Development, evaluation and effect of anionic co-ligand on the biological activity of benzothiazole derived copper(II) complexes

Article abstract of DOI:10.1016/j.jinorgbio.2020.111174

Research on development of novel metal based anti-cancer agents continues with its popularity among bioinorganic community. Benzothiazole, an important heterocyclic pharmacophore, was chosen as a valuable and useful scaffold for the synthesis of novel copper(II) complexes. Three new copper(II) complexes obtained from the synthesis of newly synthesized benzothiazole based N-(benzo[d]thiazol-2-ylmethyl)-N-methyl-2-(pyridin-2-yl)ethan-1-amine (btzpy) ligand with CuCl₂ [Cu(btzpy)Cl₂] (1), Cu(NCS)₂ [Cu(btzpy)(NCS)₂] (2), and Cu(NO₃)₂ [Cu(btzpy)(NO₃)(H₂O)]NO₃ (3) were isolated and characterized by physical and spectroscopic measurements, including single-crystal X-ray structures. The interaction of complexes 1 and 3 with calf thymus (CT)-DNA was investigated using ethidium bromide fluorescence quenching assay and weak intercalation with K_SV values of 9.8 × 10² M^?1 and 8.2 × 10² M^?1, respectively was observed. All three complexes have shown DNA cleavage of supercoiled plasmid DNA forming single nicked and double nicked forms in the presence of external reducing agents like 3-mercaptopropionic acid (3-MPA) and ascorbic acid. The water-soluble complexes 1 and 3 also show prominent hydrolytic DNA cleavage. From the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay, it was observed that complex 2 also exhibits good antioxidant properties. The cytotoxicity of complexes 1–3 was tested against the lung cancer cell line (A549) and complex 2 with -NCS moiety shows maximum activity in the micromolar range. A rationale for the observed activity is proposed in light of the other properties of these molecules.

Full text of DOI:10.1016/j.jinorgbio.2020.111174

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Development, evaluation and effect of anionic co-ligand on the
biological activity of benzothiazole derived copper(II) complexes
Surbhi Jain, Kishalay Bhar, Shreetama Bandyopadhayaya, Vikas
K. Singh, Chandi C. Mandal, Suman Tapryal, Anuj K. Sharma
PII:
S0162-0134(20)30202-6
DOI:
https://doi.org/10.1016/j.jinorgbio.2020.111174
JIB 111174
Reference:
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
7 April 2020
28 June 2020
28 June 2020
Please cite this article as: S. Jain, K. Bhar, S. Bandyopadhayaya, et al., Development,
evaluation and effect of anionic co-ligand on the biological activity of benzothiazole
derived copper(II) complexes, Journal of Inorganic Biochemistry (2020), https://doi.org/
10.1016/j.jinorgbio.2020.111174
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Development, Evaluation and Effect of Anionic Co-ligand on the Biological Activity of
Benzothiazole Derived Copper(II) Complexes
Surbhi Jaina, Kishalay Bhara, Shreetama Bandyopadhayayab, Vikas K. Singhc, Chandi C.
Mandalb, Suman Tapryalc, Anuj K. Sharmaa*
aDepartment of Chemistry, School of Chemical Sciences and Pharmacy, Central University of
Rajasthan, Bandarsindri, District Ajmer, Rajasthan, 305817, India.
bDepartment of Biochemistry, School of Life Sciences, Central University of Rajasthan,
Bandarsindri, District Ajmer, Rajasthan, 305817, India.
cDepartment of Biotechnology, School of Life Sciences, Central University of Rajasthan,
Bandarsindri, District Ajmer, Rajasthan, 305817, India.
Abstract. Research on development of novel metal based anti-cancer agents continues with its
popularity among bioinorganic community. Benzothiazole, an important heterocyclic
pharmacophore, was chosen as a valuable and useful scaffold for the synthesis of novel
copper(II) complexes. Three new copper(II) complexes obtained from the synthesis of newly
synthesized benzothiazole based N-(benzo[d]thiazol-2-ylmethyl)-N-methyl-2-(pyridin-2-
yl)ethan-1-amine (btzpy) ligand with CuCl2 [Cu(btzpy)Cl2] (1), Cu(NCS)2 [Cu(btzpy)(NCS)2] (2),
and Cu(NO3)2 [Cu(btzpy)(NO3)(H2O)]NO3 (3) were isolated and characterized by physical and
spectroscopic measurements, including single-crystal X-ray structures. The interaction of
complexes 1 and 3 with calf thymus (CT)-DNA was investigated using ethidium bromide
fluorescence quenching assay and weak intercalation with KSV values of 9.8 × 102 M-1 and 8.2 ×
102 M-1, respectively was observed. All three complexes have shown DNA cleavage of
supercoiled plasmid DNA forming single nicked and double nicked forms in the presence of
external reducing agents like 3-mercaptopropionic acid (3-MPA) and ascorbic acid. The water-
soluble complexes 1 and 3 also show prominent hydrolytic DNA cleavage. From the DPPH (2,2-
diphenyl-1-picrylhydrazyl) radical scavenging assay, it was observed that complex 2 also
exhibits good antioxidant properties. The cytotoxicity of complexes 1-3 was tested against the
lung cancer cell line (A549) and complex 2 with -NCS moiety shows maximum activity in the
micromolar range. A rationale for the observed activity is proposed in light of the other
properties of these molecules.
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1. Introduction
Cancer is the second leading cause of death in the entire world. Cancer alone caused
approximately 9.6 million deaths in 2018. 1 out of 6 people around the globe dies due to cancer
[1]. Out of all the types, breast and lung cancer are the most common types with 2.09 million
cases reported for both in the last year. The majority of the cases (~70%) are reported in low- and
middle-income countries [1]. Since the serendipitous discovery of the anticancer property of
cisplatin by Rosenberg et al. in 1964 [2] and its success in treating malignancies, there has been
a massive upsurge in the development of metal-based therapeutics. Many different metals like
Ru, Fe, Pd, Au, Ti, Ga, V, Co, etc. have been developed and used as anticancer agents [3-7].
Certain
tetrachloro(dimethylsulfoxide)imidazoleruthenium(III)), KP1019 (trans-[tetrachlorobis(1H-
indazole)ruthenate(III)]), and NKP-1339 (sodium trans-[tetrachloridobis(1H-
complexes
of
Ru
(such
as
NAMI-A
(imidazolium-trans-
indazole)ruthenate(III)]) [8-10] have reached clinical trials along with Fe based tamoxifen [11]
and Cu based casiopeína [12]. Several recent review articles highlight the latest developments
and the journey of metal based antitumor drugs [13-15]. Copper complexes induce apoptosis in
cancer cells via several mechanisms like by generating reactive oxygen species (ROS) [16, 17],
proteasome inhibition [18], targeting enzymes [19], DNA damage [20, 21], etc.; thus, making it
difficult for resistance development. Since the body has a very well-defined mechanism for
copper homeostasis, copper becomes a very suitable candidate for therapeutic purposes [22].
Several copper complexes that act as artificial nucleases have been explored for their anticancer
potential and many of them have shown exceptional activity with toxicity profiles better than the
known anticancer drugs [23]. Out of all the reported complexes, Casiopeínas®, a class of mixed
chelate copper(II) complex with a general formula [Cu(N-N)(N-O) H2O]NO3 or [Cu(N-N)(O-
O)H2O]NO3 (where, N-N = diimines as 1,10- phenanthroline, 2,2-bipyridine, or their substituted
analogues, N-O = aminoeidate and O-O = acetylacetonate, salicylaldehidate), are gaining lot of
attention as two of the complexes from this family, Casiopeína III-ia (aqua, 4,4-dimethyl-2,2’-
bipyridine, acetylacetonato copper(II) nitrate) and Casiopeína II-Gly could enter in phase I
clinical trials [24, 25]. The former is being tested against acute myeloid leukemia where it has
been shown that the complex induces fragmentation of DNA and oxidation of the base. This
suggests that the mode of action for the complex involves generation of ROS after the metal
center has been reduced [26]. Hernández-Lemus et al. demonstrated that Casiopeína II-Gly
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shows its effect by enhancing the metabolism of metals and by blocking the migration and
proliferation rate of cancerous HeLa cells [27]. Despite all the merits, no copper-based
therapeutic has been approved so far for human use. Therefore, more efforts are required towards
designing and synthesis of novel copper compounds for use as potential anticancer agents.
Over the years, it has been established that the cytotoxic activity and the nuclease ability of the
synthesized metal complexes are vastly dependent on the organic ligand framework around it
[23, 28-30]. In fact, the effect of donor atoms and structural features of the complex can affect
the lipophilicity/hydrophilicity of the compound, the preferred oxidation state of the metal center
and most importantly, the biological activity of the compounds [31]. Thus, we designed novel
N,N,N-based ligand utilizing the benzothiazole (btz) moiety, an important heterocyclic
pharmacophore. Btz and its derivatives are involved in a diverse range of pharmacological
activities such as antimicrobial, herbicidal, pesticidal, antidiabetic, anti-inflammatory,
anticonvulsant, and many more including antitumor [32, 33]. Several examples of benzothiazole
based ligands [34-40] have been reported in the literature and have been evaluated for their DNA
binding and activity against cancer cell lines. Considering the importance of Cu(II) and the
pharmacological relevance of benzothiazole based drugs, Cu(II) complexes containing
benzothiazole motifs within the ligand framework could be of valuable interest. In this lieu, we
have synthesized and characterized three novel Cu(II) complexes with benzothiazole based
ligand, N-(benzo[d]thiazol-2-ylmethyl)-N-methyl-2-(pyridin-2-yl)ethan-1-amine (btzpy), having
benzothiazole and pyridyl moieties. The ligand coordinates with metal mainly by nitrogen atoms
of pyridyl, thiazole and the alkyl nitrogen connecting the two.
Here we report the synthesis, spectroscopic characterization, single-crystal X-ray structures of
three novel Cu(II) complexes with btzpy ligand and different anionic co-ligands [Cu(btzpy)(Cl)2]
(1), [Cu(btzpy)(NCS)2] (2), and [Cu(btzpy)(NO3)(H2O)]NO3 (3). Complex 1 and 3 are water-
¯
soluble with easily dissociable -Cl¯ and -NO3 ions, whereas complex 2 with -NCS¯ ligand is
soluble in organic solvents like acetonitrile, dimethylformamide (DMF), dimethyl sulfoxide
(DMSO)) only. A comparative biological study of the complexes with DNA binding, Bovine
Serum Albumin (BSA) binding, DNA cleavage, antioxidant properties and in vitro cytotoxicity
against the lung cancer cell line (A549) is reported.
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2. Experimental
2.1. Materials and methods
All the reagents were purchased from commercial sources and used as it is without any
purification. 2-aminothiophenol was purchased from Kemphasol; chloroacetyl chloride was
purchased from Molychem; 2-methylaminoethylpyridine, Calf thymus DNA (CT-DNA),
ethidium bromide, Bovine Serum Albumin (BSA), DPPH (1,1-diphenyl-2-picrylhydrazyl),
Trolox (6-hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid) was purchased from Sigma-
Aldrich; copper chloride was obtained from Ramkem; copper perchlorate was purchased from
Alfa Aesar while all other reagents (potassium carbonate, potassium iodide, copper nitrate,
sodium sulfate, potassium thiocyanate) were purchased from CDH. All the solvents were
distilled before use. For all biological experiments, ultra-pure Milli-Q water was used. For MTT
assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) was purchased from
Himedia (MB186). All the experiments involving CT-DNA and BSA were performed in PBS
buffer (pH=7.4). The concentration of CT-DNA was calculated by measuring the absorbance at
260 nm (ε =6,600 M-1cm-1) [41]. The ratio of absorbance at 260 nm and 280 nm was 1.8-1.9,
indicating that the DNA was sufficiently protein-free. The solutions were stored at 4˚C and were
used within 5 days. E. coli DH5α cells (Invitrogen) grown overnight in incubator shaker
(Thermo scientific) at 37℃, 150 rpm were used for the isolation of plasmid pET28b (+)
(Novagen). The cells grown overnight were harvested for the isolation of bacterial plasmid
pET28b (+). MACHERY-NAGEL Nucleospin® plasmid kit was used for the isolation and
purification of the plasmid pET28b (+). The isolated plasmid pET28b (+) was used for further
analysis on 1% agarose gel. 1% agarose gel was prepared in a 1× TAE (Tris base, acetic acid and
EDTA) buffer by adding ethidium bromide (EtBr) (1 µg/mL). The samples were mixed with 6×
DNA loading dye purple (New England Biolab, UK) before loading into the respected wells.
2.2. Physical measurements
The NMR spectra for ligand were recorded on the Bruker-500 MHz spectrometer in CDCl3.
Molar conductivity was measured on a Systronics Conductivity Meter-304 at room temperature
in 1 mM acetonitrile solution for all the complexes and in double-distilled water for complex (1)
and (3). Elemental analyses (C, H, N, S analysis) were performed on a Thermo-Scientific Flash
2000 CHNS/O Elemental Analyzer and the values were within ±0.4% of the theoretical values.
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Infrared spectra were recorded as KBr pellets on a Perkin Elmer FTIR Spectrometer (Spectrum
Two) in the range 4000–400 cm−1. The UV-visible spectra were recorded on Agilent Cary-60
UV-vis spectrophotometer at room temperature in a cuvette of path length 1 cm. Fluorescence
spectra for all the studies were recorded on the Agilent Cary Eclipse spectrophotometer at room
temperature in a quartz cuvette of path length 1 cm. Cyclic voltammograms for all the complexes
(1-3) were recorded on Autolab PGSTAT 302N workstation (Eco-Chemie BV, Netherlands) at
room temperature under dinitrogen in dry acetonitrile using a standard three-electrode cell
consisting of a glassy carbon working electrode, a platinum auxiliary electrode and Ag wire as
the reference electrode. EPR was recorded on a Varian, E-112 ESR Spectrometer (for complex
1) and on a JEOL EPR Spectrometer with X & Q band (for complexes 2 and 3) at SAIF, IITB,
Mumbai in acetonitrile: propionitrile (1:3) glass at LNT (77 K). Agarose gel electrophoresis was
performed horizontal electrophoresis unit (GeNei) and run by using a Power Pac (Bio-Rad)
voltage controller system. The gels were visualized and imaged under UV light using the Gel
documentation system (DNR MiniLumi). The MTT assay was performed in 96 well flat-bottom
tissue culture plates and the absorbance was recorded at 530 nm using Spectro Star Nano Elisa
Reader (BMG Labtech). Human lung cancer cells, A549 which were procured from NCCS cell
repository, Pune, India were used for the cell culture-based experiments. The cells were grown in
Dulbecco’s Eagles Modified Medium (DMEM) (HyClone, SH30243.LS) which was
supplemented with 10% fetal bovine serum (FBS) (HyClone, SV30160.03) and was maintained
in a CO2 incubator at 5% CO2 at 37ºC.
2.3. Synthesis of ligand (btzpy) and copper complexes (1-3)
The synthetic scheme has been outlined in Scheme 1.
2.3.1. Synthesis of the ligand
2.3.1.a. Synthesis of precursor 2-chloromethylbenzothiazole: The precursor for ligand synthesis
was synthesized using the literature method [42]. The crude product obtained from this method
was purified by column chromatography using a mixture of ethyl acetate and petroleum ether
(1:9) to produce 2-chloromethylbenzothiazole as a colorless oily liquid at room temperature with
80% yield (1.2 g). Upon cooling, colorless needle-shaped crystals were obtained. 1H NMR (500
MHz, CDCl3) δ (ppm) = 7.96 (d, 1H); 7.77 (d, 1H); 7.32 (t, 1H); 7.13 (t, 1H); 4.04 (d, 2H).
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2.3.1.b. Synthesis of btzpy ligand: The ligand was synthesized by suspending K2CO3 (1 g, 0.722
mmol) in acetonitrile and 2-(2-methyl aminoethyl)pyridine (98.4 mg, 0.722 mmol) was added to
it. After 10 mins, 2-chloromethylbenzothiazole (133 mg, 0.722 mmol) and a catalytic amount of
KI (0.1 equivalents) were added to the reaction mixture. The reaction was refluxed at 90 C
overnight. The reaction mixture was filtered to remove the excess of K2CO3 and acetonitrile was
evaporated. Water (50 mL) was added to the reaction mixture and then the product was extracted
in dichloromethane (25 × 3 mL). The combined organic phases were washed with brine (20 mL)
and dried over Na2SO4. Evaporation of the organic phase yielded pure ligand as a brown viscous
liquid in 86% yield (170 mg). 1H NMR (500 MHz, CDCl3) δ (ppm) = 8.49 (d, 1H, -Hl); 7.93 (d,
1H, -Hk); 7.81 (d, 1H, -Hj); 7.55 (m, 1H, -Hi); 7.39 (m, 1H, -Hh); 7.32 (m, 1H, -Hg); 7.16 (d, 1H,
-Hf); 7.08 (t, 1H, -He); 3.99 (s, 2H, -Hd); 3.03 (d, 2H, -Hc); 2.98 (d, 2H, -Hb); 2.45 (s, 3H, -Ha).
13C NMR (125 MHz, CDCl3) δ (ppm) = 173.30 (-Co), 160.04 (-Cp), 153.29 (-Cn), 149.27 (-Cl),
136.28 (-Ci), 135.47 (-Cm), 125.71 (-Ch), 124.72 (-Cg), 123.33 (-Cf), 122.72 (-Ck), 121.69 (-Cj),
121.23 (-Ce), 59.80 (-Cd), 57.66 (-Cb), 42.71 (-Ca), 36.42 (-Cc).
2.3.2. Synthesis of [Cu(btzpy)Cl2] (1)
.
CuCl2 2H2O (103 mg, 0.6 mmol) in methanol (10 mL) was added to a stirred solution of ligand
(btzpy) (170 mg, 0.6 mmol) in dichloromethane (10 mL). After stirring the reaction at room
temperature for 24 hours, the blue colored precipitate obtained was filtered, dried and washed
with diethyl ether. The product was recrystallized in acetonitrile by slow evaporation. Dark blue
coloured crystals suitable for Single Crystal XRD were obtained. Yield: 165 mg (66 %).
Elemental analysis for C16H17Cl2CuN3S: Calcd. C, 45.99; H, 4.10; N, 10.06; S, 7.67%; found: C,
45.97; H, 4.06; N, 10.10; S, 7.89%. FTIR (KBr disc, cm-1): 3736 (m) (ʋC-H), 1697 (m) (ʋC=N
,
ʋC=C), 1513 (s), 752 (s), 710 (m). UV-Vis (λmax/nm, (ε, M−1cm−1)) in acetonitrile: 221 (2.4 × 104),
235 (2.3 × 104), 260 (1.5 × 104), >800. UV-Vis (λmax/nm, (ε, M−1cm−1))in H2O: 237 (1.5 × 104),
256 (1.4 ×104), 656 (1.1 × 102). Molar conductance, ɅM =10 Ω-1 cm2 mol-1 (1 mM solution in
acetonitrile at 25˚C); ɅM =300 Ω-1cm2 mol-1 (1 mM solution in H2O at 25˚C) (expected range for
1:2 electrolyte in H2O: 145–273 Ω-1cm2 mol-1 [43]).
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2.3.3. Synthesis of [Cu(btzpy)(NCS)2] (2)
.
Cu(NCS)2 was generated in situ by mixing Cu(ClO4)2 6H2O (260 mg, 0.70 mmol) and K(NCS)
(137 mg, 1.40 mmol) in a 1:2 ratio in methanol. The K(ClO4) generated was filtered while the
pale-yellow colored solution of Cu(NCS)2 was added to a solution of ligand (btzpy) (200 mg,
0.70 mmol) in dichloromethane (10 mL). The reaction was stirred at room temperature for 24
hours. The green-colored precipitate obtained was filtered, dried and washed with diethyl ether.
The product was recrystallized in acetonitrile by slow evaporation. Green colored crystals
suitable for Single Crystal XRD were obtained. Yield: 143 mg (44%). Elemental analysis for
C18H17CuN5S3: Calcd. C, 46.68; H, 3.70; N, 15.12; S. 20.77%; found: C, 46.49; H, 3.57; N,
15.07; S, 20.81%. FTIR (KBr disc, cm-1): 3736 (m) (ʋC-H), 2080 (m) (ʋCN (NCS)), 2040 (s) (ʋCN
(NCS)), 1513 (s), 772 (s) (ʋCS (NCS)). UV-Vis (λmax/nm, (ε, M−1cm−1)) in acetonitrile: 235 (2.2 ×
104), 255 (1.4 × 104), 405 (9.3 × 102), 713 (1.7 × 102). Molar conductance, ɅM =50 Ω-1 cm2 mol-1
(1 mM solution in acetonitrile at 25˚C).
2.3.4. Synthesis of [Cu(btzpy)(NO3)(H2O)](NO3) (3)
Cu(NO3)2 (131 mg, 0.70 mmol) in methanol (10 mL) was added to a stirred solution of ligand
(btzpy) (200 mg, 0.70 mmol) in dichloromethane (10 mL). The reaction was stirred at room
temperature for 24 hours. The blue colored precipitate obtained was filtered, dried and washed
with diethyl ether. The product was recrystallized in acetonitrile by slow evaporation. Dark blue
colored crystals suitable for Single Crystal XRD were obtained. Yield: 157 mg (46 %).
Elemental analysis for C16H19CuN5O7S: Calcd. C, 39.40; H, 3.82; N, 14.32; S, 6.56%; found: C,
39.77; H, 3.50; N, 14.72; S, 6.96%. FTIR (KBr disc, cm-1): 3736 (m) (ʋC-H), 1412 (s) (ʋC=C),
1380 (s), 1280 (vs), 1016 (m), 761 (s). UV-Vis (λmax/nm, (ε, M−1cm−1)) in acetonitrile: 233 (1.7 ×
104), 258 (1.3 × 104), 290 (5.4 × 103), 630 (1.5 × 102). UV-Vis (λmax/nm, (ε, M−1cm−1)) in H2O:
236 (2.3 × 104), 256 (1.9 ×104), 658 (1.8 × 102). Molar conductance, ɅM =110 Ω-1 cm2 mol-1 (1
mM solution in acetonitrile at 25˚C); ɅM =250 Ω-1cm2 mol-1 (1 mM solution in H2O at 25˚C)
(expected range for 1:2 electrolyte in H2O: 145–273 Ω-1cm2 mol-1 [43]).
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Scheme 1. Synthetic scheme for ligand (btzpy) and copper complexes (1-3).
2.4.Single crystal X-ray crystallographic data collection and refinement
Suitable single crystals of 1-3 for X-ray analysis were obtained by slow evaporation of a
concentrated solution of acetonitrile. Crystallographic data were collected at 296 K on Bruker
APEX-II CCD Diffractometer using graphite monochromated Mo-Kα radiation (λ = 0.71073 Å)
at USIC, The University of Burdwan, Burdwan. The structures were solved by direct methods
and refined by full-matrix least-squares methods based on F2 using SHELXT[44] and
SHELXL[45] programs. The non-hydrogen atoms were subjected to refinement by anisotropic
displacement parameters. Hydrogen atoms were given isotropic U values 1.2 times that of the
atom to which they are bonded and placed in calculated positions when possible. PLATON[46]
and OLEX2[47] programs were used for preparing materials for publication. CCDC reference
numbers for 1-3 are 1994356, 1994357 and 1994358, respectively.
2.5. Stability determination
The stability of all the copper complexes was assessed at room temperature by recording UV-
visible spectra at different time intervals from 0 to 24 hours on the Agilent Cary-60
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spectrophotometer. Solutions of complexes 1 and 3 were prepared in H2O while for complex 2,
acetonitrile was the solvent of choice due to its limited solubility in H2O.
2.6. DNA binding studies
The ethidium bromide (EtBr)-DNA adduct is highly fluorescent due to the intercalation of EtBr
in between DNA base pairs. This fluorescence can be quenched upon the interaction of another
molecule with DNA. The EtBr displacement assay could be performed for complex 1 and 3, only
as complex 2 was soluble in organic solvents like acetonitrile and DMSO and precipitated out
when added into the buffer solution. Stock solutions of DNA, EtBr, and complexes 1 and 3 were
prepared in PBS buffer of pH 7.6. Increasing concentrations of complex (0- ~100 μM) were
titrated into the EtBr-DNA solution where the final concentration of CT-DNA, as well as EtBr,
was 18 μM. The emission of all the solutions was recorded from 490 nm to 800 nm when excited
at 480 nm. The excitation and emission slit widths were kept constant throughout the experiment
at 10 nm., each. The data were analyzed using the Stern-Volmer quenching plot:
;
where Io is the fluorescence intensity of the EtBr-DNA solution, I is the fluorescence intensity of
the solution after the addition of complex, [Q] is the concentration of the complex in the cuvette
and KSV is the Stern-Volmer quenching constant.
2.7. DNA nuclease activity
The DNA cleavage ability of the synthesized complexes was determined by Agarose gel
electrophoresis. Supercoiled plasmid DNA (pET-28b) was incubated with the complex and
reducing agent (3-mercaptopropionic acid (3-MPA) or Ascorbic Acid) for 1 h at 37ºC in 10 μL
reaction volume. Complex 1 and 3 were dissolved in H2O while the stock solution of complex 2
was made in acetonitrile where the final volume of acetonitrile in the Eppendorf was ~10%. The
ratio of complex concentration to reductant concentration was 2:5 in all the reactions. The
Agarose gel electrophoresis experiment was run in 0.8% agarose gel prepared in 1× TAE buffer,
at a constant voltage of 4-5 V/cm. Ethidium bromide of concentration 1 μg/mL was added while
preparing the gel. Before loading the samples in respected wells, they were mixed with 6× DNA
loading dye purple (New England Biolab, UK).
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2.8. Protein interaction studies
The protein (BSA) binding of the complexes was evaluated using the steady-state fluorescence
quenching experiments which were performed on Agilent Cary Eclipse fluorescence
spectrometer at room temperature. 10 μM solution of BSA, prepared in PBS buffer of pH 7.6,
was used for the study. The emission spectra were recorded from 285-510 nm (λem (max.) = 350
nm) where the tryptophan residue of BSA was excited at 280 nm. Synchronous fluorescence
emission titrations at a wavelength difference of Δλ = 60 nm and Δλ = 15 nm were performed
using the same concentration of BSA with varying concentrations of the complexes. Similar to
the DNA binding studies, the protein interaction studies could also be carried out for complex 1
and 3, only. The binding constant was calculated using the Stern Volmer Equation.
2.9.Antioxidant Assay
To analyze the antioxidant properties of the synthesized complexes, DPPH (1,1-diphenyl-2-
picrylhydrazyl) radical scavenging assay was performed. DPPH is a stable radical at room
temperature and the solution is deep violet in color. When DPPH accepts an electron from
another molecule, it becomes diamagnetic and pale yellow in color. As a result, the absorption of
the solution at 517 nm is significantly quenched. To study the antioxidant activity of the
complexes, a 100 μM stock solution of DPPH in methanol was used. In a 96-well plate, 66 μL of
DPPH with different concentrations of the complexes were used and the final volume was made
up to 100 μL using methanol. The solutions were incubated for 20 mins in the dark after which
the absorption at 517 nm was recorded on a UV-visible spectrophotometer (ThermoFisher,
MultiScan Go). For control, 66 μL of DPPH diluted with 34 μL of methanol alone was used.
Trolox was used as an antioxidant standard in assay protocol. The scavenging activity (%) was
calculated using the following equation:
where, AC is the absorbance of only DPPH solution, and AS is the absorbance of DPPH in
presence of complexes.
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2.10. Cell viability assay
The synthesized copper complexes were assessed for cytotoxicity on the A549 (human lung
cancer) cell line by performing MTT cell viability assay. Approximately 5000 A549 cells/well
were seeded in a 96-well tissue culture plate. The lung cancer cells were grown in DMEM
supplemented with 10% FBS at 37ºC in 5% CO2. After 24 h of seeding, the copper complexes
were added in increasing concentrations over a range of 0.5 μM-50 μM. Ligand btzpy was also
tested for activity at higher concentrations (50 μM-250 μM). An equal volume of solvent was
added to the control wells. Since complex 2 was insoluble in aqueous media, the stock solution
of 1 mM was prepared in DMSO. The solution was then diluted to 200 μM by adding milli-Q
water. The maximum concentration of DMSO in the well plate did not exceed 5%. Control wells
with DMSO up to 20% were also set where no prominent cell death was observed. The well
plates were incubated for 48 h after the addition of the complexes. Later, MTT solution (5
mg/mL) was added to each well and the plate was further incubated for an hour in the incubator.
Afterward, the culture media was replaced with DMSO to dissolve the formed formazan crystals.
The absorbance of each well was measured at 530 nm [48, 49]. The plot of normalized
percentage viability of the cells versus log concentration was plotted and fitted using non-linear
regression of dose response-inhibition equation in graph pad prism for the calculation of IC50
values. The experiments were set up in triplicates and the values have been represented as mean
± standard deviation.
3. Results and discussion
3.1. Synthesis, stability, and characterization
The ligand (btzpy) was synthesized by a substitution reaction between 2-chlorobenzothiazole and
1
2-(2-methyl aminoethyl)pyridine (Scheme 1). The formation of the ligand was monitored by H
NMR spectroscopy. Two doublets (at 7.93; 7.81 ppm) and two triplets of benzothiazole (at 7.39,
7.32 ppm); and three -CH2 moieties present in the ligand were characteristic peaks. The -CH2
peak of 2-chloromethylbenzothiazole at 4.04 ppm shifted to 3.99 ppm upon ligation with the
amine. In addition, the DEPT NMR spectrum also showed the presence of three -CH2 groups in
the structure (ESI Figure S1-S3). The UV-visible spectrum of the ligand shows sharp π → π*
and n → π* transitions at 221 nm and 255 nm, respectively (ESI Figure S4).
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.
The complexes (1-3) were synthesized by the reaction of btzpy ligand with CuCl2 2H2O (1),
Cu(NCS)2 (2) and Cu(NO3)2 (3) in dichloromethane and methanol mixture, respectively.
.
Cu(NCS)2 was synthesized in situ by the reaction of Cu(ClO4)2 6H2O with two equivalents of
potassium thiocyanate (KNCS). Complex 1 and 3 were obtained as blue colored solids while
complex 2 was obtained as green colored solid. The complexes are highly colored and stable in
air. Complexes 1-3 are soluble in organic solvents such as acetonitrile and DMSO. Complexes 1
and 3 are additionally soluble in H2O, making them more feasible for biological studies. The
limited solubility of complex 2 in organic solvents along with precipitation upon addition of
buffer restricted the DNA and BSA binding analysis of the complex.
The elemental analyses of all three complexes show good correspondence within the accepted
range of ±0.4 (ESI Figure S5-S7). The medium intensity band in FTIR at around 710 cm-1 in
complex 1 can be assigned to the C-S stretching vibration [50]. The sharp intensity bands in the
range of 1412-1513 cm-1 in all three complexes are assigned to the C=N stretching vibration of
the benzothiazole moiety. In complex 2, the sharp intensity bands at 2040-2080 cm-1 and 772 cm-
1 are for a terminal N bound NCS for-CN and -CS stretch, respectively [51]. The presence of two
types of -NO3 groups in complex 3 can be observed from the two groups of IR frequencies
(1380, 1016 cm-1 and 1280, 761 cm-1). Also, a broad peak >3500 cm-1 shows the presence of a
H2O molecule in the system (ESI Figure S8-S10). The UV-vis spectra of the three complexes in
acetonitrile have been shown in Figure 1. The complexes show strong ligand-based π → π* and
n → π* transitions from 200-300 nm. The weaker and broader bands at wavelengths > 600 nm
are due to the d-d transition of Cu(II) [52, 53]. In addition, complex 2 showed a sharp band at
405 nm probably due to LMCT from -NCS to Cu(II) center. Further, the stability of the
complexes was assessed in suitable solvents (H2O and acetonitrile for complex 1 and 3, and only
acetonitrile for complex 2) by UV-vis spectroscopy. On careful analysis of the UV-vis spectra in
correlation with the conductivity measurements, it was observed that in complexes 1 and 3 the
anionic co-ligands dissociate when dissolved in H2O. The btzpy ligand remains intact in the
structure as the ligand bands (at wavelengths < 300 nm) can still be observed in the spectrum.
Since only slight variations in the intensities with no prominent spectral changes in the particular
solvent were observed even after 24-48 h, it indicated that the complexes are stable with no
evident hydrolysis of the btzpy ligand in the solvents used at room temperature (ESI, Figure S11-
S15).
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(a)
(b)
(c)
Figure 1(a-c). UV-vis spectra of copper complexes (1-3) recorded at 10-5 M; (Inset) 10-3 M in
Acetonitrile at room temperature.
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The EPR spectra for all the complexes were recorded in Acetonitrile at liquid N2 temperature.
Complex 1 shows an intermediate, rhombic EPR spectrum with gx (2.22) > gy (2.16) > gz (1.99)
(Figure 2). The spectrum with three g values is indicative of a penta-coordinated system with
geometry in between square pyramid and trigonal bipyramid geometries [54]. The R parameter
can be indicative of the predominant geometry, thereby showing the predominance of the dz2 or
dx2- dy2 orbital in the ground state. Here,
The value of R for complex 1 is calculated to be 2.81, i.e. greater than 1, thereby indicating that
the geometry is closer to trigonal bipyramid. The crystal structure of complex 1 shows that it has
a τ value of 0.72 which confirms the same geometry. Complexes 2 and 3 show normal axial EPR
spectra with g
g (ESI, Figure S16-S17). Complexes 2 and 3 have the g value around 2.25,
while the g values are 2.08 and 2.06, respectively. These g values signify that the geometry
around Cu2+ is square pyramidal where the ground state is the dx2-y2 orbital [55]. The EPR
spectrum of complex 3 is further split into a four-line spectrum due to unpaired electron present
in the dx2-y2 orbital. In the spectrum, three lines are well-resolved and one overlaps with the g
signal.
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Figure 2. X-band EPR spectrum of complex 1 in acetonitrile: propionitrile (1:3) glass at LNT
(77 K) (frequency 9.1 GHz and 100 kHz field) (For better viewing, the red line has been
highlighted).
Further, to analyze the Cu2+/+ redox potential, the cyclic voltammograms for all three complexes
were recorded. All the complexes show a quasi-reversible one-electron reduction for Cu2+/+
redox couple which indicates that the ligand can accommodate a Cu+ oxidation state as well
(Figure 3). The anodic peak potentials (Epa) for the Cu2+/+ couple for complexes 1-3 was
observed at ∼ 0.05 V to 0.11 V with ΔEp ranging from 110 mV to 180 mV. The E1/2 values for
complexes 1-3 were calculated to be 0.10 V, 0.16 V, and 0.2 V, respectively. The ease of
reduction of the complexes is an important aspect for the activation of the complex towards
DNA cleavage as discussed in section 3.4. Under similar conditions, the ligand (btzpy) showed
no redox activity.
(a)
(b)
(c)
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Figure 3(a-c). Cyclic voltammograms of copper complexes 1-3 at 100 mV.s-1 scan rate in dry
Acetonitrile and 0.1 M TBAP as supporting electrolyte vs. Ag wire.
3.2. X-ray crystallographic studies
The geometry of three new copper(II) compounds (1-3) and binding mode of ligand (btzpy) were
established by single crystal X-ray crystallography and are shown in Figure 4. Compounds 1 and
2 crystalize in triclinic system with P space group whereas compound 3 crystallizes in
monoclinic system with P21/n space group. The crystallographic data and refinement parameters
are summarized in Table S1. The selected bond distances and angles are given in Table S2.
(a)
(b)
(c)
Figure 4. ORTEP diagrams of 1-3 (a-c) with 50% thermal ellipsoid probability for all non-
hydrogen atoms. The hydrogen atoms were omitted for clarity.
The asymmetric unit of 1 and 2 contain [Cu(btzpy)(Cl)2] and [Cu(btzpy)(NCS)2], respectively
and that of 3 consists of a cationic moiety [Cu(btzpy)(H2O)(NO3)]+ with a nitrate counter anion.
In 1-3, the btzpy ligand acts as a tridentate N-donor chelator and coordinates to Cu(II) ion
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through pyridine N (Npy), amine N (Nam) and benzothiazole N (Nbtz) atoms with formation of a
six-membered and a five-membered chelate rings. The remaining two sites are occupied by two
¯
Cl¯ ions in 1, two NCS¯ moieties in 2, and one H2O molecule and one NO3 ion in 3. The
geometry around each penta-coordinated Cu(II) ion in 1-3 is predicted by trigonality index
parameter [τ = (-)/60;  and  are the two largest angles around Cu(II)] [56]. The values of τ
are 0.72 for 1, 0.37 for 2 and 0.10 for 3 (τ = 0 for square pyramid and τ = 1 for trigonal
bipyramid) indicating distorted trigonal bipyramidal (TBP) geometry for 1 and distorted square
pyramidal (SP) geometry for 3 around Cu(II) centre. However, Cu(II) centre in 2 adopts SP
geometry with severe distortion towards TBP geometry. The Nam atom (N1) of btzpy along with
the two Cl atoms form the equatorial plane while the Npy (N2) and Nbtz (N3) of btzpy occupy the
apical positions around TBP Cu(II) centre in 1. The values of Cu-N and Cu-Cl bond distances
are similar to that of reported compounds [57]. Further, an axial compression along Cu1-N2 and
Cu1-N3 bonds (Table S2) is noticed in 1 which is consistent with a
electronic ground state
for TBP geometry [58]. In 2 and 3, the basal plane is formed by three N atoms (N1, N2, N3) of
-
btzpy along with the N atom (N4) of NCS¯ in 2 and the O atom (O2) of NO3 in 3; while the
apical site is occupied by N atom (N5) of remaining NCS- moiety in 2 and the O atom (O1) of
water molecule in 3. The apical Cu-N/Cu-O bond distance in 2 and 3 is larger than basal Cu-
N/Cu-O distances which is consistent with the SP geometry (Table S2). A comparative account
of geometry related to some relevant penta-coordinated copper(II) compounds bound by bi-
/tridentate chelators containing benzothiazole moiety and compounds 1-3 is summarized in Table
S3.
3.3. DNA binding studies
In order to understand the interaction of complexes 1 and 3 with CT-DNA, fluorescence
titrations with ethidium bromide (EtBr) bound DNA were employed. EtBr is a well-known DNA
intercalator and is highly fluorescent when intercalated between DNA base pairs [59, 60]. In the
free state, the fluorescence of EtBr is quenched by the solvent. EtBr bound to DNA exhibits a
sharp fluorescence at around 600 nm which can be quenched upon the addition of a molecule
that interacts or intercalates with DNA. Copper complexes 1 and 3 were titrated against the EtBr-
DNA solution and a moderate quenching was observed along with a noticeable red-shift of ~3
nm (Figure 5). The studies indicate that although the complexes interact with DNA, they do not
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intercalate within the DNA strands well. The Stern-Volmer quenching constants for complex 1
and 3 were calculated to be 9.8 × 102 M-1 and 8.2 × 102 M-1, respectively (Figure 6). The absence
of planar ligand frames and extended conjugation rings (like phenanthroline, bipyridine, etc.) is
probably responsible for the similar, and weak intercalation of both the complexes.
(a)
(b)
Figure 5. Plot of fluorescence emission intensity vs. wavelength for DNA-EtBr at different
concentrations of (a) (1); (b) (3). The arrow shows the change in intensity of emission on
increasing amount of the complex from 0 to 200 μM.
Figure 6. Stern-Volmer quenching plot of DNA-EtBr for complex 1 and 3.
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3.4. DNA nuclease activity
An artificial nuclease transforms the DNA sequentially from supercoiled to single nicked and
then double nicked or linear form. Each form has a different overall structure and therefore,
moves at different rates in gel electrophoresis. The supercoiled band travels the farthest and
appears at the bottom while the single nicked form runs the shortest distance and appears at the
top. The double nicked or linear DNA appears in between these two.
The DNA cleavage activity of all three complexes (1-3) was studied with pET-28b plasmid in
presence of a fixed concentration of the complex with three equivalents of two different reducing
agents i.e. 3-MPA and ascorbic acid (AscA). The solution of complex 2 was prepared in
Acetonitrile where the final volume of Acetonitrile in the reaction vessel was around 10%. In the
control plasmid, two conformations of DNA were present as evident by the two bands (Figure 7),
where the upper band corresponds to single nicked form while the lower band corresponds to
supercoiled form. Control experiments with 3-MPA, AscA, and ligand (btzpy) alone were also
performed. Although 3-MPA showed no enhanced DNA cleavage, AscA and ligand alone
showed a slight increment in the nicked form (Lane 3-4, Figure 7). Figure 7 shows the results of
the DNA cleavage by all three complexes in the presence of 3-MPA as well as AscA. All three
complexes were able to convert the supercoiled DNA form to single nicked or linear forms. It is
noteworthy that all the complexes also show hydrolytic DNA cleavage, i.e. in the absence of any
external reducing agent. For comparison purpose, the reactivity difference in the hydrolytic DNA
cleavage for this set of compounds can be arranged as 1 > 3 > 2, with 1 showing 100% cleavage
of supercoiled form, while for the same concentration of complexes 2 and 3, 16% and 7% of the
supercoiled form is left uncleaved. The ability of these complexes to cleave DNA not only in the
presence of reducing agents but also hydrolytically makes them potent nucleases. The aqueous
solubility of complexes 1 and 3 may seem to play a role in the reactivity. It is possible that the
dissociation of -Cl and -NO3 anions from the molecular structure of 1 and 3, respectively provide
easy accessibility for the complex to interact with DNA, thus having a positive influence on the
cleavage ability.
In hydrolytic cleavage, the phosphodiester bonds are cleaved to generate smaller fragments of
DNA in the presence of water. Under ambient conditions and in absence of any catalyst, the half-
life of a phosphodiester bond in DNA is ten to a hundred billion years in neutral water. This
implies that in the absence of a catalyst, the DNA will not degrade for a very long time. In
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general, for hydrolytic cleavage, the metal complexes act as Lewis acids which can either
activate the phosphate group towards nucleophilic attack, increase the leaving group ability of
the alcohol, or activate water or hydroxide to act as a nucleophile. The most accepted mechanism
involves a nucleophilic attack by hydroxide or water at the phosphate backbone of DNA to form
a five-coordinate intermediate which is stabilized by the catalyst (metal ion in this case). This is
usually followed by cleavage of 3'-PO bond or the 5'-PO bond resulting in the scission of the
DNA strand (Scheme 2) [61].
Scheme 2. The proposed reaction mechanism for the hydrolysis of DNA [61].
On the other hand, 3-MPA, or AscA can cause oxidative DNA cleavage. Unlike hydrolytic
cleavage, the DNA fragments of oxidative cleavage cannot be re-ligated. All the four
nucleobases [62] or the deoxyribose sugar [63] can be damaged by oxidative cleavage. The
redox-active metal complexes generate the reactive intermediate species in this type of cleavage
[64-66]. The cleavage occurs in three steps: Abstraction of hydrogen, addition and electron
transfer. Scheme 3 depicts the process of oxidative cleavage after the abstraction of one
hydrogen of deoxyribose from the C-3' terminal. Guanine is the preferred site for oxidation at the
nucleic base level due to its lower oxidation potential. The hydroxyl radical reacts by adding to
the heterocyclic bases of DNA. In purines, the hydroxyl radical adds to C-4, C-5, or C-8 bond
while in pyramidines, it adds to C-5 or C-6 double bond [67].
Scheme 3. Oxidative cleavage at nucleobases [61].
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The most common mechanism for oxidative cleavage involves Fenton-type chemistry [68].
Mechanistic schemes with the external reducing agents (3-MPA and AscA) have been shown
below [69]. Both 3-MPA and AscA, denoted as RHx, can reduce Cu2+ to Cu+ which can react
with molecular oxygen leading to the production of superoxide ion radical and regeneration of
Cu2+. The radical can further combine with H+ to form H2O2 which can lead to a cascade of
reactions, thereby producing different ROS. The ROS thus generated can eventually cause the
desired DNA cleavage activity [70].
Cu2+ + RHx → Cu+ + R˙ + xH+
ˉ
˙
Cu+ + O2
⇌
Cu2+ + O2
ˉ
O2 ˙ + 2H+ → H2O2
Figure 7. Analysis of DNA cleavage by complexes 1-3 using gel electrophoresis and the relative
% amounts of different forms of DNA after 1 h of incubation at 37ºC. Lane 1: DNA control;
Lane 2: 3-MPA (500 μM) control; Lane 3: AscA (500 μM) control; Lane 4: L (btzpy) (100 μM)
control; Lane 5: 1 (100 μM) control; Lane 6: 1 (100 μM) + 3-MPA (300 μM); Lane 7: 1 (100
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μM) + AscA (300 μM); Lane 8: 2 (100 μM) control; Lane 9: 2 (100 μM) + 3-MPA (300 μM);
Lane 10: 2 (100 μM) + AscA (300 μM); Lane 11: 3 (100 μM) control; Lane 12: 3 (100 μM) + 3-
MPA (300 μM); Lane 13: 3 (100 μM) + AscA (300 μM).
3.5. Protein interaction studies
The high similarity of BSA protein with HSA protein makes it a suitable candidate to analyze the
binding of the synthesized copper complexes with serum albumins. To study the interaction of
the complexes with the biomolecule, the fluorescence of the tryptophan, tyrosine and
phenylalanine residues present in the protein has been utilized [71, 72]. The emission of the
protein can be quenched upon the interaction of the complexes due to several reasons such as the
substrate binding with the protein, conformational changes occurring in the protein as a result of
an interaction, subunit association, or denaturation of the protein [71, 73].
BSA solution was titrated with increasing concentrations of the copper(II) complexes and the
fluorescence spectra were recorded from 290-500 nm. The maximum intensity was centered
around 350 nm which decreased sequentially with negligible shift (Figure 8). The binding
parameter was calculated according to the Stern-Volmer equation (Figure 9). Both the complexes
show moderate binding to BSA with KSV values of 1.26 × 104 M-1 for 1 and 1.15 × 104 M-1 for 3.
The structural similarity of both the complexes might be a probable reason for the similar
binding constants.
(a)
(b)
Figure 8. Plot of fluorescence emission intensity vs. wavelength for BSA at different
concentrations of (a) (1); (b) (3). The arrow shows the change in intensity of emission on
increasing amount of the complex from 0 to 150 μM.
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Figure 9. Stern-Volmer quenching plot of BSA for complex 1 and 3.
3.5.1. Involvement of tryptophan and tyrosine residues in the binding
To analyze the conformational changes and the changes in the microenvironment of the BSA
protein, synchronous emission spectra were recorded at Δλ = 60 nm and Δλ = 15 nm with
increasing concentrations of complex 1 and 3 [74]. When the Δλ = 60 nm, i.e. comparatively
large, the spectra is mainly due to the tryptophan residue while when the Δλ = 15 nm, i.e.
comparatively small, then the tyrosine residue of the protein is the major contributor to the
spectra. The BSA protein has three combination parts for the drugs to bind, out of which one is
located at the sub-domain IIA which contains both tryptophan and tyrosine residue, whereas two
are located at the IIIA sub-domain which contains only the tyrosine residue [75]. As shown in
Figure 10 and 11, the fluorescence spectra for both tryptophan and tyrosine residues were
quenched significantly upon the addition of the copper complexes. The interaction of the
complexes with BSA was able to bring out changes in the microenvironment of the tryptophan
and tyrosine residues either by decreasing the hydrophobicity, increasing the polarity, or
loosening or stretching the polypeptide chains in the structure of BSA [76]. Since a clear
involvement of both the residues can be seen in the process, we can say that the binding site is
preferentially the sub-domain IIA of the protein.
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(a)
(b)
Figure 10. Synchronous fluorescence spectra of BSA with increasing concentrations of 1 at the
wavelength difference of (a) Δλ = 60 nm, (b) Δλ = 15 nm.
(a)
(b)
Figure 11. Synchronous fluorescence spectra of BSA with increasing concentrations of 3 at the
wavelength difference of (a) Δλ = 60 nm, (b) Δλ = 15 nm.
3.6. Antioxidant studies
The radical scavenging activity of all three complexes was studied using DPPH. This is a simple
and rapid test for antioxidant screening. Out of all the complexes tested, complex 2 shows major
free radical scavenging followed by complex 3 (Figure 12). Complex 2 (200 μM) shows about
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64% of scavenging of the DPPH radical while the same concentration of complex 3 shows about
23% scavenging. Complex 1 has negligible activity at this concentration. Possibly, the Cu(II)
center in the complexes gets reduced to Cu(I) while transferring one electron to DPPH, thereby
reducing it to diamagnetic species. Positive control of Trolox was also added which showed
100% scavenging activity. The antioxidant activity has a positive correlation with anticancer
activity. Excessive production of free radicals in the human body can have a great impact on the
etiology of various diseases [77-79] and therefore, compounds with antioxidant properties, i.e.
with the ability to scavenge free radicals, may be highly beneficial and relevant for therapeutic
purposes [80].
Figure 12. Free radical scavenging studies with increasing concentration of complexes 1-3.
3.7. Cell viability assay
To compare the cytotoxic activity of all three copper complexes against human lung cancer cell
line, A549, MTT assay was performed. The cytotoxicity was evaluated after 48 h of exposure of
the compounds to the cells. As can be seen from Figure 13, complex 2 is most active towards the
A549 cells among the three complexes. It shows the ability to suppress the proliferation of the
cells in a concentration-dependent manner. The IC50 value of complex 2 was calculated to be
5.45 ± 0.71 μM. The complexes 1 and 3 exhibit IC50 values of 50.8 ± 1.18 and 51.52 ± 1.17 μM,
respectively against the particular cell line used. The IC50 values of btzpy ligand was calculated
to be 91.07 ± 3.28 μM (ESI, Figure S18). The higher values of IC50 for the ligand as compared to
complexes suggest a clear involvement of the metal center for the desired activity.
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It is worth to mention here that the mechanism for copper(II) complexes-mediated cytotoxicity
may be triggered by their ability to bind and cleave DNA leading to cell cycle arrest and
apoptosis or the generation of reactive oxygen species (ROS) which also cause cell death.
Although highly detailed studies of mechanism of copper(II) complexes are scarce, few recent
examples have shown that copper(II) complexes can penetrate through cell membrane and act
via several different mechanistic pathways. Studies show that copper complexes have the ability
to enter and accumulate in cell membrane or mitochondrial membrane; some even accumulate in
the cell nuclei [81-85]. Structures of few selected complexes that support the observation of a
direct correlation of individual parameters like DNA binding, metal uptake to the observed anti-
proliferative activities have been given in ESI, Table S4.
Complex 2 has limited aqueous solubility and its conductivity data suggest that -NCS moiety
does not dissociate from Cu(II) as compared to the other two complexes 1 & 3. DNA cleavage
studies also suggest that interaction of molecule 2 with DNA and its capability to cleave it.
Since, the extent of DNA cleavage was higher by 1 & 3 as compared to 2; it is evident that either
more than one mechanism in action as far as the cell death is concerned or higher concentration
of 2 is entering into the cell due to its intact form in aqueous media. The higher potential of
complex 2 may be related to the presence of tightly bound -NCS groups which probably provide
the required hydrophobicity to enter the cells. Several Cu(II)-NCS complexes with various
ligands have been known to show different biological activities. Considering the structures of the
complexes and their cell toxicity results, it can be related that the -NCS moiety linked to Cu(II)
has a crucial role to play towards the anticancer activity of the complex.
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(a)
(b)
(c)
Figure 13. % cell viability of A549 cells after exposure to (a) 1, (b) 2, and (c) 3, for 48 h.
4. Conclusions
In this study, three mononuclear copper(II) complexes (1, 2 and 3) derived from a novel
benzothiazole based ligand (N-(benzo[d]thiazol-2-ylmethyl)-N-methyl-2-(pyridin-2-yl)ethan-1-
amine) have been synthesized and structurally characterized. Complexes 1 and 3 are water
soluble but complex 2 has very limited aqueous solubility. DNA and BSA binding with water-
soluble complexes 1 and 3 have been evaluated and the binding constants for both the complexes
are in a similar range. DNA cleavage study, antioxidant studies and cellular studies are
performed for 2 were carried out using organic solvents as vehicle. All the complexes show
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efficient DNA nuclease activity of the double-stranded pET-28b plasmid. In addition to the
oxidative DNA cleavage, complex 1 and 3 also show prominent hydrolytic DNA cleavage. In
spite of weak binding to DNA, the complexes show potent concentration-dependent nuclease
activity suggesting a different mechanism of interaction of the complexes with DNA. The major
difference in the activity of the complexes due to the difference in the co-ligand was observed in
cytotoxicity against lung cancer A549 cell lines. Only complex 2 having rhodanide (-NCS) as
co-ligand, showed good anti-proliferative activity with an IC50 value in low micromolar range.
The other two complexes have very low potency against the cell line tested. Taken together, it is
concluded that more than one mechanism is in action for the observed cell toxicity behaviors’ of
these complexes. In addition, complex 2 exhibits potential antioxidant properties that are
considered relevant for the development of therapeutic agents. The current study lays the
groundwork for the development of benzothiazole and -NCS based copper(II) complexes for
therapeutic purposes.
Conflicts of interest
There are no competing financial interests to declare.
Dedication
This manuscript is dedicated to Prof. Samaresh Bhattacharya on his 60th Birthday.
Acknowledgements
AKS acknowledges the support from UGC-BSR Research Start-up-grant (ref. no.
F.30.414/2018(BSR)), DST-SERB EMR grant (ref. no. EMR/2016/001452) and DST-INSPIRE
research grant (IFA-13, CH-97). SJ is thankful to DST-INSPIRE for Senior Research Fellowship
(IF150116), while KB acknowledges SERB for NPDF (PDF/2017/000929). SB thanks the
Central University of Rajasthan for providing the university fellowship and CCM is thankful for
the DST-RFBR grant (INT/RUS/RFBR/P-256). VKS is grateful to UGC for JRF, and ST
acknowledges DST-SERB grants (YSS/2015/000138, and EEQ/2016/000569). The authors
acknowledge the instrumental facilities at the Department of Chemistry, Central University of
Rajasthan supported by DST-FIST (ref. no. SR/FST/CSI-257/2014(C)). The authors are also
thankful to the University of Burdwan, West Bengal for single-crystal X-ray crystallographic
data collection.
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References
[1] World Health Organization, Cancer (2018). https://www.who.int/news-room/fact-
sheets/detail/cancer.
[2] B. Rosenberg, L. Vancamp, T. Krigas, Inhibition of Cell Division in Escherichia Coli by
Electrolysis Products from a Platinum Electrode, Nature. 205 (1965) 698-699.
http://doi.org/10.1038/205698a0
[3] X. Wang, Z. Guo, Towards the rational design of platinum (II) and gold (III) complexes as
antitumour agents, Dalton Trans. (2008) 1521-1532. http://doi.org/10.1039/B715903J
[4] I. Kostova, Gold coordination complexes as anticancer agents, Anti-Cancer Agents in Med.
Chem. 6 (2006) 19-32. http://doi.org/10.2174/187152006774755500
[5] K. Irena, Ruthenium Complexes as Anticancer Agents, Curr. Med. Chem. 13 (2006) 1085-
1107. http://dx.doi.org/10.2174/092986706776360941
[6] M.J. Hannon, Metal-based anticancer drugs: From a past anchored in platinum chemistry to a
post-genomic future of diverse chemistry and biology, Pure Appl. Chem. 79 (2007) 2243-2261.
http://doi.org/10.1351/pac200779122243
[7] D. Gaynor, D.M. Griffith, The prevalence of metal-based drugs as therapeutic or diagnostic
agents:
beyond
platinum,
Dalton
Trans.
41
(2012)
13239-13257.
http://dx.doi.org/10.1039/C2DT31601C
[8] D. Wernitznig, K. Kiakos, G. Del Favero, N. Harrer, H. Machat, A. Osswald, M.A. Jakupec,
A. Wernitznig, W. Sommergruber, B.K. Keppler, First-in-class ruthenium anticancer drug
(KP1339/IT-139) induces an immunogenic cell death signature in colorectal spheroids in vitro,
Metallomics (2019). http://dx.doi.org/10.1039/C9MT00051H
[9] S. Zorzet, A. Sorc, C. Casarsa, M. Cocchietto, G. Sava, Pharmacological effects of the
ruthenium complex NAMI-A given orally to CBA mice with MCa mammary carcinoma, Met.
based drugs. 8 (2001) 1-7. http://doi.org/10.1155/mbd.2001.1
[10] P.-S. Kuhn, V. Pichler, A. Roller, M. Hejl, M. Jakupec, W. Kandioller, B. Keppler,
Improved reaction conditions for the synthesis of new NKP-1339 derivatives and preliminary
investigations on their anticancer potential, Dalton Trans. 44 (2015) 659-668.
http://doi.org/10.1039/c4dt01645a
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