3-Acetyl-8-methoxy-2[H]-chromen-2-one derived Schiff bases as potent antiproliferative agents: Insight into the influence of 4(N)-substituents on the in vitro biological activity

Article abstract of DOI:10.1016/j.saa.2018.04.028

A series of 3-acetyl-8-methoxycoumarin appended thiosemicarbazones (1–4) was prepared from the reaction of 3-acetyl-8-methoxycoumarin with 4(N)-substituted thiosemicarbazides in a view of ascertaining their biological properties with the change of N-terminal substitution in the thiosemicarbazide moiety. Comprehensive characterization was brought about by various spectral and analytical methods. The molecular structures of all the compounds were determined by single crystal X-ray diffraction analysis. Binding studies with Calf thymus DNA (CT-DNA) and proteins such as Bovine Serum Albumin (BSA) and Human Serum Albumin (HSA) indicated an intercalative mode of binding with DNA and static quenching mechanism with proteins. The compounds cleaved plasmid DNA (pBR322) and acted well as free radical scavengers. A good spectrum of antimicrobial activity was observed against four bacterial and five fungal pathogens. The compounds exhibited profound antiproliferative activity on MCF-7 (human breast cancer) and A549 (human lung carcinoma) cell lines. Assay on human normal keratinocyte cell line HaCaT showed that the compounds were non-toxic to normal cells.

Full text of DOI:10.1016/j.saa.2018.04.028

Accepted Manuscript
3-Acetyl-8-methoxy-2[H]-chromen-2-one derived Schiff bases as
potent antiproliferative agents: Insight into the influence of 4(N)-
substituents on the in vitro biological activity
G. Kalaiarasi, S. Rex Jeya Rajkumar, G. Aswini, S. Dharani,
Frank R. Fronczek, R. Prabhakaran
PII:
DOI:
Reference:
S1386-1425(18)30333-0
doi:10.1016/j.saa.2018.04.028
SAA 15985
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 17 January 2018
Revised date:
Accepted
date:
11 April 2018
13 April 2018
Please cite this article as: G. Kalaiarasi, S. Rex Jeya Rajkumar, G. Aswini, S. Dharani,
Frank R. Fronczek, R. Prabhakaran , 3-Acetyl-8-methoxy-2[H]-chromen-2-one derived
Schiff bases as potent antiproliferative agents: Insight into the influence of 4(N)-
substituents on the in vitro biological activity. The address for the corresponding author
was captured as affiliation for all authors. Please check if appropriate. Saa(2017),
doi:10.1016/j.saa.2018.04.028
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ACCEPTED MANUSCRIPT
3-Acetyl-8-methoxy-2[H]-chromen-2-one derived Schiff bases as potent
antiproliferative agents: Insight into the influence of 4(N)-substituents on
the in vitro biological activity
G. Kalaiarasi,a S. Rex Jeya Rajkumar,b G. Aswini,a S. Dharani,a Frank R. Fronczek,c
a*
and R. Prabhakaran
a Department of Chemistry, Bharathiar University, Coimbatore 641 046, India.
b Department of Biosciences and Technology, Karunya University, Coimbatore 641 114,
India.
c Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USA
Abstract
A series of 3-acetyl-8-methoxycoumarin appended thiosemicarbazones (1-4) was prepared
from the reaction of 3-acetyl-8-methoxycoumarin with 4(N)-substituted thiosemicarbazides in
a view of ascertaining their biological properties with the change of N-terminal substitution in
the thiosemicarbazide moiety. Comprehensive characterization was brought about by various
spectral and analytical methods. The molecular structures of all the compounds were
determined by single crystal X-ray diffraction analysis. Binding studies with Calf thymus
DNA (CT-DNA) and proteins such as Bovine Serum Albumin (BSA) and Human Serum
Albumin (HSA) indicated an intercalative mode of binding with DNA and static quenching
mechanism with proteins. The compounds cleaved plasmid DNA (pBR322) and acted well as
free radical scavengers. A good spectrum of antimicrobial activity was observed against four
bacterial and five fungal pathogens. The compounds exhibited profound antiproliferative
activity on MCF-7 (human breast cancer) and A549 (human lung carcinoma) cell lines. Assay
on human normal keratinocyte cell line HaCaT showed that the compounds were non-toxic to
normal cells.
Key words: Coumarin Schiff Bases; Spectroscopy; X-ray crystallography; DNA/protein
binding; Antimicrobial studies; Anticancer activity.
Corresponding author Tel.: +91-422-2428319; Fax: +91-422-2422387; E-Mail:
rpnchemist@gmail.com (R. Prabhakaran)

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Introduction
The chemistry of heterocyclic compounds is always been a promising field of research, from
which numerous applications could be accounted. Stemming from their pharmacological and
physical properties, coumarins and their derivatives have emerged as attractive synthetic
targets. A number of organic compounds containing this coumarin scaffold are reported to
exhibit antidepressant [1], antitumor [2], anti-inflammation [3], antimicrobial [4], antiviral
[5], anticoagulant properties [6] and found to inhibit Aβ peptide aggregation [7]. Substitution
affects the activity of coumarin to a greater extent. For example, structure activity
relationship studies denote that the presence of an alkyloxy group at 7th position increases the
potential to reduce plasma alkaline tranferase level in hepatitis and inhibition of caspase-3
activation [8]. In particular, 7-hydroxy-4-methyl coumarin derivatives possess varied biological
properties including neuroleptic, antitubercular, anti-HIV, antimicrobial, antineoplastic and
antihelmintic [9,10]. Coumarin derivatives target a number of pathways in cancer cells such
as cell cycle arrest, antimitotic activity, inhibition of kinase, angiogenesis, heat shock protein
(HSP90), telomerase, carbonic anhydrase, etc [11]. On the other hand, coumarin fused
heterocycles exhibit unprecedented properties and function effectively as antioxidants [12].
More than 1300 coumarin derivatives have been identified till date [13]. A report on natural
and synthetic coumarins made by Fylaktakidou et al [14] and Bansal et al [3] depicts their
potential as anti-inflammatory and antioxidant agents. Zacharski et al demonstrated that
warfarin ((RS)-4-hydroxy-3-(3-oxo-1-phenylbutyl)-2H-chromen-2-one), a coumarin containing
compound, can prolong the survival of cancer patients [15]. These reports provide a
comprehensive discussion on the importance of coumarin derivatives in the current research
scenario. Moreover, condensation of substituted coumarins with thiosemicarbazides results in
the enhancement of their biological activity [16]. As such, both the thiosemicarbazides and
coumarins have their own capability in exhibition of pharmaceutical properties [17-20]. In
order to get a deep insight into their biological activity, we made a systematic study on the
derivatives of 3-acetyl-8-methoxycoumarin with four different substituted thiosemicabazides.
Results and Discussion
Synthesis and Characterization
The preparative route for 3-acetyl-8-methoxycoumarin-4(N)-substituted thiosemicarbazones
(1-4) are given in Scheme 1 and formation of the compounds was confirmed by spectral
1
techniques (IR, UV-Vis and H NMR) and single crystal X-ray diffraction studies. The

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compounds were stable to light and air and dissolve in common organic solvents such as
methanol, acetonitrile, dichloromethane, chloroform, ethanol, dimethylformamide and
dimethylsulfoxide. The compounds were stable in aqueous solutions, which was confirmed
by using UV-Visible spectroscopic techniques (Fig. S1).
OCH3
OCH3
S
O
O
O
O
H
R
METHANOL
H2N
H
N
N
N
H
R
O
H
N
C
S
N
R=H
1
R=CH3
R=C2H5
R=C6H5
2
3
4
Scheme 1. Synthesis of the compounds (1-4)
Spectroscopic studies
The IR and UV-Visible spectral details have been discussed in the supporting information.
1
In the H-NMR spectra of the compounds 1-4 (Fig. S2-S6), a signal appeared at δ 2.23-2.32
ppm due to the N=C-CH3 proton [21] and a singlet observed at δ 10.32-10.86 ppm
corresponding to the hydrazinic proton N(2)–H [22]. The signals due to C5, C6 and C7
protons appeared as a multiplet in the region of δ 7.15-7.59 ppm while the C4 proton
accounted for a sharp signal centered at δ 7.95-8.47 ppm [23]. The signals due to –OCH3
group protons observed as a singlet at δ 3.90 ppm [22] and NH2 protons for 1 appeared as two
broad singlet at δ 8.39 and δ 8.44 ppm. In the spectra of 2, 3 and 4 the terminal –NH protons
observed as a quartet, triplet and singlet at δ 8.48-8.51, δ 8.47-8.50 and δ 10.15 ppm
respectively. The methyl group and methylene group of protons observed in the expected region.
X-Ray crystallography
The molecular structures of the compounds 1–4 have been determined by single crystal X-ray
diffraction method and the ORTEP drawings are shown in Fig. 1-4. The details concerning
the data collection and structure refinement of the compounds are summarized in Table 1 and
selected bond distances and bond angles of the compounds 1–4 are given in Table S1 in the
supporting information.

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Crystal structure of compounds 1–4
The compounds 1 and 2 crystallized in monoclinic P21/c (1), orthorhombic Pbca (2) space
group respectively and triclinic symmetry with P-1 space group for the compounds 3 and 4.
The crystal structure of the compounds 1-4 showed that the thiocarbonyl sulphur (S1) and
imine nitrogen N(1) atom in the thiosemicarbazone are trans to each other across the C(11)-
N(2) bond. The structural arrangement corresponds to the E-isomer. The bond distances and
bond angles in the compounds 1-4 agree well with the values observed for other reported
Schiff bases and coumarin derivatives [21-26] where the C(11)=S(1) group found as thione
form with the bond length of 1.6868(5) Å, 1.6847(10) Å, 1.6839(5) Å and 1.6813(11) Å for
1, 2, 3 and 4 respectively. Further, no large charge delocalization was detected.
In compound 1, O(1) oxygen atom of the first molecule is engaged in intermolecular
hydrogen bonding with one of the hydrogen atoms of the amino group of the second
molecule O(1)…H(31)-N(3), whereas the O(3) oxygen atom of the first molecule is engaged
in intermolecular hydrogen bonding with second hydrogen atom of the amino group in
second molecule. In addition, the O(1) and O(3) oxygen atoms of the second molecule makes
a bond with the hydrogen atoms of the amino group of the next molecule and this
intermolecular hydrogen bonding at both the end led to a layer structure (Fig. 5; Table 2). In
compound 3 we found the donor–acceptor distance (3.034 and 3.034 Å) corresponding to the
N(1)-O(1) and O(1)-N(1) bond between the two molecules (Fig. 6; Table 2).
Fig. 1. ORTEP diagram of [8MAC-tsc)] (1) (thermal ellipsoid at 30% probability level)

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Fig. 2. ORTEP diagram of [8MAC-mtsc)] (2) (thermal ellipsoid at 30% probability level)
Fig. 3. ORTEP diagram of [8MAC-etsc)] (3) (thermal ellipsoid at 30% probability level)

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Fig. 4. ORTEP diagram of [8MAC-ptsc)] (4) (thermal ellipsoid at 30% probability level)
Fig. 5. ORTEP diagram of [8MAC-tsc)] (1) with hydrogen bonding
Fig. 6. ORTEP diagram of [8MAC-etsc)] (3) with hydrogen bonding

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Table 1. Crystallographic data of the compounds 1-4
Identification code
[8MAC-tsc)](1)
[8MAC-mtsc)](2) [8MAC-etsc)] (3) [8MAC-ptsc)](3)
Empirical formula
Formula weight
C13H13N3O3S
291.32
C14H15N3O3S
305.35
C15H17N3O3S
319.38
C19H17N3O3S
367.41
Temperature
Wavelength
Crystal system
Space group
90.0(5) K
0.71073 Å
Monoclinic
P21/c
90.0(5) K
0.71073 Å
Orthorhombic
P bca
90.0(5) K
0.71073 Å
Triclinic
90.0(5) K
0.71073 Å
Triclinic
P -1
P-1
Unit cell dimensions
A
B
C



4.7341(18) Å
13.5129(5)Å
20.2518(8) Å
90
7.4705(2) Å
17.8863(6) Å
21.2458(7) Å
90
8.0293(3) Å
9.5230(4) Å
10.8690(4) Å
71.7983(18)
70.982(2)
73.884(2)
731.96 Å3
2
7.1312(4) Å
9.5534(4) Å
13.4948(6) Å
107.481(2)
102.918(3)
94.324(3)
844.603 Å3
2
96.2640 (18)
90
90
90
Volume
Z
1287.8 Å3
4
2838.86 Å3
8
Density
Absorption
coefficient,
F(000)
Crystal size
1.503 Mg/m3
0.263 mm-1
1.429 Mg/m3
0.242 mm-1
1.449 Mg/m3
0.238 mm-1
1.445 Mg/m3
0.217 mm-1
608
1280
336
384
0.33  0.26  0.25 0.29  0.23  0.14 0.33  0.25  0.19 0.26  0.14  0.07
mm3
mm3
mm3
mm3
2.044 to 40.259
2.26 to 33.41

range for data 1.815 to 40.27 
1.917 to 33.183
collection
Limiting indices
-8 h 6, -24 k -7h 11, -27 k -14h14, -17k -11h10, -14k
23, -36 l 36
27, -27 l 32
17, -19 l 19
14, -20 l 20
Independent
reflections
55783
R(int)=0.0295)
29446
(R(int)=0.0295)
31467
R(int)=0.0172)
12109
(R(int)=0.0268)
Absorption correction multi-scan
Refinement method
multi-scan
multi-scan
multi-scan
Full-matrix least- Full-matrix least- Full-matrix least- Full-matrix least-
squares on F2
squares on F2
squares on F2
squares on F2
Data/Restarints/Param 55783/0/192
eters
29446/0/199
31467/0/208
12109/0/243
Goodness-of-fit on F2 1.073
1.026
1.045
1.030
Final
R
indices R1
=
0.0276, R1
=
0.0364, R1
=
0.0309, R1
=
0.0428,
wR2= 0.0828
R1 = 0.0320,
wR2 = 0.0864
wR2= 0.0985
R1 = 0.0489,
wR2 = 0.0919
wR2= 0.0875
R1 = 0.0354,
wR2 = 0.0909
wR2= 0.1053
R1 = 0.0624,
wR2 = 0.1149
[I>2(I)]
R indices (all data)

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Table 2. Hydrogen bonds for compounds 1 and 3 [Å and °]
D–H⋯A
d(D–H)
d(H⋯A)
d(D⋯A)
<(DHA)
[8MAC-tsc)]
O(1)…H(31)-N(3)
O(3)…H(32)-N(3)
N(3)-H(31)…O(1)
N(3)-H(32)…O(3)
0.879
0.854
0.879
0.854
2.845
2.217
2.845
2.217
2.986
3.061
2.986
3.061
72.27
169.82
72.27
169.82
Symmetry operation: (x, y, z); : (-x, ½+y,1/2-z); (-x, -y, -z); (x, ½-y,1/2+z);
[8MAC-etsc)]
O(1) …N(1)
N(1) …O(1)
3.034
3.034
Symmetry operation: (x, y, z); (-x, -y, -z);
DNA binding studies
UV–Vis absorption spectral titrations
The electronic absorption spectra of compounds 1–4 (25 µM) in the absence and presence of
CT-DNA (2.5–25 µM) are depicted in Fig. 7. The absorption bands of the compounds
exhibited hypochromism of about 9.11, 13.89, 21.72 and 37.22 % with a blue shift of 2-5 nm
at around 318-325 nm for compounds 1, 2, 3 and 4 respectively. In order to quantitatively
determine the magnitude of the binding strength of the compounds with CT-DNA, the
intrinsic binding constant Kbin was calculated from the ratio of slope to the y intercept in plots
of [DNA]/[εa−εf] versus [DNA] (Fig. 8) and the values are found to be 2.2683±0.291 x105,
2.8738±0.372x105, 3.9088±0.310x105 and 1.2048±0.286x105. The obtained binding constant
values, bathochromic shift and hypochromism showed that the compounds 1-4 behaved as
potent binders to DNA through intercalation and the order of binding affinity of the
complexes with CT-DNA is 3 > 2 > 1 > 4. These results are comparable with earlier reports
describing the intercalative mode of various intercalators [26,27].

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0.3
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
2
1
0.2
ISOBESTIC POINT
ISOBESTIC POINT
0.1
0.0
250
300
350
400
450
500
250
300
350
400
450
WAVELENGTH(nm)
WAVELENGTH(nm)
0.25
0.20
0.15
0.10
0.05
0.00
3
0.4
4
0.3
0.2
0.1
0.0
ISOBESTIC POINT
ISOBESTIC POINT
300
400
500
250
300
350
400
450
500
550
WAVELENGTH (nm)
WAVELENGTH(nm)
Fig.7. Absorption titration spectra of compounds (1-4) (25 µM) with increasing
concentrations (2.5-25 µM) of CT-DNA (tris HCl buffer, pH 7.2)
Compound 1
Compound 2
Compound 3
Compound 4
0.0035
0.0030
0.0025
0.0020
0.0015
0.0010
0.0005
0.0000
0
5
10
15
20
25
[DNA]
Fig. 8. Binding isotherms of the compounds 1-4 with CT-DNA

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EB-DNA quenching studies
The competitive ethidium bromide displacement studies have been undertaken to gain deep
insight into the interaction of the compounds with CT-DNA. The ability of a compound to
affect the fluorescence intensity of an EB–DNA adduct is a reliable tool for the measurement
of its affinity towards DNA. Relative binding of the compounds with EB-bound to CT-DNA
is depicted in Fig. 9. On increasing the concentration of the compounds (10-100 µM) to CT-
DNA pretreated with EB ([DNA]/[EB] = 1), quenching of the initial emission intensity was
observed about 17.62, 19.16, 18.90 and 15.63 % together with a red shift of 2-3 nm for 1, 2, 3
and 4 respectively.
For a better quantitative understanding of the behavior of EB-DNA fluorescence suppression
by coumarin Schiff bases, the magnitude of quenching was analyzed by the Stern-Volmer
equation and the quenching constant determined by linear regression plot graph of (Iₒ/I)
versus [Q] (Fig. 10) and the calculated quenching constant was found to be 3.24±0.009x103,
1.82±0.005x103, 2.05±0.004x103 and 5.77±0.002x103 for 1, 2, 3 and 4 respectively. The data
showed that DNA-bound EB can be more readily replaced by the compounds and the
obtained results concluded the intercalative mode of binding of the compounds with DNA.
400
400
1
350
2
350
300
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150
150
100
100
50
50
0
0
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600
650
700
750
550
600
650
700
750
WAVELENGTH (nm)
WAVELENGTH (nm)

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400
350
300
250
200
150
100
50
350
3
4
300
250
200
150
100
50
0
0
550
600
650
700
750
550
600
650
700
750
WAVELENGTH (nm)
WAVELENGTH (nm)
Fig. 9. The emission spectra of the DNA–EB system (λexc= 515 nm, λem= 530–750 nm), in
the presence of compounds 1-4. [DNA] = 10 µM, [compound] = 10–100 µM, [EB] = 10 µM.
The arrow shows the emission intensity changes upon increasing compound concentration
Compound 1
1.35
1.30
1.25
1.20
1.15
1.10
1.05
1.00
Compound 2
Compound 3
Compound 4
0
20
40
60
80
100
[Q]
Fig. 10. Stern−Volmer plot of the fluorescence titration of compounds (1-4) (10-100 µM)
with DNA-EB (10 µM)

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Viscosity measurements
As a mean to investigate the binding mode of the compounds with DNA, viscosity
measurements were carried out. A classical intercalation mode causes a significant increase
in the viscosity of DNA solution due to an increase in the separation of base pairs at
intercalation sites and hence an increase in overall DNA length [28,29]. As it is seen in
Fig. 11, the viscosity of DNA (100 µM) increased slowly with increasing the concentration of
compounds (20-100 µM) which showed that the coumarin Schiff bases bound to CT-DNA by
intercalation mode which were encouraged by electron donating substitution on the N-
terminal nitrogen of the compound and the increasing order of viscosity of CT-DNA by the
compounds is 3 (NH-ethyl) > 2 (NH-methyl) > 1 (NH-hydrogen) > 4 (NH-phenyl).
Compound 1
Compound 2
Compound 3
Compound 4
1.45
1.40
1.35
1.30
1.25
1.20
1.15
1.10
1.05
1.00
0.95
0.0
0.2
0.4
0.6
0.8
1.0
1/[R]= [Compound]/[DNA]
Fig. 11. Effect of the compounds (1-4) on viscosity of CT-DNA
Plasmid DNA pBR322 cleavage studies
Attempts were made to determine the chemical nuclease activities of the compounds by using
supercoiled plasmid DNA pBR322 in Tris−acetic acid−ethylenediamine tetraacetic acid
buffer pH 7.2 at 37 °C by 1.5 % agarose gel electrophoresis without any external agents. The
cleavage efficiency was measured by determining the ability of the compound to convert the
supercoiled DNA (SC Form) to linear circular form or nicked form (NC Form). All the
compounds exhibited double-strand DNA cleavage to generate the LC form before

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converting all of the SC form to NC form through single-strand break. From Fig. 12, we
inferred that the compounds showed potent nuclease activity in the order of 3 > 2 > 1≈ 4,
which may be due to the change in the electron accepting or donating ability pertaining to the
N-terminal substituent of the compound. The observed result pattern is concise with their
DNA binding affinity.
Fig. 12. Gel electrophoresis diagram showing the cleavage of supercoiled pBR322 DNA by
compounds 1-4 in 5% DMSO and 95% 5 mM Tris−HCl/50 mM NaCl buffer at pH 7.2 and
37 °C with an incubation time of 2 h. Lanes M: Marker; Lane C1: compound 1 (50 µM).
Lane C2: Compound 2 (50 µM); Lane C3: Compound 3 (50 µM); Lane C4: Compound 4 (50
µM. Forms SC, NC, and LC are supercoiled, nicked circular, and linear circular DNA,
respectively.
Protein Binding Studies
The binding of compounds 1–4 with HSA and its homologue BSA was investigated by
fluorescence emission spectroscopy, since the albumin solution exhibits an intense emission
band (λex = 290 nm) at λem,max = 345 nm (for HSA) and 346 nm (for BSA) which is assigned
to the existence of tryptophans. The emission titration studies have been performed at room
temperature using BSA (10 μM)/ HSA (10 μM) with increasing concentrations of compounds
1–4 (0−100 μM) in the range 290−500 nm (Fig. 13-14). Addition of the above test
compounds to BSA solution resulted in a significant decrease in the fluorescence intensity of
BSA at 346 nm, up to 62.65, 44.51, 74.87 and 49.84 % of the initial fluorescence intensity of

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BSA with blue shift of 2-5 nm for 1, 2, 3 and 4 respectively. Addition of the compounds to
HSA solution resulted in a quenching of the emission band of HSA at 345 nm up to 48.89,
36.03, 47.13, 40.55 of the initial fluorescence intensity together with a blue shift of 2-4 nm
for 1, 2, 3 and 4 respectively (Fig. 13-14). From the above observations, we may conclude
that definite interaction is taking place between the compounds and serum albumins.
The absorption spectra of BSA/HSA in the absence and presence of compounds are given in
Fig. S7. In the presence of compounds 1-4, the absorbtion intensity of BSA/HSA exhibited
hypochromism with a 2-3 nm of hypsochromic shift, indicating a static quenching mechanism of
the serum albumins by the compounds.
By using Stern Volmer quenching equation, the values of the Stern Volmer quenching constant
(KSV) and the quenching constant (Kq) for compounds interacting with serum albumins (BSA
or HSA) were calculated [30]. The Stern Volmer quenching constant results follows the order
3 > 2 > 1 > 4 and the observed Ksv values are comparable to those reported for thiosemicarbazone
compounds [27]. The quenching constant values for the quenching of the serum albumins by
the compounds (kq ≈ 1012 M-1 s-1) suggested the good binding affinity of the compounds with
serum albumins through static quenching mechanism [30]. The binding constant Kbin and
number of binding site (n) can be calculated from the Scatchard equation and are given in
Table 3 (Fig. 16), from these values we knew that the compounds 1-4 showed strong binding
affinity with serum albumins. The obtained binding constant and quenching constant values
revealed that the compounds bind to both the albumins in the following order 3 > 2 > 1 > 4
and the results are consistent with the trend followed by them in their DNA binding profile.
This observed fashion can be explained on the basis of hydrophobicity of the compounds. As
seen from the results, complex 3 with the enhanced hydrophobicity showed the best binding
ability [31]. The obtained quenching constant and binding constant values of these coumarin
Schiff bases agree well with those reported for Schiff bases [26,27].

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500
400
300
200
100
0
900
4
800
700
600
500
400
300
200
100
0
3
300
350
400
450
300
320
340
360
380
400
420
440
WAVELENGTH (nm)
WAVELENGTH (nm)
Fig. 13. The emission spectra of BSA (10 µM; λexc= 280 nm; λemi= 346 nm) in the presence
of increasing amounts of compounds 1-4 (10–100 µM). The arrow shows the emission
intensity changes upon increasing compound concentration

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350
300
250
200
150
100
50
1
2
300
250
200
150
100
50
0
0
300
320
340
360
380
400
420
440
320
340
360
380
400
420
440
WAVELENGTH (nm)
WAVELENGTH (nm)
350
4
350
300
250
200
150
100
50
300
250
200
150
100
50
3
0
0
300
320
340
360
380
400
420
440
300
320
340
360
380
400
420
440
WAVELENGTH (nm)
WAVELENGTH (nm)
Fig. 14. The emission spectra of HSA (10 µM; λexc= 280 nm; λemi= 346 nm) in the presence
of increasing amounts of compounds 1-4 (10–100 µM). The arrow shows the emission
intensity changes upon increasing compound concentration
Compound 1
Compound 2
Compound 3
Compound 4
STERN VOLMER PLOT
2.0
1.9
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1.0
4.2
4.0
3.8
3.6
3.4
3.2
3.0
2.8
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
B
STERN VOLMER PLOT
Compound 1
Compound 2
Compound 3
Compound 4
A
0
20
40
60
80
100
0
20
40
60
80
100
[Q]
[Q]
Fig. 15. A) Stern−Volmer plot of the fluorescence titration of the compounds (1-4) (10-100
µM) with BSA (10 µM). B) Stern−Volmer plot of the fluorescence titration of the compounds
(1-4) (10-100 µM) with HSA (10 µM)

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Compound 1
Compound 2
Compound 3
Compound 4
SCATCHARD PLOT
A 0.6
Compound 1
Compound 2
Compound 3
Compound 4
SCATCHARD PLOT
0.0
-0.2
-0.4
-0.6
-0.8
-1.0
-1.2
0.4
B
0.2
0.0
-0.2
-0.4
-0.6
-0.8
-1.0
-1.2
-1.4
-5.0
-4.8
-4.6
-4.4
-4.2
-4.0
-5.0
-4.8
-4.6
-4.4
-4.2
-4.0
log [Q]
log [Q]
Fig. 16. A) Scatchard plot of the fluorescence titration of the compounds (1-4) (10-100 µM)
with BSA (10 µM). B) Scatchard plot of the fluorescence titration of the compounds (1-4)
(10-100 µM) with HSA (10 µM)
Table 3. Stern Volmer quenching constant (Ksv), Quenching constant (kq), binding
constant (Kbin) and number of binding sites (n) for the interactions of compounds 1-4
with BSA/HSA
Compounds
Stern-Volmer
quenching constant
Ksv/M-1
Quenching
Constant (kq)
M-1s-1
Binding constant
Kbin/M-1
n
BSA
1
2
3
4
1.724±0.032x104
0.890±0.019x104
3.095±0.016x104
0.950±0.039x104
1.724±0.032x1012
0.890±0.019x1012
3.095±0.016x1012
0.950±0.039x1012
2.790±0.059x104
3.195±0.019x104
6.514±0.091x104
1.884±0.086x103
1.0543±0.013
1.0421±0.045
1.007±0.020
0.8198±0.019
HSA
1
2
3
4
0.933±0.020x104
0.578±0.018x104
0.898±0.014x104
0.681±0.014x103
0.933±0.020x1012
0.578±0.018x1012
0.898±0.014x1012
0.681±0.014x1012
4.661±0.020x103
4.927±0.020x103
9.395±0.014x103
3.692±0.014x103
0.9177±0.021
0.9784±0.021
1.0030±0.015
0.9326±0.015

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Conformational Investigation
In the synchronous fluorescence spectra of the tyrosine residue of both the serum albumins,
the addition of the compounds to the serum albumin solution showed hypochromism with
negligible shift in the emission wavelength (Fig. S8-S9). Synchronous fluorescence spectra at
Δλ = 60 nm of BSA and HSA solution exhibited a decrease in fluorescence intensity up to
43.23-50.00 % and 36.20-49.54 % at 340 nm with significant blue shift for compounds 1-4
respectively (Fig. S10-S11). The obtained results showed that the test compounds effectively
affect the microenvironment of the tryptophan and tyrosine micro regions.
Three-dimensional fluorescence spectra analysis
To further study the nature of microenvironmental changes occurring in serum albumins
during interaction with the compounds, 3D fluorescence spectroscopic studies have been
carried out. Fig. 17-18 shows the changes encountered in 3D emission spectra of serum
albumins in the absence and presence of compounds and their corresponding characteristic
parameters are given in Table S2. On adding the compounds to serum albumins, the
fluorescence intensity of Rayleigh first order scattering peak increased, which is due to the
compound formation of serum albumins with our compounds leading to an increase in the
diameter of the macromolecule which in turn resulted in the enhancement of scattering effect
[32]. The emission intensity of peak ‘B’ corresponding to the tryptophan and tyrosine
residues decreased with slight blue shift. The obtained results revealed that conformational
changes and the molecular micro-environment of serum albumins occurred after interaction
with the compounds.

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Fig. 17. Three-dimensional fluorescence spectra of BSA in the absence and presence of
compounds (1-4) (pH 7.4, 298 K, [BSA] =10 µM, [Compound] =10 µM)

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Fig. 18. Three-dimensional fluorescence spectra of HSA in the absence and presence of
compounds (1-4) (pH 7.4, 298 K, [HSA] =10 µM, [compound] =10 µM)

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In vitro Antioxidant activity
The compounds which exhibit radical scavenging activity are receiving much attention
because they possess interesting anticancer, anti-ageing and anti-inflammatory activities [33].
The analysis of the DPPH radical scavenging ability of 3-acetyl-8-methoxy-coumarin-4(N)-
substituted thiosemicarbazones have been carried out along with the standard Vitamin C.
The results showed that the activity of the compounds (68.45-92.67 µM) is higher than that
of the standard 98.72±1.50 µM (Fig. 19; Table 4), in which compound 3 shows the best
DPPH scavenging activity. In phosphomolybdenum assay, the antioxidant capacity is
expressed as the number of equivalents of ascorbic acid (Table 5). A comparison of the
radical scavenging activity of compounds 1–4 with that of the reported Schiff base
compounds may reveal that coumarin appended thiosemicarbazones 1–4 act as potent radical
scavengers [22,34,35].
20 
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
40 
60 
80 
100 
DPPH RADICAL SCAVENGING ASSAY
0
STD
C 1
C 2
C 3
C 4
Fig. 19. DPPH scavenging activity of compounds (1-4). Error bars represent the standard
deviation of the mean (n=3)

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Table 4. The DPPH radical scavenging activity of the compounds 1-4
COMPOUNDS
Ascorbic acid
1
IC50 VALUE (µM)
98.72±1.50
89.79±1.23
2
3
4
77.23±0.93
68.45±0.87
92.67±1.60
Table 5. Estimation of Total antioxidant capacity of compounds 1-4
Compounds
µg Ascorbic acid equivalents/ml
1
2
3
4
37.74±0.57
39.12±0.68
43.53±0.74
34.42±0.39
Antimicrobial activity
In vitro antimicrobial activities of the compounds 1–4 were examined with few fungi
A. niger, A. fumigatus C. albicans, C. tropicalis and T. rubrumi and some pathogenic bacteria
such as S. aureus, S. paratyphi, P. aeruginosa and S. pneumonie, and the results are
expressed as the zone of inhibition and minimum inhibitory concentration (MIC) (Fig. 20-21;
Table S3-S6). Ketaconazole and Gentamicin were used as the positive controls for fungi and
bacteria respectively and their MIC values are also given for comparison purpose. The
control disc with negative control (10 % aqueous DMSO) exhibited no zone of inhibition.
From the zone of inhibition values, we knew that antimicrobial activity of the compounds
was increased while increasing the concentration of the compounds. In antibacterial studies,
compounds 3 and 4 showed similar minimum inhibitory concentrations on S. aureus followed
by 2 and 1. Compound 4 was more active on P. aeruginosa, S. pneumonie and S. paratyphi
and the activity of the compounds in the order of 4 > 3 > 2 > 1. 4(N)-phenyl substituted
coumarin thiosemicarbazones stood out as the best when employed against the five different
fungal species and the activity follows the order of 4 > 3 > 2 > 1. When tested against C.
albicans complex 4 stood out as good followed by the complexes 2, 3 and 1 respectively. Due
to the structural variations of compounds and variation on the group of microorganisms, the
compounds (1-4) exhibited different degree of activity [36]. The activities of these

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compounds were comparable to the thiazolyl coumarin derivatives reported by Hasnah et al
[37,38] and some other reported Schiff bases, showing that our compounds showed
significant activity [27,35,37-41].
140
S. aureus
S. Pneumonie
P. Aeruginosa
S. paratyphi
120
100
80
60
40
20
0
Compound 1
Compound 2
Compound 3
Compound 4
STD
Fig 20. Anti bacterial activity of compounds (1-4). Error bars represent the standard deviation
of the mean (n=3)
ANTIFUNGAL STUDIES
120
100
80
60
40
20
0
T. rubrum
A. niger
A. fumigatus
C. tropicalis
C. albicans
Compound 1
Compound 2
Compound 3
Compound 4
STD
Fig. 21. Anti fungal activity of compounds (1-4). Error bars represent the standard deviation
of the mean (n=3)

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Cytotoxicity studies
Antiproliferative Studies - Cancer Cell Growth Inhibition
Our synthesized compounds act as potent DNA/Protein binders and free radical scavengers,
which prompted us to evaluate the possible antiproliferative effects of the 3-acetyl-8-
methoxy-coumarin appended Schiff bases 1-4 with the breast cancer cells (MCF-7) and lung
cancer cell lines (A549) using MTT assays. The dose dependent cell death inducing ability of
the compounds has been investigated by using the percentage of cell viability versus
compound concentration plot (Fig. 22-23), which showed that our compounds differentially
inhibit the tumor cell viability, depending on the dose applied. On increasing the
concentration, our compounds 1-4 exhibited a higher decrease in the cell viability. The results
were analyzed by means of cell inhibition expressed as IC50 values and are given in Table 6.
As shown in Table 6, IC50 values for the test compounds ranged from 11.31 µM to 12.65 µM
with both the cell lines. The results showed that the compounds 1-4 exhibited significant
cytotoxic activities than the standard drug cisplatin against the MCF-7 and A549 cells. The
antiproliferative activity of the compounds follows the order cisplatin (16.79±0.08) < 4
(12.19±0.20) ≈ 1 (12.15±0.23) < 2 (11.77±0.29) < 3 (11.31±0.15) in human breast cancer cell
lines (MCF-7). In human lung cancer cells (A549), the compounds exhibited anticancer
activity in the order of cisplatin (15.10±0.05) < 4 (12.65±0.16) < 1 (12.48±0.14) < 2
(11.98±0.19) < 3 (11.46±0.14).
Electron withdrawing phenyl substituent at terminal nitrogen possesses somewhat poor
inhibitory activity compared to other compounds (1-3), which is in agreement with its lower
binding propensity towards DNA. Alkyl substituted at terminal nitrogen atom containing
compounds showed improved anticancer activity. On the basis of results, the anticancer
activity of the compounds has been arranged in the order 3 > 2 > 1 > 4. To investigate the
selectivity of the compounds for cancer cells rather than normal cell lines, compounds were
screened for their activity on the human normal keratinocyte cells (HaCaT), the results
confirmed the non-toxic nature of the compounds. Cytotoxic nature of our compounds are
superior to those reported Schiff base compounds against MCF-7 cells and A549 cells [27,42,43].

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CELL PROLIFERATION MTT ASSAY
Control
2.5 M
5 M
100
80
60
40
20
0
10 M
20 M
40 M
STD MCF-7 C1 MCF-7 C2 MCF-7 C3 MCF-7 C4 MCF-7 STD A549 C1 A549
C2 A549
C3 A549
C4 A549
Fig. 22. The compounds 1-4 and Cisplatin inhibit MCF-7 and A549 cells proliferation in a dose
dependent manner. MCF-7 and A549 cells were treated with different concentrations of
compounds for 48 h, the cell viability was determined and the results were expressed as
percentage cell viability with control. Results shown are mean, which are three separate
experiments performed in triplicate
Control
2.5 M
5 M
CELL PROLIFERATION MTT ASSAY
100
80
60
40
20
0
10 M
20 M
40 M
Cisplatin
C 1
C 2
C 3
C 4
Fig. 23. The newly compounds 1-4 and Cisplatin inhibit HaCaT cells proliferation in a dose
dependent manner. HaCaT cells were treated with different concentrations of compounds for
48 h, the cell viability was determined and the results were expressed as percentage cell
viability with control. Results shown are mean, which are three separate experiments
performed in triplicate.

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Table 6. The IC50 values for the human breast cancer cell line MCF-7, human lung
carcinoma cancer cell line A549 and human normal keratinocyte cells (HaCaT) with the
compounds 1-4 for 48h
Compounds
IC50 values (μM)
A549
MCF-7
HaCaT
>40
Cisplatin
16.79±0.08
12.15±0.23
11.77±0.29
11.31±0.15
12.19±0.20
15.10±0.05
12.48±0.14
11.98±0.19
11.46±0.14
12.65 ±0.16
1
2
3
4
>40
>40
>40
>40
LDH assay
LDH assay act as an indicator of cell membrane integrity. It is a stable cytoplasmic enzyme,
which released into the culture medium due to the loss of membrane integrity resulting from
apoptosis of cells. To further investigate the drug induced cytotoxicity of cancer cells [44],
LDH assay have been carried out with cancer cell lines such as A549 and MCF-7. These
cancer cells were treated with the IC50 concentration of the compounds 1-4 for a period of
48 h, a considerable increase in the level of LDH released into the culture medium was
observed (Fig. 24). The compounds showed good level of LDH leakage in A549 and MCF-7
cells compared to cisplatin. These results authenticated that the effectiveness of the
compounds in inducing cell death by collapsing the membrane integrity. The order of
increased induction of LDH release by the compounds was found to be 3 > 2 > 1 > 4. The
coumarin appended thiosemicarbazones showed potent activity when compared with the
earlier reports [45].

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A549
MCF-7
LACTATE DEHYDROGENASE (%)
80
70
60
50
40
30
20
10
0
Control
Cisplatin Compound 1 Compound 2 Compound 3 Compound 4
Fig. 24. Percentage of lactate dehydrogenase released by the human cancer cell lines A549
and MCF-7 after an incubation period of 48 h with compounds 1-4. Error bars represent the
standard mean error (n= 6).
Nitric oxide assay
A well-known short-lived free radical Nitric oxide produced non-enzymatically by NOS,
causes damage in most of the biomolecules, including protein and DNA. Nitric oxide has
been shown to directly inhibit methionine adenosyl transferase, leading to glutathione
depletion and its reaction with superoxide generates the strong oxidant peroxynitrite, which
can initiate lipid peroxidation or cause a direct inhibition of the mitochondrial respiratory
chain [46]. In the present study NO release by the compounds 1-4 was evaluated using A549
and MCF-7 cells. From the results, it is concluded that the compounds were found to release
more NO than the control and cisplatin (Fig. 25), in which compound 3 was more potent in
increasing the level of NO in the culture medium followed by 2, 1 and 4. The results of the
present study revealed the significant anticancer potential of the compounds (1–4) and our
results are significant when compare to the reported results [45].

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70
60
50
40
30
20
10
0
Nitric Oxide Release
A549
MCF-7
Control
Cisplatin
Compound 1 Compound 2 Compound 3 Compound 4
Fig. 25. Nitrite released (nmoles) by the human cancer cell lines A549 and MCF-7 after an
incubation period of 48 h with compounds 1-4. Error bars represent the standard mean error (n= 6).
Conclusion
Four coumarin appended thiosemicarbazone derivatives (1-4) were synthesized and
characterized by using analytical and spectral techniques. The respective crystal structures
were resolved by means of X-ray diffraction studies. The compounds intercalatively bound to
CT-DNA, as supported by EB displacement studies and viscosity measurements. Protein
binding studies indicated that the quenching mechanism is static and the micro environmental
changes occurred has been confirmed by 3D fluorescence experiment. Similar to the reported
coumarin derivatives, the compounds were able to scavenge the free radicals and inhibit the
growth of all the tested bacterial and fungal pathogens. The outcome from the MTT assay
revealed the anticancer potential of the compounds, with an activity greater than cisplatin.
Combining the overall results, it is evident that the biological activity of the compounds
follows the pattern 3 > 2 > 1 > 4 and the promising results obtained from the biological
studies suggested that the compounds can act as good probes for further exposure in
pharmaceutical applications.

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Experimental section
Materials
Analar or chemically pure grade reagents were used and solvents were purified/dried
according to standard procedures [47]. The compounds such as 3-Acetyl-8-
methoxycoumarin-4(N)-substituted thiosemicarbazones were prepared with
a
slight
modification of the literature procedures [23,37,38,48]. The techniques used for the
characterization of the compounds are given in supporting information.
Preparation of 3-acetyl-8-methoxy-2H-chromen-2-one [37]
3-Acetyl-8-methoxycoumarin was prepared from 3-methoxysalicylaldehyde (1.22 g, 1 mmol)
and ethylacetoacetate (1.95 g, mmol) in the presence of catalytic amount of piperidine as
described in standard literature methods [37]. Yield = 95 %. Mp. 123 °C; UV-Vis (DMSO),
-1
λmax (ε): 316 (21,770) nm (dm3mol−1cm−1); IR (ν, cm ): ν(C=O lactone) 1731, ν(C=O acetyl
1
group) 1683. H NMR (400 MHz, DMSO-d6, δ ppm, J Hz): δ 8.62-8.62 (d, J=1.6, 1H, C3-H),
δ 7.47-7.49 (dd, J= 0.8, 6.6, 1H, C5-H), δ 7.31-7.35 (m, 1H, C6-H), δ 7.41-7.43 (dd, J= 0.8,
6.2, 1H, C7-H), δ 3.91 (s, 3H, -OCH3), δ 2.57 (s, 3H,-CH3).
Synthesis of ((1E)-1-(1-(8-methoxy-2-oxo-2H-chromen-3yl)ethylidene) thiosemicarbazide)
[8MAC-tsc] (1) [37]
3-acetyl-8-methoxy-2H-chromen-2-one (1 g, 4.58 mmol) was dissolved in 20 cm3 methanol.
To this, methanolic solution (30 cm3) of thiosemicarbazide (0.417 g, 4.58 mmol) was added
and to this, catalytic amount of glacial acetic acid was added and the mixture was refluxed for
2 h with continuous stirring. A yellow compound precipitated was collected by filtration,
washed well with cold methanol and dried under vacuum. The compound was recrystallized
from DMF-Methanol (1:9 v/v) to yield yellow single crystals suitable for X-ray analysis.
Yield: 76 %. M.p: 201 °C. FT-IR (ν, cm−1) in KBr: ν(C=O lactone) 1713, ν(C=N) 1612, ν(-
NH2) 3344, ν(-NH) 3187, ν(C=S) 770. UV-Vis (DMSO), λmax (ε): 335 (14,472) nm
1
(dm3mol−1cm−1). H NMR (400 MHz, DMSO-d6, δ ppm, J Hz): δ 7.95 (s, 1H, C4-H), δ 7.28-
7.32 (m, 3H, Ar-H), δ 3.90 (s, 3H,-OCH3), δ 2.23 (s, 3H,-CH3), δ 10.43 (s, 1H, NH-C=S),
δ 8.39 & 8.44 (2s, 2H, -NH2).
The very similar method was used to prepare the following compounds.