New copper(II) complexes of pyridine-2-carboxaldehyde-N-(2-pyridyl)hydrazone and 2-hydroxy-1-naphthaldehyde-semicarbazone: Synthesis, characterization, DNA binding and antimicrobial activity studies

Article abstract of DOI:10.14233/ajchem.2020.22827

Two new copper(II) complexes of pyridine-2-carboxaldehyde-N-(2-pyridyl)hydrazone [Cu(PCPH)(H2O)2](NO3)(H2O)2 (1) and 2-hydroxy-1-naphthaldehyde-semicarbazone [Cu(II)(HNSC)H2O]·NO3·H2O (2) have been synthesized and characterized by spectroscopic techniques and single crystal X-ray diffraction study. Complex 1 crystallized as square pyramidal coordination complex in triclinic crystal system while complex 2 crystallized as square planar complex in monoclinic crystal system. EPR spectral patterns are of normal order of energy levels, i.e. x²-y² >> z² > xy > xz, yz, with partial covalent character. Both copper(II) complexes were found to be groove binding to calf-thymus DNA and showed activity against E. coli, S. aureus, B. cereus and E. faccium.

Full text of DOI:10.14233/ajchem.2020.22827

Asian Journal of Chemistry; Vol. 32, No. 11 (2020), 2783-2792
A
SIAN
J
OURNAL OF HEMISTRY
C
https://doi.org/10.14233/ajchem.2020.22827
New Copper(II) Complexes of Pyridine-2-carboxaldehyde-N-(2-pyridyl)hydrazone and
2-Hydroxy-1-naphthaldehyde-semicarbazone: Synthesis, Characterization,
DNA Binding and Antimicrobial Activity Studies
1
1,*
1
1
OINAM U-WANG , RAJ KUMAR BHUBON SINGH , USAM IBOTOMBA SINGH , RAMINA ,
1
2
3
THOUDAM SURCHANDRA SINGH , TOKA SWU and CH. BRAJAKISHORE SINGH
1Department of Chemistry, Manipur University, Canchipur, Manipur-795003, India
2Department of Chemistry, Pondicherry University, Kalapet, Puducherry-605014, India
3Department of Biotechnology, Institute of Bioresources and Sustainable Development, Imphal-795001, India
*Corresponding author: E-mail: bhubonsingh@gmail.com
Received: 26 May 2020;
Accepted: 17 July 2020;
Published online: 28 October 2020;
AJC-20102
Two new copper(II) complexes of pyridine-2-carboxaldehyde-N-(2-pyridyl)hydrazone [Cu(PCPH)(H2O)2](NO3)(H2O)2 (1) and 2-hydroxy-
1-naphthaldehyde-semicarbazone [Cu(II)(HNSC)H2O]·NO3·H2O (2) have been synthesized and characterized by spectroscopic techniques
and single crystal X-ray diffraction study. Complex 1 crystallized as square pyramidal coordination complex in triclinic crystal system
while complex 2 crystallized as square planar complex in monoclinic crystal system. EPR spectral patterns are of normal order of energy
levels, i.e. x2-y2 >> z2 > xy > xz, yz, with partial covalent character. Both copper(II) complexes were found to be groove binding to calf-
thymus DNA and showed activity against E. coli, S. aureus, B. cereus and E. faccium.
Keywords: Copper(II), Hydrazone, Semicarbazone, DNA binding.
times more potent than tetracycline against E. coli HB101/
pBR322, a bacterial strain resistant to tetracycline and Pd(II)
doxycycline complex is two times more potent than doxycy-
cline against the same resistant strain [10].
INTRODUCTION
sp2 carbon rich flat aromatic rings are almost ubiquitous
in medicinal chemistry [1]. It is due to the well characterized
aromatic synthetic paths [2]. However, majority of aromatic
moieties are heteroaromatic rings with polar surfaces [3] and
amine and amide functionalities outside the ring structure have
been added to enhance solubility [1], lower solubility and higher
lipophilicity, leading to poorer efficacy and greater toxicity
[2], remains a challenge for drugs containing aromatic moieties
[4]. Also, the development of drug resistance and escape of
microorganisms from the conventional antimicrobials call for
new agents [5] and medicinal chemists have directed efforts
toward metal-based drugs.
The formation of complexes increases the bioavailability
of the metal ion or the ligand drug or both [6]. Many metal
complexes of quinolone antibiotics, which inhibit bacterial DNA
gyrase and topoisomerase IV required for DNA replication [7],
viz. ciprofloxacin, norfloxacin and ofloxacin have been reported
to possess enhanced activity rather than antibiotic alone [8,9].
Palladium(II) tetracycline complex has been reported sixteen
Metal complexes of ligands containing aromatic moieties,
can bind to DNA via intercalation and minor groove binding
[11].Among such ligands, aryl-/heteroaryl-/N-heteroaryl hydra-
zones have received significant attention due to their ligation
property [12]. In continuation of our research on biologically
active transition metal coordination complexes [13-20], we
herein report the syntheses, spectroscopic characterization,
crystal structures, DNA-binding study and antimicrobial activity
of copper(II) complexes of pyridine-2-carboxaldehyde-N-(2-
pyridyl)hydrazone [Cu(PCPH)Cl(H2O)](NO3)(H2O)2 (1) and 2-
hydroxy-1-naphthaldehyde semicarbazone [Cu(II)(HNSC)-
H2O]·NO3·H2O (2).
EXPERIMENTAL
All the chemicals were purchased from Sigma Aldrich
and Himedia and used as received without further purification.
Elemental (C, H, N) analysis were carried out using Perkin
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2784 U-wang et al.
Asian J. Chem.
Elmer 2400 II ElementalAnalyzer. FTIR spectra (KBr pellets,
4000-400 cm-1) were recorded on a Shimadzu FTIR 8400S
spectrophotometer. UV-vis spectra as well as the absorption
titration studies were recorded on Perkin Elmer Lambda 35
UV/VIS spectrophotometer. Perkin-Elmer LS 55 Fluorescence
spectrophotometer was used in ethidium bromide-DNA fluore-
scence quenching experiment. Cyclic voltammetry measure-
ments were carried out using a CH602C Electrochemical
Analyzer againstAgCl/Ag (saturated KCl) reference electrode.
JEOL, JES-FA200 ESR spectrometer was used to record EPR
spectra at RT and LNT. Sherwood scientific magnetic suscep-
tibility balance calibrated with mercury(II)tetrathiocyanato-
cobaltate(III) was used to measure RT magnetic susceptibi-
lities. Viscosity measurements in DNA-binding studies were
carried out using Ostwald’s viscometer immersed in a thermo-
stated water bath at 298 K. The viscosities (η) of samples were
determined using the equation:
tion correction were performed with CrysAlisPro, Version 1.
171.36.21 [21]. The structures were solved using SHELX-2008
[22] and refined with full-matrix-least-squares on F2. Empirical
absorption correction using spherical harmonics were
implemented in SCALE3 ABSPACK scaling algorithm.
Primary atoms were located by structure-invariant direct method.
Hydrogen atom sites were inferred from neighboring sites.
Molecular structure and crystallographic illustrations were
prepared using OLEX-2-1.3 [23].
DNA-binding studies: For predicting the mode of ct-DNA
binding of the newly synthesized complexes, electronic absorp-
tion titrations, fluorescence quenching experiment, cyclic
voltammetric measurements and viscosity measurements were
carried out.
Antimicrobial activity study:A Gram-negative and three
Gram-positive bacteria and a species each of yeast and fungus
were used for the antimicrobial assays. The strains were Esche-
richia coli (EC) ATCC-11229, Staphylococcus aureus (SA)
ATCC-11632, Bacillus cereus (BC) MTCC-430, Enterococcus
faecium (EF) ATCC-35667, Candida albicans (CA) ATCC-
10231 and Aspergillus niger (AN) ATCC-16888. Microbial
growth inhibitory potentials of the compounds were determined
by Agar diffusion method and minimum inhibitory concen-
tration (MIC) were determined by serial dilution method.
η = (t – to)/to
where to is the flow time of buffer alone and t is the flow time
of ct-DNA solutions with increasing concentrations of complex.
Synthesis of pyridine-2-carboxaldehyde-N-(2-pyridyl)-
hydrazone (PCPH) and [Cu(PCPH)(H2O)2](NO3)(H2O)2 (1):
The PCPH ligand was prepared by refluxing pyridine-2-carbo-
xaldehyde (1 mmol, 0.09 mL) and 2-hydrazinopyridine
dihydrochloride (1 mmol, 0.18 g) in 20 mL methanol for 2 h.
To the resulting yellow colour solution was added copper(II)
nitrate trihydrate (1 mmol, 0.24 g) solution in 10 mL ethanol
drop-wise and stirred overnight. On keeping the filtrate for
slow evaporation, single crystals suitable for X-ray diffraction
study were collected after 2 weeks. Colour: dark green.Yield:
0.30 g (70.58%). m.p.: (°C): 250-252. Anal. calcd. (found) %
for C11H16N5O9Cu: C, 30.98 (30.95); H, 3.75 (3.70); N, 16.43
(16.40)%. UV-vis [H2O, λmax, nm (ε)]: 231 (100), 272 (100),
640 (2.7). FT-IR (KBr, νmax, cm-1): 3163 (-NH-), 1687 (C=N).
Magnetic moment (27 ºC, µB): 1.72. ΛM (water, 25 °C, S cm2
mol-1): 70.0.
RESULTS AND DISCUSSION
The reported copper(II) complexes were synthesized by
Schiff base condensation followed by reaction with copper(II)
nitrate (Fig. 1). The complexes are soluble in water and other
polar solvents. The molar conductance of copper(II) complexes
1 and 2 in water were found to be 70.0 and 64 S cm2 mol-1,
respectively showing 1:1 electrolytic nature for both complexes.
H
N
NH2
O
H2N
H
O
Synthesis of 2-hydroxy-1-naphthaldehyde-semicarba-
zone (HNSC) and [Cu(II) (HNSC)H2O]·NO3·H2O (2): The
HNSC ligand was synthesized by refluxing 2-hydroxy-1-naph-
thaldehyde (2 mmol, 0.34 g) and semicarbazide hydrochloride
(2 mmol, 0.22 g) in 20 mL methanol for 2 h. To the resulting
yellow colour solution was added copper(II) nitrate trihydrate
(2 mmol, 0.482 g) solution in 20 mL ethanol dropwise and
stirred for 2 h. On keeping the filtrate for slow evaporation
single crystals of [Cu(II)(HNSC)H2O]·NO3·H2O (2) suitable
for X-ray diffraction studies were collected after two weeks.
Colour: dark green.Yield: 0.70 g (89.86%). m.p.: 218-221 ºC.
Anal. calcd. (found) % for C12H14N4O7Cu: C, 36.90 (36.94);
H, 3.55 (3.59); N, 14.34 (14.36). UV-vis [H2O, λmax, nm (ε)]:
291 (60), 363 (87), 585 (4.1). FT-IR (KBr, νmax, cm-1): 3333,
3193 (-NH2), 3134 (-NH-), 1683 (C=O), 1663 (C=N). Magnetic
moment (27 °C, µB): 1.73. ΛM (water, 25 °C, S cm2 mol-1): 64.0.
Crystallographic data collection and refinement: X-ray
crystallographic data were collected on Xcalibur, Eos diffracto-
meter equipped with graphite monochromatized MoKα
radiation (λ = 0.7107 Å) at 298K. Data reduction and absorp-
O
HO
HN
H2N
N
N
MeOH
Reflux, 2 h
MeOH
H
N
N
H
H2N
H
N
O
O
N
Cu
N
N
OH2
Cu(NO3)2 3H2O, EtOH
Cu(NO3)2.3H2O
EtOH
H
H
N
H
N
N
H2N
N
N
N
O
O
Cu
Cu
(1)
NO3- (H2O)
NO3- (H2O)2
OH2
OH2
H2O
(2)
Fig. 1. Representative equations for syntheses of Cu(II) complexes 1 and 2

Vol. 32, No. 11 (2020) Synthesis, Characterization, DNA Binding and Antimicrobial Activity Studies of New Copper(II) Complexes 2785
FTIR, UV-vis and EPR spectroscopic studies
orbital [24]. The k|| is greater than k⊥ (k|| > k⊥), showing the
possibility of out-of-plane π bonding in the complex [25]. G
value greater than 4 indicates normal order of energy levels,
i.e. x2-y2 >> z2 > xy > xz, yz [26]. The EPR spectrum of complex
2 obtained in DMF at LNT (Fig. 3), also shows similar charac-
teristics with 14% overlapping between ligand orbital and
metal d-orbital and normal order of energy levels (Table-1).
FTIR studies: The FTIR absorption bands of copper(II)
complex 1 at 3136 cm-1 can be assigned to ν(-NH-) vibrations.
The ν(C=N) bands are observed at 1687 cm-1. Bands at 524
and 468 cm-1 are assigned tentatively to ν(Cu-N) vibrations
[20].
The FTIR absorption bands of compound 2 at 3333 and
3193 cm-1 are assigned to ν(NH2) vibrations. The band at 3134
cm-1 is assigned to –NH- stretching vibration. The ν(C=O) and
ν(C=N) bands are observed at 1683 cm-1 and 1653 cm-1. Bands
at 565 cm-1 are assigned tentatively to Cu-N vibration and 450,
425 cm-1 to Cu-O vibrations [20].
UV-vis studies: UV-visible spectrum of Cu(II) complex 1
shows broad d-d bands at 640 nm (15625 cm-1, ε = 2.7 M-1
cm-1) characteristic of square base copper(II) complexes. The
spectrum shows ligand to metal charge transfer transitions at
272 and 231 nm (ε = 100 M-1 cm-1). UV-vis spectrum of Cu(II)
complex 2 shows d-d band at 585 nm (17094 cm-1, ε = 4.1 M-1
cm-1) and LMCT transitions at 363 (ε = 87 M-1 cm-1) and 291
nm (ε = 59 M-1 cm-1) [20].
A⊥ = 6.6 G
A|| = 233 G
3230 G
2530 G
20000
10000
0
g⊥ = 2.02
-10000
-20000
-30000
-40000
g|| = 2.17
EPR studies: The EPR spectrum of complex 1 obtained
in DMF at LNT (Fig. 2), is of characteristic mononuclear
copper(II) complex. The trend g|| > g⊥ > 2.003 indicates axial
200
220
240
260
280
300
320
340
Field (mT)
2
2
symmetry with unpaired electron in dx [24]. The g|| value
-y
Fig. 3. EPR spectrum of Cu(II) complex 2 obtained from frozen DMF
was found to be less than 2.38 indicating significant covalent
2
solution at LNT
character in metal-ligand bonding. Bonding parameters (α ,
2
2
β , γ ) and orbital reduction factors (k|| and k⊥) were calculated
from EPR parameters (Table-1). The in-plane σ covalency
X-ray diffraction studies of complexes 1 and 2: Crystal
data and structure refinement are given in Table-2. The complex
1 crystallizes in space group P-1 of triclinic crystal system as
[Cu(PCPH)(H2O)2](NO3)(H2O)2 in which the copper(II) is in
a distorted square pyramidal structure (Table-2, Fig. 4). In the
complex, the ligand PCPH functions as a tridentate ligand
bonded through N12(pyridine), N3(imine) and N14(pyridine)
forming five membered chelate rings. Three nitrogen atoms
of PCPH (N12N3N14) and a oxygen atom (O5) of water mole-
cule occupy four coordination sites of the square plane while
a water molecule, bonded through O6 atom, occupies the apical
site. Selected bond lengths and bond angles of complex 1 are
given in Table-3. The imine C1-N3 bond (1.37(10) Å) is shorter
than C10-N2 single bond (1.57(11) Å) and pyridine C16-N12
bond (1.40(14) Å). The metal nitrogen bond lengths are nearly
the same [Cu1-N3 (1.95(8) Å), Cu1-N12 (1.97(9) Å, Cu1-
N14 (2.00(8) Å)]. The basal bond angle N12-Cu1-N14 =
163.5(3)º is larger than N3-Cu1-O5 = 158.8(3)º. The structural
parameter τ, which is given by (larger basal angle-smaller basal
angle)/60, is 0.08 showing distortion from perfect square pyra-
mid geometry (τ = 0 for perfectly square pyramidal and τ = 1
for perfectly trigonal bipyramdal geometry) [27].
2
parameter α value was found to be 0.42 suggesting that there
is about 58% overlapping between ligand orbital and metal d-
A⊥ = 6.6 G
300
200
100
A|| = 90 G
g⊥ = 2.02 G
0
-100
-200
g|| = 2.12
-300
-400
-500
250 260 270 280 290 300 310 320 330 340 350
Field (mT)
Fig. 2. EPR spectrum of Cu(II) complex 1 obtained from frozen DMF solution
at LNT
TABLE-1
EPR BONDING PARAMETERS OF COPPER(II) COMPLEXES 1 AND 2 IN DMF AT 77 K
Complex
Ed-d (nm)
640
585
g||
gav
A|| (G)
90
233
k||
G
6
8.5
g⊥
2.02
2.02
A⊥(G)
6.6
6.6
α2
0.42
k⊥
0.44
0.45
β2
1.26
γ2
1.04
2.12
2.17
2.05
2.07
0.53
0.66
1
2
0.86
0.76
0.52
α2 = -(A||/0.036) + (g|| - 2.0023) + 3(g⊥ - 2.0023)/7 + 0.04; k||2 = (g|| - 2.0023) Ed-d/8λo; k⊥2 = (g⊥ -2.0023) Ed-d/2λo; k||=α2β2 and k⊥= α2γ2; G = (g||-
2)/(g⊥-2). Here λo is one electron spin orbit coupling constant and is equal to 828 cm-1.

2786 U-wang et al.
Asian J. Chem.
TABLE-2
CRYSTAL DATA AND DETAILS OF STRUCTURE REFINEMENT OF COPPER(II) COMPLEXES 1 AND 2
Complex 1
1575942
C11H16N5O9Cu
425.83
Complex 2
918868
C12H14N4O7Cu
389.81
CCDC
Empirical formula
Formula weight
T (K)
293
298
Crystal system
Space group
a (Å)
b (Å)
c (Å)
α (°)
β (°)
Triclinic
P-1
6.84(5)
10.22(7)
12.06(8)
88.61(6)
82.40(6)
89.72(6)
835.14(10)
2
Monoclinic
P21/n
11.80(7) Å
6.67(4) Å
19.05(14) Å
90.00
102.63(7)°
90.00
1484.1(3)
4
γ (°)
V (Å3)
Z
calc (g cm-3)
1.693
1.367
1.745
1.518
ρ
µ (mm-1)
F000
436.0
796.0
Crystal size/mm3
Radiation
0.34 × 0.034 × 0.04; block
MoKα (λ = 0.71073 Å)
7.58 to 50
0.19 × 0.11 × 0.06; block
MoKα (λ = 0.71073 Å)
6.4 to 50
2θ range for data collection/°
Index Ranges
Reflections collected
-8 ≤ h ≤ 8, -12 ≤ k ≤ 12, -13 ≤ l ≤ 14
7598
-14≤ h ≤14, -8 ≤ k ≤ 8, -22≤ l ≤ 22
7186
Indipendent reflections
Data/restraints/parameters
Goodness-of-fit, S(F2)
Final R indexes [I>=2σ (I)]
Final R indexes [all data]
Largest diff. peak/hole/e Å-3
4536 [Rint = 0.0248, Rsigma = 0.0379]
4536/3/473
1.040
R1 = 0.0344, wR2 = 0.0894
R1 = 0.0400, wR2 = 0.0945
0.40/-0.39
2593 [Rint = 0.0496, Rsigma = 0.0637]
2593/0/228
1.094
R1 = 0.0508, wR2 = 0.1144
R1 = 0.0796, wR2 = 0.1282
0.72/-0.34
C46
C41
C52
C47
N4
C17
C33
N14
C43
C27
O5
N1
C49
N18
O6
C20
C10
N2
Cu1
N3
Cu2
C13
O2
C51
N12
C28
O9
O38
N7
O36
N11
C35
C16
C31
C50
C1
C45
C48
C44
O32
O21
O7
O8
O37
O34
N15
O42
O10
(a)
(b)
Fig. 4. (a) Crystallographic diagram of Cu(II) complex 1 with ellipsoids of 50% probability, (b) Packing diagram of Cu(II) complex 1 along a axis
The complex 2 crystallizes in space group P21/n of mono-
clinic crystal system as [Cu(II)(HNSC)H2O]·NO3·H2O in which
the copper(II) is in a square planar structure (Table-2, Fig. 5).
In the complex, the ligand HNSC functions as a tridentate ligand
bonded through O1(amide), N1(imine) and O2(phenoxide)
forming five membered and six chelate rings. The fourth coor-
dination site is occupied by O2(water). The +2 charge of the
copper atom is balanced by the phenoxide ion in the coordi-
nation sphere and a nitrate counter ion. Selected bond lengths
and bond angles are given in Table-4. The C-O bond lengths

Vol. 32, No. 11 (2020) Synthesis, Characterization, DNA Binding and Antimicrobial Activity Studies of New Copper(II) Complexes 2787
O4
O5
N3
N4
C12
O1
O6
N2
N1
Cu1
C11
C1
O3
C9
O2
O7
C2
C3
C10
C5
C8
C7
C4
C6
(a)
(b)
Fig. 5. (a) Crystallographic diagram of Cu(II) complex 2 with ellipsoids of 50% probability, (b) Packing diagram of Cu(II) complex 2 along b axis
TABLE-3
in amide [C12-O1, 1.26 (5) Å] is shorter than that in phenoxide
[C2-O2 1.33 (5) Å]. In semicarbazone N3-C12-N2-N1=C11
fragment, the bond lengths N3-C12 (1.31 (6) Å), C12-N2 (1.36
(5) Å), N2-N1 (1.37 (5) Å) and N1-C11 (1.30 (5) Å) are nearly
the same. The metal-donor atom bond lengths are Cu1-O1
(1.95 (3) Å), Cu1-O2 (1.88 (3) Å), Cu1-O3 (1.93(4) Å) and
Cu1-N1 (1.90 (3) Å). The bond angles around the metal center
O2-Cu1-O1 (175.45 (13)°), N1-Cu1-O3 (173.64 (17)°), O2-
Cu1-O3 (92.92 (17)°), N1-Cu1-O1 (82.42 (14)°) etc. show
distortion in square planar geometry of the complex. The bond
angles around C12 atom, N3-C12-N2 (118.5 (4)°), N2-C12-
O1 (118.5 (4)°) and O1-C12-N3 (122.9°) (Table-4).
SELECTED BOND LENGTHS (Å) AND
BOND ANGLES (°) OF COPPER(II) COMPLEX 1
Selected bonds Bond length (Å) Selected bond Bond angle (°)
Cu1-N3
Cu1-N12
Cu1-N14
Cu1-O5
Cu1-O6
C1-N3
C10-N2
N2-N3
C16-C1
C16-N12
C16-C44
1.95 (8)
1.97 (9)
2.00 (8)
1.96 (7)
2.28 (8)
1.37 (10)
1.57 (11)
1.33 (12)
1.45 (13)
1.40 (14)
1.34 (15)
N3-Cu1-O6
N12-Cu1-O6
N14-Cu1-O6
N3-Cu1-N14
N3-Cu1-N12
N12-Cu1-N14
N3-Cu1-O5
N3-Cu1-O6
N12-Cu1-Cl5
O5-Cu1-O6
O5-Cu1-N14
108.2 (3)
95.9 (3)
90.6 (3)
81.9 (3)
82.0 (3)
163.5 (3)
158.8 (3)
108.2 (3)
95.3 (3)
93.1 (3)
99.1 (3)
DNA-binding study
Electronic absorption titration: Electronic absorption
titrations were carried out in Tris buffer solution (pH = 7.2).
The absorbance ratio of ct-DNA solution in buffer at 260 and
280 nm was found to be 1.9, suggesting that the ct-DNA was
satisfactorily free from protein [28]. The titrations were carried
out by maintaining a constant concentration of the complex (5 ×
10-4 M) and varying the concentration of ct-DNA solution
added.The absorbance for each addition of ct-DNA was recorded
subsequently. The intrinsic-binding constant, Kb, for complexes
1 and 2 were determined from eqn. 1:
TABLE-4
SELECTED BOND LENGTHS (Å) AND
BOND ANGLES (°) OF COPPER(II) COMPLEX 2
Selected bonds Bond length (Å) Selected bond Bond angle (°)
Cu1-O1
Cu1-O2
Cu1-O3
Cu1-N1
C12-O1
C2-O2
N1-N2
N2-C12
N3-C12
N1-C11
C11-C1
C1-C2
1.95 (3)
1.88 (3)
1.93 (4)
1.90 (3)
1.26 (5)
1.33 (5)
1.37 (5)
1.36 (5)
1.31 (6)
1.30 (5)
1.42(5)
1.39 (6)
1.42 (6)
1.36 (6)
1.41 (6)
1.42 (6)
O2-Cu1-O1
O2-Cu1-O3
O2-Cu1-N1
O3-Cu1-O1
N1-Cu1-O1
N1-Cu1-O3
C12-O1-Cu1
C2-O2-Cu1
N2-N1-Cu1
C11-N1-Cu1
C11-N1-N2
Cl2-N2-N1
O1-C12-N2
N3-C12-N2
O1-C12-N3
N1-C11-C1
175.45 (13)
92.92 (17)
93.01 (14)
91.61 (18)
82.42 (14)
173.64 (17)
112.4 (3)
127.2 (3)
112.2 (2)
128.0 (3)
119.9 (4)
114.4 (3)
118.5 (4)
118.5 (4)
112.9 (4)
124.4 (4)
[DNA] [DNA]
1
=
+
(1)
εa − εf εb − εf Kb (εb − εf )
where, [DNA] is the concentration of DNA base pair which
was calculated using molar absorption coefficient value 6600
M-1cm-1 for DNA at 260 nm [29], the apparent absorption coeffi-
cients εa, εf and εb corresponds to Aobs/[complex], extinction
coefficients of complex in free and bound state, respectively.
A plot of [DNA]/(εa – εf) vs. [DNA] was used to calculate Kb
from the ratio of slope and intercept [30].
C2-C3
C3-C4
C5-C10
C5-C6

2788 U-wang et al.
Asian J. Chem.
Complex 1 (500 µM) in Tris buffer solution (pH = 7.2)
shows charge transfer transitions at 272 nm Fig. 6a. The absor-
ption spectra of complex 1 in the presence of ct-DNA (0-100
µM) displayed hypochromism at 272 nm and hyperchromism
at 260 nm. The intrinsic-binding constant was calculated using
absorbance values at 272 nm. The presence of isosbestic points
in the spectra indicates the presence of more than one absorbing
species in solution. Complex 2 (500 µM) in Tris-buffer solution
(pH = 7.2) shows charge transfer bands at 347 and 265 nm
Fig. 6b. The spectra in the presence of increasing concentration
of ct-DNA (0-100 µM) displayed hypochromism at 347 nm
and hyperchromism at 265 nm. The absorbance values at 347
nm were used to calculate intrinsic binding constant. The spectral
patterns of complexes 1 and 2 were analogous to previously
reported complexes whose interaction mode with DNA is non-
intercalative and groove binding [16-18]. The intrinsic-binding
constant, Kb for each complex was determined from the plot of
[DNA]/(εa – εf) versus [DNA] using eqn. 1. The slope to inter-
cept ratio of the curves gave intrinsic-binding constant Kb of
2.4 × 104 M-1 for complex 1 and 8.7 × 104 M-1 for complex 2.
The Kb values are lower than those observed for classical inter-
calators (ethidium-DNA, 1.4 × 106 M-1 in 40 mM Na+ ion
concentration in 25 mM Tris-HCl (pH = 7.9) at 37 ºC [28-
31]). The higher Kb value for complex 2 could be due to
naphthalene moiety. The Kb values suggest weaker DNA-
binding affinity of both Cu(II) complexes 1 and 2 than the
classical intercalators [28-32]. The binding modes in both
Cu(II) complexes were non-partially intercalative and groove
or surface binding.
prepared. Appropriate dilutions of the stock solutions were
made for each experiment. In a typical experiment, 1.5 mL
each of ethidium bromide solution was transferred into a series
of vials. 25 µL each of ct-DNA (A260nm = 2) were added to the
ethidium bromide solutions. To the DNA-EB solutions in
different vials, different volumes of complex (0, 10, 20, 30,
etc., µM) were added.A total volume of 2.5 mL in each solution
was maintained by adding additional volume of buffer. The
solutions were equilibrated for 15 min. The variation in the
fluorescence intensities with increasing concentration of
complex were recorded at 605 nm over a spectra range of 500
to 750 nm using an excitation wavelength of 546 nm. The
apparent binding constant Kapp was calculated from eqn. 2:
KEB[EB] = Kapp[Complex]
(2)
where, KEB = 1.0 × 107 M-1, [EB] = 2 µM and [Complex] = the
molar concentration of complex at 50% reduction of fluore-
scence [33]. The classical Stern-Volmer quenching constants
(KSV) were calculated for each complex using the plot of linear
Stern-Volmer equation:
Io
=1+ Ksvr
(3)
I
where, Io and I are the fluorescence intensities of DNA-EB in
the absence and presence of the Cu(II) complex, respectively
and r is the concentration ratio of Cu(II) complex to DNA
[34,35]. Ksv value was obtained from the slope of a linear plot
of Io/I vs. r [35].
Ethidium bromide (EB) is a DNA intercalator [36] which
gives significant fluorescence emission when intercalated
between the adjacent DNA base pairs [32,35]. The extent of
fluorescence quenching for ethidium bromide bound to DNA
was used to study the extent of binding between the molecule
and DNA [33,35,37]. Fig. 7a-b show the reductions in the
fluorescence emission intensities of EB-DNA (2 µM) with
increasing concentration of Cu(II) complexes 1 and 2 (0-100
µM) recorded in Tris buffer solution (pH = 7.2). Using eqn. 2
Fluorescence quenching experiment: Stock solution of
protein free (A260 nm/A280 nm = 1.9 [28]) ct-DNA in 5 mM Tris
buffer (pH = 7.2) was freshly prepared and the concentration
was calculated as 2 × 10-4 M using molar absorption coefficient
value 6600 M-1 cm-1 of DNA at 260 nm [29]. Stock solutions
of Cu(II) complexes 1 and 2 in water (2 × 10-4 M) and stock
solution of ethidium bromide (EB) in water (3.3 µM) were
8
2.0
11
2.2
7
(a)
(b)
10
1.8
6
2.0
9
5
1.8
1.6
8
4
1.6
1.4
7
3
1.4
6
2
1.2
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
5
0
5
10 15 20 25 30 35 40 45
[DNA] × 10–6
M
1.0
0.8
0.6
0.4
0.2
0.0
20 25 30 35 40 45 50 55 60 65
[DNA] × 10–6
M
200 220 240 260 280 300 320 340 360 380 400
Wavelength (nm)
180 200 220 240 260 280 300 320 340 360 380 400 420
Wavelength (nm)
Fig. 6. (a) Electronic absorption titration of complex 1 (5 × 10-4 M) with ct-DNA (0-100 µM) in Tris-buffer (pH = 7.2); Inset: plot of [DNA]/
(εa – εf) versus [DNA]. (b) Electronic absorption titration of complex 2 (5 × 10-4 M) with ct-DNA (0-100 µM) in Tris-buffer (pH = 7.2);
Inset: plot of [DNA]/(εa – εf) versus [DNA]

Vol. 32, No. 11 (2020) Synthesis, Characterization, DNA Binding and Antimicrobial Activity Studies of New Copper(II) Complexes 2789
600
3.5
(a)
(b)
3.0
2.5
2.0
1.5
1.0
0.5
3.0
2.8
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
600
500
400
300
200
100
0
500
400
300
200
100
0
0
2
4
6
8
10 12
0
2
4
6
8
10 12 14 16 18 20
[Complex]/[DNA]
[Complex]/[DNA]
540 560 580 600 620 640 660 680 700 720
Wavelength (nm)
500
550
600
650
700
750
Wavelength (nm)
Fig. 7. (a) Fluorescence emission spectra of ethidium bromide-ctDNA in Tris-HCl buffer (pH = 7.2) with increasing concentration of Cu(II)
complex 1 (λex = 546 nm); Inset: Fluorescence quenching curve of EB-DNA by Cu(II) complex 1. (b) Fluorescence emission spectra of
ethidium bromide-ct-DNA in Tris-HCl buffer (pH = 7.2) with increasing concentration of Cu(II) complex 2 (λex = 546 nm); Inset:
Fluorescence quenching curve of EB-DNA by Cu(II) complex 2
the apparent-binding constant Kapp were calculated and found
to be 3.5 × 105 M-1 and 3.7 × 105 M-1 for both Cu(II) complexes
1 and 2, respectively. The observed values of Kapp suggested
the complexes were binding toward ct-DNA [33]. The
quenching of EB-DNA fluorescence by the complexes is in
good agreement with the linear Stern-Volmer equation (eqn.
3) [32]. The slope of the linear plot Io/I vs. r (r = [Complex]/
[DNA]) gave quen-ching constant KSV values of 2.35 and 2.57
for Cu(II) comp-lexes 1 and 2, respectively. The values suggest
competitive inhibition caused by minor groove binding of the
complexes, releasing some free ethidium bromide from DNA-
EB complex and blocking potential interaction sites of ethidium
bromide [32,35,38].
KCl) within +1.4 V to –1.4 V at a scan rate of 0.05 V/s in Tris
buffer (pH = 7.2) in the absence of DNA gave two reduction
peaks at 0.18 and –1.12 V, (Fig. 8a) which may be assigned to
Cu(II)/Cu(I) and Cu(I)/Cu(0) reductions. The oxidation peaks
in the reverse scan, observed at 0.18 and 0.75V, were assigned
to Cu(0)/Cu(I) and Cu(I)/Cu(II) oxidations, respectively. In
the presence of ct-DNA (200 µM), a reduction peaks at –1.24
V and an oxidation peak at 1.12 V were observed. The peaks
were tentatively assigned Cu(II)/Cu(I) couples. The formal
potential E1/2 values for Cu(II)/Cu(I) couple were observed as
0.18 V and -0.01 V in the absence and presence of DNA,
respectively (Table-5).
From the wavy cyclic voltammogram of Cu(II) complex
2 (200 µM; Tris buffer pH = 7.2; +1.4 to –1.4V; 0.02 V/s) vs.
AgCl/Ag (saturated KCl) in the absence of DNA two reduction
Cyclic voltammetry: Room temperature cyclic voltam-
mogram of Cu(II) complex 1 (200 µM) vs.AgCl/Ag (saturated
0.00003
(a)
(b)
0.00003
0.00002
0.00001
0.00000
-0.00001
-0.00002
Complex 2
Complex 1
0.00002
Complex 2 + DNA
Complex 1 + DNA
0.00001
0.00000
-0.00001
-0.00002
-0.00003
1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 -0.2 -0.4 -0.6 -0.8 -1.0 -1.2 -1.4
Potential (V)
1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 -0.2-0.4-0.6-0.8-1.0-1.2-1.4
Potential (V)
Fig. 8. (a) Cyclic voltammogram of Cu(II) complex 1 (200 µM) against AgCl/Ag (saturated KCl) electrode within +1.4 V to –1.4 V at a scan
rate of 0.05 V/s in the absence (red) and presence (black) of ct-DNA (2 × 10-4 M). (b) Cyclic voltammogram of Cu(II) complex 2 (200
µM) against AgCl/Ag (saturated KCl) electrode within +1.4 V to –1.4 V at a scan rate of 0.02 V/s in the absence (red) and presence
(black) of ct-DNA (2 × 10-4 M)

2790 U-wang et al.
Asian J. Chem.
TABLE-5
CYCLIC VOLTAMMETRY DATA FOR COMPLEXES 1 AND 2 IN THE ABSENCE AND PRESENCE OF ct-DNAa
Complex
EPC (V)
iPC × 106 (A)
EPA (V)
iPA × 106 (A)
iPA/iPC
E1/2 (V)
∆E (V)
+0.18
-1.12
-1.24
+0.29
-1.05
+0.28
-1.12
3.39
7.91
5.56
4.72
17.31
+0.18
+0.75
+1.12
+0.37
+0.86
+0.28
+0.86
-2.75
-10.30
-13.70
-4.65
-24.35
-1.52
0.0
1.87
2.36
0.08
1.91
0.0
0.81
1.30
2.46
0.98
1.40
0.36
1.03
0.18
-0.18
-0.01
0.33
-0.11
0.28
1
1 + DNA
2
4.16
19.39
2 + DNA
-20.13
1.98
-0.13
aEPA, iPA and EPC, iPC are anodic and cathodic peak potentials and peak currents; ∆E = EPA – EPC and E1/2 = (EPA + EPC)/2.
peaks at 0.29 and –1.05 V (Fig. 8b) were assigned to Cu(II)/
Cu(I) and Cu(I)/Cu(0) reduction processes. The corresponding
oxidation peaks in the reverse scan were observed at 0.37 and
0.86 V. In the presence of DNA the Cu(II) complex 2 showed
reduction peaks at 0.28 V (Cu(II)/Cu(I)) and -1.12 V (Cu(I)/
Cu(0)). In the reverse scan oxidation peaks were observed at
0.28 V (Cu(0)/Cu(I)) and 0.86 V (Cu(I)/Cu(II)). The formal
potential E1/2 values for Cu(II)/Cu(I) couple were observed as
0.33 V and 0.28 V in the absence and presence of DNA,
respectively (Table-5). The E1/2 values for Cu(I)/Cu(0) in the
absence and presence of DNA were -0.11 V and -0.13 V,
respectively (Table-5).
For both Cu(II) complexes, the peak separation values and
peak current ratios show that the redox processes are not rever-
sible. As the DNA and the respective ligands do not show any
redox processes it may be concluded that the redox processes
are due to the presence of metal centers. The observed experi-
mental result suggests that the presence of DNA strongly affects
the redox processes of metal centers in both complexes.Among
the three kinds of binding modes of small molecules to DNA,
if the formal potential E1/2 is shifted to more positive value the
interaction mode is intercalative binding, while E1/2 shifted to
more negative value the binding mode is electrostatic [35]. It
has been observed that the shift in E1/2 values in presence of
DNA from that in the absence of DNA are toward more negative
values, suggesting the complexes bind to ct-DNA by non-
intercalative surface or groove-binding mode through electro-
static interaction.
Viscosity measurement:Viscosity measurement of DNA
solution with increasing concentrations of Cu(II) complex is
useful for determining the binding mode of interaction of
Cu(II) complex with DNA. The effects of small molecules on
viscosity of DNA solution are as follows: Intercalative binding
causes an increase in viscosity; non-classical intercalative binding
causes a lowering in viscosity and surface or groove binding causes
no change in viscosity of ct-DNA solution [16]. When visco-
sities of DNA solutions with increasing concentrations of comp-
lexes 1 and 2 were measured the relative viscosities (η/ηo)
were found to be unchanged suggesting that complexes 1 and 2
binds with DNA via non-intercalative surface or groove binding.
organisms tested were Escherichia coli (EC) ATCC-11229,
Staphylococcus aureus (SA) ATCC-11632, Bacillus cereus
(BC) MTCC-430, Enterococcus faecium (EF) ATCC-35667,
Candida albicans (CA) ATCC-10231 and Aspergillus niger
(AN) ATCC-16888.
Preparation of inoculum: Bacterial inoculums were
prepared by growing cells in Luria Bertani Broth (HiMedia)
for 24 h at 37 ºC. On the other hand, the inoculums of yeast
and fugus strains were transferred directly into potato dextrose
agar (HiMedia) plates and incubated at 25 ºC for 48 h. They
were maintained on agar slant at 4 ºC and sub cultured on a
fresh appropriate agar plates 24 h prior to any antimicrobial
test. The Muller Hinton Broth (MHB) was used for the mini-
mum inhibition concentration (MIC).
Determination of microbial growth inhibitory poten-
tial: The microbial growth inhibitory potentials of both Cu(II)
complexes were determined by using the agar diffusion method
[39] with some modification. Sterile Petri plates (90 mm) were
prepared with 20-25 mL of sterile Luria Bertani Agar (LBA)
(Himedia) and potato dextrose agar (PDA) (Himedia) and
allowed to solidify. 100 µL of adjusted test cultures (108 CFU/
mL) was swabbed on the top of the solidified media and allowed
to dry for 10 min on the plates. Solidified plates were punched
to make well of 6 mm diameter with the help of sterile coke
borer. Next, 30 and 20 µL of each compound dissolved in
dimethyl sulfoxide (DMSO; 5 mg/mL) were added into the
wells. Gentamycin (5 µg/well) and nystatin (10 µg/well) (RA)
1 mg/mL in sterile distilled water were used as positive control
for the bacteria and yeast, respectively.A well containing only
DMSO (30 µL) was used as a negative control. Culture plates
were incubated at 37 ºC for 24 h for bacteria and at 25 ºC for
48 h for yeast. At the end of the incubation, the diameters of
the inhibition zone formed around the wells were measured in
millimeter. The zone inhibition was increased with the increase
in concentration of the compound and thus exhibiting concen-
tration dependent activity.All tests were performed in triplicate
(Table-6).
Determination of minimum inhibitory concentration
(MIC): The MIC of Cu(II) complexes and reference antibiotic
(RA) were determined as follows; the test sample was dissolved
in 10% (v/v) DMSO/MHB to give a final concentration 500
µg/mL. This was serially diluted two-fold to obtain concen-
tration ranges of 0.40-50 µg/mL. Each concentration (100 µL)
was added in a well (96-well micro plate) containing 100 µL
of inoculum.An inoculum density of 1 × 106 cfu/mL of bacterial
Antimicrobial activity study
Tested microorganism: For the antimicrobial assays a
Gram-negative and three Gram-positive bacterial and a species
each of yeast and fungus were used. The strains of micro-

Vol. 32, No. 11 (2020) Synthesis, Characterization, DNA Binding and Antimicrobial Activity Studies of New Copper(II) Complexes 2791
TABLE-6
MICROBIAL GROWTH INHIBITORY POTENTIAL OF COMPOUNDS AND THE REFERENCE ANTIBIOTICS
Diameter of zone of inhibition (mm)/each well = 5 mm; Tested microorganisms
Gram-negative
Tested
compounds
Concentration
(µg/well)
Gram-positive bacteria
Fungus
Yeast
bacteria
Escherichia
coli
Enterococcus
faccium
Staphylococcus
aureus
Aspergillus
niger
Candida
albicans
Bacillus cereus
PCPH
Complex 1
HNSC
150
100
150
100
150
100
150
100
5
8
7
9
7
7
6
8
7
10
8
18
16
16
14
18
15
8
6
10
7
10
8
15
13
10
8
14
11
14
12
16
15
Not active
Not active
Not active
Not active
Not active
Not active
Not active
Not active
Not active
18
Not active
Not active
Not active
Not active
Not active
Not active
Not active
Not active
Not active
20
Complex 2
Gentamycin
Nystatin
DMSO
18
–
22
–
18
–
20
–
10
30 µL
Not active
Not active
Not active
Not active
Not active
Not active
TABLE-7
MINIMUM INHIBITORY CONCENTRATION (µg/mL) OF THE COMPOUNDS AND REFERENCE ANTIBIOTIC (RA)
Tested microorganisms
Tested
Gram-negative bacteria
Gram-positive bacteria
Fungus
Yeast
compounds
Enterococcus
faccium
Staphylococcus
aureus
Aspergillus
niger
Candida
albicans
Escherichia coli
Bacillus cereus
PCPH
Complex 1
HNSC
Complex 2
Gentamycin
Nystatin
50
12.5
>50
12.5
5
25
6.12
25
6.12
5
25
12.5
50
6.12
5
25
12.5
50
6.12
5
Inactive
Inactive
Inactive
Inactive
Inactive
10
Inactive
Inactive
Inactive
Inactive
Inactive
10
–
–
–
–
strain was prepared in normal saline (9 g/L) by comparison
with a 0.5 MacFarland turbidity standard with appropriate
dilution [40]. The final concentration of DMSO in the well
was less than 3%. The negative control well consists of 100
µL of MHB and 100 µL of the standard inoculum [41]. The
plates were covered with sterile plate sealer, then agitated to
mix the contents of the wells using a plate shaker and incubated
at 37 ºC for 24 h. The assay was repeated three times. The
MIC of sample was detected following additional (40 µL) of
0.2 mg/mL nitroblue tetrazolium chloride (NBT) (Himedia)
to microplate wells and incubated at 37 ºC for 30 min [42].
Viable microorganisms reduced the yellow to black. MIC was
defined as the lowest sample concentration of drug at which
there was no visible growth of the organism.
1 and 2 have higher bacterial growth inhibitory potential than
the corresponding ligand. The MIC for copper(II) complexes
1 and 2 in case of Staphylococcus aureus, Bacillus cereus and
Enterococcus faccium were comparable with standard antibiotic.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge UGC, New Delhi,
India for financial support.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interests
regarding the publication of this article.
REFERENCES
Conclusion
1. S.D. Roughley and A.M. Jordan, J. Med. Chem., 54, 3451 (2011);
https://doi.org/10.1021/jm200187y
2. M. Aldeghi, S. Malhotra, D.L. Selwood and A.W.E. Chan, Chem. Biol.
Drug Des., 83, 450 (2014);
https://doi.org/10.1111/cbdd.12260
3. T.J. Ritchie, S.J.F. Macdonald, R.J. Young and S.D. Pickett, Drug
Discov. Today, 16, 164 (2011);
https://doi.org/10.1016/j.drudis.2010.11.014
4. T.J. Ritchie and S.J.F. Macdonald, Drug Discov. Today, 14, 1011 (2009);
https://doi.org/10.1016/j.drudis.2009.07.014
5. H.W. Boucher, G.H. Talbot, J.S. Bradley, J.E. Edwards, D. Gilbert,
L.B. Rice, M. Scheld, B. Spellberg and J. Bartlett, Clin. Infect. Dis.,
48, 1 (2009);
The present work reports the synthesis, crystal structure,
spectroscopic and DNA-binding and antimicrobial activity
studies of pyridine-2-carboxaldehyde-N-(2-pyridyl)hydrazone
(PCPH) Cu(II) complex [Cu(PCPH)Cl(H2O)](NO3)(H2O)2 (1)
and 2-hydroxy-1-naphthaldehyde-semicarbazone (HNSC)
Cu(II) complex [Cu(II)(HNSC)H2O]·NO3·H2O (2). Electronic
absorption titration experiment, EB-DNA fluorescence quenching
experiments, cyclic voltammetric experiments and viscosity
measurements suggests that both Cu(II) complexes (1 and 2)
might be non-intercalative surface or groove binder to ct-DNA.
Agar diffusion method showed that both copper(II) complexes
https://doi.org/10.1086/595011

2792 U-wang et al.
Asian J. Chem.
6. F. Gao, P. Yang, J. Xie and H. Wang, J. lnorg. Biochem., 60, 61 (1995);
https://doi.org/10.1016/0162-0134(95)00002-6
23. O.V. Dolomanov, L.J. Bourhis, R.J. Gildea, J.A.K. Howard and H.
Puschmann, J. Appl. Cryst., 42, 339 (2009);
7. S. Correia, P. Poeta, M. Hébraud, J.E. Capelo and G. Igrejas, J. Med.
Microbiol., 66, 551 (2017);
https://doi.org/10.1107/S0021889808042726
24. D. Kivelson and R. Neiman, J. Chem. Phys., 35, 149 (1961);
https://doi.org/10.1063/1.1731880
https://doi.org/10.1099/jmm.0.000475
8. V. Uivarosi, Molecules, 18, 11153 (2013);
25. I.M. Procter, B.J. Hathaway and P. Nicholls, J. Chem. Soc. A, 1678
(1968);
https://doi.org/10.3390/molecules180911153
9. M. Imran, J. Iqbal, S. Iqbal and N. Ijaz, Turk. J. Biol., 31, 67 (2007).
10. W. Guerra, E. de Andrade Azevedo, A.R. de Souza Monteiro, M.
Bucciarelli-Rodriguez, E. Chartone-Souza, A.M.A. Nascimento, A.P.S.
Fontes, L. Le Moyec and E.C. Pereira-Maia, J. Inorg. Biochem., 99,
2348 (2005);
https://doi.org/10.1039/j19680001678
26. J.R. Wasson and C. Trapp, J. Phys. Chem., 73, 3763 (1969);
https://doi.org/10.1021/j100845a034
27. A.W. Addison, T.N. Rao, J. Reedijk, J. van Rijn and G.C. Verschoor, J.
Chem. Soc., Dalton Trans., 1349 (1984);
https://doi.org/10.1016/j.jinorgbio.2005.09.001
https://doi.org/10.1039/DT9840001349
11. R. Rohs, I. Bloch, H. Sklenar and Z. Shakked, Nucleic Acids Res., 33,
7048 (2005);
28. M.E. Reichmann, S.A. Rice, C.A. Thomas and P.A. Doty, J. Am. Chem.
Soc., 76, 3047 (1954);
https://doi.org/10.1093/nar/gki1008
https://doi.org/10.1021/ja01640a067
12. A.-M. Stadler and J. Harrowfield, Inorg. Chim. Acta, 362, 4298 (2009);
https://doi.org/10.1016/j.ica.2009.05.062
13. M.J. Borah, R.K. Bhubon Singh, U.B. Sinha, T. Swu and P.J. Borah, J.
Chem. Crystallogr., 42, 67 (2012);
29. J. Marmur, J. Mol. Biol., 3, 208 (1961);
https://doi.org/10.1016/S0022-2836(61)80047-8
30. A. Wolfe, G.H. Shimer Jr. and T. Meehan, Biochemistry, 26, 6392 (1987);
https://doi.org/10.1021/bi00394a013
https://doi.org/10.1007/s10870-011-0205-5
14. W.B. Devi, R.K.B. Singh, J.P. Jasinski and J.A. Golen, Inorg. Chem.
Commun., 21, 163 (2012);
31. J.B. LePecq and C. Paoletti, J. Mol. Biol., 27, 87 (1967);
https://doi.org/10.1016/0022-2836(67)90353-1
32. J. Liu, T. Zhang, T. Lu, L. Qu, H. Zhou, Q. Zhang and L. Ji, J. Inorg.
Biochem., 91, 269 (2002);
https://doi.org/10.1016/j.inoche.2012.05.006
15. U.I. Singh, R.K. Bhubon Singh, M.J. Borah, J.P. Jasinski and J.A. Golen,
J. Struct. Chem., 54, 116 (2013);
https://doi.org/10.1016/S0162-0134(02)00441-5
33. M. Lee, A.L. Rhodes, M.D. Wyatt, S. Forrow and J.A. Hartley,
Biochemistry, 32, 4237 (1993);
https://doi.org/10.1134/S0022476613010162
16. M. Nandy, D.L. Hughes, G.M. Rosair, R.K.B. Singh and S. Mitra, J.
Coord. Chem., 67, 3335 (2014);
https://doi.org/10.1021/bi00067a011
34. J.R. Lakowicz and G. Weber, Biochemistry, 12, 4161 (1973);
https://doi.org/10.1021/bi00745a020
https://doi.org/10.1080/00958972.2014.964697
17. R.K.B. Devi, S.P. Devi, R.K.B. Singh, R.K.H. Singh, T. Swu, W.R.
Devi and C.H.B. Singh, J. Coord. Chem., 67, 891 (2014);
https://doi.org/10.1080/00958972.2014.902449
18. N. Shantibala Devi, L. Jaideva Singh, S. Pramodini Devi, R.K. Bhubon
Singh, R.K. Hemakumar Singh, B. Rajeswari and R.M. Kadam, J. Mol.
Struct., 1076, 411 (2014);
35. Y. Li, Y. Wu, J. Zhao and P. Yang, J. Inorg. Biochem., 101, 283 (2007);
https://doi.org/10.1016/j.jinorgbio.2006.10.004
36. W.D. Wilson, L. Ratmeyer, M. Zhao, L. Strekowski and D. Boykin,
Biochemistry, 32, 4098 (1993);
https://doi.org/10.1021/bi00066a035
37. B.C. Baguley and M. Le Bret, Biochemistry, 23, 937 (1984);
https://doi.org/10.1021/bi00300a022
https://doi.org/10.1016/j.molstruc.2014.08.005
19. R.N. Patel, Y.P. Singh, Y. Singh, R.J. Butcher, M. Zeller, R.K.B. Singh
and O. U-wang, J. Mol. Struct., 1136, 157 (2017);
https://doi.org/10.1016/j.molstruc.2017.01.083
38. D.L. Boger, B.E. Fink, S.R. Brunette, W.C. Tse and M.P. Hedrick, J.
Am. Chem. Soc., 123, 5878 (2001);
https://doi.org/10.1021/ja010041a
20. O. U-Wang, R.B. Singh, W.B. Devi, U.I. Singh, R.B. Devi, O.B. Devi,
R. Shahani and T. Swu, Inorg. Nano-Met. Chem., 49, 363 (2019);
https://doi.org/10.1080/24701556.2019.1661457
21. Agilent Technologies, CrysAlisPro, version 1.171.36.21; CrisAlis 171.
Net, 10 Mead Road, Oxford Industrial Park,Yarnton, Oxfordshire, U.K.
(2012).
39. C. Perez, M. Pauli and P. Bazerque, Acta Biol. Med. Exp., 15, 113 (1990).
40. V. Kuete, D.C. Fozing, W.F.G.D. Kapche, A.T. Mbaveng, J.R. Kuiate,
B.T. Ngadjui and B.M. Abegaz, J. Ethnopharmacol., 124, 551 (2009);
https://doi.org/10.1016/j.jep.2009.05.004
41. J.R. Zgoda and J.R. Porter, Pharm. Biol., 39, 221 (2001);
https://doi.org/10.1076/phbi.39.3.221.5934
22. G.M. Sheldrick, Acta Crystallogr. A, 64A, 112 (2008);
https://doi.org/10.1107/S0108767307043930
42. V. Kuete, B. Ngameni, C.C.F. Simo, R.K. Tankeu, B.T. Ngadjui, J.J.M.
Meyer, N. Lall and J.R. Kuiate, J. Ethnopharmacol., 120, 17 (2008);
https://doi.org/10.1016/j.jep.2008.07.026