Copper complexes containing thiosemicarbazones derived from 6-nitropiperonal: Antimicrobial and biophysical properties

Article abstract of DOI:10.1016/j.saa.2017.04.057

A series of four thiosemicarbazones from 6-nitropiperonal along with the corresponding copper complexes were synthesized. The biophysical characteristics of the complexes were investigated by the binding to DNA and human serum albumin. The binding to DNA

Full text of DOI:10.1016/j.saa.2017.04.057

Accepted Manuscript
Copper complexes containing thiosemicarbazones derived from
6
-nitropiperonal: Antimicrobial and biophysical properties
Floyd A. Beckford, Kelsey R. Webb
PII:
S1386-1425(17)30327-X
DOI:
doi: 10.1016/j.saa.2017.04.057
SAA 15110
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date:
Revised date:
Accepted date:
6 November 2016
20 March 2017
18 April 2017
Please cite this article as: Floyd A. Beckford, Kelsey R. Webb , Copper complexes
containing thiosemicarbazones derived from 6-nitropiperonal: Antimicrobial and
biophysical properties. The address for the corresponding author was captured as
affiliation for all authors. Please check if appropriate. Saa(2017), doi: 10.1016/
j.saa.2017.04.057
This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. 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.

ACCEPTED MANUSCRIPT
1
Copper complexes containing thiosemicarbazones derived from 6-nitropiperonal:
antimicrobial and biophysical properties
Floyd A. Beckford* and Kelsey R. Webb
Department of Natural Sciences, The University of Virginia’s College at Wise, 1 College
Avenue, Wise, VA 24293
*
Corresponding author: fab5b@uvawise.edu, Ph: (276) 376-4657, Fax: (276) 376-4603

ACCEPTED MANUSCRIPT
2
Abstract
A series of four thiosemicarbazones from 6-nitropiperonal along with the corresponding copper
complexes were synthesized. The biophysical characteristics of the complexes were
investigated by the binding to DNA and human serum albumin. The binding to DNA is moderate;
4
-1
the binding constants run from (0.49 – 7.50) x 10 M . In relation to HSA, the complexes
5
-1
interact strongly with binding constants on the order of 10 M . The complexes also display
antioxidant behavior as determined by the ability to scavenge diphenylpicrylhydrazyl (dpph) and
nitric oxide radicals. The antimicrobial profiles of the compounds, tested against a panel of
microbes including five of the ESKAPE pathogens (Staphylococcus aureus, MRSA, Escherichia
coli, Klebsiella pneumoniae, MDR, Acinetobacter baumannii, Pseudomonas aeruginosa) and
two yeasts (Candida albicans and Cryptococcus neoformans var. grubii), are also described.
The compounds contain a core moiety that is similar to oxolinic acid, a quinolone antibiotic that
targets DNA gyrase and topoisomerase (IV). The binding interaction between the complexes
and these important antibacterial targets were studied by computational methods, chiefly
docking studies. The calculated dissociation constants for the interaction with DNA gyrase B
(from Staphylococcus aureus) range from 4.32 to 24.65 M; the binding was much stronger to
topoisomerase IV, with dissociation constants ranging from 0.37 to 1.27 M.
Keywords: Thiosemicarbazone; Antioxidant; Antibacterial; Molecular docking

ACCEPTED MANUSCRIPT
3
1. Introduction
The development of organometallic and inorganic complexes for medicinal use is a field
of emerging significance due to the diversity of coordination chemistry that is available.
Frequently used for cancer treatment, cisplatin and its many derivatives have demonstrated the
successful capabilities of organometallics; however, there is a crucial need for the development
of alternatives to these drugs because of their harmful side effects, such as nephrotoxicity and
platinum resistance [1]. Copper, which is a trace metal responsible for many important biological
functions in the body, has been recognized for its many biological activities, such as antioxidant,
antibacterial, antimicrobial, and antitumor behaviors, thus making it a promising candidate for
anticancer as well as for other medicinal uses.
The combination of certain transition metals and ligands has been shown to have
synergistic effects by either enhancing and/or resulting in new biological activities in the
complexes. Thiosemicarbazone (TSC) ligands have attracted the attention of many researchers
due to their antitumor, antibacterial, and antiviral properties [2-5]. Recently, the idea of
incorporating biological components into the ligands themselves has led to the development of
new and encouraging anticancer complexes [6]. The biologically active compound 6-
nitropiperonal (NP) is known to possess antitumor and antibacterial properties, making it a
structure of considerable interest for continued study. This research presents four novel ligands
constituted of 6-nitropiperonal and various thiosemicarbazide units that were incorporated into
the synthesis of four novel copper(II) 6-nitropiperonal thiosemicarbazone (Cu NP-TSC)
complexes.
There is a variety of cellular targets that are vital to consider when investigating the
biological mechanisms for metal complexes. This is in order to improve the efficacy as well as
selectivity of the complexes. Human serum albumin (HSA), the most abundant protein in human
blood plasma, is a carrier protein of particular interest for drug delivery because it is nontoxic
and binds to a variety of compounds [7]. Since HSA plays a vital role in the transportation of
drugs, it is important to understand the interactions present between the drug and protein. In
this research, we have investigated the ability of the Cu NP-TSC complexes to bind to this
protein. In addition to HSA, deoxyribonucleic acid (DNA) is another potentially significant
biological target. For the development of less toxic yet successful metal complexes for medicinal
use, it is also vital to understand the interactions and mechanisms between the metal complex
and DNA. Although some copper complexes have been reported in literature to induce
apoptosis [8], there is not much information currently known about the general interactions of

ACCEPTED MANUSCRIPT
4
copper complexes with DNA. This paper investigates experimentally the interactions that the
novel Cu NP-TSC complexes exhibit with DNA.
In addition, other biological targets of interest were theoretically explored. Two specific
enzymes, DNA gyrase B and Topoisomerase IV, were utilized in computational studies with the
complexes. These two enzymes play important roles in DNA replication and cellular growth [9].
Therefore, the ability to inhibit these enzymes is of great interest for many antibacterial studies.
Computational studies were carried out for the four complexes by docking to these enzymes in
order to assess their potential for medicinal use.
2. Experimental
2.1 Material and Methods
Analytical or reagent grade chemicals were used throughout. All the chemicals including
solvents were obtained from Sigma-Aldrich or other commercial vendors and used as received.
Microanalyses (C, H, N) were performed by Galbraith Laboratories, (Knoxville, TN). Infrared
-1
spectra in the range 4000 – 500 cm were obtained using the UATR accessory or in KBr discs
on a Perkin Elmer Spectrum 100 FTIR spectrophotometer. Absorption spectra were recorded on
a Perkin Elmer Lambda 25 spectrophotometer. Mass spectrometric measurements were carried
out with an Advion expression-L CMS. Conductivity measurements were made on a Accumet
AB200 conductivity meter. Cyclic voltammetric (CV) data were collected on a Bioanalytical
-3
Systems Inc. Epsilon potentiostat on a C3 cell stand at 296 K. DMSO solutions (1 x 10 M)
containing 0.1 M tetrabutylammonium hexafluorophosphate as the supporting electrolyte. The
measurements were carried out with a three-electrode system consisting of a platinum working
electrode, a platinum wire auxiliary electrode and a Ag/AgCl reference electrode. The thermal
analysis measurements were made on a Perkin Elmer TGA4000 instrument.
2.2 Syntheses
The ligands were synthesized according to a standard method [10,11]. Briefly, equimolar
amounts of the aldehyde and the appropriate alkyl-substituted thiosemicarbazide were
suspended in ethanol containing a few drops of glacial acetic acid, and the pale yellow
suspension heated at reflux for 4h. After cooling to ambient temperature, the resulting bright
yellow suspension was filtered and the solid washed with ethanol and air-dried. In all cases,
near quantitative yields were obtained.
+
1
npTSC: Yellow solid. ESI-MS (CH CN) m/z [M+H] [Observed (Calcd.)]: 268.2 (268.3). H NMR
3
(
400 MHz ,DMSO-d ):  6.26 (s, 1H, H ), 7.60 (s, 1H, H ), 8.04 (s, 1H, H ), 8.25, 8.32 (s, 2H, H ),
6
e
d
f
a

ACCEPTED MANUSCRIPT
5
1
3
8
1
3
8
.47 (s, 1H, H ), 11.65 (s, 1H, H ); C NMR (100 MHz, DMSO-d ):  178.3 (C ), 151.9 (C ),
c
b
6
1
5
-1
48.6 (C ), 143.2 (C ), 137.4 (C ), 125.7 (C ), 105.9 (C ), 104.8 (C ), 103.6 (C ). IR (cm ): 3417,
7
2
9
3
8
4
6
229, 3152, 3100, 2948, 2795, 1601, 1538, 1517, 1474, 1383, 1322, 1266, 1100, 1036, 928,
85, 835, 825.
+
1
MnpTSC: Yellow solid. ESI-MS (CH CN) m/z [M+H] [Observed (Calcd.)]: 283.0 (283.3). H
3
NMR (400 MHz ,DMSO-d ):  6.27 (s, 1H, H ), 7.61 (s, 1H, H ), 8.01 (s, 1H, H ), 8.47 (s, 1H, H ),
6
e
d
f
c
13
8
.69 (s, 1H, H ), 11.72 (s, 1H, H ); C NMR (100 MHz, DMSO-d ):  177.8 (C ), 151.8 (C ),
a
b
6
1
5
1
48.5 (C ), 143.1 (C ), 136.8 (C ), 125.8 (C ), 105.6 (C ), 104.8 (C ), 103.7 (C ), 30.9 (-CH ). IR
7
2
9
3
8
4
6
3
-1
(
cm ): 3414, 3375, 3120, 2945, 2897, 2776, 1611, 1567, 1538, 1522, 1480, 1324, 1264, 1103,
038, 929, 887, 818.
1
+
1
EnpTSC: Yellow solid. ESI-MS (CH CN) m/z [M+H] [Observed (Calcd.)]: 297.0 (297.3). H
3
NMR (400 MHz ,DMSO-d ):  6.27 (s, 1H, H ), 7.61 (s, 1H, H ), 8.01 (s, 1H, H ), 8.47 (s, 1H, H ),
6
e
d
f
c
1
3
8
1
1
1
.69 (s, 1H, H ), 11.72 (s, 1H, H ); C NMR (100 MHz, DMSO-d ):  176.8 (C ), 151.8 (C ),
a
b
6
1
5
48.5 (C ), 143.2 (C ), 136.9 (C ), 125.7 (C ), 105.8 (C ), 104.8 (C ), 103.7 (C ), 38.3 (-CH ),
7
2
9
3
8
4
6
2
-1
4.5 (-CH ). IR (cm ): 3376, 3345, 3129, 2916, 1614, 1538, 1500, 1480, 1324, 1268, 1221,
3
101, 1034, 928, 892, 882, 819, 801.
+
PnpTSC: Bright yellow solid. ESI-MS (CH CN) m/z [M+H] [Observed (Calcd.)]: 345.1 (345.4).
3
1
H NMR (400 MHz ,DMSO-d ):  6.28 (s, 1H, H ), 7.65 (s, 1H, H ), 8.21 (s, 1H, H ), 8.60 (s, 1H,
6
e
d
f
H ), 10.25 (s, 1H, H ), 12.05 (s, 1H, H ), 7.53 (d, 2H, phenyl ring), 7.46 (m, 2H, phenyl ring),
c
a
b
1
3
7
1
1
9
.24 (m, 1H, phenyl ring) ; C NMR (100 MHz, DMSO-d ):  176.4 (C ), 151.8 (C ), 148.7 (C ),
6
1
5
7
43.4 (C ), 138.9 (C ), 125.4 (C ), 106.2 (C ), 104.9 (C ), 103.7 (C ), {136.0, 128.1, 126.4,
2
9
3
8
4
6
-
1
25.6} (phenyl ring). IR (cm ): 3412, 3286, 2926, 1614, 1596, 1547, 1480, 1328, 1269, 1032,
24, 872, 816.
The complexes were synthesized as follows: The ligand (2 molar equivalents) was suspended
in approximately 20 mL of methanol and the suspension heated to boiling. To the boiling mixture
was added, dropwise, a solution of CuCl 2H O (one molar equivalent) in 5 mL methanol. The
2
2
yellow mixture was heated at reflux for 2½ - 3h during which time it became a bright orange
color. The reaction mixture was allowed to cool to room temperature and then filtered to collect
a yellow solid. The product was washed with methanol and then a small amount of ether,
followed by drying at the vacuum line.
[
Cu(npTSC) ]3H O, 1.
Yield: yellow solid, 185 mg (73 %). Elemental analysis for
2
2
C H CuN O S ; Calc.: C 33.15, H 3.09, N 17.18. Found: C 33.58, H 2.37, N 16.36. ESI-MS
18
20
8
11
2

ACCEPTED MANUSCRIPT
6
+
-1
(
CH CN) m/z [M + H] [Observed (Calcd)]: 599.0 (599.0). IR (cm ): 3436, 3212, 3152, 2981,
3
1599, 1585, 1484, 1407, 1282, 1161, 1070, 943, 883, 842, 823, 782, 730.
[
Cu(MnpTSC) ]3H O, 2.
Yield: yellow solid, 120 mg (50 %). Elemental analysis for
2
2
C H CuN O S ; Calc.: C 35.32, H 3.56, N 16.48. Found: C 35.64, H 3.03, N 15.59. ESI-MS
20
24
8
11
2
+
-1
(
CH CN) m/z [M + H] [Observed (Calcd)]: 627.0 (627.1). IR (cm ): 3354(w), 3342, 3153, 2978,
3
2
929, 2876, 1623, 1533, 1473, 1416, 1299, 1265, 1219, 1158, 1087, 1047, 1018, 943, 891, 840,
12, 785, 729.
8
[
Cu(EnpTSC) ]2H O, 3.
Yield: yellow solid, 150 mg (62 %). Elemental analysis for
2
2
C H CuN O S ; Calc.: C 38.29, H 3.80, N 16.24. Found: C 38.56, H 3.37, N 15.48. ESI-MS
22
25
8
10
2
+
-1
(
CH CN) m/z [M + H] [Observed (Calcd)]: 655.1 (655.1). IR (cm ): 3309, 3129, 3046, 2985,
3
1
622, 1593, 1541, 1531, 1442, 1416, 1398, 1304, 1259, 1194, 1069, 1017, 936, 889, 841, 783,
33.
7
[
Cu(PnpTSC) ]1½ H O, 4. Yield: yellow solid, 110 mg (47 %). Elemental analysis for
2
2
C H CuN O S ; Calc.: C 46.36, H 3.24, N 14.42. Found: C 46.32, H 3.03, N 14.43. ESI-MS
30
25
8
9.5
2
+
-1
(
CH CN) m/z [M + H] [Observed (Calcd)]:751.2 (751.23). IR (cm ): 3309, 3129, 3046, 2985,
3
1
622, 1593, 1541, 1531, 1442, 1416, 1398, 1304, 1259, 1194, 1069, 1017, 936, 889, 841, 783,
33.
7
2.3 Anti-oxidant assays
DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging assay: The assay was conducted in a 96-well
format. The total volume of each well was 250 L. The concentrations of the metal complex
varied from 2 to 500 M and 150 M of dpph was added. The reactions were incubated at 25 C
for 30 minutes in the dark (shielded from light) before reading the absorbances at 520 nm. For
comparisons ascorbic acid (AA) and butylated hydroxytoluene (BHT)) were incorporated onto
the plates along with blanks and controls. The control was a solution of DPPH and the blank is
the solvent.
Nitric oxide (NO) scavenging assay: The assay was carried out in a 96-well plate with the total
well volume being 300 L. A 100 L aliquot of sodium nitroprusside (SNP) in phosphate buffer,
was incubated with the appropriate amount of metal complex (0 – M) for 1½ h at room
temperature under light. After this period of time, 100 L of Griess’s reagent (1% sulfanilamide
and 0.1% naphthylethylenediamine in 2.5% phosphoric acid) was added to each well. The

ACCEPTED MANUSCRIPT
7
mixture was then incubated at room temperature for 10 min before the absorbance was read at
560 nm.
2.4 Antimicrobial assay
The antimicrobial activity of the complexes and ligands were assay using three protocols.
Ligands: The ligands were tested against five bacteria and two yeasts using the following
procedure carried out by the CO-ADD initiative of the University of Queensland’s Institute for
Molecular Bioscience. The organisms tested were bacteria Staphylococcus aureus, MRSA
(
ATCC 43300), Escherichia coli (ATCC 25922), Klebsiella pneumoniae, MDR (ATCC 700603),
Acinetobacter baumannii (ATCC 19606), Pseudomonas aeruginosa (ATCC 27853); the yeasts
were Candida albicans (ATCC 90028) and Cryptococcus neoformans var. grubii, (H99; ATCC
208821). Inhibition of bacterial growth was determined measuring absorbance at 600 nm
(
OD600), using a Tecan M1000 Pro monochromator plate reader. For the yeasts, growth
inhibition of C. albicans was determined measuring absorbance at 530 nm (OD530), while the
growth inhibition of C. neoformans was determined measuring the difference in absorbance
between 600 and 570 nm (OD600-570), after the addition of resazurin (0.001% final concentration)
and incubation at 35 °C for additional 2 h. The absorbance was measured using a Biotek
Synergy HTX plate reader. For all the microbes, the percentage of growth inhibition was
calculated for each well, using the negative control (media only) and positive control (bacteria
without inhibitors) on the same plate as references. The significance of the inhibition values was
determined by Z-scores, calculated using the average and standard deviation of the sample
wells (no controls) on the same plate. Samples with inhibition value above 80% and abs(Z-
Score) above 2.5 for either replicate (n=2 on different plates) were classed as actives. Samples
with inhibition value between 50-80% and abs(Z-Score) above 2.5 for either replicate (n=2 on
different plates) were classed as partial actives. Negative inhibition value mean that the growth
rate (or OD600) is higher compared to the control (bacteria only, set to 0% inhibition).
Complexes: For the determination of inhibition zones, the disk diffusion antibacterial screenings
were performed using Miller-Hinton Agar (MHA) for all the bacterial strains. The appropriate
amount of the complexes was dissolved in DMSO to make of a solution that was approximately
2
mM. A suspension of the bacterial cultures with exponentially growing cells (100 - 200 μL) was
adjusted to the equivalent of a 0.5 McFarland Turbidity standard using sterile saline solution.
The suspension was swabbed over the entire surface of the plated-medium using cotton swabs.
Sterile paper disks (6 mm in diameter) were saturated with 30 μL of the compounds and placed
onto the plate. One disk, saturated with 30 μL of DMSO, was placed on the plate to serve as a

ACCEPTED MANUSCRIPT
8
negative control. A disk saturated with 15 μL of 4.67 mM ampicillin was used as a reference
standard to determine the sensitivity of each strain. The inoculated plates were incubated
upside-down at 37 °C for 18–24 h. The antibacterial activity was measured as the diameter
(
mm) of the clear zone of growth inhibition. Each plate was run each in duplicate.
The in vitro antimicrobial activity of the four complexes was tested against S. aureus, E.
coli, K. pneumoniae, P. aeruginosa, E. faecalis and B. cereus. The bacteria were maintained on
nutrient agar and cultured in Mueller Hinton Broth and compounds were dissolved in DMSO.
The percent inhibition was determined from a microdilution method. Approximately 50-mL of
bacteria culture was incubated overnight at 37.0 C to produce exponentially growing cells. This
8
was then diluted to yield a suspension containing 1.5 x 10 CFU/mL (based on comparison of
the turbidity to a 0.5 McFarland standard). Subsequently, 200 L of this bacteria mixture were
then inoculated into a sterile 96-well microplate. Various amounts of the compounds were
added to the wells to give predetermined concentrations ranging from 20 – 180 M. The well
absorbances (at 620 nm) were recorded on a Fisher Scientific Multiskan MCC microplate reader
immediately after inoculation. The microplates were then incubated for 24 h at 37.0 C, and the
absorbance read again. Solvent, negative and positive growth controls were included on each
plate. Ampicillin was used as a standard comparison. Each plate contained three replicates of
each concentration. The percent inhibition was calculated from the equation below.


Abs
bacteria
Abs


bacteriacomplex
%
inhibition 
x100




Abs
bacteria
2.5 DNA-interaction studies
All the experiments involving the interaction of the complexes with DNA were carried out
in TRIS buffer (5 mM Tris, 50 mM NaCl, pH 7.20). The solutions of DNA were made by
dissolving the nucleic acids in the buffer. After an appropriate dilution, the concentration (per
nucleotide phosphate) was determined spectrophotometrically using the molar absorption
-1
-1
coefficient of 6600 M cm at 260 nm [12]. The solutions were deemed to be pure, (free of
protein), since a ratio of absorbance at 260 and 280 nm was  1.8 [13]. The DNA stock
solutions were stored at 4 C and used within 4 days after their preparation. Purified water (18.2
S) was used in all the experiments.
2.5.1 Absorbance titration experiments

ACCEPTED MANUSCRIPT
9
Electronic absorption titrations were carried out at room temperature to investigate the
binding between the complexes and ct-DNA. A constant concentration of the complexes (1.0 x
-5
1
0 M) was treated with aliquots of a stock concentrated solution of the DNA in a 1-cm quartz
cell. A sample of buffer in the reference cell was titrated in the same manner simultaneously.
2.5.2 Fluorescence titration experiments
In the ethidium bromide (EB) fluorescence displacement experiment, a buffered solution
that contained 10 M DNA pre-treated with EB (0.66 M), was titrated with a concentrated
solution of the complex. After each addition, the solution was stirred at the appropriate
temperature for 5 minutes before the fluorescence measurement was taken by exciting at 520
nm and measuring the emission spectra from 530 – 700 nm.
2.6 Reaction with human serum albumin (HSA)
For the fluorescence titration, a similar procedure to the EB displacement experiments
was done. Solutions of HSA (fatty acid-free) were prepared in TRIS buffer (50 mM Tris, pH 7.40,
100 mM NaCl) and stored in the dark at 4 C. The protein concentration was determined
4
-1
-1
spectrophotometrically and calculated using the molar absorptivity of 3.6 x 10 M cm at 280
nm [14]. In the experiments, a 3.0 mL solution of HSA (5 M) was titrated with aliquots of a
concentrated solution of the complex. The complex concentration in these mixtures ranged from
3
to 30 M. The fluorescence spectra of the solutions were obtained by exciting at 295 nm and
measuring the emission spectra from 300 – 500 nm.
3. Discussion
3.1 Synthesis and characterization
The compounds used in this study have been characterized by elemental analysis as
well as NMR (ligands), infrared and electronic absorption spectrophotometry. In addition, cyclic
voltammetry, thermogravimetric analysis as well as conductivity measurements, were performed
on the metal complexes. The ligands were synthesized by the acid-catalyzed condensation of
the thiosemicarbazide with the appropriate aldehyde in refluxing ethanol and were obtained as
bright yellow solids. The complexes were synthesized by refluxing the ligands with the copper
salt in methanol (Fig. 1). The complexes were also obtained as yellow solids. The compounds
are soluble in solvents such as DMSO and acetone but not very soluble in alcohols.

ACCEPTED MANUSCRIPT
1
0
The proposed structural formulations of the ligands are consistent with their NMR
spectra. All the ligands have a number of common features and they are reflected in the proton
and carbon spectra. The benzodioxole moiety is of course present in all the ligands and the
signals arising from this moiety is indeed common to all. For the most part, npTSC, MnpTSC
and EnpTSC show very similar chemical shifts while PnpTSC show a little variation. In the
proton NMR, the –CH group (H ) show at 6.26 ppm in all the ligands. The azomethine proton
2
e
(
H ), is present at
b
Fig. 1. Structure of the ligands and complexes synthesized in the study.
approximately 11.70 ppm for npTSC, MnpTSC and EnpTSC while occurring at 12.05 ppm for
the PnpTSC compound. The presence of this peak confirms that the ligands are in the thiol
tautomeric
form in solution. The aldehydic proton (H ) resonate at 8.47 ppm (8.60 ppm for PnpTSC) and
c
the other protons on the benzene ring (H , H ) are consistently at 7.61 ppm and 8.04 ppm
d
f
respectively (8.20 ppm for PnpTSC). The amine proton (H ) has distinct behavior. In npTSC, we
a
observe a singlet for each of the two protons (at 8.25 and 8.32 ppm). This is not quite
unexpected as thioamides display hindered rotation about the (S=C)(NH ) bond. This is a
2
consequence of the bond possessing some p-character with the end-result being that the two
protons are magnetically non-equivalent. For MnpTSC and EnpTSC, this proton shows at 8.69
ppm but is found at a much higher frequency (10.25 ppm) for PnpTSC. For the substituent

ACCEPTED MANUSCRIPT
1
1
groups on the amine nitrogen, the protons show at what would be considered typical for those
groups.
13
The C NMR supports the proton data. In all the ligands there is a very high frequency
peak near 177 ppm which is easily attributable to the thiocarbonyl (C=S) group. This group
seems relatively insensitive to the substitution on the adjacent amine nitrogen, (unlike the
protons). The signals for the other carbons in the piperonal fragment occur in typical regions.
For example, the –CH group (C ) will has its peak near 104 ppm, the high frequency being a
2
6
consequence of being sandwiched between two electronegative oxygen atoms. The carbons of
the amine substituent groups are also typical.
Like an imine or a typical ketone, a thiosemicarbazone may display two tautomeric
forms. Consequently, thiosemicarbazone ligands can coordinate as a neutral species or as a
monoanionic species. We conclude from the characterization data that the ligands are obtained
as the thiol tautomer; however, they are deprotonated when they coordinate to the metal center.
The complexes are therefore neutral compounds. This was verified by conductivity
measurements on 1 mM DMSO solutions of the complexes. The values determined were 13.37
S/cm, 16.26 S/cm, 14.30 S/cm and 14.15 S/cm for 1, 2, 3 and 4, respectively. They confirm
the non-electrolytic nature of the complexes [15]. (Electrospray ionization) mass spectrometry
on the complexes also supports the formulation of the complexes as neutral species. Peaks
+
were observed for the complexes as follows: 1, m/z 599.0 (calc. 599.04), [M+H] ; 2, m/z 627.09
+
+
(
calc. 627.0), [M+H] ; 3, m/z 655.1 (calc. 655.14), [M+H] ; and, 4, m/z 751.23 (calc. 751.2),
+
[
M+H] . Further evidence for the composition of the complexes comes from spectroscopic
methods. The infrared spectrum of the complexes has the telltale signatures of chelation
through the azomethine nitrogen and the thiol sulfur [16]. The UV-vis spectrum of the complexes
indicates coordination through the sulfur as well (Fig. S1, Electronic Supplementary Information,
ESI). The ligands show a band at 325 nm which we interpret to be the result of the *
transition of the -C=S group. This band shift to higher energies by as much as 6 nm in the
spectra of the complexes. The shoulder that appears in the spectra of the ligands near 387 nm
completely disappears in that for the metal complexes. The cyclic voltammogram for the
complexes (Fig. 2) show one irreversible oxidation peak. The peak, which is somewhat ill-
defined for 1 and 3, occur at potentials of 1.25 V and 1.20 V, respectively. For 2 and 4, the peak
is at 1.20 V and 1.15 V respectively. The values remained essentially constant when the scan
rate was changed from 100 mV/s to 200 mV/s.
3.2 Thermal analysis measurements

ACCEPTED MANUSCRIPT
1
2
The thermal behavior of the complexes was investigated by thermogravimetric analysis.
Fig. 3 shows the heating profile of the complexes when heated from 40 C – 900 C. It appears
from the figure that there are three significant events happening in this range. Of particular
interest are the first two since it might be suggested that the third has not been completed
before the end temperature. The differences between the complexes is a matter of when the
events start and the transition temperature of the particular event. For convenience, we have
divided the thermogram into two parts: 200 – 335 C and 350 – 530 C. The transition
temperatures for the first (major) stage are 263.32 C, 256.77 C, 261.63C and 251.61 C for 1,
2, 3 and 4 respectively. It is obvious from the figure that complex 1 has the sharpest transition –
starting at 263.01 C and
-3
Fig. 2. Cyclic voltammograms for all four complexes measured in (1 x 10 M) DMSO (vs.
Ag/AgCl).
ending at 264.10 C. In this range, the material lost 36.7 weight percent. The transitions for the
other complexes are not as sharp with ranges of 19 C (20.7 weight %) for 2, 12 C (23.0 weight
%) for 3, and 28 C (22.2 weight %) for 4. We propose that the transition in this event is the
result of the melting of the complexes. This is supported by the fact that the weight loss of the
complexes does not correlate well with decomposition of the complexes with concurrent loss of
a ligand. The second minor stage is even more unexplainable. The events are obvious but not
very well defined. With our analysis methodology, the derived transition temperatures range

ACCEPTED MANUSCRIPT
1
3
from 512 C – 524 C with one exception, 360.67 C for 3. The weight loss in these transitions
does not exceed 9% (range: 3.7 – 8.8).
3.3 Structure description
The calculated structures of the complexes were computed by density functional theory
*
using the B97X-D method and a basis set of 6-31G . This was done using Spartan 14 [17] and
*
in this software package, this mentioned basis set corresponds to 6-31G on atoms smaller than
krypton and LANDL2DZ on atoms larger than krypton. Fig. 4 shows the structure calculated for
complex 1. We describe this geometry as a severely distorted tetrahedron around the copper
center.
Fig. 3. TGA curves of the four complexes heated under a nitrogen atmosphere.
The coordinating fragment of the thiosemicarbazone ligands is at angle of 122.89 to each
other. Some selected geometric parameters from the calculations are shown in Table 1.
Amongst all four complexes, the overall shape is not significantly different, suggesting that the
alkyl group on the amine nitrogen has no bearing on the geometry at the copper center. The
benzodioxole moiety of the ligands is essentially flat; the oxygen of the nitro groups are twisted
out of the plane by 21.86 and 32.74 in the two cases. This is seen in the free ligands as well.
Relative to the coordinating fragment, this moiety is out of plane by 18.09 in one ligand and

ACCEPTED MANUSCRIPT
1
4
26.39 in the other. The two ligands are close to each other and this might have an effect on the
coordinating ability to macromolecules.
Fig. 4. Calculated structure of complex 1 along with graphical representations of the highest
occupied (HO) and lowest unoccupied (LU) molecular orbitals (MO)
Table 1 Selected calculated geometrical parameters for the complexes
1
2
3
4
Bond distances (Å)
Cu(1)-S(1)
2.246
2.246
1.980
1.980
2.249
2.246
1.987
1.976
2.246
2.244
1.986
1.975
2.244
2.242
1.983
1.974
Cu(1)-S(2)
Cu(1)-N(1)
Cu(1)-N(5)
Bond angles ()
S(1)-Cu(1)-N(5)
S(2)-Cu(1)-N(1)
S(2)-Cu(1)-N(5)
S(1)-Cu(1)-N(1)
S(1)-Cu(1)-S(2)
N(1)-Cu(1)-N(5)
147.15
147.21
86.61
149.65
147.19
86.92
149.52
146.84
86.93
149.43
147.86
86.93
86.61
86.40
86.42
86.31
107.51
97.29
103.18
100.59
103.34
100.64
103.25
100.37

ACCEPTED MANUSCRIPT
1
5
As might be expected, complex 5 is larger than 1 by about 1.5 times. Other (calculated)
quantitative structure activity relationship parameters are essentially the same; for instance,
there are zero hydrogen bonded donors and sixteen hydrogen acceptors in each complex. The
frontier molecular orbitals (FMO) of a coordination complex play an important role in its
reactivity. The specific HOMO and LUMO for the complexes are quite similar and Fig. 4 show
those for 1. The HOMO (-7.5415 eV) is heavily localized on the coordination sphere around the
metal ion with the s and p orbitals on N2 and p orbitals on S1 the major orbitals; the six-
x
z
membered ring of the benzodioxole part (including the nitro groups) of the ligand appears to
bear most of the LUMO (-0.73336 eV). This is applicable to all the complexes. The negative
energies of all the FMOs indicates the stability of the complexes [18]. The energy gap between
HOMO and LUMO, (E), suggests the chemical reactivity of compounds. The presence of the
alkyl group substituted onto the amine nitrogen, appears to have some influence on the
electronic characteristics of the complexes. E decreases as the substituent changed from –H
to –C H . For 1, E was 6.81 eV and that decreased to 6.39 eV in the complex bearing the
6
5
methyl-substituted ligand. The differences for 2, 3 and 4 were subtler, with E = 6.37 eV and
6.33 eV for 3 and 4, respectively.
3.4 Electronic absorption titration
By using UV-Vis spectroscopy, it is possible to examine the strength of the binding
interactions of metal complexes with DNA [19-21]. A cursory look at the structure of the
thiosemicarbazone ligands used in the complexes would preclude the binding of the complexes
to DNA via an intercalative mode. As mentioned below, small molecules can bind to DNA via an
intercalation mode. This mode of binding is usually favored by the presence of a planar,
extended fused aromatic (polyaromatic) ligand. To intercalate into DNA, a planar molecule
slides between two base pairs without disrupting the hydrogen bonds and typically involve
significant p-electron overlap. For metal complexes, binding modes to DNA may be more
complicated than for simple organic molecules. For instance, intercalation may be initiated by
entry to the DNA via the grooves. Consequently, it may be that for our situation neither
intercalation, nor groove binding are unambiguous or independent concepts. Ordinarily,
complexes which adopt this method of binding generally have electronic absorption bands that
show bathochromic changes relative to the free complex and also display hypochromism

ACCEPTED MANUSCRIPT
1
6
[
22,23]. For our complexes, the calculated structures (Fig. 4) suggest that the
thiosemicarbazone moieties are relatively planar, the methylene of the dioxole ring obviously
3
being sp and the nitro groups slightly out of the plane. So we investigated the possible binding
modes for the complexes via a titration where the changes in the absorption of solutions with a
-4
fixed complex concentration (10 M), were measured upon addition of aliquots of a
concentrated solution of ct-DNA. Fig. 5 shows the absorption spectra of complex 1 upon titration
with ct-DNA. There is a 13% decrease in the absorption at the wavelength of maximum
absorption (294 nm). A small red shift of three nanometers accompanies this decrease. The
other complexes exhibit similar behavior with % hypochromicity of approximately 8% and
basically no wavelength change (figures shown in the Supplementary Information). To calculate
the binding strength, equation 1 [19], was used to calculate the intrinsic binding constant Kb.
[
DNA] [DNA]
1


ε ε
ε ε K (ε ε )
b b
a
f
b
f
f
(
1)
where  ,  and  corresponds to the molar absorptivities of the metal complex after each
a
f
b
addition of ct-DNA, for the free metal complexes and for the metal complexes when completely
bound. The binding constants K are calculated from the plots of [DNA]/( -  ) versus [DNA]
b
a
f
taking the ratio of the slope to the intercept. These values are shown in Table 2. The binding
4
-1
constant are on the order of 10 M which, compared to a classic organic-type intercalator,
characterizes them as moderate binders. The nature of the results suggests that the complexes
do not interact with DNA via intercalation or at least this type of interaction is weak.
Fig. 5. Left: Electronic absorption spectral changes of complex 1 on titration with ct-DNA. [Cu] =
25 M, [DNA] = 0, 5, 10, 15, 20, 25 and 30 M. Arrow indicates the change upon increasing
DNA concentration. Right: Plot of [DNA]/(ε –ε ) vs. [DNA]
a
f
3.5 Ethidium bromide competition experiment

ACCEPTED MANUSCRIPT
1
7
As mentioned above small molecules may interact with DNA by binding in the grooves,
intercalating between the base pairs or by electrostatically interacting with the phosphate
backbone if charged. One experiment commonly used to investigate a potentially intercalative
Table 2 Stern-Volmer quenching along with binding parameters for the interaction of the
complexes with ct-DNA
Ethidium bromide titration
UV-vis titration
4
-1
4
11
-1 -1
4
-1
Complex
Kapp x 10 M
KSV x 10
K x 10 M s
K x 10 M
q
b
1
2
3
4
1.26
0.825
1.20
2.03
0.753
1.34
9.24
3.42
6.20
3.82
7.50
0.485
2.40
0.881
0.842
4.26
mechanism is the ethidium bromide displacement experiment. Both DNA and ethidium bromide
EB) are non-emissive in aqueous solution; however, when they interact, the EB intercalates
(
into the DNA molecule generating an adduct that is fluorescent. This fluorescence may be
quenched if an added compound can displace the EB from the binding sites on the DNA
(
returning to the non-emissive states). We can see from Fig. 6 that all the complexes reduced
the fluorescence of the EB-DNA solution (indicated by the arrow).
Fig. 6. Left: Fluorescence spectra of the EB-DNA complex in the absence and presence of
increasing amounts 1,  = 520 nm, [EB] = 0.33 M, [DNA] = 10 M, [M] (M): 0 – 50 in 5 M
ex
increments. Arrow indicates the change upon increasing complex concentration. Right: Stern-
Volmer plots for all the complexes

ACCEPTED MANUSCRIPT
1
8
Using the Stern-Volmer equation (equation 2), the quenching constant for the various
complexes can be calculated.
F0

1K [Cu]1K  [Cu]
SV
q 0
F
(
2)
F0 and F are the fluorescence intensities of the ethidium bromide-DNA solution in the absence
and presence of the metal compound. KSV is the Stern-Volmer quenching constant. The
expected linearity of the plot (F /F vs. [Cu]) was observed for all three complexes with
0
4
-1
quenching constants on the order of 10 M (Table 2). A linear Stern-Volmer plot is generally
indicative of a single class of fluorophores, all equally accessible to quencher. This is not
unexpected in our system as the complex is essentially breaking up a fluorescent adduct
consisting of an intercalated EB molecule. The mechanism of this process can be collisional or
through complex formation. Collisional quenching of fluorescence occurs when the quencher
bumps into the fluorophore and deactivates the excited state. Static quenching occur as a result
of the formation of a non-fluorescent ground-state complex between the fluorophore and
quencher.
It should be noted that static quenching also results in linear Stern-Volmer plots and
therefore the observation of a linear Stern-Volmer plot does not prove that the fluorescence was
quenched via a collisional mechanism. Both mechanisms can be described by the Stern-Volmer
equation but the KSV would be best described as an association constant in the static case. [24]
Intensity measurements alone cannot differentiate dynamic or static processes; the true type of
quenching can be shown using lifetime measurements [24]. In our case, we can certainly
speculate that static quenching is indeed a possibility by looking the bimolecular quenching
constant, K in equation 2. Values of this parameter larger than the diffusion-controlled limit
q
usually indicate some type of binding interaction. Table 2 also display the values of K which is
q
built into the KSV and reflects the degree of quenching efficiency or the accessibility of the
fluorophores to the quencher. The size of the values, calculated by using  = 22 ns [25], are
0
11
-1 -1
10
-1 -1
about 10 M s which is larger than the limiting value of 10 M s [24].
To assess the strength of the binding, equation 3 was employed to calculate the
apparent binding constant.
KEB[EB]
Kapp

[Cu]50%
(
3)
6
-1
In this equation, KEB is the binding constant for ethidium bromide, taken as 1.2 x 10 M , [26]
and [Cu]50% is the concentration of the complex that causes a 50% reduction of the initial
4
-1
fluorescence. These values are shown in Table 2 and are on the order of 10 M which suggest

ACCEPTED MANUSCRIPT
1
9
that the complexes are weak to binders when one compares them to a classical intercalator. A
6
-1
typical intercalator like ethidium bromide has a binding constant of 3.0 x 10 M [27]. This is not
unexpected, as the complexes do not have the structural characteristics that are common for
intercalators. The similarity in values for KSV and Kapp lend support for the idea that complex
formation is a major player in the quenching of the fluorescence.
3.6 Interaction with human serum albumin
As the principal extracellular protein of the circulatory system, human serum albumin (HSA)
serves as the major transporter of drugs as well as endogenous compounds. It has been shown
that major pharmacokinetic parameters of drugs can be significantly altered because of their
binding to HSA. Since HSA serves as a transport carrier for drugs, it is important to study the
interactions of a potential drug with this protein. In this study, we investigated the binding of the
complexes to HSA using fluorescence spectroscopy. HSA has a well-known structure consisting
of a single polypeptide chain. The secondary structure has three homologous domains, each
consisting of two subdomains. Generally speaking, the main binding sites of the protein, site I
and site II, are located on subdomains IIA and IIIA. The primary structure of the protein has a
single tryptophan residue at position 214 (site I) along with a number of tyrosine and
phenylalanine residues. All three amino acids are emissive but tryptophan >> tyrosine >
phenylalanine with respect to fluorescent intensity [24]. Consequently, most of the intrinsic
fluorescence of HSA is due to the single tryptophan residue. While tyrosine residues may
contribute significantly to the protein fluorescence, by appropriate choice of the exciting
wavelength it is theoretically possible to isolate the tyrosine fluorescence. In addition, the
wavelength of the emitted light is a good indication of the environment of the fluorophore.
Tryptophan residues that are exposed to an aqueous environment, have maximal fluorescence
at a wavelength of about 340-350 nm; residues in the interior of the proteins, fluoresce at about
330 nm. Therefore, the fluorescence titration is a good investigative method for binding as this
emission can be attenuated by a small molecule interacting at or near the tryptophan; this amino
acid unit is quite susceptible to changes in its micro-environment.
Fig. 7(a) show the fairly large reduction in fluorescence intensity associated with a
solution of HSA upon addition of aliquots of complex 1. All the complexes display this behavior
(
ESI). This is indicative of some sort of binding interaction at or near the tryptophan residue. It
was also observed that there is a small red shift of about 0 - 6 nm for the complexes. It is
therefore likely that the interaction of the complexes with the protein has resulted in changes in
the secondary structure of the protein. It appears that the tryptophan residue is becoming more

ACCEPTED MANUSCRIPT
2
0
exposed to the polar environment. The strength of the binding may be quantified using the
Stern-Volmer equation (4).
F0

1K τ [Cu]1K [Cu]
q
0
D
F
,
(4)
K is the bimolecular quenching constant and τ is the lifetime of the fluorophore in the absence
q
0
of complex. The Stern-Volmer quenching constant is given by K = K τ a representation of the
D
q 0
Fig. 7. Emission spectra of HSA in the absence and presence of increasing amounts of 1,  =
ex
295 nm, [HSA] = 5.0 M and [1] (M): 0 – 22.5 in 2.5 M increments.
KSV if the quenching is known to be dynamic. KSV is the measure of the effectiveness of the
complex as a quencher.
As seen in Fig. 8, the Stern-Volmer plot shows a distinct upward curvature. For protein
solutions having homogeneous emission, upward curvature has been observed with a wide
variety of quenchers [24]. In fact, the fluorescence of most proteins is likely heterogeneous [29].
Since linearity of a Stern-Volmer plot is usually associated with the fluorophore possessing a
single binding site or multiple accessible binding sites, we suggest that deviation from linearity is
due to the occurrence of combined quenching sites and/or the existence of non-equivalent
(
different or same (accessible or non-accessible)) binding. Combined quenching implies that

ACCEPTED MANUSCRIPT
2
1
static quenching can occur simultaneously to the dynamic quenching mechanism that governs
the Stern-Volmer plot. The static contribution may be calculated from the following equation:
Fig. 8. Stern-Volmer plots for the interaction of the complexes HSA.
F0

(1K [Cu])(1K [Cu])
D
S
F
(
5)
In this equation, K and K are the dynamic and static quenching constants. This is a modified
D
S
form of the Stern-Volmer equation applicable when one is considering the same population of
fluorophores [24].


Fo

-1
Kapp

1 [Cu]
F0
F


 F

1K [Cu]
where
(6)
app
A plot of Kapp versus [Cu] yields a straight line with an intercept (I) of K + K and a slope (S) of
D
S
K K . Manipulation of these relationships, gives a quadratic equation
S
D
2
KS – K I + S = 0
(7)
S
We analyzed the data for the complexes according to this equation (5) [24] (Fig. 9). In all cases,
solutions to the quadratic equation are complex roots. Generally speaking, a completely
unambiguous assignment of KD and K requires fluorescence lifetime measurements [24].
S
Typically, for proteins the quenching constants are nearly identical [28].

ACCEPTED MANUSCRIPT
2
2
Fig. 9. Plot of the Stern-Volmer analysis according to equation (5)
As mentioned above, tryptophan amino acid residues have higher quantum yields than
the two other fluorescent amino acids. Despite this, tyrosine, because it is typically present in
larger numbers, may contribute significantly to the protein fluorescence. In the case of HSA with
the eighteen tyrosine residues, one cannot completely eliminate tyrosine emission. Therefore, it
is possible that there is more than one population of fluorophores accessible to the metal
complexes and each residue can be differently accessible. In the situation where there is one
population of fluorophores, one accessible and the other inaccessible to the quencher, deviation
from linearity of the Stern-Volmer relationship is also observed [24]. Under these circumstances,
the binding constant can be obtained from a modified Stern-Volmer (MSV) analysis [28].
F0
1
1
f


F-F0 fK[Cu]
(
8)
This allows estimation of the apparent binding constant K for the process, from the ratio of the
slope to intercept of a plot of F /(F – F ) vs. 1/[Cu]. This plot is shown in Fig. 10. The calculated
0
0
5
-1
5
-1
5
-1
5
-1
binding constants are 1.99 x 10 M , 2.02 x 10 M , 3.45 x 10 M and 1.89 x 10 M for 1, 2, 3,
and 4, respectively. These values indicate that the complexes are strong binders to the protein.

ACCEPTED MANUSCRIPT
2
3
Fig. 10. Plot of the modified Stern-Volmer equation
Another possible reason for the upward curvature may be that with increasing binding as
the concentration of the complexes increased, a change in the conformation of the protein
results in the exposure of previously inaccessible fluorophores [30,31].
3.7 Anti-oxidant assays
Free radicals, particularly reactive oxygen species (ROS), can have a deleterious effect
on cellular processes by, for instance, targeting biomolecules such as nucleic acids. The
adverse effects will arise if the cell’s natural anti-oxidant machinery is compromised so that it is
incapable of neutralizing excess free radicals. There are a number of methodologies available
for estimating the anti-oxidant capability of metal complex. One popular method is based upon
the use of the stable free radical diphenylpicrylhydrazyl (dpph). This radical can be reduced via
a reaction with a hydrogen atom donor. The reaction can be monitored by measuring the
change in absorbance of the reaction mixture at 520 nm. This is the wavelength of maximum
absorbance for the dpph molecule, that results from delocalization of the unpaired electron. The
scavenging capacity was calculated from the following equation (9):

Acontrol Asample

SC%
x100%




Acontrol
(
9)

ACCEPTED MANUSCRIPT
2
4
Acontrol is the absorbance of the control and Asample is the absorbance of the sample. All the
absorbances should be corrected for the blank. Fig. 11 show the variation of the scavenging
capacity with the concentration of the complexes. Generally speaking, there is a saturation
effect at the higher concentrations. The activities of the complexes are not spectacular; at the
highest concentration studied, percent scavenging capacity is only 13-21%. Complex 2 showed
the highest scavenging ability.
Fig. 11. Radical scavenging ability of the complexes by dpph assay (left) and nitric oxide assay
(
right)
Nitric oxide (NO) is another free radical that is an important chemical mediator with a
variety of physiological functions. Like the ROS, NO exhibit these effects at low concentrations.
At higher concentrations, it too can cause biological damage to tissues and organs. A number of
disease conditions can then result. Because it is a potent vasodilator, sodium nitroprusside,
Na [Fe(CN) NO], is a drug used to treat a variety of medical conditions including hypertension.
2
5
The mode of action of this drug is the basis of the assay, which is the use of an aqueous
solution that at physiological pH spontaneously generates nitric oxide. This nitric oxide interacts
with oxygen to produce nitrite ions, the amount of which can be estimated using Griess’ reagent.
Consequently, the presence of any species that can scavenge the nitric oxide radical will result
in reduced production of nitrite ions and a decrease in the absorption of such a mixture at 550
nm. The scavenging capacity was determined as above for dpph. The activity, from the
standpoint of scavenging capacity, is higher against the nitric oxide radical relative to dpph.
Complexes 1 and 3 show the highest activity of approximately 40% at the highest
concentrations.

ACCEPTED MANUSCRIPT
2
5
3.8 Antimicrobial assays
The antimicrobial activity of the compounds was studied with three protocols. The
ligands were tested against five ESKAPE pathogens – S. aureus (MRSA), E. coli, K.
pneumoniae (MDR), A. baumannii, and P. aeruginosa. These organisms are so called because
they can ‘escape’ the common antibacterial regimens. Table 3 shows the results of the
biological assays.
Table 3 Antimicrobial profile of the ligands and complexes
%
inhibition
npTSC
29.38
-8.98
-0.72
4.44
MnpTSC
17.16
-12.93
-7.59
EnpTSC PnpTSC
Staphylococcus aureus (MRSA)
Escherichia coli
Gram (+)
Gram (-)
Gram (-)
Gram (-)
Gram (-)
Yeast
12.92
-8.70
4.74
27.76
-5.23
19.90
7.86
Klebsiella pneumoniae (MDR)
Acinetobacter baumannii
Pseudomonas aeruginosa
Candida albicans
-9.14
-4.09
1.44
-19.37
9.81
2.27
1.57
2.75
3.58
1.03
Cryptococcus neoformans
Yeast
-13.48
-39.33
-37.11
-39.72
1
2
3
4
Pseudomonas aeruginosa
Klebsiella pneumonia
Staphylococcus aureus
Escherichia coli
21.9
39.5
15.8
34.7
92.2
35.0
2.86
54.7
51.5
6.25
46.3
34.2
45.8
17.8
75.4
Enterococcus faecalis
Bacillus cereus
93.1
17.6 (131)
69.7 (117)
64.3 (118)
1
, 2, 3, and 4 were tested at 22.9 M, 20.0 M, 20.0 M and 19.0 M respectively for P.
aeruginosa and K. pneumoniae. For the other bacteria, the tested concentrations were 21.7
M, 20.0 M, 19.4 M and 19.6 M for 1, 2, 3, and 4 respectively. For B. cereus the numbers

in parentheses are the concentrations at which the complex was tested.
It is obvious that the ligands are not active (defined as > 80%) against these bacteria with the
best compound being npTSC and PnpTSC against S. aureus. Negative inhibition values mean

ACCEPTED MANUSCRIPT
2
6
that the growth rate is higher compared to the control (bacteria only, set to 0% inhibition). The
complexes were tested against four of the same bacteria in addition to B. cereus. The first set of
experiments involved determining the susceptibility of the bacteria to the complexes using the
disc diffusion method and measuring the zones of inhibition. The only bacterial strain that
displayed any growth inhibition was S. aureus. (Fig. 12). The inhibition zones were 16 mm, 10
mm, 11 mm and 10 mm for 1, 2, 3, and 4, respectively. This would classify the bacteria as
resistant. In comparison, ampicillin, a clinically used antibiotic, exhibited a 39 mm zone of
inhibition. The vehicle for the complexes, DMSO, did not inhibit growth of the bacteria.
Fig. 12. Antibacterial activity of the complexes on the growth on S. Aureus as measured by the
disc diffusion method.
To determine if the concentration on the discs was insufficient to cause inhibition, an experiment
to determine the percent inhibition was done using a microdilution method. The results of this
study are presented in Table B. It was observed that there is no pattern to the reactivity and an
IC50 value could not be calculated. The values represent the inhibition at the lowest tested
concentration. A possible reason for this is the absorption characteristics of the complexes. At
the high or even medium concentrations, the absorption of the complexes at 600 nm (the
wavelength of the analysis), is significant enough to affect that from the growing bacteria. This
distorted the measurements leading to a lack of precision at these concentrations.
It is not exactly clear why the complexes or the ligands are not biological active.
However, we can speculate on some possible reasons. Drug-likeness, the likelihood that a

ACCEPTED MANUSCRIPT
2
7
compound will have useful therapeutic activity, is a qualitative concept used in drug design and
is predicted from the molecular structure. The quantitative structure activity relationships
(
QSAR) attempt to correlate biological activity with the physiochemical properties of a drug. The
properties are any structural, physical or chemical property of the drug. Probably the most
common of these properties is lipophilicity, which is represented by the partition coefficient log
P. Two other properties that are related to each other are molar refractivity (represented as
CMR: c = calculated) and the topological polar surface area (tPSA). The molar refractivity
represents size and polarizability of a molecule. It is also related to the London dispersive forces
that act in the drug-receptor interaction. The size of the molecule is also important. Most drugs
enter cell membranes by passive transport and the rate of passive transport depends on the
size of the molecules. The topological polar surface area (tPSA) is a convenient measure of the
molecular polar surface area and is correlated with passive molecular transport through
membranes and other drug transport properties. We have calculated, (using ChemDraw), the
values for these three parameters for the thiosemicarbazone ligands. We assume that the
complexes will have somewhat similar properties, likely bigger values since they include the
metal ion. As is expected, the size of the ligands increases with alkyl substitution on the amine
3
3
3
3
nitrogen. The CMR values are 6.945 cm /mol, 7.407 cm /mol, 7.873 cm /mol and 9.456 cm /mol
for npTSC, MnpTSC, EnpTSC and PnpTSC respectively.
Generally speaking, drugs should be designed with the lowest possible log P. For oral
bioavailability, this should be in the range −0.4 to +5.6. The calculated log P values for the
ligands were 0.22, 0.65, 0.86 and 2.21 for npTSC, MnpTSC, EnpTSC and PnpTSC respectively.
This pattern seems reasonable given the differences between the methyl, ethyl and phenyl
groups with respect to polarity. Compounds with higher log P values are non-polar, with lower
water solubility. The complexes should have higher log P values since there are two ligands and
this might be the real reason for their poor antimicrobial activity – their poor aqueous solubility.
This is likely true for the other properties as well.
The tPSA for the compounds show an interesting pattern. npTSC has a calculated tPSA
2
2
of 120.68 Å and the other three have the same value of 106.69 Å . It is interesting to note that
the tPSA is actually smaller for the larger molecules. According to Veber’s rule [32], these
values should be from 40 to 130. The complexes are expected to be bigger than the individual
ligands so the CMR and tPSA values are likely to be unfavorable. In fact, the calculated (from
2
2
2
the DFT geometry optimization) volume for the complexes are 170.68 Å , 144.20 Å , 143.47 Å
2
and 141.25 Å for 1, 2, 3 and 4 respectively. The values for the bigger complexes are at the top
limit of drug-likeness. Veber’s rules also suggest that molecules with hydrogen bonding

ACCEPTED MANUSCRIPT
2
8
capability (acceptors and/or donors) greater than ten would have poor oral bioavailability. All the
complexes possess sixteen hydrogen bond acceptors.
In addition to poor physiochemical characteristics, it is possible that the compounds do
not significantly interact with common microbial biological targets. To investigate this possibility,
we have computationally (molecular docking) studied the interaction of the complexes with two
macromolecules that are targets for a variety of clinically used antibacterials.
3.9 Molecular docking interaction of complexes with biological targets
The thiosemicarbazone ligands used in this study, with their benzodioxole moiety, has a
high degree of structural similarity to oxolinic acid, which is a member of the quinolone class of
antibiotics. The quinolone antimicrobials targets DNA gyrase (topoisomerase II) and
topoisomerase IV (Topo IV) [33-35]. These enzymes are related bacterial type II
topoisomerases that have significant roles in DNA replication and cellular growth; they catalyze
topological changes in the bacterial genome. The two enzymes have somewhat different roles
[
36] but are essential for bacterial growth. They also have susceptibilities to antibiotics that are
species dependent [9]. Despite the poor antibacterial profile of the ligands and complexes, we
thought it worthwhile to explore, through molecular docking models, to what extent the
complexes can bind to these important enzymes. We expected that useful information could be
obtained by looking at the binding constants along with the QSAR properties of the complexes.
The molecular docking simulations were performed using the X-ray crystallographic structures
of the DNA gyrase of Staphylococcus aureus, obtained from the Protein Data Bank (code 3G75)
along with that of the parE subunit of topo IV obtained from the same source (code 3LNU). In
the preparation of the topo II protein, the co-crystallized inhibitor was first removed from the
structure before docking with the complexes. In both cases, the runs were carried out on a rigid
(
protein) molecule. The optimized geometric forms of the complexes were used in the docking
simulations. The individual interactions were studied in order to obtain more detailed
information. An examination of the information in Table 4 below reveals the general trend that
the complexes bind stronger to the Topo IV than the DNA gyrase B. It is also interesting to note
that compound 4 binds the strongest to the DNA gyrase B and Topo IV out of all four
complexes, which is reflected by the most negative binding energy and the lowest dissociation
constant. A look at the binding interactions between all four complexes and the macromolecule
of interest provides further insight into the observed trends.
All of the complexes docked with DNA gyrase B, has the –NO group on at least one of
2
the TSC ligands playing an important role in the binding process. Figure A shows such

ACCEPTED MANUSCRIPT
2
9
interactions for complex 4. (The other complexes are shown in the ESI). The –NO group is
2
involved in conventional hydrogen bonding with a variety of amino acid groups present within
the enzyme. For example, complex 1 exhibits a total of three conventional hydrogen bonding
interactions: two between the –NO on the first TSC ligand and the –NH of TYR192 and one
2
between the –NO and the –NH group of ARG217. Both sets of interactions are 1.63 Å.
2
2
Complex 2 displays two conventional hydrogen bonding interactions between the –NO on
2
TSC1 and the protonated –NH group of LYS170 (2.08 Å and 1.70 Å). 3 displays a single
2
conventional hydrogen bonding
Table 4 Calculated binding energies and dissociation constants (kI) for the complexes with DNA
gyrase B and Topoisomerase IV
Macromolecular Target →
Compound ↓
DNA gyrase B
Topoisomerase IV
Binding
Binding
kI
kI
Energy
Energy
1
2
3
4
-7.05
-6.68
-6.29
-7.25
6.79
12.61
24.65
4.82
-8.06
-8.04
-7.3
1.23
1.27
4.42
0.37
-8.79
*Binding energies are reported in kJ/mol and dissociation constants are in micromolar units.
interaction between the –NO of TSC1 and the –NH of THR171 (1.78 Å). Lastly, for 4, which is
2
the complex demonstrating the strongest binding to DNA, there are a total of three conventional
hydrogen bonding interactions. One of these interactions is between an oxygen on the –NO of
2
TSC2 and a hydrogen on the five membered ring of HIS143 (1.93 Å). The other two interactions
are between the two oxygens on the –NO group of TSC1 and two hydrogens on the protonated
2
–
NH group of LYS170 (2.32 Å and 2.33 Å). It is observed that with increasing amount and
2
strength of hydrogen bonding interactions between the –NO group on the TSC ligand, the
2
binding with DNA gyrase B becomes more favorable.
Other interactions between the complexes and DNA gyrase B are worth noting. In
addition to conventional hydrogen bonding, the aromatic portion of the piperonal moiety present
in the TSC ligand also show various interactions that can explain the binding energies. For