate s value (0.29) compared to 0.0 for perfect square pyramid and 1.0 for a
trigonal bipyramid [39]. The S(11) atom also forms a bridge to Zn(1) atom with dis-
tance of 2.846(13) Å which occupies an axial position. Therefore, the dithiocarbamate
0
2
ligands are bonded to the Zn(1) and Zn(1) atoms in a l ,ΠS,S-chelating-bridging
2
manner, forming a dinuclear structure.
The Zn–S bond lengths range between 2.334 and 2.373 Å. The bite angle S11-Zn(1)-
ꢂ
ꢂ
S13 is 69.0 while for S41-Zn(1)-S43 is 75.6 . The N14-C12 and N44-C42 bond lengths
are 1.320 Å and 1.327 Å, respectively; these values are intermediary to that of the C–N

JOURNAL OF COORDINATION CHEMISTRY
13
Table 2. Structure refinement and crystallographic data for complex A2.
Formula
Formula weight
Crystal system
Space group
a (Å)
40 48 4 2 8
C H N Zn S
972.04
Triclinic
P-1
8.6272(7)
b (Å)
c (Å)
11.3893(15)
11.7159(16)
86.487(9)
1087.5(2)
1
b (Å)
3
V (Å )
Z
ꢁ3
q
calc (g cm
)
1.484
T(K)
l(Mo Ka) (mm
150(2)
1.521
ꢁ
1
)
F(000)
Crystal size (mm )
Index ranges
504
3
0.27 ꢃ 0.23 ꢃ 0.23
ꢁ12 ꢇ h ꢇ 8, ꢁ15 ꢇ k ꢇ 15, ꢁ15 ꢇ l ꢇ 16
Reflections collected
Independent reflns.
No. of parameters
Reflections with I > 2
7670
6066
246
3893
0.0698
r
(I)
a
a
b
b
R
R
1
, wR
, wR
2
[I >2s(I)]
[all data]
1
2
0.1144, 0.1749
0.961
2
Goodness of fit on F
Largest difference in peak and hole (eÅ
ꢁ
3
)
1.194, ꢁ0.979
P
P
P
P
2
2
2 2 1/2
2
2
2
2
R
2
1
¼
jjFojꢁjFcjj/ jFoj. R
2
¼ f[ w (Fo – Fc )/ w (Fo ) ]g , w ¼ 1/[r (Fo ) þ (xP) ], where P¼(Fo
þ
2
Fc )/3.
ꢂ
Table 3. Selected molecular dimensions (Å and ) in [C H N Zn S ], A2.
4
0
48
4
2 8
Bond length
Bond angle
Zn(1)–S(13)
Zn(1)–S(11)
Zn(1) –S(11)
2.334(12)
2.373(13)
2.846(13)
2.339(12)
2.453(14)
1.733(4)
1.720(16)
1.749(4)
1.722(4)
1.320(5)
1.327(5)
S(11)–Zn(1)–S(13)
S(11)–Zn(1)–S(11)
S(13)–Zn(1)–S(41)
S(41)–Zn(1)–S(11)
S(13)–Zn(1)–S(43)
S(41)–Zn(1)–S(43)
S(11)–Zn(1)–S(43)
104.8(4)
87.9(4)
137.2(5)
114.8(4)
104.6(3)
75.6(4)
111.6(5)
69.0(4)
96.2(4)
160.3(5)
160.3(5)
0
Zn(1)–S(41)
Zn(1)–S(43)
S(43)–C(42)
S(13)–C(12)
S(11)–C(12)
S(41)–C(42)
N(14)–C(12)
N(44)–C(42)
0
S(11)– Zn(1) –S(13)
0
S(11)– Zn(1) –S(41)
0
S(11)– Zn(1) –S(43)
0
Zn(1)–S(11)–Zn(1)
(
1.47 Å) and C ¼ N (1.28 Å), indicating the partial double bond character between C–N
bond of the ligands. The C–S bond lengths 1.722 to 1.749 Å in the dithiocarbamate
complexes are significantly shorter than the C–S single bond length (1.81 Å) due to p
delocalization over the four membered rings.
In views of various types of intramolecular C–Hꢀꢀꢀp interactions (Figure 5(A)), the
structure of complex A2 is further stabilized by weak C–Hꢀꢀꢀp interaction (3.955 Å)
between the C–H hydrogen atom of a phenyl ring and the p electrons of other phenyl
ring. Aromatic methylene protons C–Hꢀꢀꢀp interaction (3.343 Å) and methylene protons
(
adjacent to nitrogen atom) of alkyl chain C–Hꢀꢀꢀp interaction is observed (3.326 Å).
A specific weak interaction C–HꢀꢀꢀS (Figure 5(B) and Table 4) apparently takes place
between the hydrogen atom of the methyl and benzyl group with the thioureide sul-
fur. The continuation of definite interactions C–HꢀꢀꢀS is manifested by irregular down-
field shift of the signal from the related proton [40]. The interatomic SꢀꢀꢀH distance

1
4
V. K. MAURYA ET AL.
Figure 4. ORTEP diagram of [C H N Zn S ] (A2) showing atomic numbering scheme.
40
48
4
2 8
Figure 5. A. Perspective views of various types of intramolecular C–Hꢀꢀꢀp interactions in complex
A2. B. C–HꢀꢀꢀS and CꢀꢀꢀH interactions in complex A2.
ꢂ
Table 4. Weak intermolecular interaction ((Å) and ) in [C H N Zn S ], A2.
4
0
48
4
2 8
D-HꢀꢀꢀA
d(D-H)
d(HꢀꢀꢀA)
2.947
2.889
d(DꢀꢀꢀA)
3.781
3.799
<(DHA)
C(32)–H(32B)ꢀꢀꢀS(11)
0.960
0.930
145.98
166.31
C(25)–H(25)ꢀꢀꢀS(41)
corresponding to the equilibrium geometry for S11ꢀꢀꢀH32B and S41ꢀꢀꢀH25 were found
to be 2.94 and 2.88 Å, respectively, where van der Waals radii was found smaller than
the sum of the hydrogen and sulfur atoms. C–HꢀꢀꢀS bond angle was found to be
ꢂ
ꢂ
1
45.9 and 166.3 in S11ꢀꢀꢀH32B and S41ꢀꢀꢀH25, respectively. Thus, in complex A2, the
DTC groups coordinate the metal center in a bidentate fashion, forming two chelate
rings in a distorted square pyramidal geometry, and its point group was found to be
C1 (Figure 5(A)); no other symmetry element was detected in the complex, according
to the group theory.

JOURNAL OF COORDINATION CHEMISTRY
15
3.6. Growth inhibition assays of complexes
In spite of profound interest in the DTC transition metals as chemotherapeutic agents
for the treatment of various carcinomas, very little ponderance has been given to
investigate their antibacterial and antibiofilm properties [13, 28, 36, 41]. However, the
literature documents some antibacterial activity of some Pd(II)-DTC; however, meager
information is found in the literature about their antibiofilm activities. By the mid-
1990s, it became clear that our victory over pathogens was an illusion, with resistance
spreading faster than the discovery of new antibiotics/antimicrobials. Infectious disease
is often untreatable, even when caused by a pathogen that is not resistant to antibiot-
ics [5]. This is the essential paradox of chronic infections. In most cases, chronic infec-
tions are accompanied by the formation of biofilms, which seems to indicate the
source of the problem. Even National Institute of Health claims the involvement of
biofilms in around 80% of all bacterial infections [7, 42]. This leads to the growing
number of morbidity and mortality in consequence of biofilm/bacterial infections in
individuals. Vancomycin, teicoplanin, daptomycin, and linezolid are the first choice for
the clinical treatment of aggressive gram-positive infections; however, they have pro-
nounced nephrotoxicity, etc. Similarly, for the treatment of gram-negative pathogens,
Meropenem, imipenem, amikacin, etc. are the drugs of choice but they all have their
complications. Some quantum hybrids using MoS and curcumin have also been fabri-
2
cated but they still need further validations by pharmacokinetic and pharmacodynam-
ics studies [43]. In the current scenario of rapidly growing cases of resistant infections,
more effective and less toxic complexes/drugs are required. In the present work, the
antibacterial and antibiofilm activities of complexes A1–A4 were performed in terms
of disc diffusion assay, MIC, growth rate analysis and tissue culture based semi-
quantitative antibiofilm assay. The complex, which showed promise in both the activ-
ities including the antitumor activity, was further studied. TCP based antibiofilm assay
and fluorescence microscopy study were also performed.
3.6.1. Preliminary screening
Initial screening of complex A2 showed its prominence as effective antimicrobial
against S. aureus and E. coli. The results suggest dose dependent killing of S. aureus
(Supplementary Figure S26). As the concentration is increasing by 10 units per disc as
we move from A3–A2–A1–A4 the resultant zone of inhibition is also increasing in the
case of S. aureus. After incorporation of 40 mg in disc A4, we noted maximum zone of
inhibition (29 mm). However, in the case of E. coli, the trend was different. At concen-
ꢁ
1
trations 30 and 40 mg disc , the zone of inhibition was the same (28 mm). 30 mg con-
centration was found to be more effective for E. coli.
3.6.2. MIC Determination
MIC of compounds was determined against four different bacterial strains (2 clinical
and 2 control strains) using the broth micro dilution method. The results show that
the compounds exhibited antimicrobial activity against all the tested bacterial strains
ꢁ
1
(
Table 5) and the MIC ranged from 1 to >256 mg mL . For comparison, MIC values of
Vancomycin (for gram-positive isolates) and Meropenem (for gram negative) are also
listed therein.

1
6
V. K. MAURYA ET AL.
Table 5. Minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration
ꢁ
1
(
MBIC) of Zn(II) dithiocarbamate complexes in (mg mL ).
MIC/MBIC
Staphylococcus aureus,
MRSA (764/2018)
MIC/MBIC
Staphylococcus aureus,
MIC/MBIC
Escherichia coli
(2042/2018)
MIC/MBIC
Escherichia coli,
ATCC 25922
Compounds
ATCC 25923
Vancomycin/Meropenem
A1
4/2
64/32
1/0.5
32/32
16/8
256/128
1/0.5
128/64
KL1
A2
KL2
A3
KL3
A4
>256/>256
16/8
>256/256
16/4
>256/>256
32/16
>256/>256
4/1
>256/128
4/2
>256/128
16/4
>256/>256
128/32
>256/>256
>256/>256
>256/>256
256/128
>256/>256
64/32
>256/128
>256/256
>256/256
128/64
KL4
>256/128
128/256
>256/>256
>256/>256
Table 6. Biofilm inhibition in terms of percent reduction in biofilm biomass.
Bacterial strains
% reduction in biofilm biomass
S. epidermidis ATCC 35984
E. coli ATCC 25922
S. aureus 764/2018
54.44
100
94.71
8
41.54
91.46
81.53
4
33.1
78.12
68.25
2
22.34
61.24
44.61
1
12.43
8.03
21.13
11.37
0.25
49.84
31.22
0.5
ꢁ1
Complex A2 conc. (mg mL
)
Table 7. Bacterial growth rate pre- and post-treatment with complex A2.
ꢁ
1
ꢁ1
Growth rate (h
)
Growth rate (h
)
Bacteria
(before treatment)
(after treatment)
S. epidermidis (ATCC 35984)
E. coli (ATCC 25922)
S. aureus (1156/2018)
0.471 ± 0.0048
0.3589 ± 0.0071
0.4344 ± 0.006068
0.3814 ± 0.0421
0.1896 ± 0.0534
0.1772 ± 0.0253
3.6.3. Tissue culture plate-based antibiofilm assay
Complex A3 triggered almost complete degradation of the E. coli (ATCC 25922) and S.
ꢁ
1
aureus (764/2018) biofilms at concentration 8 mg mL whereas biofilm formation by S.
epidermidis (ATCC 35984) was found to be resistant to complex A3 at comparable con-
centration of the complex (Table 6). Interestingly, more than 75% of biofilm biomass
formed by both S. aureus and E. coli was inhibited by A2, at concentrations 2 and 4 mg
ꢁ
1
mL , respectively (Supplementary Figure S25). For comparison, MBIC values of
Vancomycin (for gram-positive isolates) and Meropenem (for gram-negative) are also
listed in Table 5.
3.6.4. Bacterial growth rate analysis
The mean growth rates of the S. epidermidis (ATCC 35984), E. coli (ATCC 25922), and
ꢂ
S. aureus (764/2018) were significantly different in BHI broth at 37 C (p ꢇ 0.001)
(
(
Table 7). The treatment with complex A2 has significantly reduced the growth rate
p ꢇ 0.001) (Table 7).
3
.6.5. Fluorescent microscopy
The effect of complex A2 on 72-h old biofilms of S. aureus (764/2018) and E. coli
2042/2018) was investigated by fluorescent microscopy. In agreement with the broth
microdilution, S. aureus 764/2018 showed an intense green staining of sytox green,
(

JOURNAL OF COORDINATION CHEMISTRY
17
Figure 6. Fluorescent micrographs depicting the effect of compound A2 on 72 h old biofilms of S.
aureus (764/2018) and E. coli (2042/2018) utilizing sytox green. Panel A depicts S. aureus while
panel B depicts E. coli.
indicating the marked damage in the cell membrane that was caused by complex A2.
This intense green-color uptake as shown in Figure 6 clearly unveils that complex A2
ꢁ
1
fostered bacterial death at the concentration as low as 4 mg mL by altering the bac-
terial membrane.
Although these complexes of Zn(II) are not much investigated for their antibacterial
and antibiofilm activities, some other dithiocarbamate derivatives are investigated for
antibacterial activities. Kang et al. documented the antibacterial activity of pyrrolidine
dithiocarbamate. They reported that S. aureus was inhibited at MIC of 30 mM, whereas
a much higher concentration was needed to inhibit the growth of E. coli completely
(
MIC ¼ 400 mM) [44]. Another report from Onwudiwe et al. using some mixed ligand
complexes of Zn(II) dithiocarbamate documented some moderate to good antimicro-
bial activity against gram positive and gram-negative bacterial strains. MICs of their
complexes ranged between 50 and 75 mg mL against S. aureus [45]. In our present
ꢁ
1
ꢁ
1
report, we documented the MIC against MSSA and MRSA to be 4 and 16 mg mL
.
Besides, they did not report the mechanism of killing. We herein report the biocom-
patible, antibacterial, and antibiofilm agent with activity against the cell membrane.
However, due to different lipophilicity, they may disorganize the lipoprotein leading to
the cell death. Our report is in consonance with Tweedy’s theory, which states that
metal complexes show increased activity than their ligands due to the increased
p-electron delocalization throughout the chelate ring of ligands. Chelation leads to
reduction in metal ion’s polarity and increases chelates lipophilic nature that ultimately
favors microbial cell membrane lipid layer permeation as evidenced by our fluores-
cence microscopic data [46].
3.6.6. Elucidation of antitumor activity
The antitumor activities of the complexes are assessed utilizing SRB assay against
HCT116 cells. Complex A2 had no significant influence on cell viability in the said
tested range, which can easily be perceived from the observation that at exposure
ꢁ
1
dose of 2 mg mL , cell viability was around 97% which remained 12% when exposed

1
8
V. K. MAURYA ET AL.
ꢁ
1
to 256 mg mL . Unlike complex A2, the exposure to complex A3 had significant cyto-
toxic effects over HCT116 cells in a dose-dependent manner as evidenced by the
ꢁ
1
observation that upon exposure to 2 mg mL concentration, around 96% cell viability
ꢁ
1
was noted but no sooner the concentration was raised to 256 mg mL ; cell viability
found reduced to <1%. After exposure for 48 h, the GI50 and LC50 values for the com-
ꢁ
1
plexes were estimated. For complex A2, it was 71.25 and 229.92 mg mL , respectively,
whereas for complex A3, the GI and LC50 were found to be 41.15 and 133.73 mg
5
0
ꢁ
1
ꢁ1
mL , respectively. The results indicated that 16 mg mL of complexes A2 and A3
inhibited the cell proliferation by approximately 12% and 47%, respectively, signifying
ꢁ
1
that complex A3 is dramatically more effective than complex A2. At the 256 mg mL
concentration, complex A2 inhibited cell proliferation by ꢅ88%, whereas complex A3
virtually resulted in complete inhibition of tumor cell proliferation. The results
obtained were in consonance with the phase contrast microscopic observations as
shown below (Supplementary Figure S27). However, the report by Fregona et al. docu-
ments that one of the synthesized Zn–DTC complexes, [Zn(DMDT) ], had IC as low
2
50
as ꢅ0.9 lM in human ovarian and cervix carcinoma cell lines (2008 and A431) [25].
The growing spread of drug resistance among bacteria makes it prudent to look for
specific antibacterial agents with better biocompatibility and lower toxicity to the host
and the environment. Metals are indispensable for the living system for survival.
However, metal toxicity is also obvious among them. As both zinc and dithiocarba-
mates interact with living cells, we expect pronounced activity by bonding them
together to rejuvenate the ligand-based metal complexes (potential drug candidates)
class to answer this claim, representing an alternative therapeutic route. We were
more interested in the mechanistic underpinnings of such complexes as antibacterial
agents and we observed the effects on bacterial cell membranes. The number and
nature of the organic groups bonded to the metal center influence the toxicity
towards microorganisms, which, in general, decreases in the order A2 > A4 > A1 > A3.
However, the order of toxicity was found dependent on the bacteria and varied from
strain to strain. The toxicity of our synthesized complexes correlates with their ability
to fit into the target/receptor site, their ability to undergo reversible redox reactions at
suitable potentials, as well as total molecule surface (TSA). Hence, complex A2 which
is benzyl and ethyl substituted should be more toxic to the bacterial system than A4
(methyl group at para position), A1 (isopropyl group at para position of benzene ring,
methyl group at methylene carbon of aryl group), and A3 (isopropyl group at para
position and methoxy group at para position).
However, the order of toxicity was found reversed when assayed over HCT116
colon cancer cells. We propose that the toxic effects are intra-cellular over HCT116
cells, following transport through the cell membrane; a correlation definitely exists
between toxicity and lipophilicity. Complex A3 is more lipophilic and hence it was
found more active. To fit on the membrane region of the bacteria snugly, very com-
pact ligands are needed. Complex A2 fitted well in our requirement window that
behaved like a high-spin complex more susceptible to reversible redox processes.
However, complex A3 behaved like a low-spin complex owing to the presence of two
major electron-donating groups (isopropyl group at para position and methoxy group
at para position). It is also probable that as the aryl-substituents become larger in

JOURNAL OF COORDINATION CHEMISTRY
19
complex A3 that facilitated its entry in HCT116 cells more readily through the lipo-pro-
teinous cell membrane because of its higher hydropathy index. The high toxicity over
HCT116 cells can be understood in terms of chelation theory, which states that, upon
complexation, the polarity of the metal ion gets reduced that increases the lipophilic-
ity of the metal complexes, facilitating them to cross the cell membrane easily. Thus,
the observed decline in bactericidal activity with larger aryl substituents is not due to
a decreasing supply of agent to the cell but due to increased lipophilicity.
4. Conclusion
The current study reports the synthesis and characterizations of four new dithiocarba-
mate ligands and their complexes of general formula [M(L) ]. Complex A2 crystallizes
2
in triclinic system with space group P-1 having distorted square pyramidal geometry,
stabilized by weak C–Hꢀꢀꢀp and C–HꢀꢀꢀS intramolecular interactions. The photophysical
behavior evaluations revealed that upon excitation with 233–269 nm wavelengths, all
the synthesized Zn(II) complexes exhibited an emission band ꢅ308 nm with the Stokes
shift in the 76–79 nm range, originating from the metal perturbed metal-to-ligand
charge-transfer transition. The rationale of synthesis was pertaining to its wide medical
application(s). However, we restricted our study to the bacteria and the tumor cell. By
the mid-1990s, it became obvious that our victory over bacteria was an enigma with
resistance spreading faster than the development of new therapeutic modalities.
Bacterial biofilms are infectious lesions that bring about nosocomial infections.
National Institutes of Health (NIH) reports about 65% of all microbial infections and
80% of all chronic infections to be associated with biofilms. Bacteria develop resistance
through many means, of which, biofilm is more complex and threatening due to evo-
lution of persisters and persister-like populations. The current study reports that com-
plex A2 is able to combat both planktonic and biofilm dwelling modes in a non-toxic
biocompatible range. Similarly, complex A3 was potentially inhibitory/killing to the
colon cancer cell HCT116. However, the detailed toxicity profiling of the said com-
plexes are still needed before translating them as a drug.
Acknowledgements
We are thankful to Department of Chemistry, Institute of Sciences, Banaras Hindu University
Varanasi for their Instrumentals facilities. We are also thankful to Prof. N. Singh, Department of
Chemistry, Institute of Sciences, BHU Varanasi for his fruitful suggestions.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
We sincerely acknowledge the DST-PURSE grant sanctioned to Department of Microbiology, IMS,
BHU. VKM, AKS are also acknowledging University Grant Commission (UGC), New Delhi for
Senior Research Fellow Ref. No: 22/12/2013 (II) EU-V and 22/06/2014 (i) EU-V, respectively, and

2
0
V. K. MAURYA ET AL.
SY acknowledges Council of Scientific and Industrial Research, New Delhi for financial assistance
in the form of Junior research fellow ref. no. [1121630870 vide 18/12/2016 (ii) EU-V].
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