DNA-binding, -cleavage and antimicrobial investigation on mononuclear Cu(II) Schiff base complexes originated from Riluzole

Article abstract of DOI:10.1016/j.molstruc.2021.131002

Two mononuclear metal complexes, [Cu(L₁)₂] (1) and [Cu(L₂)₂] (2) of the respective Schiff bases, HL₁ = 2-((E)-(6-(trifluoromethoxy)benzo[d]thiazol-2-ylimino)methyl)-4-methoxyphenol and HL₂ = 2-((E)-(6-(trifluoromethoxy)benzo[d]thiazol-2-ylimino)methyl)-4,6-dibromophenol were synthesized and well characterized by analytical and various spectroscopic techniques like elemental analysis, NMR, mass spectrometry, IR, UV, ESR and thermogravimetric analyses. These spectral studies gave a square planar geometry for both the complexes. These complexes underwent DNA investigation against calf thymus DNA and supercoiled pBR322 DNA. The complexes bound the DNA through an intercalation mode and the binding affinity order follows as 1 > 2 > HL₂ > HL₁. Both complexes show good cleavage ability against double-stranded pBR322 DNA under oxidative and photolytic conditions. In vitro antimicrobial study resulted in both complexes have shown marked biocidal potential compared to respective free ligands.

Full text of DOI:10.1016/j.molstruc.2021.131002

Journal of Molecular Structure 1244 (2021) 131002
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
DNA-binding, -cleavage and antimicrobial investigation on
mononuclear Cu(II) Schiff base complexes originated from Riluzole
Aveli Rambabu, Sreenu Daravath, Dasari Shiva Shankar, Shivaraj∗
Department of Chemistry, Osmania University, Hyderabad, Telangana 500007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Two mononuclear metal complexes, [Cu(L1)2] (1) and [Cu(L2)2] (2) of the respective Schiff bases,
HL1 = 2-((E)-(6-(trifluoromethoxy)benzo[d]thiazol-2-ylimino)methyl)-4-methoxyphenol and HL2 = 2-((E)-
(6-(trifluoromethoxy)benzo[d]thiazol-2-ylimino)methyl)-4,6-dibromophenol were synthesized and well
characterized by analytical and various spectroscopic techniques like elemental analysis, NMR, mass spec-
trometry, IR, UV, ESR and thermogravimetric analyses. These spectral studies gave a square planar geom-
etry for both the complexes. These complexes underwent DNA investigation against calf thymus DNA and
supercoiled pBR322 DNA. The complexes bound the DNA through an intercalation mode and the binding
affinity order follows as 1 > 2 > HL2 > HL1. Both complexes show good cleavage ability against double-
stranded pBR322 DNA under oxidative and photolytic conditions. In vitro antimicrobial study resulted in
both complexes have shown marked biocidal potential compared to respective free ligands.
Received 20 March 2021
Revised 16 June 2021
Accepted 28 June 2021
Available online 1 July 2021
Keywords:
Schiff base
Mononuclear metal complex
DNA binding
DNA Cleavage
Antimicrobial activity
© 2021 Elsevier B.V. All rights reserved.
1. Introduction
Schiff base metal complexes, especially synthesized from tran-
sition metals, occupied a pride position in inorganic biochem-
Riluzole [1], commercial name Rilutek, a bicyclic and hetero-
cyclic compound, used to treat a certain type of nerve disease
named Amyotrophic Lateral Sclerosis. It contains a benzothiazole
molecule as a core unit which has numerous biological applica-
tions [2,3]. Though it is a good drug, still being considered as a
prominent one to get more biological applications by the modi-
fication of its original frame [4]. A lot of benzothiazole-originated
Schiff bases came to the limelight with their flexible synthesis pro-
cedure and higher biological potency [5,6]. The substituent ‘triflu-
oromethoxy group’ of Riluzole plays an important role in enhanc-
ing the biological potency. Hence, it is thought worthwhile to fo-
cus on synthesizing the different Schiff bases using this Riluzole
molecule. The azomethine group of Schiff base offers an opportu-
nity for stupendous biological activities such as antimicrobial, an-
titumor and herbicidal activities [7,8]. Schiff bases having hetero-
cyclic moieties as core units particularly with nitrogen, oxygen and
sulpher atoms are easily soluble in common organic solvents be-
cause of more polarizable nature of those hetero atoms [9]. Triflu-
oromethoxy group has high lipophilic nature and molecules con-
taining such groups exhibit a remarkable enhancement in their in
vivo uptake and transport in biological systems [10].
istry. Cisplatin, for instance, and its analogs carboplatin, oxaliplatin
and nedaplatin were proved to be better chemotherapeutic drugs
which bind DNA covalently, used in the treatment of various car-
cinomas [11]. But, the platins’ covalent binding mode causes un-
wanted side effects like hepatotoxicity, nephrotoxicity, neurotoxic-
ity, ototoxicity and gastrointestinal reaction [12]. Hence, the bio-
chemists’ focus was shifted to develop non-platins using other
transition metals which should have the non-covalent binding abil-
ity, minimum side effects and maximum curative potential. Copper
is the one, among transition metals, which gets more attention and
being investigated by biochemists. Palaniandavar [13] and Serment-
Guerrero [14] have already been stated that the copper complexes
are the best alternatives to Cisplatin.
DNA is the primary intracellular target for the drugs that are
used in the treatment of various carcinomas. An obvious under-
standing of Drug-DNA interaction mechanism helps the researchers
to design the potential and specific DNA targeted drugs. For this
purpose, according to literature statistics, a lot of metal complexes
having various oxidation states, various substituents, of different
geometries and of different sizes have been and being investigated.
The Schiff base-transition metal complexes have a great tendency
to interact with DNA because of their cationic character and
three-dimensional structural profiles. They can bind DNA through
various distinct modes of non-covalent interactions that are in-
tercalative, groove and external electrostatic binding [15]. A large
number of intercalatively bound Schiff’s base metal complexes
∗
Corrosponding author.
E-mail address: srajkavadi@gmail.com (Shivaraj).
https://doi.org/10.1016/j.molstruc.2021.131002
0022-2860/© 2021 Elsevier B.V. All rights reserved.

A. Rambabu, S. Daravath, D.S. Shankar et al.
Journal of Molecular Structure 1244 (2021) 131002
having different moieties as core units have been reported earlier
from our laboratory [16–20]. In continuation of our investigation,
the Riluzole’s promising biological and other features emerge
us to focus on the design and synthesis of biologically active
metal complexes derived from Riluzole originated Schiff’s bases,
2-((E)-(6-(trifluoromethoxy)benzo[d]thiazol-2-ylimino)methyl)−4-
methoxyphenol and 2-((E)-(6-(trifluoromethoxy)benzo[d]thiazol-2-
ylimino)methyl)−4,6-dibromophenol. These were further inves-
tigated for their DNA binding and cleavage ability by various
spectroscopic and gel electrophoresis methods.
2-((E)-(6-(trifluoromethoxy)benzo[d]thiazol-2-
ylimino)methyl)−4,6-dibromophenol (HL2): Yellow solid. Yield:
68%. M.p. 204 °C. 1H NMR (400 MHz, CDCl3): δ 13.04 (s, 1H), 9.26
(s, 1H), 7.99 (d, J = 9.0 Hz, 1H), 7.86 (s, 1H), 7.75 (s, 1H), 7.64
(s, 1H), 7.40 (d, J = 9.0 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ
168.3, 165.4, 157.6, 149.8, 146.6, 140.4, 135.9, 135.1, 124.3, 120.9,
119.9, 114.4, 112.6, 112.2, 111.3; MS (ESI): m/z 497 (M + 4); IR
(KBr)(cm−1): ν(OH) 3431, ν(HC=
1587, ν(C–O) 1199; UV (DMSO)
max/nm(cm−1): 260 (38,461), 342 (29,239).
N
)
λ
2.3.2. Synthesis of metal complexes
2. Experimental
Mononuclear Cu(II) complexes (1 and 2) were synthesized by
adding the methanolic solution of copper acetate monohydrate
[Cu(AcO)2. H2O] to the methanolic solution of HL1 / HL2 in 1:2 M
ratio. The reaction mixture was refluxed on an oil bath for 6 h by
maintaining the temperature at 60–70 °C. The precipitate formed
was filtered off and washed thoroughly with methanol and cold
water, then dried in a desiccator over anhydrous CaCl2.
2.1. Materials and instrumentation
The required chemicals and solvents were purchased from Finar,
HiMedia, Merck and Sigma-Aldrich Pvt Ltd. The solvents were well
purified using the standard protocols [21]. The calf thymus and su-
percoiled pBR322 DNA were purchased from Genei, Bangalore and
stored at 4 °C.
[Cu(L1)2] (1): Dark blue solid. Yield: 78%. m.p.: 130 °C. MS (ESI):
m/z 797 [M]+. Anal. Calc. (C32H20CuF6N4O4S2): C, 48.15; H, 2.53; N,
7.02; S, 8.03; Cu, 7.96. Found: C, 47.96; H, 2.32; N, 6.88; S, 7.92; Cu,
7.88. IR (KBr) (cm−1): ν(HC= 1571, ν(C–O) 1163, ν(M–O) 580, ν(M–N)
CHNS estimation for all compounds was carried out on Perkin
Elmer 240C elemental analyzer. NMR spectra of ligands were
recorded on Bruker 400 MHz NMR instrument using TMS (tetram-
ethylsilane) as a standard. ESI-MS data were recorded on VG AU-
TOSPEC mass spectrometer. IR spectra were recorded on a Shi-
madzu IRPrestige-21 spectrophotometer using KBr pellets. Elec-
tronic spectra were obtained from Shimadzu UV−Vis 2600 spec-
trophotometer. Magnetic susceptibilities of metal complexes were
obtained from Gouy balance model 7550 using Hg[Co(NCS)4] as
a standard. GBC Avanta 1.0 AAS estimated the metal content of
each complex. Polmon instrument (Model No. MP-96) provided the
melting points data. JESFA200 ESR spectrometer (JEOL-Japan) pro-
vided ESR spectral information under liquid nitrogen temperature.
Thermograms with a temperature range of 27−1000 °C were ob-
tained from Shimadzu TGA-50H instrument under a dynamic ni-
trogen atmosphere with a heating rate of 10 °C min−1. Ethidium
bromide displacement assay was performed on a JASCO spectroflu-
orometer FP-8500. Ostwald viscometer, Vensil made, offered vis-
cosity measurement data.
N
)
446. UV–Vis (DMSO) λmax/nm (cm−1): 272 (36,764), 302 (33,112),
590 (16,949). μeff (BM): 1.78. ESR: g = 2.29, g┴ = 2.06, G = 4.62.
ꢀ
[Cu(L2)2] (2): Dark blue solid. Yield: 74%. m.p.: 204 °C. MS
(ESI): m/z 1052 [M + 4]. Anal. Calc. (C30H12CuBr4F6N4O4S2): C,
34.20; H, 1.15; N, 5.32; S, 6.09; Cu, 6.03. Found: C, 34.04; H, 1.02;
N, 5.10; S, 6.11; Cu, 6.15. IR (KBr) (cm−1): ν(HC= 1626, ν(C–O)
N
)
1152, ν(M–O) 572, ν(M–N) 420. UV–Vis (DMSO) λmax/nm (cm−1):
260 (38,461), 421 (23,752), 589 (16,977). μeff (BM): 1.89. ESR:
g
= 2.28, g┴ = 2.07, G = 4.15.
ꢀ
2.4. DNA binding
UV absorption titration is a prominent technique to investigate
the binding mode of synthesized compounds against DNA. Here,
absorption titrations for prepared HL1, HL2, and their complexes
1 and 2 were performed at RT in TAE buffer (40 mM Tris base,
20 mM acetic acid and 1 mM EDTA) by the test compound’s con-
centration fixed at 10 μM, while ascending the calf thymus-DNA
concentration from 0 to 10 μM in successive portions. The prepa-
ration procedure of respective DNA and test compounds’ stock so-
lutions were given in our previous literature [22]. While perform-
ing titrations, the same quantities of DNA were added to both test
compound and reference solutions to avoid the absorbance of DNA
itself. The spectrum was recorded after the resultant DNA-treated
solution was kept for incubation for 5 min for each titration. The
data obtained from whole titrations were helpful to calculate the
binding constant (Kb) values with the help of the following equa-
tion [23], by that the estimation of binding affinity of the test com-
pounds can be done.
2.2. Synthesis of Riluzole
(2-amino-6-(trifluoromethoxy)benzothiazole) (i)
The starting material (i) was prepared according to the method
reported early [4].
2.3. Synthesis of Schiff base ligands and their metal complexes
2.3.1. Synthesis of Schiff bases, HL1 and HL2
Both the Schiff bases were synthesized by adding the 20 ml of
methanolic solution of 5–methoxy salicylaldehyde (10 mM) / 3,5-
di–bromo salicylaldehyde to 20 ml of hot methanolic solution of
Riluzole in 1:1 M ratio with continuous stirring and refluxing at
60–70 °C temperature on an oil bath for 24 h. The orange / yellow
colored precipitate was formed, filtered off and recrystallized from
methanol.
DNA / ε − ε = DNA / ε − ε + 1/K ε − ε
(1)
Where, [DNA] is the concentration of DNA base pairs, and εa, εf,
[
] ( [ ] (
)
)
(
)
a
f
b
f
b
b
f
and εb are the extinction coefficients of test compound at a given
concentration (Aobs/[compound]), in its free and fully DNA-bound
forms, respectively. Kb is the binding constant, gets from the plot
of [DNA]/(εa – εf) versus [DNA].
To get further support of the binding mode, fluorescence quench-
ing titrations were also done for these compounds against EB–DNA
(EB: 12.5 μM and DNA: 125 μM) adduct in TAE buffer by tak-
ing the concentration ratio as 1:10 EB to DNA. This titration in-
volves the EB–DNA adduct concentration was kept constant while
increasing the test compounds’ concentration as of 0–60 μM. The
quenching titrations against the adduct were recorded in the range
of 520–750 nm by setting the excitation wavelength as 510 nm.
2-((E)-(6-(trifluoromethoxy)benzo[d]thiazol-2-ylimino)methyl)−4-
methoxyphenol (HL1): Orange solid. Yield: 72%. M.p. 130 °C. 1H
NMR (400 MHz, CDCl3): δ 11.78 (s, 1H), 9.22 (s, 1H), 7.95 (d,
J = 9.0 Hz, 1H), 7.71 (s, 1H), 7.36 (d, J = 8.7 Hz, 1H), 7.11 (dd,
J1 = 3.0, J2 = 9.0 Hz, 1H), 7.0 (d, J = 9.0 Hz, 1H), 6.96 (d,
J = 3.0 Hz, 1H), 3.81 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 170.0,
167.5, 156.7, 152.7, 149.9, 146.3, 135.4, 124.1, 123.8, 120.6, 119.2,
118.7, 117.7, 115.6, 114.3, 55.8; MS (ESI): m/z 369 (M + H+); IR
(KBr)(cm−1): ν(OH) 3433, ν(HC= 1568, ν(C–O) 1167; UV (DMSO)
N
)
λ
max/nm(cm−1): 252 (39,682), 333 (30,030), 415 (24,096).
2

A. Rambabu, S. Daravath, D.S. Shankar et al.
Journal of Molecular Structure 1244 (2021) 131002
Table 1
Characteristic IR, electronic spectral and magnetic moment data.
IR frequencies (cm−1
)
UV–Vis (nm) and magnetic data
Compound
ν(OH)
ν(
ν(C–O)
ν(M–O)
ν(M–N)
π–π∗
π–π∗
d–d
μeff (BM)
=
C
N
)
HL1
HL2
1
3433
3431
–
1568
1587
1571
1626
1167
1199
1163
1152
–
–
252
260
272
260
333
342
302
–
–
–
–
–
–
–
580
572
446
420
590
589
1.78
1.89
2
–
From obtained spectra, the quenching efficiency with the help of
following Stern-Volmer equation [24] and as a consequence, the
binding affinity of test compounds was determined.
3. Results and discussion
The ligands (HL1and HL2) and their copper complexes (1 and
2) were synthesized in a conventional manner [25], stable at RT
and non-hygroscopic. Ligands are soluble in all common organic
solvents except in water, whereas complexes are soluble only in
DMSO. All prepared compounds are structurally characterized by
various analytical and spectroscopic methods.
Io/I = 1+K
Q
SV[ ]
(2)
Where, Io and I are the emission band intensities in the pres-
ence and absence of the quencher, respectively, [Q] is the quencher
concentration and KSV is the Stern-Volmer quenching constant.
Besides, to further evaluate the binding mode, Viscometric titra-
tions were carried out with Ostwald viscometer at 30 °C. 200 μM
fixed DNA concentration was taken to which variable concentra-
tions (0–200 μM) of the test compound was added consecutively.
The flow times were recorded with a digital timer and each sample
was ran for triplicate to get an average flow time. Data obtained
were plotted as (η/ηo)1/3 versus [test compound]/[DNA], where η
and ηo are the viscosity of DNA in the presence of the test com-
pound and alone, respectively. Viscosity values were calculated
from the experimental flow time of DNA treated solutions (t) cor-
rected for buffer alone (to), η = (t-to)/to.
3.1. Analytical and spectral analysis
The HL1 and HL2 gave good NMR spectra (Fig. S1) with proton
peaks of the hydroxyl group at 11.78 and 13.04 ppm, and azome-
thine at 9.22 and 9.26 ppm, respectively. These ligands, in mass
spectrometry, gave molecular ion peaks at m/z 369 (M + H+) and
m/z 497 (M + 4) given in Fig. S2, and tallied with NMR spectra.
Upon coordination of these ligands with copper acetate salt, the
IR spectra (Fig. S3) of complexes 1 and 2 exhibited characteristic
peaks at ν(HC= 1571 and 1626 cm−1 which were in ligands as
N
)
1568 and 1587 cm−1 at lower frequency region, confirming that
the azomethine nitrogen has participated in coordination. And, the
hydroxyl group peaks, in ligands, appeared at ν(OH) 3433 and 3431
cm−1 are well disappeared in complexes, indicating that the hy-
droxyl group participated in coordination. The peak at 3448 cm−1
in spectrum of complex 2 is due to lattice water molecule not from
coordinated water molecules. This is further confirmed by absence
of rocking and wagging bands in the range of 750–850 cm−1. Fur-
thermore, two new non-ligand peaks, in complexes, appeared at
ν(M–O) 580 and 572 and ν(M–N) 446 and 420 is further confirmation
of both hydroxyl and azomethine groups have participated in coor-
dination. This is further supported by UV–visible spectra (Fig. S4)
recorded in DMSO, in which the bands, in complexes, are shifted to
higher or lower frequency region, and at high concentration, char-
acteristic broad d–d transition bands appear at 590 (1) and 589 (2)
in the visible region. The magnetic moment values, for these com-
plexes, are 1.78 and 1.89 BM by which a squareplanar geometry is
tentatively proposed. The IR, UV–Vis and magnetic moment data
are given in Table 1. EPR spectroscopy (Fig. S5) further supports
the geometry with the ‘g’ parameters’ trend, g// > g┴ > ge which is
2.5. DNA cleavage
To investigate the DNA cleavage ability of complexes 1 and 2,
the pBR322 DNA was used and the experiment was performed by
agarose gel electrophoresis technique with (Oxidative cleavage) and
without (Photolytic cleavage) using H2O2. DNA was diluted in TE
(10 mM Tris–HCl and 1 mM Na2EDTA) buffer to make a stock solu-
tion. The DNA (with H2O2 for oxidative cleavage) was treated with
variable concentrations of each complex and followed by dilution
with Tris–HCl/NaCl buffer(5:50 mM, pH 7.2). The DNA treated sam-
ples were incubated for 2 h at 37 °C and followed by addition of
bromophenol blue (2 μL). The respective aliquots were loaded in
the wells of 1% agarose gel which was placed in TAE (Tris-acetate-
EDTA) buffer (pH 8.0) containing tray and the whole set up was
subjected to electrophoresis at 70 V for 45 min. The resultant gel
was well stained with ethidium bromide and photographed under
UV light using the BIO-RAD Gel documentation instrument.
2
2
2.6. Antimicrobial activity
the indication of the unpaired electron is localized in dx
orbital
–y
2
with B1g as the ground state [26]. Thermograms of complexes 1
and 2 are given in Fig. S6, and showed thermal stability up to 200
°C which is the obvious indication of the absence of coordinated
water molecules. Complex 2 showed slight degradation at 80 °C
due to the lattice water molcules. Gradual decomposition of ligand
moiety around the metal ion occurred from 200 to ~700 °C, and
above this temperature, the thermograms gave a horizontal line
because the complexes remain as Cu–O residue after complete de-
composition of the organic moiety.(Scheme 1 and Table 2)
The antimicrobial screening was performed for all prepared
compounds against bacterial (B. amyloliquefaciens, S. aureus, E.
coliand P. aeruginosa) and fungal (M. phaseolina and S. rolfsii) organ-
isms by paper disk technique using the nutrient agar broth. Strep-
tomycin and Mancozeb were the standard antibiotics for bacterial
and fungal organisms, respectively. The concentration of each test
compound was made about 500 μg/mL of DMSO to make a stock
solution. Agar broth was inoculated with the selected microorgan-
isms and left overnight to develop the culture. 6 mm of sterilized
Whatman filter paper discs that were soaked in the test solution,
were placed on the culture impregnated agar plates and incubated
for 24 h at 37 °C. On incubation, the growth of the inoculated mi-
croorganisms was badly affected by the diffusion of the test solu-
tion. Then, the extent of the zone of inhibition was measured.
In complex 2, there is a certain chances of steric hindrance es-
pecially from Br atoms which can effect the geometry around the
metal center. In this case, steric hindrance may cause to distortion
from perfect square planar geometry. But based on other spectro-
scopic and analytical data, the geometry is tentatively confirmed
as squareplanar rather than others. This is strongly supported by
3

A. Rambabu, S. Daravath, D.S. Shankar et al.
Journal of Molecular Structure 1244 (2021) 131002
Scheme 1. Schematic representation of Schiff bases and their copper(II) complexes.
Fig. 1. Absorption spectra of HL1, HL2 and complexes, 1 [Cu(L1)2] and 2 [Cu(L2)2] in the absence (dashed line) and presence (solid lines) of increasing concentrations of CT-
DNA in Tris–HCl buffer (pH 7.2). Conditions: [Test compound] = 10 μM, [DNA] = 0–10 μM. Arrow (↓) indicates the absorbance changes upon increasing DNA concentration.
Inset: linear plot for the calculation of the intrinsic binding constant, Kb for DNA binding.
the article published by our team, in which highly steric hindered
3,5-diiodo substituted compound got squareplanar geometry with
slight distortion [27]. And this is further supported by mass spec-
trum (Fig. S2) of complex 2 which gave [M + 4] peak at m/z value
of 1052 due to isotopic abundance of two Br atoms. And this m/z
value is in accordance with four coordinated metal center.
3.2. Biochemistry
The above prepared ligands and their complexes are subjected
to various biological studies such as DNA binding, cleavage and an-
timicrobial activity study, and the resultant data that represent how
they react and affect the DNA and microorganisms.
4

A. Rambabu, S. Daravath, D.S. Shankar et al.
Journal of Molecular Structure 1244 (2021) 131002
Fig. 2. Emission spectra of EB-DNA system in the absence and presence of HL1, HL2 and complexes, 1 [Cu(L1)2] and 2 [Cu(L2)2]. [EB] = 12.5 μM, [DNA] = 125 μM, [Test
compound] = 0, 10, 20, 30, 40, 50 and 60 μM, respectively; λex = 510 nm. The arrow ↓ indicates the intensity changes on increasing the test compound concentration.
Fig. 4. Oxidative cleavage of pBR322 DNA (0.2 μg/μL) at 37 °C in TAE buffer
(40 mM Tris base, 20 mM aceticacid and 1 mM EDTA) by the complexes 1 (a) and
2 (b). Lane 1: DNA control, Lane 2: DNA + H2O2 (1 mM), Lane 3–8: DNA + H2O2
(1 mM) + 1 or 2 [Test compound] = 20, 30, 40, 50, 60 and 70 μM, respectively.
3.3. DNA binding and cleavage study
The binding study was evaluated by three methods, Electronic
absorption (Fig. 1), Fluorescence quenching and Viscometric titra-
tions. Electronic absorption spectroscopy, for the synthesized com-
Fig. 3. Effect of successive incremental amounts of EB, complexes 1 and 2 on the
pounds, gave n–π∗ bands in the range of 310–340 nm at which
relative viscosity of CT-DNA at 30
0.1 °C.
the data was taken for calculating the binding constant values af-
ter successive addition of calf thymus-DNA by increasing the con-
centration itself at each interval. Upon increasing the concentra-
tion, the n–π∗ band of the test compound showed hypochromism
and slightly bathochromism that means the test compound did
bind the DNA intercalatively. Using the Eq. (1), the binding con-
Table 2
Kb and KSV values for HL1, HL2, 1 and 2.
Compound
Kb/M–1 (UV–Vis)
KSV/M–1
HL1
3.06
6.28
5.93
1.09
0.02 × 104
0.01 × 104
0.01 × 105
0.01 × 105
2.14
1.87
6.85
1.65
0.02 × 103
0.01 × 104
0.02 × 105
0.01 × 105
stant values are calculated and found as 3.06
0.02 × 104 M−1
HL2
0.01 × 104 M−1 (HL2), 5.93
0.01 × 105 M−1 (1)
[Cu(L1)2] (1)
[Cu(L2)2] (2)
(HL1), 6.28
and 1.09
0.01 × 105 M−1 (2), respectively. This data indicates
that the complexes show higher binding efficiency than the parent
5

A. Rambabu, S. Daravath, D.S. Shankar et al.
Journal of Molecular Structure 1244 (2021) 131002
Viscometric titration results, shown in Fig. 3, also followed the
order of consistency obtained from above both titrations which
strongly support the intercalation mode of interaction. In this titra-
tion, the binding mode of prepared compounds was explained by
plotting a graph between the coordinates of relative specific vis-
cosity of DNA and concentration of test compound. The continuous
raising in the concentration of test compounds leads to an elonga-
tion in the helix as the amount of intercalating agents greatly in-
creases in between the base pairs, as a consequence the DNA vis-
cosity enhances. This enhancement of viscosity indicates the extent
of binding affinity of test compounds towards DNA.
Fig. 5. Photoinduced cleavage of pBR322 DNA (0.2 μg/μL) at 37 °C in TAE buffer
by the complexes 1 (a) and 2 (b) using UV irradiation of wavelength 345 nm. Lane
1: DNA control, Lane 2–7: DNA + 1 or 2 [Test compound] = 20, 30, 40, 50, 60 and
70 μM, respectively.
The cleavage ability of a test compound can be monitored by
the conversion of double-stranded circular DNA (Form-I) to nicked
circular (Form-II) and/or linear form (Form-III), briefly given in pre-
vious articles [20,25]. Figs. 4 and 5 illustrate the lane 1 or DNA
control (absence of test compounds) retains its Form-I as it is with
no cleavage in both methods. Lane 2 containing DNA + H2O2, in
oxidative cleavage, didn’t show Form-II or Form-III, where the free
H2O2 does not show any effect on DNA in absence of the com-
plex. The lanes 3–8 (2–7 in photolytic cleavage) having the different
aliquots of DNA-pretreated samples exhibit the Form-II and Form-
III cleavages in both cleavage methods. Both complexes exhibited
good cleavage efficiency with no big difference against DNA in both
the mechanistic ways.
ligands. The complex 1 possessed a bit of higher binding efficiency
that may be due to the playing key role of methoxy group while
complex binding the DNA which is supported by our previously re-
ported articles, 2.21 0.02 × 104 M−1 and 3.38 0.03 × 105 M−1
for bromine substituted complexes and 1.90 0.01 × 104 M−1 and
2.92 0.02 × 105 M−1 for methoxy substituted complexes [27,28].
However, the binding efficiency of complex 1 itself is lower than
that of classical intercalator like ethidium bromide (Kb = 7 × 107
M
−1) [29].
Further evaluation of the binding mode of test compounds
was performed by Fluorescence quenching titrations. EB is a pla-
nar cationic dye and sensitive fluorescence probe which gives a
weak fluorescent peak in Tris–HCl buffer by the quenching effect
of water molecules presented in the buffer. When we add DNA, EB
binds to it to form the EB-DNA adduct which emits very intense
fluorescence. Such fluorescence enhancement of the adduct can be
quenched by adding other DNA binding agents that compete and
replace the EB from the adduct [13]. Fig. 2, in this study, illustrated
the emission spectra of EB-DNA adduct in the absence and pres-
ence of test compounds that are non-fluorescent. Each successive
addition of a test compound dramatically degraded the intensity
of the EB-DNA adduct at around 595 nm. This might be due to
the replacement of the EB molecule by the test compound from
the intercalating site of the DNA sequence. This confirms the test
compounds involved in the intercalation mode of binding towards
DNA. The splendid quenching mechanism obeyed the linear Stern-
Volmer equation and gave the linearity as shown in the inset plot.
The Stern-Volmer quenching constants have been estimated using
3.3.1. Antimicrobial study
The inhibiting ability of the HL1, HL2 and their complexes over
the growth of microbial species is illustrated in Fig. 6 and the data
are given in Table 3. Both parent ligands have very little activ-
ity whereas corresponding metal complexes exhibited moderate to
good activity compared to the standard drugs used in this study.
The activities of solvent DMSO and copperacetate monohydrate salt
are also investigated. The DMSO didn’t show any effect against
microbials but the copper acetate salt exhibited very less activity
against few microbials like Gram-negative bacterium, E. coli and
fungus, S. rolfsii. Upon the coordination, in general, the futile or
less active compounds become more active, this can be explained
on the basis of Overtone’s concept and Chelation theory [31,32].
Both the complexes showed the same potentiality with no big dif-
ference against both kinds of microorganisms, bacterial and fun-
gal, suggesting the effect of the substituents (–OCH3 of HL1 and
–Br of HL2) is very less or almost nil. In the previous study of
some isoxazole and benzothiazole derivatives, from our laboratory,
the –OCH3 group containing compounds exhibited slightly higher
potency than the compounds having –Br substituent [19,27]; it
might be due to the key role of the methoxy group in increas-
ing the lipophilic nature of the metal complex. Contrary to this, in
the Eq. (2), and data found to be 2.14
0.02 × 104 M−1 (HL1),
1.87
1.65
0.01 × 103 M−1 (HL2), 6.85
0.01 × 105 M−1 (1) and
0.02 × 105 M−1 (2) which are in order of consistency with
the results of electronic absorption study.
Fig. 6. A plot of the zone of inhibition (mm) of HL1, HL2 and corresponding complexes 1 and 2 tested against bacterial (a) and fungal strains (b).
6

A. Rambabu, S. Daravath, D.S. Shankar et al.
Journal of Molecular Structure 1244 (2021) 131002
Table 3
Antimicrobial data for HL1, HL2, complexes 1 and 2 at 500 μg/ml concentration.
Bacteria (mm)
Fungi (mm)
Gram-positive bacteria
B. amyloliquefaciens
Gram-negative bacteria
M.
S.
Compound
phaseolina
rolfsii
S. aureus
E. coli
P. aeruginosa
HL1
3.0
3.0
7.0
6.0
0
0.26
0.22
0.28
0.30
4.0
4.0
7.0
7.0
0
0.20
4.0
4.0
9.0
8.0
0
0.24
0.22
0.20
0.20
4.0
3.0
6.0
6.0
0
0.22
0.22
0.18
0.22
4.0
2.0
8.0
6.0
0
0.16
0.22
0.28
0.24
4.0
5.0
8.0
7.0
0
0.18
0.20
0.26
0.22
HL2
0.24
0.18
0.20
1
2
DMSO
[Cu(CH3COO)2.H2O]
Streptomycin
Mancozeb
0
0
1.0
10.0
–
0.18
0
0
1.0
–
0.14
15.0
–
0.18
11.0
–
0.14
0.22
10.0
–
0.26
–
15.0
0.18
14.0
0.16
the present investigation, the substituents on ligands or complexes
didn’t show such a huge potential difference. The whole results re-
flected that none of the complexes is more potent against the mi-
crobials used in this study compared to commercial antibiotics like
Streptomycin and Mancozeb.
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2021.131002.
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