Synthesis, characterization and bioactivity studies of new dithiocarbazate complexes

Article abstract of DOI:10.1039/d0nj01187h

A new compound of the dithiocarbazate family, 2,2′-((disulfanediylbis((ethylthio)methylene))bis(hydrazin-2-yl-1-ylidene))bis(2-oxo-1,2-dihydro-3H-indol-3-ylidene), and its five metal complexes are synthesized. All compounds are characterized by elemental and mass analysis, spectroscopic (FT-IR,¹H-NMR and¹³C-NMR) and TGA techniques. The crystal structure of the synthesized compounds is determined by single crystal X-ray diffraction analysis, which shows that the ligand acts as a tridentate chelate coordinated to Zn, Co and Ni ions in a distorted octahedral fashion and also as bidentate to Pt and Pd ions in a distorted square planar geometry. In order to investigate the compounds’ cytotoxicity, two human cancer cell lines of HeLa and MCF-7 are used and compared to the normal cell line of CHO. The clinical drug cisplatin is used as the standard drug. Fluorescence spectrophotometry is used to study the interaction of the compounds with human serum albumin (HSA) and parameters such as the Stern-Volmer quenching constant (K_SV), the number of binding sites (n), association constants (K_a) and free energy changes (ΔG) at 298 K are calculated.

Full text of DOI:10.1039/d0nj01187h

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Synthesis, characterization and bioactivity studies of new dithiocarbazate
complexes
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Zahra Yekke-ghasemi,a Mohammad Ramezani,b Joel T. Maguec and Reza Takjoo*a
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a Department of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran.
b Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences,
Mashhad, Iran.
c Department of Chemistry, Tulane University, New Orleans, LA 70118, USA.
Reza Takjoo
Associate Professor of Inorganic Chemistry
Department of Chemistry, Faculty of Sciences, Ferdowsi University of Mashhad, 91775-1436,
Mashhad, Iran.
Phone: +98 (51) 38805536
Fax: +98 (51) 38975457
E-mail: r.takjoo@um.ac.ir, rezatakjoo@yahoo.com
Homepage: http://r.takjoo.profcms.um.ac.ir/
ORCID ID: orcid.org/0000-0001-9969-9779 http://orcid.org/0000-0001-9969-9779
Researcher ID: E-5740-2015 http://www.researcherid.com/rid/E-5740-2015
1

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Abstract
A new compound of dithiocarbazate family, 2,2'-
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((disulfanediylbis((ethylthio)methylene))bis(hydrazin-2-yl-1-ylidene))bis(2-oxo-1,2-dihydro-3H-
indol-3-ylidene) and its five metal complexes are synthesized. All compounds are characterized
by elemental and mass analysis, spectroscopic (FT-IR, 1H-NMR and 13C-NMR) and TGA
techniques. Crystal structure of the synthesized compounds is determined by single crystal X-ray
diffraction analysis which shows the ligand acts as a tridentate chelate coordinated to Zn, Co and
Ni ions in a distorted octahedral fashion and also as bidentate to Pt and Pd ions in a distorted
square planar geometry. In order to investigate the compounds cytotoxicity, two human cancer
cell lines of Hela and MCF-7 are used and compared to normal cell line of CHO. The clinical
drug, cisplatin is used as the standard drug. Fluorescence spectrophotometry is used to study the
interaction of the compounds with human serum albumin (HSA) and parameters such as Stern–
Volmer quenching constant (KSV), the number of binding sites (n), association constants (Ka) and
free energy changes (ΔG) at 298 K are calculated.
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Keywords
Dithiocarbazate, X-ray crystallography, MTT assay, HSA-binding, Fluorescence
spectrophotometry.
1. Introduction
Cancer is a disease that occurs at the cell nucleus and affects the gene, but is not inherited and can
be acquired. The importance of metal complexes in biology and their role in controlling certain
diseases is found by scientists through understanding the operation mechanism of metalloproteins
and the role of metals in proteins active site. The most well-known complex of these kind is the
cisplatin (cis-diaminedichloroplatinum(II), cis-DDP or cisplatin). Researches show that this
complex is able to interfere with the DNA strand and its configuration prevents the DNA from
replicating.[1,2]
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Despite the profound effect of cisplatin in treatment of many cancers, today its use is limited. The
influence of cisplatin on some kind of cancers is very low or is almost ineffective.[3] In addition,
drug-resistant is one of the most important barriers to cisplatin action which can be inherent or
adventitious in the cell. For example, glutathione tripeptide (GSH, Glutathione) is the most
common intracellular non-protein thiol that is used as a protective agent against toxic substances
and oxidants. Coordination of cisplatin to GSH through thiols leads to inactivation of cisplatin
(and also GSH), thereby destroying its antitumor properties. Therefore, cisplatin side effects can
further appear, such as renal and neurological toxicity, bone marrow problems, hearing loss and
nausea. Today, in order to reduce side effects of the platinum drugs on nephrons, sulfur-containing
molecules are used in chemotherapy. Nitrogenous ligands are applied in metallodrugs synthesis to
improve the solubility of the drug in water.[4,5]
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Dithiocarbazate ligands, due to their special structure and presence of nitrogen and sulfur, show
extensive biological activity, including anticancer, antifungal, and antibacterial effects.[6] The
dithiocarbazate ligand is the product of hydrazine reaction with CS2 in the presence of a base such
as KOH and in all these compounds the NH2NHCSS- functional group is common.[7] In fact, these
compounds can attach to metal ions as bidentate chelates through azomethine nitrogen and
thioamide sulfur atoms to form a five-membered chelate ring. To increase the number of donor
atoms in the ligand structure, coordinated groups at suitable positions should be added. Knowledge
of metal ions coordination and redox reactions (electron or atom transfer), have a very important
role in the treatment of cancers.[8,9] Complexes based on main transition metals have recently
attracted attention because they can adopt numerous geometries including square-planar, square-
pyramidal, trigonal-bipyramidal and octahedral depending on the coordination number of the
metal ion. Although all these geometries are not available in organic drug compounds because
carbon cannot exceed coordination number of four, presence of the transition metal ions allows
this variation in molecular structure.[10,11]
Proteins and nucleic acids with negative charges are suitable ligands for binding to metal ions.
Since the transfer of drug into the body is carried out by the serum albumin protein, binding of a
ligand to albumin has been studied over the past 50 years. For example, investigation of the
interaction between the palladium complex and the human albumin serum shows there are 23
binding sites on the protein for the palladium complex. When all the protein surface is covered
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with palladium compounds, the protein loses its structure almost completely. The denaturation
process of protein structure is a cooperative process and hydrophobic interactions play a major
role in the process of linkage.[12,13]
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The goal of the present investigation is the synthesis and characterization of some novel
dithiocarbazate complexes in order to have more anticancer properties and fewer side effects than
cisplatin. Therefore, in the design process of the ligand, isatin is used for its biological properties
that include a range of actions in the brain tissue and the protection against certain types of
infection.[14,15] Cytotoxic activity of the compounds against Hela and MCF-7 cancer cells is
evaluated by MTT assay and compared to normal CHO cell. Moreover, fluorescence spectroscopy
is used as a powerful technique to study the ligand-protein interaction and to understand the
antitumor effects of the compounds more.
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2. Experimental
2.1. Materials and methods
All other material and solvents were purchased and used without purification. Melting points with
an electrothermal 9300 digital melting point apparatus were obtained. IR spectra in the region 400-
4000 cm−1 were recorded with a Thermos Nicolet model of Avatar 370 FT-IR. Elemental analyses
(CHNS) were performed with a Thermo Finnigan Flash 1112EA elemental analyzer. The EI-mass
spectra were carried out using a 5975C VL MSD with Tripe-Axis Detector instrument at 70 eV.
1H- and 13C-NMR spectra of compounds were recorded on a Bruker FUM-300 spectrometer using
DMSO-d6 as solvent. TGA analysis was performed with a TGA-50 SHIMADZU instrument at a
heating rate of 10 ºC/min under air atmosphere from ambient temperature to 950 ºC.
For biology studies, human serum albumin (HSA 99%, fatty acid free) from Sigma Chemical
Company, RPMI-1640 medium and fetal bovine serum (FBS) from GIBCO (Gaithersburg, USA),
penicillin and streptomycin from Biochrom AG (Berlin, Germany) were purchased.
2.2.
Synthesis
of
2,2'-((disulfanediylbis((ethylthio)methylene))bis(hydrazin-2-yl-1-
ylidene))bis(2-oxo-1,2-dihydro-3H-indol-3-ylidene (L)
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Potassium dithiocarbazide was obtained by increasing the dropwise of carbon disulfide (7.75 g,
102 mmol) to hydrazine hydrate (5.00 g, 100 mmol) in the present of potassium hydroxide (6.00
g, 107 mmol), water (6 mL) and 25 mL of cold ethanol with stirring and cooling in an ice-salt bath
for one hour. Ethyl bromide (10.35 g, 94.98 mmol) was added to the above mixture over 30
minutes. A two-phase mixture was obtained with adding of cold distilled water (60 mL) to the
solution. Isatin (7.03 g, 40.37 mmol) solution in 30 mL of 75% ethanol, at room temperature, was
added to the separated organic phase. The resulting orange precipitate was filtered off, washed
with water and dried in a vacuum desiccator over silica gel.
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Orange block-like crystals were obtained after recrystallization from ethanol, Yield: 5.887 g,
23.4% (based on the mass of the oily organic phase), m.p.: 210 °C. Anal. Calc. for C22H20N6O2S4
(528.38 g mol-1): C, 49.98; H, 3.81; N, 15.90; S, 24.26. Found: C, 49.47; H, 4.18; N, 16.54; S,
24.84%. IR (KBr), cm-1: ν(N―H) 3185 m, ν(C=O) 1688 s, ν(C=N) 1621 s, ν(N―N) 1149 w. Mass
spectrometry, m/z (%): 267 (4), 263 (85), 235 (100), 145 (84), 131 (83), 90 (87), 77 (95), 30 (86).
1H-NMR (300 MHz, DMSO-d6, 295 K) δ ppm: 1.335 (t, 6H, J = 7.39 Hz, CH3), 3.248 (q, 4H, J =
7.40 Hz, CH2), 1.075 (t, 3H, J = 6.99 Hz, CH3), 3.473 (q, 2H, J = 6.70 Hz, CH2), 6.940 (td, 2H, J
= 7.80, 0.80 Hz, H-4 and H-15), 7.089 (td, J = 7.59, 0.97 Hz, 2H, H-5 and H-16), 7.405 (td, J =
7.73, 1.27 Hz, 2H, H-6 and H-17), 7.519 (dd, J = 7.57, 1.14 Hz, 2H, H-3 and H-14), 11.346 (s, 2H,
H-N1), 13.906 (s, 2H, H-N2). 13C-NMR (75.6 MHz, DMSO-d6, 296 K) δ ppm: 13.88 (C11); 28.49
(C10); 19.02 (C23); 59.53 (C24); 111.86 (C2); 123.22 (C3); 121.54 (C4); 132.80 (C5); 119.70
(C6); 143.77 (C7); 135.89 (C8); 162.83 (C9), 200.47 (C1).
2.3. Synthesis of the metal complexes
A general method of preparation for all metal complexes was employed.
The appropriate metal salt (1 mmol) was dissolved in ethanol (4 mL) and mixed with a boiling
solution of the L (2 mmol) in ethanol (4 mL). The mixture was then heated on a water bath for 1
hour. The product that formed was filtered off, washed with ethanol and dried in a desiccator over
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2.3.1.
Synthesis
of
bis(S-ethyl-2-((2-oxo-1,2-dihydro-3H-indol-3-
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ylidene)dithiocarbazato)zinc(II) (1)
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The complex 1 was synthesized by reaction of L and Zn(OAc)2.2H2O. Suitable crystals for X-ray
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analysis were obtained from recrystallization of the precipitate in ethanol.
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Red column-like crystals, Yield: 0.180 g, 84% (based on metal salt). m.p. > 300 °C. Anal. Calc.
for C22H20N6O2S4Zn (594.05 g mol-1): C, 44.48; H, 3.39; N, 14.15; S, 21.59. Found: C, 44.73; H,
3.70; N, 13.24; S, 20.29%. IR (KBr), cm-1: ν(N―H) 3195 m, ν(C=O) 1700 s, ν(C=N) 1611 s,
ν(N―N) 1095 w. 1H-NMR (300 MHz, DMSO-d6, 295 K) δ ppm: 1.429 (t, 6H, J = 7.39 Hz, CH3),
3.226 (q, 4H, J = 7.40 Hz, CH2), 7.058 (d, 2H, J = 7.83, H-3 and H-14), 7.208 (t, J = 7.60 Hz, 2H,
H-4 and H-15), 7.506 (t, J = 7.69 Hz, 2H, H-5 and H-16), 8.104 (d, J = 7.53 Hz, 2H, H-6 and H-
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17), 11.284 (s, 2H, H-N1). C-NMR (75.6 MHz, DMSO-d6, 296 K) δ ppm: 14.63 (C11); 27.35
(C10); 112.51 (C2); 128.15 (C3); 123.90 (C4); 134.78 (C5); 116.33 (C6); 144.79 (C7); 141.61
(C8); 166.67 (C9), 198.04 (C1).
2.3.2.
Synthesis
of
bis(S-ethyl-2-((2-oxo-1,2-dihydro-3H-indol-3-
ylidene)dithiocarbazato)cobalt(II) (2)
The complex 2 was synthesized by reaction of L and Co(OAc)2.4H2O. Suitable crystals were
grown by slow evaporation of the mother solution for 2 days in the fridge.
Dark orange crystals, Yield: 0.143 g, 68% (based on metal salt). m.p. > 300 °C. Anal. Calc. for
C22H20CoN6O2S4 (587.62 g mol-1): C, 44.97; H, 3.43; N, 14.30; S, 21.82. Found: C, 44.92; H, 3.44;
N, 11.82; S, 19.28%. IR (KBr), cm-1: ν(N―H) 3174 m, ν(C=O) 1671 s, ν(C=N) 1615 s, ν(N―N)
1114 w.
2.3.3.
Synthesis
of
bis(S-ethyl-2-((2-oxo-1,2-dihydro-3H-indol-3-
ylidene)dithiocarbazato)nickel(II) (3)
The complex 3 was obtained by reaction of Schiff base and Ni(OAc)2.4H2O. Suitable crystals were
grown by slow evaporation of the mother solution for 6 days in the fridge.
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Dark orange column-like crystals, Yield: 0.156 g, 71 % (based on metal salt). m.p. > 300 °C. Anal.
Calc. for C22H20NiN6O2S4 (587.39 g mol-1): C, 44.99; H, 3.43; N, 14.31; S, 21.83. Found: C, 45.40;
H, 3.57; N, 12.78; S, 22.76%. IR (KBr), cm-1: ν(N―H) 3170 m, ν(C=O) 1672 s, ν(C=N) 1615 s,
ν(N―N) 1124 w.
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2.3.4.
Synthesis
of
bis(S-ethyl-2-((2-oxo-1,2-dihydro-3H-indol-3-
ylidene)dithiocarbazato)platinum(II) (4)
The complex 4 was obtained by reaction of L and K2Pt(S(CH3)2)2. Suitable crystals were obtained
from recrystallization of the precipitate in acetonitrile.
Dark red column-like crystals, Yield: 0.250 g, 90% (based on metal salt). m.p. > 300 °C. Anal.
Calc. for C22H20PtN6O2S4 (723.77 g mol-1): C, 36.51; H, 2.79; N, 11.61; S, 17.72. Found: C, 37.77;
H, 2.38; N, 10.04; S, 20.97%. IR (KBr), cm-1: ν(N―H) 3194 m, ν(C=O) 1720 s, ν(C=N) 1612 s,
ν(N―N) 1064 w. 1H-NMR (300 MHz, DMSO-d6, 295 K) δ ppm: 1.444 (m, 9H, CH3 cis/trans),
3.358 (m, 6H, CH2 cis/trans), 6.683 (d, J = 7.87 Hz, 1H, H-6 and H-17 trans), 6.794 (d, J = 7.79
Hz, 2H, H-6 and H-17 cis), 6.882 (td, J = 7.69, 0.98 Hz, 1H, H-4 and H-15 trans),7.069 (td, J =
7.70, 1.03 Hz, 2H, H-4 and H-15 cis), 7.396 (td, J = 7.77, 1.24 Hz, 1H, H-3 and H-14 trans), 7.476
(td, J = 7.71, 1.28 Hz, 2H, H-3 and H-14 cis), 8.130 (d, J = 7.75 Hz, 1H, H-5 and H-16 trans),
8.234 (d, J = 7.96 Hz, 2H, H-5 and H-16 cis), 10.458 (s, 1H, H-N1, trans), 10.916 (s, 2H, H-N1,
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cis). C-NMR (75.6 MHz, DMSO-d6, 296 K) δ ppm: trans: 14.70 (C11); 30.01 (C10); 111.05
(C2); 128.39 (C3); 121.47 (C4); 135.36 (C5); 117.80 (C6); 151.08 (C7); 144.48 (C8); 158.95 (C9),
185.13 (C1); cis: 15.18 (C11); 30.62 (C10); 111.15 (C2); 130.36 (C3); 122.90 (C4); 135.45 (C5);
119.27 (C6); 152.78 (C7); 145.33 (C8); 163.22 (C9), 188.34 (C1).
2.3.5.
Synthesis
of
bis(S-ethyl-2-((2-oxo-1,2-dihydro-3H-indol-3-
ylidene)dithiocarbazato)palladium(II) (5)
The complex 5 was obtained by reaction of L and PdCl2. Suitable crystals were obtained from
recrystallization of the precipitate in acetonitrile.
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Red plate-like crystals, Yield: 0.157 g, 73% (based on metal salt). m.p. > 300 °C. Anal. Calc. for
C22H20PdN6O2S4 (635.08 g mol-1): C, 41.61; H, 3.17; N, 13.23; S, 20.19. Found: C, 41.62; H, 2.95;
N, 10.99; S, 20.29%. IR (KBr), cm-1: ν(N―H) 3194 m, ν(C=O) 1721 s, ν(C=N) 1614 s, ν(N―N)
1056 w. 1H-NMR (300 MHz, DMSO-d6, 295 K) δ ppm: 1.428 (t, 6H, J = 7.30 Hz, CH3), 3.300 (q,
4H, J = 7.28 Hz, CH2), 6.823 (d, 2H, J = 7.77 Hz, H-5 and H-16), 7.074 (td, 2H, J = 7.71, 1.05
Hz, H-4 and H-15), 7.473 (td, 2H, J = 7.71, 1.30 Hz, H-3 and H-14), 8.122 (dd, 2H, J = 7.82, 1.28
Hz, H-5 and H-16), 10.924 (s, 2H, H-N1). 13C-NMR (75.6 MHz, DMSO-d6, 296 K) δ ppm: 14.62
(C9); 29.93 (C11); 111.26 (C2); 130.31 (C3); 122.97 (C4); 135.32 (C5); 118.21 (C6); 151.11 (C7);
144.76 (C8); 163.59 (C10), 188.00 (C1).
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2.4. X-ray Crystallography
Suitable crystals of L, L' and 1-5 were attached on polymer loops with a drop of heavy oil and
placed in a cold nitrogen stream on a Bruker Smart APEX CCD diffractometer. Full spheres of
data were collected under control of the APEX3 program suite.[16] The raw data were converted to
F2 values with SAINT[16] and empirical absorption corrections as well as merging of equivalent
reflections were performed with SADABS[16]. The structures were solved by direct methods
(SHELXT[17]) and refined by full-matrix, least-squares procedures (SHELXL[18]). Hydrogen atoms
attached to carbon were placed in idealized positions with displacement parameters tied to those
of the attached atoms. In the case of L, H-atoms attached to oxygen was placed using an AFIX 147
instruction. For N―H, fixed the bond lengths at 0.91 Å. All were included as riding contributions
with isotropic displacement parameters tied to those of the attached atoms. Crystals and refinement
details are presented in Table 1.
2.5. Biology studies
2.5.1. Cytotoxicity activity
The cytotoxic activity of compounds was determined by MTT assay against the human tumor cell
lines Hela and MCF-7 and the normal cell line CHO. In order to compare the cytotoxic properties
of these compounds with commonly used drugs in chemotherapy, the cytotoxicity of cisplatin was
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also studied as a reference. So, 25 cm2 tissue culture flasks were used to grow the cells in an
incubator at 37 ºC in a humidified atmosphere consisting of 5% CO2 and 95% air. A culture
medium of RPMI 1640, 10% (v/v) fetal bovine serum, 100 (U/ml) penicillin, and 100 μg/mL of
streptomycin were used to maintain the cells. 100 µL volumes of cell solutions were seeded into
96-well plates containing diverse concentrations of L and its complexes and the mixtures incubated
for 48 h. After 48 h, 20 µL of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide] solution (final MTT concentration 5 mg/ml in phosphate buffered saline) was added and
the samples incubated for 4 h. Finally, 100 µL of DMSO is added to each well to dissolve the
purple formazan formed. The absorbance (A) of all samples at 570 nm wavelength was measured
by an ELISA plate reader (TECAN infinite M200, Switzerland) at 570/630 nm. Experimental
conditions were performed in four replicates. IC50 is concentration required to reduce the
absorbance by 50% compared to the controls after exposure to the test sample for 72 h. The IC50
values of each test sample were determined by the dose dependence of surviving cells. The
statistical significance of data was determined by using GraphPad Prism 8.0 software (GraphPad
Software, Inc.).
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2.5.2. HSA-binding studies
When a fluorescent specimen is present, direct detection of the fluorometer is possible using a
spectrometer that is considered for excited and observed appropriate wavelengths. The intrinsic
fluorescence intensity of tryptophan-214 amino aside of human albumin protein in the presence of
different concentrations of compounds (0-16 μM) and 15 μM concentration of protein in a Tris
buffer (pH 7.4) with a spectrophotometer device at a temperature of 298 K were measured. The
exact concentration of HSA was determined spectrophotometrically using the molar extinction
coefficient of 35700 M-1cm-1. Samples were prepared by dissolving each compound in DMSO
with final concentration of 1% of the total volume. The thickness of the quartz cell in the test, 1
cm and the width of the slit used for excitation and emission states were 5 and 5, respectively. The
excitation wavelength was 280 nm and the emission wavelength were recorded range of 285-500
nm.
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3. Results and Discussion
5
6
7
8
The L and complexes are obtained through the mentioned methods in Section 2. The schematic
synthesis of compounds is presented in Scheme 1. All compounds characterized by spectroscopic
(FT-IR, 1H-NMR and 13C-NMR) and TGA techniques. The crystal structure of the compounds is
determined by single crystal X-ray diffraction analysis as discussed below.
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12
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20
21
22
23
24
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32
33
34
35
36
37
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46
47
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49
50
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53
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60
H
H
N
SK
N
S
KOH
EtOH
Et-Br
KBr
H2N
H2N
CS2
H2O
H2N
NH2 .H2O
S
S
S
N
HN
N
HN
S
O
O
M=Zn,
M
O
O
S
Co,
N
Ni
NH
N
S
S
N
HN
N
Pd
S
S
t,
O
O
M'=P
S
N
NH
N
N
HN
N
S
S
O
M'
N
O
S
N
NH
S
Scheme 1. The schematic synthesis of the ligand and complexes
3.1. IR and NMR studies
The IR spectrum of the free ligand (L) do not display the ν(SH) band at ∼2500 cm–1 (Fig. SI 1a).
Also, ν(C=S) band appears at 1222 cm–1 that confirm the L in the solid state remains in the thioketo
form.[19]
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The ν(NH) band of isatin fragment of the L is detected at 3185 cm–1. A high intensity band
observed at 1688 cm–1 can be assigned to ν(–C=O) fragment of the amide within the lactam ring[20]
that for the complexes show red-shift in relating to the free ligand and confirm the binding of
ligand to the metal via amido oxygen.[21] The ν(–C=N) band in dithiocarbazates observed at ∼1600
cm–1. Because of the conjugation of the double bond with the ring and the amido oxygen, the
vibration transformed to the high frequencies and observed at 1621 cm–1.[22] This imine band shows
a red-shift after complexation compared to the free ligand, which confirms the coordination of the
ligand to the metal through azomethine nitrogen atom.[23] The shift of ν(–C–S) band in the free
ligand (1040 cm–1) to lower frequencies after complexation also indicates coordination of the
sulfur atom to the metal ion (Fig. SI 1b-f).[24] The blue-shift of N–N band in complexes regard to
the free ligand (1149 cm–1) is another evidence of coordination of the ligand to metal.[25]
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21
22
23
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35
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45
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The 1H- and 13C-NMR spectra of compounds are carried out in DMSO-d6 (Fig. SI 2a-d and SI 3a-d
respectively). The ring protons signals of compounds are observed as multiplet signals in the range
of 6.5–8.2 and 111-145 ppm in the 1H and 13C-NMR spectra respectively.[26-28]
1
In the H-NMR spectrum of the L, the sharp signals at 11.35 and 13.90 ppm respectively are
assigned to isatin (-N1―H) and azomethine (-CH―N) protons. The signal of isatin proton (-
N1―H) shifts up-field in the complexes and in the case of Pt complex the satellites appear around
this signal correspond to the coupling of this proton with 195Pt isotope at a ratio of 1:4:1.[8,29] For
Pt complex, the average ratio of the cis and trans isomers signals indicates the presence of %28 of
the trans isomer to %72 of the cis isomer. The absence of azomethine proton signal in the spectra
of the complexes confirms that coordination occurs through the azomethine nitrogen atom. The
triplet and quartet signals correspond to the ethyl protons which are observed in the ranges 1.2-1.4
and 3.2-3.5 ppm with 6 (CH3) and 4 (CH2) integrals respectively.[30] Also, the ligand is
accompanied by one ethanol molecule that its protons resonated in 1.07 and 3.47 ppm respectively
as quartet and triplet.
In the 13C-NMR spectra of the L, amidic carbon signal of isatin resonates at 200 ppm which shifts
up-field in Zn complex and confirms coordination through the amido oxygen atom. Because of the
low solubility of 2 and 3, 13C-NMR spectra were not obtain for them (See Experimental section
for more details about the other carbons).
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3.2. Thermogravimetric analysis
7
8
9
TGA and DTG curves of complexes 1-5 (Fig. SI 4a-e) are carried out over a temperature range
from room temperature up to 950 °C. TGA and DTG curves of all complexes except Zn complex
show two thermal decomposition stages.
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The data of thermogravimetric analysis show that decomposition of 1 occurs in three steps (two
fast and one slow stages). The degradation of isatin fragment of the L' occurs in the first and second
steps with a mass loss of 42.66% (Calc. 44.15%) at a temperature range of 255-455 °C. In the last
step, the L' residue is removed by further heating to 947 °C (34.83% mass loss; Calc. 39.40%).
Finally, ZnO remains as the final product with a mass of 19.20% (Calc. 21.80%).[31]
For complexes 2-5, thermal decomposition occurs through two stages. The organic part of these
complexes are removed in two steps and a final product remains. In 2, CoO2 with mass of 14.49%
(Calc. 15.47%)[32] and in 3, NiO with a mass of 15.33% (Calc. 15.47%) remain as the final
product.[33,34] For 4 and 5, Pt metal and PdS[35,36] remains as the final products with a mass of
27.23% (Calc. 26.98%) and 20.00% (Calc. 21.80%) respectively.
3.3. X-ray crystal structures
Molecular structure and atom-numbering scheme for the L, L' and all five complexes are
determined. The L' crystals obtained as the only product in reaction of L with vanadium metal.
Crystal data and structure refinement details are tabulated in Table 1.
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Table 1. Crystal and refinement details for the ligands and complexes 1-5
L
L'
1
C22H20N6O2ZnS4
1920804
2
C22H20CoN6O2S4
1920805
3
C22H20N6NiO2S4
1920806
587.39
4
5
C22H20N6O2PdS4
1920808
635.08
Chemical formula
C46H46N12O5S8
1920802
C11H11N3OS2
1920803
C22H20N6O2PtS4
1920807
CCDC No.
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13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
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31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
Mr (g/mol)
1103.43
265.35
594.05
587.61
723.77
Crystal system
Triclinic
Orthorhombic
P212121
Monoclinic
C2/c
Monoclinic
P21/c
Monoclinic
C2/c
Orthorhombic
Pbca
Monoclinic
P2/c
Space group
1
P
Unit cell (Å, °)
a
8.4393(16)
11.765(2)
14.287(3)
86.938(3)
81.286(3)
74.585(2)
1351.6(4)
1
4.5172(2)
13.5286(5)
19.8833(7)
90
17.0306(6)
15.5145(6)
10.2986(4)
90
15.820(4)
15.920(4)
10.324(2)
90
16.812(2)
15.539(2)
10.2873(14)
90
22.002(5)
10.401(3)
22.111(5)
90
8.555(2)
21.607(6)
13.272(4)
90
b
c
α
β
90
113.411(2)
90
108.978(3)
90
112.258(2)
90
90
98.413(4)
90
γ
V (Å3)
Z
90
90
1215.10(8)
4
2497.10(17)
4
2459.0(10)
4
2487.2(6)
4
5060.(2)
8
2426.9(11)
4
Mo/Kα
Mo/Kα
(λ = 0.71075 Å)
1.450
Cu/Kα
Mo/Kα
(λ = 0.71075 Å)
1.587
Mo/Kα
(λ = 0.71075 Å)
1.569
Mo/Kα
(λ = 0.71075 Å)
1.900
Mo/Kα
(λ = 0.71075 Å)
1.738
Radiation type
(λ = 0.71075 Å)
1.356
(λ = 1.54178Å)
1.580
ρ (g cm-1)
μ (mm-1)
T (K)
0.386
3.871
4.775
1.071
1.149
5.909
1.143
100(2)
150(2)
552
150(2)
100(2)
100(2)
100(2)
2816
95(2)
F(000)
574
1216
1204
1208
1280
No. of refls.
R1 [I ≥ 2σ (I)]
R1 (all data)
wR2
12852
9302
17812
23075
11535
45382
0.0402
0.0785
0.0799
22752
0.0624
0.0461
0.0473
0.1162
0.0757
0.0405
0.0622
0.0942
0.0421
0.0686
0.1003
0.0630
0.0909
0.1537
0.1036
0.1100
0.1506
0.1783
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GOF on F2
1.011
1.094
1.042
0.987
0.994
1.005
0.982
3.075, -1.803
Largest diff.
peak/hole (e Å−3)
0.676, -0.561
1.406, -0.411
0.906, -0.706
0.912, -0.411
0.744, -0.304
2.293, -0.769
6
7
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23
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X-ray crystallography data reveal that L is constructed by connection of the two monomer ligands
(L') through S―S sulfur atoms (Fig. 1). This bond length is 2.047(1) Å which is close to the length
of the bond in other similar compounds.[36-38] Also, mean planes of dithiocarbazate fragment (N2-
N3-C9-S1-S2 and N5-N6-C20-S3-S4) form a dihedral angle of 88.85º indicating that the two L'
fragments are almost perpendicular to each other. Selected bond lengths and angles are listed in
Table 2.
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48
49
50
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53
54
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56
57
58
59
60
Figure 1. The molecular structure of L and L'. Displacement ellipsoids at the 50% probability
level.
In the structure of the free ligand (L), an ethanol molecule is connected to L via O3i―H3Ai···O1
(2.111(2) Å) hydrogen bond (symm. code i: -x,1-y,1-z) (Fig. 2).
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Figure 2. The hydrogen bonding of the ethanol molecule to the free ligand (L).
The L molecules connect to each other through N4―H4A···O2ii (1.894(4) Å) (symm. code ii: -x,-
y,1-z) hydrogen bonds and form one-dimensional chain in the direction of [-1 -2 1] vector. The
C5―H5···S4iii (symm. code iii: 2-x,1-y,-z) hydrogen bonds create the second dimension in
direction of [-1 1 0]. Finally, the third dimension expands along the [-1 0 0] vector that is through
the C15―H15···O3iv (symm. code iv: 1+x,y,z) hydrogen bond (Fig. SI 5a-c).
The S―S bond of the L breaks through the complexation reaction and the initial structure of the
free ligand (L') appears as in figure 1. An intramolecular hydrogen bonding of N3―H3A···O1
(1.978(6) Å) which creates a six-membered ring consists of O1-C1-C8-N2-N3-H3A exists in the
structure with 1(6) graph set (Fig. 1). This ring is completely planar and is aligned with the isatin
S1
aromatic ring.
The Lꞌ is planar and only the ethyl group with an 84.62º angel is located perpendicular to the
dithiocarbazate fragment plan (N2-N3-C9-S1-S2). The hydrogen bond of N1―H1···Ov (1.911(4)
Å) (symm. code v: 1/2+x,-1/2-y,1-z) along the a direction creates a one dimensional ribbon. This
ribbon simultaneously develops the second and third dimensions through C11―H11B···O1vi
(2.511(6) Å) (symm. code vi: 1-x,1-y,1-z) interaction in the bc plane (Fig. SI 5d-e).
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The Lꞌ as a bidentate ligand chelates to the Pt and Pd through the azomethine nitrogen and the
thioamide sulfur atoms in 4 and 5 respectively. However, the L' as a tridentate chelate coordinates
to Zn, Co, Ni through the isatin amidio oxygen, the azomethine nitrogen and the thioamide sulfur
atoms in 1-3. In the process of L' to metal coordination, a 180° rotation occurs around the N2―N3
bond which alter the configuration of the N2 and S1 atoms from cis to trans in L' regard to
complex. Also, C9=S1 and C9―N3, double and single bonds in L' convert to single and double
bonds after coordination respectively. N2―N3 bond in 4 and 5 is longer than 1-3.
The complexes 1-3 are six-coordinated and exhibit a distorted octahedral configuration, while 4
and 5 are four-coordinated and exhibit a distorted square planar geometry. Selected bond lengths
and angles are listed in Table 2.
Table 2. Selected bond lengths (Å) and angles (º).
L
L'
1
2
3
4
5
25
C9―S1
26
1.720/1.722(3)
1.724/1.717(2)
1.331/1.334(3)
1.301/1.304(3)
1.246/1.244(3)
0.91
1.655(4)
1.719(6)
1.325(8)
1.286(8)
1.229(7)
0.91
1.704(3)
1.345(3)
1.297(3)
1.233(3)
0.91
1.729/1.727(6)
1.303/1.321(7)
1.302/1.308(7)
1.230/1.223(6)
0.91
1.724/1.728 (5)
1.296/1.297(6)
1.288/1.295(5)
1.221/1.214(6)
0.91
C9―N3
27
1.297/1.289(4)
1.356(5)
28
C8―N2
1.284/1.281(4)
1.305(5)
29
O1―C1
30
1.225/1.221(4)
1.242(5)
N1―H1
0.81/0.92(4)
0.94(5)
31
32
N2―N3
1.396/1.384(3)
1.351(5)
1.366(6)
1.507(8)
2.061(5)
1.362/1.360(2)
1.492/1.488(3)
2.0635(19)
1.354(3)
1.495(3)
1.999(2)
1.389/1.373(6)
1.514/1.505(7)
2.063(4)
1.387/1.400(5)
1.496/1.516(6)
2.143(4)
33
C1―C8
1.514/1.511(4)
1.511(4)
34
M1―N2
-
-
35
36M1―N5
-
-
2.0573(18)
2.058(5)
2.135(4)
37
38
M1―S1
-
-
2.3505(16)
2.3591(7)
2.3584(7)
2.2609(14)
2.2569(15)
-
2.2347(13)
2.2458(13)
-
M1―S3
-
-
2.3707(7)
39
M1―O1
-
-
-
-
2.530(5)
-
2.2414(16)
2.2180(18)
-
40
41
M1―O2
2.3037(15)
42
C8―N2―N3
113.5(2)
109.7(3)
121.2/121.5(2)
100.78(16)
-
116.8(3)
120.5(3)
119.7(3)
101.92(19)
-
118.9(5)
113.0(5)
129.6(4)
103.4(3)
145.4(3)
119.49/118.69(18)
110.82/118.69(17)
128.37/127.80(17)
104.08 (11)
119.6(2)
111.1(2)
127.88(19)
103.34(13)
165.58(12)
114.2/112.3(4)
113.1/114.3(4)
124.9/121.9(4)
104.9(3)
113.2/112.5(4)
112.5/112.7(4)
126.5/126.7(4)
103.0(2)
43
N2―N3―C9
44
45
N3―C9―S1
C9―S2―C10
163.01(7)
102.04(18)
110.97(14)
-
-
83.82/115.69(13)
81.21/113.58(5)
107.15(6)
82.04/167.61(13) 81.37/164.70(11)
170.42/82.45(13) 166.01/81.86(10)
-
-
-
-
-
-
-
-
-
105.52/80.66(5)(5)
78.85/91.98(6)
86.02/78.16(6)
-
78.47(18)
-
80.60(7)
-
-
-
-
-
53
O2―M1―N2/N5
54
O1―M1―S1/S3
55
56
57
58
59
60
88.42/154.19(12)
158.95/91.69(4)
90.49/161.75(5)
-
-
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O2―M1―S1/S3
S1―M1―S1
-
-
-
-
-
92.79/156.58(5)
102.46(3)
-
-
-
113.58(9)
100.56(4)
95.37(6)
87.03(5)
6
7
8
9
All the mononuclear complexes consist of the deprotonated L' ligand and metal ions in the +2
oxidation state. Coordination of each tridentate ligand to the metal creates two five-membered
chelate rings in complexes 1, 2 and 3 (Fig. 3). The mean planes of these chelating rings are not
planar and therefore reveals coordination of Lꞌ to the central metal is accompanied by steric strain.
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48
49
50
51
52
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56
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59
60
Figure 3. The molecular structure of complexes 1-3, showing displacement ellipsoids at the 50%
probability level.
In 1, 2 and 3, the ligands are perpendicular (82.65˚, 81.40˚ and 84.53˚ respectively) to each other
and the three dentate ligands are meridionally disposed to O and S trans located. The coordinated
oxygen and sulfur atoms of the two ligands are in the cis position, while the nitrogen atoms are
trans to each other.
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In these three complexes, one-dimensional chains expand in the c direction through pairwise
hydrogen bonds of N1―H1···O1/O2 and C3―H3···N3.in 1, 2 and 3 which can create a nine-
membered ring N1-H1-O1-M1-N2-N3-H3-C3-C2 with 1(9) graph set (Fig. SI 5f-h). The
S1
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hydrogen bond of C11―H11A···C11 form the second dimension along the a axis in all the three
complexes which has the longest and shortest in 3 (2.927(3) Å) and 1 (2.799(9) Å) respectively
(Fig. SI 5i-k). Likewise, the third dimension is completed through C5―H5···S1 interaction in the
b direction and the length of this bond is the longest in 3 (3.100(7) Å) and shortest in 2 (3.045(9)
Å) (Fig. SI 5l-n).
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The mononuclear complexes of four-coordinate platinum and palladium with bidentate ligands of
SN, ON and OS have 50 and 25% cis isomers, respectively (CSD; version 5.40, updated to
November 2018).[39] In complexes 4 and 5 (Fig. 4), cis orientation of thioamide sulfur and
azomethine nitrogen atoms to each other is the result of the bidentate ligand coordination to the
metal ion similar to the previous report.[40]
The largest and smallest bonding angles around the central metal in 4 belong to N5―M1―S1 and
N2―M1―S1 by 170.42(13)˚ and 82.04(13)˚ and in 5 is 166.01(10)˚ and 81.37(11)˚, respectively.
Also, the longest and shortest bonding distances around the central metal are M1―S1/S3
(2.2609(14), 2.2569(15) Å), M1―N2/N5 (2.063(4) in 4, (2.058(5) Å) and M1―S1/S3
(2.2347(13), 2.2458(13) Å), M1―N2/N5 (2.143(4), 2.135(4) Å) in 5. This variation in bond angles
reveals the distortion of a planar structure.
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Figure 4. The molecular structure of complexes 4 and 5 showing displacement ellipsoids at the
50% probability level.
The one-dimensional chain expands through the hydrogen bonds of N1―H1···O2 through the b
and a directions in 4 and 5 respectively and the length of this hydrogen bond in each of the two
complexes is almost equal (Fig. SI 5o-p). Moreover, C3―H3···S2 interaction participates in
development of the one-dimensional chain in 4. The second dimension for Pt compound expands
through the hydrogen bonds of C21―H21B···O2 and C11―H11A···S4 in the a direction and
through the hydrogen bonds of C22―H22B···O1, C9―H9B···S3 and C4―H4···S1 in the b
direction for Ps complex (Fig. SI 5q-r). Finally, the third dimension expands through C4―H4···S1
and C5―H5···C16/C17 hydrogen bonds for the Pt complex and also C22―H22A···S2 hydrogen
bond for the Pd complex both in the c direction (Fig. SI 5s-t).
3.4. Cytotoxicity studies
The cytotoxicity effect of compounds evaluated against cancer cells of Hela and MCF-7. So
cytotoxicity effect of the compounds in against normal cell of CHO also evaluated (see graphs in
Figs. SI 6a-c). The in vitro cytotoxicity evaluation results of compounds are summarized in Table
3.
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Table 3. IC50 values (Mean ± SDa (μM)) of compounds against three of cell lines.
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IC50 ± SD (μM)a
9
Cell lines
Hela
MCF-7
CHO
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L
4.069±0.79
6.967±0.67
3.403±0.67
5.645±0.74
2.518±0.53
3.912±0.72
3.929±0.65
7.397±0.77
9.375±0.66
5.353±0.77
5.927±0.72
9.328±0.81
6.346±0.86
6.622±0.54
1.939±0.69
8.351±0.77
8.397±0.67
10.38±0.73
3.671±0.66
6.543±0.69
5.931±0.23
1
2
3
4
5
cisplatin
a Data are presented as mean ± SD (standard deviation). All experiments were independently performed at least four times.
The cytotoxicity evaluation of compounds reveals that coordination of the L to the metal ions has
a dominant influence on cytotoxicity. To be more precise, coordination of ligand to metal ion
improve cytotoxic effect of complexes. Only, compound 1 does not show good effect against all
three cell lines. Also, 3 against Hela cell line and 4 against MCF-7 and CHO cell lines poorly
acted.
Cisplatin as a standard drug is selected to compare cytotoxic properties of compounds. The results
indicate that compound 2, 3 and 5 have more cytotoxic effect compare to cisplatin in all three cell
lines and so show better in vitro therapeutic index than cisplatin against all the three cell lines.
Compound 4 has better effect only against Hela cell line than cisplatin (See the pictures referring
to the color change of compounds during the MTT experiment in Figs. SI 6d-f).
3.5. Study of fluorescence quenching of HSA by dithiocarbazate complexes
Formation of the ligand-protein complex is one of the interesting subjects in drug discovery and
an effective technique which is applied in this study is the fluorescence spectroscopy. Human
serum albumin (HSA) is known as the most abundant protein in the blood plasma and also as a
drug carrier, so it is selected to investigate the interaction of drugs and macromolecules.[41]
Since fluorescence spectroscopy is useful for identifying organic compounds containing one or
more aromatic functional groups, therefore, aromatic parts of HSA include phenylalanine,
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tyrosine, and tryptophan have absorbance only in the ultraviolet region of the electromagnetic
spectrum.[42]
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It is characterized that most of fluorescence emission of HSA comes from Tryptophan (Trp)
residues that is located in the hydrophobic cavity of the subdomain IIA and is called Sudlow’s
drug-binding site I region.[43] The fluorescence spectrum of Trp-214 is highly influenced by the
surrounding environment and therefore is useful for studying the structural changes around it.[44]
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The changes in emission of HSA fluorescence in the region of 290-500 nm at exited state
wavelength (280 nm) were recorded in the absence and presence of different concentrations of
complexes at a temperature of 298 K (Figs. SI 7a-e). The effect of compounds on the fluorescence
intensity of HAS is investigated and for L' is pictured in figure 5 that indicates a decrease in the
fluorescence intensity of HSA with increasing concentration of L at 290 K without changing the
maximum and the shape of the peaks. The experiments show that the L quench the fluorescence
peak of HSA significantly even before its concentration become equal to that of HSA and so
fluorescence quenching effect was due to the formation of non-fluorescent complex.
1000
800
600
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200
0
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430
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Wavelenght (nm)
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Figure 5. Fluorescence quenching spectra of HSA in the presence of increasing concentration of
L'. [HSA] = 15 μM, [L] = 0 - 16 μM, λex = 280 nm, T = 298 K.
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Fluorescence quenching can be induced by two mechanisms of dynamic and static quenching that
dynamic quenching process refers to collisional interaction between quencher and excited state
molecule of the fluorophore and static quenching is resulted from the formation of a ground state
complex between the fluorophore and the quencher. In order to confirm the quenching mechanism
of compounds, the results from fluorescence measurements can be used to estimate the binding
constant using classical Stern–Volmer (SV) equation:[45]
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퐹0
Eq. 1
= 1 + 퐾푆푉[푄] = 1 + 퐾푞휏0 [푄]
퐹
where F0 and F are the steady-state fluorescence intensities in the absence of a compounds and in
its presence at [Q] concentration respectively. Kq is the quenching rate constant of the biomolecule,
τ0 is the average lifetime of HSA without the quencher and its value is 10−8 s, and KSV is the Stern–
Volmer quenching constant.
The shape of the SV plot is curved (Fig. 6a) and so indicate that the quencher has created dynamic
and static quenching (Figs. SI 7f-j).
30
a)
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0
0
5
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15
[Q] Х 10 ⁻⁶M
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Figure 6. a) Stern-Volmer plot for quenching of HSA (15 μM) fluorescence by L' at different
퐹0
concentrations (λex = 280 nm, λem = 346 nm),b) Plot of ― 퐹 vs 1/[L2-] for HSA in the presence
퐹0
of increasing concentrations of L at T = 298 K (λex = 280 nm, λem = 346 nm), c) Double-log plots
for the fluorescence quenching of the HSA by L.
For the positive deviation cases a modified SV equation was used and for negative deviation cases
the following equation was used:[46]
퐹0
1
1
1
Eq. 2
=
+
퐹0 ― 퐹
푓푎퐾푆푉 [푄]
푓푎
where fa is the fraction of accessible fluorescence while the other parameters are as the same as
those defined for classical Stern-Volmer equation (Eq. 1).
The values fa and KSV can obtained from graph of figure 6b. The graph is linear and Kq is more
than 2×1010 M-1S-1 therefore indicates that the fluorescence quenching process is of static
quenching and the role of dynamic quenching is almost insignificant. The KSV values, calculated
from Eq. 2 for L and complexes 1-5 (Fig. SI 7 k-o) are collected in Table 4.
Table 4. Quenching constants for the interaction of HSA with L and 1-5 complexes.
L
1
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3
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5
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KSV (×10-5 M-1)
0.48±0.07
0.75±0.02 0.37±0.09
0.98±0.05 1.34±0.1
1.14±0.03
Kq (×10-13 M-1S-1) ±SDa
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a Data are presented as mean ± SD (standard deviation). All experiments were independently performed three times
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For static quenching interaction, ka and the number of binding sites (n) can be calculated using Eq.
3:[47]
퐹0 ― 퐹
Eq. 3
푙표푔
= log 퐾 + 푛푙표푔[푄]
푎
(
)
퐹
퐹0 ― 퐹
퐹
From the plot of log
versus log[Q] (Fig. 6c), the number of binding sites (n) and association
(
)
constant (Ka) values were calculated for L and complexes (Figs. SI 7 p-t) from the slope and the
intercept on the Y-axis respectively (Table 5).
From the values of n, it may be inferred that there is a single binding site on HAS for complexes
and the values of Ka is indicating the static type of quenching mechanism.
Table 5. Values of Ka, n and ΔG (kJmol-1) parameters for systems of (compounds + HSA) at T = 298 K.
L
1
2
3
4
5
Ka (×10-5 M-1)±SDa
2.39±0.3 1.55±0.04 1.2±0.02
0.72±0.03 1.51±0.06 1.63±0.08
n
1.40
1.54
1.34
1.30
1.15
1.34
ΔG
-42.1
-41.0
-40.3
-39.1
-40.9
-41.1
a Data are presented as mean ± SD (standard deviation). All experiments were independently performed three times
The thermodynamic parameters for reaction between a ligand and a protein are the main evidence
for confirming the binding forces. Generally, there are essentially four types of non-covalent
interactions include hydrogen bonds, van der Waals forces, electrostatic and hydrophobic bond
interactions which play a key role in ligand binding to proteins. The value of the free energy change
(∆G) was calculated using Equation 4.
Eq. 4
Δ퐺 = ― 푅푇푙푛퐾푎
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where Ka is the binding constant obtained from Equation 3 and R is the universal gas constant and
indicates the spontaneity of the binding process of peptide to human albumin.[48]
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The calculated values for ΔG show the binding process is always spontaneous as demonstrated by
the negative values of ΔG (Table 3).
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4. Conclusion
We represent here synthesis and X-ray crystallography characterization of a new compound (L)
and five metal complexes of the dithiocarbazate family. X-ray diffraction analysis shows L is a
four dentate ligand N2O2 incorporating S―S bond which breaks during the complexation reaction
and L' forms. L' acts as a tridentate ONS donor ligand with Zn, Co and Ni metals. However, L' is
a bidentate NS donor ligand toward Pt and Pd. The interaction studies between the synthesized
compounds and HSA by fluorescence spectroscopy, revels that the intrinsic fluorescence of Trp-
214 residue is quenched in the presence of each compound. The quenching intensity is increased
with increasing the concentration of compounds at 298 K and quench the fluorescence peak of
HSA before their concentrations become equal to that of HSA. Also, the fluorescence data
indicates a good interaction characterized by K∼1.5×105 M−1 and n∼1. Moreover, the findings
reported herein show that compounds binds to HSA quenched the intrinsic fluorescence emission
of protein through static quenching mechanism. The negative values regard to ∆G confirm the
spontaneity of the processes. The cytotoxicity evaluation of compounds against the Hela and MCF-
7 cell lines shows that coordination of ligand to the metal ion improves the cytotoxic effect of
compounds compared to the clinical drug, cisplatin.
Acknowledgements
This work was supported by the Ferdowsi University of Mashhad (3/39699-1394/11/11) and Buali
Research Institute. Authors thank Tulane University for support of the Tulane Crystallography
Laboratory.
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