Structural and biological study of synthesized anthraquinone series of compounds with sulfonamide feature

Article abstract of DOI:10.1080/07391102.2018.1552198

1, 4 and 5, 8-Positions as well as type of functionalities on these positions at anthraquinone-9, 10-dione are proposed to be significant for anticancer activity. Therefore, keeping this into consideration, a series of 1-substituted anthraquinone-based compounds are designed, synthesized, characterized and biologically evaluated for anticancer activity. The structure of synthesized compounds is confirmed by spectroscopic analysis, i.e. 1D (¹H and ¹³C) nuclear magnetic resonance (NMR), electrospray ionization-mass spectrometry (ESI-MS) studies and Fourier transform infrared (FT-IR) tools. Synthesized 1-substituted anthraquinone compounds showed cytotoxic effect against human breast cancer cell line (MCF-7), human prostate cancer cell line (PC-3) and Hela derivative human cell line (Hep 2C) (Hela derivative) cell lines. All the compounds showed mild antibacterial property in comparison to standard antibiotic streptomycin against Gram + ve and –ve bacteria. They also exhibit mild antifungal activity. In vitro calf thymus (ct)-DNA binding studies of synthesized series using UV–visible absorption spectra measurement and fluorescence tools indicate partial intercalative mode of binding. Electronic properties of synthesized analogues and mitoxantrone are compared using highest occupied molecular orbital–lowest occupied molecular orbital (HOMO–LUMO) calculation. Low energy gap between HOMO and LUMO of 1-substituted anthraquinone compounds indicates the highly charged structure of the molecules in comparison to mitoxantrone, and the same is proposed to be responsible for comparable cytotoxic activities of the synthesized 1-substituted anthraquinone molecules. Docking interaction of synthesized 1-substituted anthraquinone compounds and i-motif sequence indicates intercalative mode of binding of compounds with telomeric junction. Communicated by Ramaswamy H. Sarma.

Full text of DOI:10.1080/07391102.2018.1552198

Journal of Biomolecular Structure and Dynamics
ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsd20
Structural and biological study of synthesized
anthraquinone series of compounds with
sulfonamide feature
Pamita Awasthi, Manu Vatsal & Anjali Sharma
To cite this article: Pamita Awasthi, Manu Vatsal & Anjali Sharma (2018): Structural and biological
study of synthesized anthraquinone series of compounds with sulfonamide feature, Journal of
Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2018.1552198
To link to this article: https://doi.org/10.1080/07391102.2018.1552198
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Structural and biological study of synthesized anthraquinone series of
compounds with sulfonamide feature
a
*
a
a
Pamita Awasthi , Manu Vatsal , Anjali Sharma
a
Department of Chemistry, National Institute of Technology, Hamirpur, 177005, Himachal Pradesh,
India.
Corresponding author address: Department of Chemistry, National Institute of Technology, Hamirpur,
177005, HP, India,Tel: 01972-254140
E-mail: pamitawasthi@gmail.com
1

ABSTRACT
1
1
, 4 and 5, 8- positions as well as type of functionalities on these positions at anthraquinone-9,
0-dione are proposed to be significant for anticancer activity. Therefore, keeping this in to
consideration, series of 1-substituted anthraquinone based compounds are designed, synthesized,
characterized and biologically evaluated for anticancer activity. The structure of synthesized
1
13
compounds are confirmed by spectroscopic analysis i.e. 1D ( H and C) NMR, ESI-MS studies,
FT-IR tools. Synthesized 1-substituted anthraquinone compounds showed cytotoxic effect
against MCF-7, PC-3 and Hep2C (Hela derivative) cell lines. All the compounds showed mild
antibacterial property in comparison to standard antibiotic streptomycin against Gram +ve and –
ve bacteria. They also exhibit mild antifungal activity. In-vitro ct-DNA binding studies of
synthesized series using UV-Visible, Fluorescence tools indicates partial intercalative mode of
binding. Electronic properties of synthesized analogues and Mitoxantrone are compared using
HOMO-LUMO calculation. Low energy gap between HOMO-LUMO of 1-substituted
antharaquinone compounds indicates the highly charged structure of the molecules in
comparison to mitoxantrone and same is proposed to be responsible for comparable cytotoxic
activities of the synthesized 1-substituted anthraquinone molecules. Docking interaction of
synthesized 1-substituted anthraquinone compounds and i-motif sequence indicates intercalative
mode of binding of compounds with telomeric junction.
Keywords: Anthraquinone; Sulfonamide; Mitoxantrone; HOMO-LUMO; Docking; Cytotoxicity;
Antibacterial.
1
. Introduction
Anticancer class of drugs includes large number of molecules which are divided on the basis of
their mechanism of action like-alkylating agents, antimetabolites, anthracycline class of
antibiotics, hormonal agents and signal transduction inhibitors (Beziane, Teerme, & Vanelle,
2
005; Nor et al, 2013; Adachi et al, 2014; Krapcho, Getahun, Avery, Vargas, & Hacker, 1991;
Guidi et al, 1994; Zuravka, Sosic, Gatto, & Goottlich, 2015). Anthracycline class of compounds
are widely found in nature. The naturally occurring anthraquinones compounds carry a broad
spectrum of bioactivities, such as antibacterial, antifungal, anticancer alongwith antioxidant
activities (Zhang et al, 2011). The anthraquinone chromophore is an important structural feature
2

of the anthracycline class of antibiotics. Further anthracyclines are proved to be most effective
for cancer treatment and use to treat cancers such as leukemia, lymphoma, breast uterine,
ovarian, bladder and lung cancer. Some anthracycline class of drugs like daunorubicin,
adriamycin, epiadriamycin and mitoxantrone are presently used in chemotherapy, however,
major side effect associated with them is - cardiotoxicity. Mitoxantrone proposed to be less
cardiotoxic then adriamycin and daunomycin (Murdock, Child, Fabio, & Angier, 1979).
Mitoxantrone has symmetrical structure consist of tricyclic planar anthraquinone chromophore
with [(aminoethyl) amino]-ethanol side chain. Large number of analogues of mitoxantrone have
been synthesized and biologically evaluated for their anticancer activity (Bailly, Routier, Bernier,
&
Waring, 1996; Sissi et al, 1999; Collier, & Neidle, 1988; Cheng-Zee, Robert, & Cheng, 1978;
Cairns, Michalitsi, Jenkins, & Mackay, 2002; Routier, Bernier, Catteau, Riou, & Bailly, 1998;
Horn et al, 2000). Further anthraquinone series of compound having amido linkage are reported
in literature and interestingly some of these compounds showed good biological activity (Zagotto
et al, 1997). Compounds with 1, 4 dihydrogen having longer side chains at 5, 8 position on
anthraquinone ring showed better intercalative activity along with cytotoxicity at micro molar
concentration. Further incorporation of amino acid in the side arms at 5,8 position on
anthraquinone ring; followed the similar mechanism of inetraction like that of Mitoxantrone
(Zagotto, Sissi, Gatto, & Palumbo, 2004; Zagotto, Supino, Favini, Moro, & Palumbo, 2000; S-
Aliabadi, Tabarzadi, & Zarghi, 2004). Analogues of 1-hydroxy derivative of anthraquinone
showed significant anticancer activities against various cell lines. Further epoxidation of
anthraquinone increases the cytotoxicity (Wei, Wu, Chung, Won, & Lin, 2000). Also
modification of ketonic functionality of anthraquinone ring also reported in literature. Most of 9-
substituted compounds are cytotoxic but lower than the Mitoxantrone (Konopa, 2001; Huang,
Chiu, Lu, & Yuan, 2005).
Anthraquinone derivatives are proposed to be telomerase inhibitor and clinically proven to be the
active site of drug interaction (Freccero et al, 2014; Chung et al, 2014; Balasubramanian, &
Neidle, 2009). Telomeric activity is very high in cancer cells however low or nearly non-
noticeable in normal cells as per literature review. Further -SO -NH- feature have been reported
2
to bear anticancer activity. This feature has been proposed to show CDK2 inhibitory property
and arrest cell cycle. Number of compounds with SO -NH- feature are synthesized and shown to
2
be active against various cancer cell lines (Supuran, Briganti, Tilli, Chegwidden, & Scozzafava,
2
001; Fukuoka et al, 2001; Ella, Ghorab, Heiba, & Soliman, 2011). Further this feature has been
3

reported to show significant multifunctional activities (Alsughayer, Elassar, Mustafa, & Sagheer,
011; Tan et al, 2011).
2
In present research paper we have incorporated 1-substituted sulfonamide functionality along
with various amino acids i.e glycine, valine, phenylalanine and 훽-alanine in side chain on the
anthraquinone chromophore. All molecules are synthesized in our laboratory under normal
temperature pressure condition. The structure of all the molecules are confirmed by FT-IR, ESI-
13
MS, PMR and C-NMR spectrophotometric tools. All the 1- substituted synthesized compounds
S1-S8) are tested against MCF-7, PC-3 and Hela cancer cell lines. Likewise, compounds are
(
also evaluated for their antibacterial, antifungal and antioxidant activity. Majority of 1-
substituted compounds of the series are observed to be cytotoxic in comparison to Mitoxantrone.
Detailed in-vitro binding study of 1-substituted synthesized molecules (S1-S8) has been carried
out in comparison to Mitoxantrone using UV-visible and spectrofluoremetric tools. The study
confirmed the partial intercalative mode of binding of 1-substituted synthesized molecules.
Quantum chemical calculation indicates that synthesized 1-substituted molecules are highly
charged in comparison to mitoxantrone which might suppose to facilitate DNA binding
interactions at cellular level. Further, telomeric intercalation activity is confirmed by docking
study on d (TC5).
2
. Material and methods
The compounds under investigation (S1-S8) were synthesized in our laboratory. The chemicals
used for synthesis were purchased from Sigma-Aldrich with a stated purity of 97%. All solvents
were distilled and dried, as per set methods and procedures, before use. Reactions were
monitored by TLC performed on silica gel plates, the spots were located by iodine. Purity was
checked in TLC with solvent system of benzene and methanol giving single spot. Melting points
were uncorrected and measured in open capillary tubes on Digital Melting/Boiling point
apparatus. The FT-IR spectrum of the compounds were collected in the frequency region of
-1
4
000-500 cm using KBr pellet recorded on PERKIN ELMER spectrophotometer. The spectrum
-1
-1
was recorded at room temperature, with a scanning speed of 8 cm min with the spectral
-1 1
resolution of 8 cm . H NMR spectra were recorded in CDCl and DMSO on a BRUKER
3
AVANCE II 400 NMR spectrometer. Chemical shifts are given in ppm at 294.7 K. Mass spectral
studies are carried out on ESI-MS instrument of Waters Micromass Q-Tof Micro; equipped with
electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) sources
having mass range of 4000 amu in quadrupole and 20000 amu in ToF.
4

2
.1. General procedure for the synthesis of (S1-S8) compounds
A mixture of benzene sulfonyl chloride (6.35ml, 0.05mol), different amino acids (0.05 mol) and
°
NaOH sol (1N, 200ml) was stirred together at 45-50 C for 4 hours. A clear solution was obtained
°
and cooled to 5 C and treated with conc. HCl to make it slightly acidic (pH-6.5). Benzene
sulfonyl amino acid (A) was separated out as white crystals. Further benzene sulfonyl amino
acids was stirred for 20 min. with the freshly prepared thionyl chloride (5.0g) and then refluxed
for 3 hours to give acid chloride of benzene sulfonyl amino acid. Benzene sulfonyl amino acid
chloride (0.003M) and 1-amino anthraquinone (0.004M) was then taken in dry benzene (1N),
°
stirred together at 35-40 C for 6-8 hours. It was then poured into ice- water mixture and extracted
with chloroform. The extract was washed with excess of water. The solvent was then removed to
leave a residue which was recrystallized from diethyl ether to give final product (B). The purity
and reaction progress of the compound was checked by TLC (Vatsal, Devi, & Awasthi, 2018).
The procedure is same for p-toluenesulfonyl amino acids. [Supplementary Fig. I (1a) and
characterization (1b)].
2
.2. Cytotoxicity assay
2
.2.1. In vitro cytotoxicity analysis: [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium
bromide] (MTT) - based cytotoxicity assay against Hep2C.
The cytotoxic effects of synthesized compounds were carried out against Hep 2C Heal derivative
(human cervix carcinoma) cell lines which were determined using MTT and compared with
control. The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented
with 10% fatal calf serum, penicillin (100 units/ml) and streptomycin (100μg/ml) in a 5% CO2–
humidified atmosphere incubator. The cells were plated in a 96-well plate and treated with
different concentrations of the compounds for 12 h in a CO incubator (37ºC, 5% CO and 90%
2
2
relative humidity). The cytotoxicity was measured by adding 20μl of freshly prepared MTT
solution (5 mg/mL in PBS, sterile filtered) to each well. Culture plates were gently stirred
at150rpm for 5 min, to thoroughly mix the MTT into the media and incubated for another 3 h in
dark at 37ºC to allow metabolization of MTT. The purple formazan crystals were resuspended by
adding 5mg/ ml in methanol to each well. The absorbance was read at 570 nm in a
spectrophotometer. The cell death was calculated as follows:
Cell death = 100 − [(test absorbance/control absorbance) × 100].
5

2
.2.2 SRB (Sulforhodamine B)assay against MCF-7(Human breast cancer cell line) and PC-3
(Human prostate cancer cell line).
Cells were inoculated into 96 well microtiter plates in 100 µL. After cell inoculation, and before
addition of experimental drugs plates were incubated at 37°C, 5% CO , 95 % air and 100 %
2
relative humidity for 24 h. Experimental drugs were dissolved in dimethyl sulfoxide at 100mg/ml
and diluted upto 1mg/ml using water. At the time of drug addition, an aliquot of frozen
concentrate (1mg/ml) was thawed and diluted to 100 μg/ml, 200 μg/ml, 400 μg/ml and 800 μg/ml
with complete medium containing test article. Aliquots of 10 µl of these different drug dilutions
were added to the appropriate microtiter wells already containing 90 µl of medium, resulting in
the required final drug concentrations i.e.10 μg/ml, 20 μg/ml, 40 μg/ml, 80 μg/ml.
Further, plates were incubated under standard conditions for 48 hours. Cells were fixed in situ by
the gentle addition of 50 µl of cold 30 % (w/v) TCA (final concentration, 10 % TCA) and
incubated for 60 minutes at 4°C. The supernatant was discarded and plates were washed five
times with tap water and air dried. Sulforhodamine B (SRB) solution (50 µl) at 0.4 % (w/v) in 1
%
acetic acid was added to each of the wells followed by 20 minutes incubation at room
temperature. Residual dye was removed by washing five times with 1 % acetic acid. The plates
were air dried and stain was eluted with 10 mM trizma base. Absorbance was checked at a
wavelength of 540 nm (SRB) with 690 nm reference (Mitoxantrone) wavelength. Percent growth
was calculated for test wells relative to control wells. Percentage growth inhibition was
calculated as: [Ti/C] x 100 % (Vanicha Vichai & Kanyawim Kirtikara, 2006; Skehn et al, 1990).
2
.2.3. Cell cycle analysis
Cell culture was trypsinized, and washed with phosphate buffered saline (PBS) and incubated at
5
a density of 5X10 cells/well with 5 mL of complete medium in 6 well plate. Cells were grown in
Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and
1
% antibiotic-antimycotic. Test samples dissolved in DMSO (Dimethyl sulfoxide) to make 10
mg/mL stock solution. Finally working concentration made by farther dilution of stock solution
in complete medium and several dilutions of the test compounds in 1 mL of complete medium
were added. Alone cells (without treatment) used as control. The plates were then incubated at
◦
3
7 C for 12 to 24 hours in 5% CO incubator. After Incubation period, both adherent and floating
2
cells were harvested and centrifuged at 200 g for 5 min. Pellet were washed with phosphate
buffer saline and again centrifuged at 200g for 5 min. After that, cell pellets were resuspended in
6

7
0% EtOH and fixed for 2h at 4 °C. Fixed cells were washed with PBS (14 mL) and centrifuged
at 200g for 5 min. Cell pellets were resuspended in propidium iodide solution ( 50μg/mL in 0.1
sodium citrate, 0.1 % Triton-X 100 and 20µg/ml RNaes A) for 30 min in dark. Samples were
%
processed using an Amis flow cytometer. The distribution of cells in each phase of the cell cycle
was calculated using IDEA software.
2
.3. Preparation of solution for antibacterial and antifungal study
All the chemicals and solvents used in the present work were of analytical grade. A phosphate-
buffer saline (PBS) was used as a diluents and prepared by dissolving 1.5mM disodium
hydrogen phosphate, 0.5mM sodium phosphate, 10mM NaCl and 0.25mM EDTA at pH=7.1
using double distilled water. The Mueller-Hinton agar growth medium was nutrient broth for
antibacterial assay and Potato dextrose agar for antifungal study. Streptomycin was used as an
internal standard for Minimum inhibitory concentration in antibacterial assay. 200μl
concentration range of streptomycin was prepared using PBS as an internal standard. The
addition of 2μL microbes grown in nutrient broth and 2μL of solutions of varying concentrations
of each compound dissolved in methanol to 96 microtiter plates. Six bacterial species, 3 gram-
positive (Salmonella. aureus, Salmonella epidermidis, and Salmonella. citreus) and 3 gram
negative (Escherichia. coli, Klebscilla. pneumonia and Shigella. flexneri) were used as target
organisms in antibacterial assay and two fungi Candida albicans and Mucor sps. for antifungal
activites. The plates were incubated and autoclaved.
2
.3.1. Antibacterial activity assay
The nutrient agar and nutrient broth medium was prepared and then it was autoclaved. The agar
medium poured in to the petriplates in a laminar flow and the media was left as it is overnight.
The bacterial strains were spread over the media and left this for half an hour. The media
containing the bacterial strains was punched with the help of the sterilized puncher. The
compounds was dissolved in methanol were put inside the punched holes. The petri plates were
left overnight in BOD incubator. The plates were incubated for 24 hours at 37˚C then diameter of
zone of inhibition was measured to know the antibacterial activity of the samples. The
antibacterial study was carried out using a modified agar well diffusion method in order to check
the MIC measurement and diameter of zone of inhibiton. Antibacterial activity of compound can
be determined against 6 pathogenic strains. The activity was determined at a concentration of
1
mg/ml in methanol against 3 gram-positive, Staphylococcus aureus, Salmonella epidermidis,
and Salmonella citreus and 3 gram-negative Escherichia coli, Klebsiclla pneumonia and Shigella
7

flexneri. The microplate assay for measuring antibacterial activity was adapted by incorporating
2
00μl concentration range of streptomycin as an internal standard (40μg/ml).
2
.3.2. MIC (Minimum Inhibihitory concentration) measurements
MIC is the lowest concentration to inhibit any kind of visible bacterial growth on culture plates
by an antibacterial agent which will inhibit the visible growth of a microorganism after 24h
incubation at 37 ºC. The most commonly employed methods are the tube dilution method and
agar dilution methods. This procedure is a standard assay for antimicrobials. Minimum inhibitory
concentrations are important in diagnostic laboratories to confirm resistance of microorganisms
to an antimicrobial agent and also to monitor the activity of new antimicrobial agents. The MIC
was determined by the broth micro dilution bioassay in 96-well micro titer polystyrene plates;
where in six bacterial species, 3 gram-positive (S. aureus, S. epidermidis, and S.citreus) and 3
gram negative (E. coli, K. pneumonia and S. flexneri) were used. The calculation of MICs
involves semiquantitative test procedure. It gives an approximate value of minimum
concentration of an antibacterial agent required to prevent bacterial growth. The serial dilution
method was used for the determination of MICs of these compounds. The method involves the
addition of 2μL microbes grown in nutrient broth and 2μL of solutions of varying concentrations
of each compound dissolved in methanol to 96 microtitre plates containing 200μL of nutrient
broth each. The end result of the test is the minimum concentration of the compound that gives a
clear solution, i.e. no visible growth after 24 h in a BOD incubator at 37 ºC.
2
.3.3. Antifungal study
The aim of study is to assess the antifungal activity of anthraquinone based compounds and to
determine the zone of inhibition of synthesized compounds (S1-S8) on fungal strains i.e.
Candida albicans and Mucor species. The samples were dissolved in methanol. Aliquots
(10mg/mL) of the each sample were added to assay. The flasks containing 100 mL of sterile
growth medium potato dextrose agar (PDA). After vortexing, 50 mL were poured into Petri
dishes. The assay was performed by placing a 0.7 cm diameter plug of growing mycelium in the
centre of a Petri dish. Three replicates were run simultaneously. The zone of growth inhibition
was measured after 24 hours at an incubation of 30˚C.
2
.4. Superoxide scavenging ( Superoxide Dismutase assay)
For antioxidant study by SOD (Superoxide dismutase assay) the reaction mixture containing 1
ml of alkaline DMSO (1 ml DMSO in 10 mM NaOH) and drug (200μg/ml), 100μl of Nitroblue
8

Tetrazolium (NBT -1mg/ml) was added. NBT was purchased from Hi-Media laboratories.
Inhibition of the photo reduction of nitro blue (NBT) was performed to measure the SOD
activity. The photo chemically excited riboflavin was first reduced by methionine into a
semiquinone, which donated an electron to oxygen to form a superoxide source. The superoxide
readily converted the NBT into a purple formazan product. As a result, the SOD activity was
inversely related to the amount of formazan formation. MTX was used as a standard. Superoxide
radical is generated when sodium hydroxide was added to air saturated DMSO. The generated
superoxide remains stable in solution, which can reduce NBT into formazan dye at room
temperature and this formazan can be measured at 570 nm. Briefly, to there action mixture
containing 1 ml of alkaline DMSO (1 ml DMSO in 10mM NaOH) and drug (200μg/ml), 100μl
of NBT (1mg/ml) was added. The scavenging activity was calculated as follows:
Superoxide radical scavenging effect (%) = [A – A / A ] ×100
0
1
0
Where A was the absorbance of the control and A was the absorbance in the presence of the
0
1
sample.
2
.5. Spectrophotometric parameters
2
.5.1. Materials
UV–Visible absorption spectra measurement was carried out on T80+UV/Vis spectrometer
Shimadzu). The absorption spectra were recorded in wavelength range of 200-800nm.
Fluorescence experiments were performed using RF-5301 PC spectrofluorophotometer
Shimadzu).
.5.2. Sample preparation for UV-Visible and fluorescence spectroscopic studies
(
(
2
Calf thymus (ct) DNA and other chemicals for buffer preparation such as sodium dihydrogen
phosphate, disodium hydrogen phosphate were purchased from Sisco research laboratories. The
buffer was prepared by dissolving 1.5mM disodium hydrogen phosphate, 0.5mM sodium
phosphate, 10mM NaCl and 0.25mM EDTA at pH=7.1 using double distilled water (400 ml).
The concentration of ct-DNA was spectroscopically determined by molar extinction coefficient
-1
-1
DNA = 6600M cm at 257 nm. The stock solution of mitoxantrone and synthesized series (S1-
S8) was prepared by dissolving 1mg of synthesized molecules and MTX in 1ml DMSO. 10µL
of each i.e. mitoxantrone and synthesized compounds of S1-S8 series is used to each different
concentration of DNA for further study.
2
.6. Computational studies
9

2
.6.1. Gaussian protocol
The computational work was carried on Gaussian 98W and Gauss view 6 software tools in order
to study the electronic properties of synthesized analogues in comparison to mitoxantrone. The
quantum chemical calculations were fully optimized at Density functional theory level of
approximation. The optimized HOMO-LUMO structure of analogs were computed by Density
functional theory methods with 6-31G d,p. DFT is a widely used approach to compute the
geometrical structure of atoms or molecules at electronic level. The hybrid density functional in
DFT is B3LYP which stands for Becke, 3-parameter, Lee-Yang-Parr. This method is a low cost
method which provides good results. The energy and dipole moment of the optimized structures
are obtained from DFT methods.
2
.6.2. Autodock
Molecular docking was performed using Autodock 4.0 software http://autodock.scripps.edu/.
The receptor in docking process is i-moif sequence d (TC ) downloaded from RCSB protein
5
data bank bearing PDB code (225d) i.e. the tetrameric DNA structure with protonated cytosine:
cytosine base pairs. Molecules were designed using chem sketch (www.acdlabs.com) and saved
as mol file. DNA sequence was optimized for energy minimization in Swiss PDB Viewer
(SPDBV) version 4.1.0. DNA sequence was loaded and stored as 225d.pdb. The mol file of
synthesized analogue was also loaded as pdb in Open BabelGUI. The grid map files were
selected directly with 40x40x40 dimensions for searching of ligand with active site of DNA
sequence. This way grid parameter was created. Followed by genetic algorithm, the docking
parameter used Lamarckian genetic algorithm to predict best fit conformation in docking
process. Docking is carried out using Cygwin interface and results were analysed in Pymol 27
software (ww.pymol.com).
3
. Result and discussion
3
.1. Cytotoxic study
MCF-7 (Human Breast Cancer cell line), PC-3 ( Human Prostate Cancer cell line) and Hep 2C (
Hela derivative Human cell line) were used with the purpose to confirm the concentration of
synthesized 1-substituted (S1-S8) compounds required to cause the damage. Cell lines were
treated with different concentration of S1 to S8 series of synthesized compounds for a fixed
hours. Mitoxantrone showing cytotoxic effect at lower concentration (1-5µg/ml) for all the cell
lines (K-Chyla, Jedrzejczak, Skierski, Kania, & Jozwiak, 2005). 1-substituted anthraquinone
1
0

series (S1-S8) compounds showed poor performance for PC-3 cell lines. However little
inhibition to MCF-7 cell lines at higher concentration of S2, S3 and S5 compounds has been
observed (Fig.1a and 1b) and (Table 1a and 1b). Interestingly S-series showed better action
against Hep 2C cell lines. The synthesized compounds S1 and S3 were cytotoxic at 9µg/ml while
S4 was cytotoxic at 1.008µg/ml i.e. better than mitoxantrone at 2.5 µg/ml.
3
.2. Cell cycle analysis
Cell cycle analysis of S3 and S4 compounds of S series against Hela cells for 24 hours produced
interesting results. It is well established that mitoxantrone arrests G1 and G2 phases at cell cycle
progression and ultimately inhibit the cell growth (Khan, Lal, Kimar, & Khan, 2010). Further it
is also confirmed that mitoxantrone also promotes the arrest of S-phase of cell cycle. G1 and G2
are the growth phases in cell cycle analysis whereas S-phase is a synthetic phase where in DNA
replication and DNA synthesis takes place. Hence cell goes apoptosis upon treatment with MTX
due to inhibition at cell growth phases G1 and G2 alongwith inhibiting the DNA replication/
duplication process (S phase). Direct attack on cell regulatory protein is suggested. Further it
play vital role in controlling the regulation of G2/M and G1 phases. S4 in accordance to
cytotoxic studies on Hep 2C cell lines showing better effect over mitoxantrone i.e. 1.008µg/ml
and 2.5 µg/ml respectively (Table 2). Like wise S3/S1 is showing inhibiting action at higher
concentration i.e. 9 µg/ml than MTX 2.5 µg/ml as per cytotoxic study.
We have carried out flow cytometric study of S4 and S3 compounds only as S3 and S1 are
showing almost similar cytotoxic effects. Concentrations of S3 and S4 compounds were taken 10
µg/ml and 5µg/ml respectively as per cytotoxic studies. After examining the data for S4 (Fig. 2)
good number of cells are arrested in G /G phase i.e. initial phase of cell cycle. Hence all the
0
1
chemical activities i.e. increase in supply of proteins, doubling in number of cell organelles etc is
countered. Biosynthetic activity is very high during G phase which seems to be droped by 5 %
1
in comparison to control. However the activites in G phase increase by~4.5% i.e. cell
2
accumulation takes place in G phase. 0.1 % cells arrest is observed in S-phase i.e. inhibiting
2
DNA replication mechanism. Similarly for S3 different pattern is observed i.e. variation at G1
and G phase are 1.3 and 0.1 % respectively in comparison to control. Interestingly S pahse is
2
reduced by 1.9 % in comparison to control; indicating the arrest at DNA synthesis i.e. DNA
duplication and replication. Hence both the molecules S4 and S3 deregulate the cell cycle which
is the primary condition for any drug candidate to be cytotoxic. Further as per literature study G1
and G check points are mandatory for tumor cell to go for uncontrolled proliferation. Both the
2
1
1

molecules S4 and S3 arrest G and G phase by disturbing biochemical process alongwith
1
2
disturbance at S phase i.e. affecting the DNA synthesis/ replication. But some difference in the
mechanism of operation of S4 and S3 has been observed during the progression of cell cycle
like:


G /G phase were highly disturbed by S4.
1 2
S phase was highly disturbed by S3
It concluded the different mechanism of operation of both the molecules (S3 and S4). Though
basic structure of both the compounds i.e. anthraquinone ring and 1-substituted position of side
arm is same but small variation in the structural features of side arm affects the cytotoxicity and
mechanism of the operation of S3 and S4 at cellular level.
3
.3. Antibacterial study and Antifungal studies
Two features (i) -SO NH and (ii) -NH-CO are reported to be important for antibacterial and
2
antifungal action. Number of compounds/ drugs are presently in market bearing the said feature ,
act as an antibacterial and antifungal agents. Synthetic series (S1-S8) is tested against Gram +ve
as well as Gram –ve strains in comparison to streptomycin (Table 3). Analogues of (S1-S8) are
found to be more active on K. pneumonia (Supplementary Fig. II and III; and Supplementary
Table I) in comparison to streptomycin. Lack in universality in antibacterial action in comparison
to standard drug could be due to hydrophilicity and inability to penetrate in to cell wall of
bacteria along with long chains at position 1 on the anthraquinone ring. Likewise the S1 to S8
series showed mild antifungal activity against Mucor species and Candida albicans (Kang, Fong,
&
Tsang, 2010) (Supplementary Table II). Hence it is not only the structure features but there
position and orientation in a molecule is vital for important biological activities. This requires
more detailed structural study while keeping into consideration the pattern shown by S1-S8
series in the said study.
3
.4. Antioxidant study
It has been reported by researchers that use of anthraquinone class of drugs in chemotherapy is
limited due to their peroxidating activity and production of the free radicals. The activity is
initiated by the oxy-reductase enzymes at cellular level which is the main cause of cardiotoxicity.
Anthraquinone class of drugs have admiration towards these enzymes as it is likely to go for one
.
electron transfer reaction and produce O radicals (oxygen radicals). Comprehensive study have
2
1
2

been carried out and reported in literature on natural and synthetic anthraquinones class of
compounds with diversed structural features to check their ability and essential requirement to
undergo redox reactions. It has been confirmed that functional group present over the
anthraquinone ring favours or disfavours the oxidation-reduction/ free radical formation (
Tarasiuk et al, 1998; Alderton, Gross, & Green, 1992; Reszka, Hartley, Kolodziejczyk, & Lown,
1
989). However mitoxantrone, a standard refrence drug taken in present study, showed poor
affinity towards NADH dehydrogenation reaction despite of presence of OH groups at 1and 4
position and unmodified carbonyl group. Mechanistic reason is not clear but it could be due to
presence of aromatic amino group with long chains at 5, 8 substituted positions on the ring.
Highest level of flexibility and steric hindrance with protein molecule could be the reason.
Therefore, we have carried out SOD analysis of synthesized series (S1-S8) to evaluate the
potential to scavange O2 radical production. Interestingly synthesized 1-substituted
.
anthraquinone molecules (S1-S8) were ineffective in stimulating free radical (O ) oxygen
2
production (Supplementary Table III).
3
.5. Spectroscopic confirmation of binding of (S1-S8) compounds with DNA.
3
.5.1. UV-Visible spectral studies
Various studies, biochemical as well as biophysical, has confirmed the intercalative mode of
binding of mitoxantrone with receptor site i.e. DNA. Mitoxantrone exhibit maximum absorption
at 608 and 660 nm and successive addition of ct-DNA leads to hypochromic and bathochromic
effect. Intercalative mode of binding is proposed.
Likewise interaction of synthesized 1-substituted anthraquinone molecules (S1-S8) with ct-DNA
has been carried out and K (binding constant) and ∆퐺 (free energy of binding) has been
b
calculated according to Benesi-Hildebrand equation:
퐴0
∈ꢀ
∈ꢀ
1
= ∈
+∈
(1)
퐴−퐴0
퐻ꢁꢀ−∈ꢀ 퐻ꢁꢀ−∈ꢀ 퐾 [퐷푁퐴]
푏
A and A are the absorbances of compounds in the absence and presence of DNA,
0
respectively, and  and 
are the respective absorbance coefficients. K is the association/
b
G
H-G
binding constant. A plot of A /A-A versus 1/ [DNA] was constructed, linear fitting of data
0
0
yielded binding constant (Fig. 3). The values (Table 4) obtained out of experiments are in range
4
5
with the values reported for interactions of anthracycline molecules with DNA (K≈ 10 to 10
-1
M ) (Li, Ma, Yang, Guo, & Yang, 2005). All synthesized molecules (S1-S8) show absorption
maxima at 420-425 nm. Upon comparing with mitoxantrone, having two maxima at 608-660 nm,
1
3

is due to two types of functionalities i.e. OH and NH at 1,4 and 5,8 position respectively. S1-S8
compounds are only 1-substituted and maxima at lower 휆 could be due to one NH group directly
attached to anthraquinone ring along with O=S=O group and absence of OH auxochrome at 1, 4
position of anthraquinone ring. Continuous decrease in absorbance with red shift has been
observed for all the molecules (S1-S8) with increasing concentration of DNA. This hypochromic
effect is due to interaction between electronic states of (S1-S8) molecules and base pairs of
DNA. Slight red shift (~9 ꢂ ꢃ4푛푚) in maxima could be due to lack of functional group
interaction in internal hydrophobic environment of DNA. All the synthesized molecules (S1-S8)
series shows hypochromic effect with red shift upon binding with DNA. This behavior is an
indication of partial intercalative mode of binding (Pasternack, Gibbs & Villafranca, 1983;
Krugh & Reinhardt, 1975; Lu, Wang, Lv, Zhang, & Liu, 2010). Slightly different pattern of
absorption maxima is observed for S5 and S6 molecules. The additional structural feature
incorporated in S5 and S6 is CH group at para position of benzene ring A. In case of S5 though
3
intense hypochromic effect with bathochromic shift is observed where as in S6 at high
concentration of DNA sharp bathochromic effect has been observed. The only structural
difference in S5 and S6 is additional –CH group at (C ) of (Structure 1). CH group at para
2
n
3
position on ring A in S5 could be having some interactions due to fixing of –NH–SO - group or
2
hydrophobic character leading to steric interactions and allowing -NH–SO - group for additional
2
interaction with DNA molecule. However in case of S6 initially, if allowed, some polar
interaction between the charge functionality of the molecule with DNA bases could be there but
at high concentration it was lost. Upon comparing the K , the value does not show much
b
difference. However ∆퐺 value for S6 is more than S5. Therefore it confirms some additional
interaction of molecule S6 with DNA and finally stabilizing the molecule. But to our surprise
same behaviour could not be observed for S7 and S8 where p-substituted –CH is in position at
3
ring A. Upon comparing the structure of S2 and S6 the only difference is para –CH group at
3
ring A while comparing S6 with S7, S5, S8 an additional –CH group is incorporated in the side
2
arm which might have induced extra flexibility to the side chain and ultimately dissimilar
behavior of S6 in terms of absorbance maxima and ∆퐺 value.
3
.5.2. Fluorescence spectra
Interaction of synthesized compounds (S1-S8) with ct-DNA has been investigated by
fluorescence spectroscopic study in order to confirm the intercalative mode of interaction and
effect of different structural feature on behavior of molecules (S1-S8) with DNA. The data is
1
4

compared with interaction of Mitoxantrone-DNA complex. Mitoxantrone show an excitation
maximum at 596nm and emission maxima at 680 nm (Li, Ma, Yang, Guo, & Yang, 2005).
We have carried out experiment by 10µM solution of (S1-S8) series and MTX in phosphate
buffer solution. λexc for S1-S8 molecules observed at 390 nm and λem was in range of 560-565
nm. Similarly for MTX λ was 600-602nm. Quenching effect is observed in S1-S8 molecule
em
upon binding to DNA. This confirms the hydrophobic interaction of chromophore with DNA
bases. In literature, it is proposed that there is lack of substituent interaction of side chain of
mitoxantrone with the solvent i.e. water molecules upon binding to DNA and confirms the
intercalative mode of binding. In S1-S8 series, no doubt that quenching is shown by all the
molecules (S1-S8) i.e. decrease in fluorescence intensity accompanied with shifting toward
higher wavelength (~ 6nm) indicates partial intercalative mode of interaction upon DNA
binding. Slight deviation in fluorescence spectra of synthesized compound S1-S8 seems to be
due to difference in type of side chain (structure 1 and 2) as red shift is observed at lower D/P
ratio. However fluorescence quenching mechanism for S4, S6 and S7 was different.
Fluorescence emission spectra of S6 was entirely different from rest of the molecules. Though
quenching was observed in fluorescence intensity of S6 but emission spectra is moving fast
towards higher 훌 with rise in DNA concentration. This indicates some additional
mode/interaction of ligand with acceptor as observed in absorption spectra (Fig. 3). Though Ksv
and K and ∆G values could not add much on this but their values shows partial intercalative
q
mode of binding (Table 4). However behaviour of S1-S8 series molecule in emission spectra
clearly indicates that extension of side arm with more hydrophobic groups along with polar
functionality i.e. –NH–SO - would leads to additional flexibility at side chain and ultimately
2
additional interactions (Awasthi, Dogra, & Barthwal, 2013; Khan, Islam, Yennamalli, Sultan,
Subbarao and Khan, 2008 ).
The fluorescence quenching constant, K is evaluated using Stern-Volmer equation:
sv
F /F= 1+ K [DNA]
(2)
0
sv
Where F and F are the fluorescence intensities in the absence and presence of DNA respectively,
0
[
DNA] is total concentration of ct-DNA, K is Stern Volmer quenching constant (a measure of
sv
quenching efficiency by DNA). K is obtained by plot of F /F versus [DNA]. Using a typical
sv
0
-9
fluorescence life time of 0.2x10 s; the corresponding biomolecular quenching constant K is
q
1
5

calculated. The binding constant K and binding stoichiometry (n) of the compound has been
b
determined using following equation:
Log (F –F)/F= log K + n log [Q]
(3)
0
b
Where Q is quencher concentration i.e. [Q] = [DNA].
Further the total binding energy changes were calculated according to standard Gibbs equation:
0
∆
G = - RT ln Kb
(4)
where R and T refers to gas constant and temperature in kelvin respectively. All the parameters
are mentioned in (Table 5) and fluorescence spectra are shown in (Fig. 4). All molecules (S1-S8)
series showing linear straight line graph of Stern-Volmer plot. Therefore, only one type of
quenching process is operational i.e. either static or dynamic (Lakowicz, pp. 239-240; Zhong,
Yu, Huang, Ni, & Liang, 2001). S4 is showing better cytotoxic activity in comparision to
Mitoxantrone and fluorescence spectrum of S4 differs than rest (S1, S2, S3, S5, S6, S7 and S8)
as well as Mitoxantrone. Quenching is slow and shift in fluorescence intensity toward right i.e. at
higher wavelength. Therefore, in addition to partial intercalative mode of interaction, molecule is
also undergoing additional mode of interaction with DNA molecule by involving it polar side
chains bearing -NH–CO - and -NH–SO - functional groups (Dogra, Awasthi, Nair, & Barthwal,
2
2
2
013).
3
.6. Molecular modelling
.6.1. Electronic properties of synthesized (S1-S8) and MTX
3
In order to correlate the binding mechanism and cell killing potential of S1-S8 series with their
electronic properties; detailed quantum chemical study has been carried out using DFT/6-31G
(d,p) method. Molecular orbitals and atomic charges are calculated. Results were compared with
MTX. The highest occupied molecular orbital (HOMO) represents the ability to donate an
electron while lowest unoccupied molecular orbital (LUMO) represents the ability to accept an
electron hence corresponds to ionization potential and electron affinity respectively. The
HOMO-LUMO calculation has been carried out and graphical representation of movement of
charge, dipole moment and energy indicates the large electronic difference between synthesized
series (S1-S8) (Fig. 5). For the stability of the structure of the molecule, energy gap between the
HOMO and LUMO orbitals is an important parameter. The molecule with smaller energy gap
have higher reactivity and low kinetic stability and considered to be soft molecule (Shukla,
1
6

Yadava, & Roychoudhury, 2015). Energy gap (a.u.) in MTX is higher than rest of synthesized
compounds of the series (S1-S8). At the same time dipole moment of synthesised series (S1-S8)
is higher than MTX (Table 6). Therefore it can be concluded that due to small difference in
HOMO-LUMO of S1-S8 series along with high dipole moment; synthesized molecules (S1-S8)
can be easily reduced. At the same time due to instability in the conformation might lead to fall
in cytotoxicity in comparison to MTX. As per electronic study S1 is quite comparable with
mitoxantrone in terms of energy gap but S3 and S4, are claimed to be comparable to MTX in
terms of ct-DNA binding and cytotoxic study (Table 1a). Moreover upon comparing the dipole
moment of synthesized molecules (S1-S8) it is interesting to note that since all the synthesized
molecules (S1-S8) differs at R and R (Structure1); the dipole moment of S1 and S5 ( R = H and
1
2
1
R = CH ) is comparable to MTX, whereas rest of compounds with ( R = H and R = CH ) i.e.
2
3
1
2
3
S3 and S7; S2 and S6; S4 and S8 large variation in dipole moment is observed. In majority of
cases, X component of dipole moment is going –ve to +ve and adding up in the total resultant
value (Fig. 5). However variation in R , specifically, in S4, addition of CH group in the side
2
2
chain at position 1 raised the direction of electrical dipole value to +8.2 which is completely
different from MTX. Interestingly S4 is more cytotoxic at lesser micro molar concentration than
MTX i.e. 1.008휇g/ml and 2.5휇g/ml respectively (Table 1a). Therefore, some additional/different
mechanism of operation seems to be followed while drug interacting with receptor.
3
.6.2. Molecular docking details
Computational docking tool is used to understand the binding interaction of synthesized
molecules (S1-S8) with telomeric sequence. The binding energy, binding interactions, hydrogen
bond length (A˚) are listed in (Supplementary Table IV). The orientation and hydrogen bond
length is shown in (Fig. 6). It is well known that mitoxantrone interact with DNA via interfering
the telomeric activity. They have also shown to inhibit telomeric activity (Barthwal, Tripathi,
Pradeep, 2015).; Searle, & Balkwill, 2006; Phan, & Mergny, 2002; Kim et al, 1994, Tripathi, S.,
Pradeep, P, Tarikere & Barthwal Ritu, 2016). Telomere are proposed to be “biological clock” in
order to determine the proliferative activity of the human cells (Cairns, Michalitsi, Jenkins, &
Mackay, 2002). Mitoxantrone and various synthesized analogue has been studied to intercalate at
telomeric junction of the DNA (Neidle, & Parkinson, 2002; Waller, Sewitz, Hsu, &
Balasubramanian, 2009; Yang, & Issa, 2003). Based on these lines we have carried out docking
study of synthesized series (S1-S8) along with mitoxantrone on with d (TC ). Data is tabulated
5
1
7

in (Supplementary Table IV). Not much of the difference in ∆G (Kcal/mol) could be observed.
b
However variation in K can be seen for synthesized series (S1-S8) and MTX. Therefore it is
i
concluded that synthesized series (S1-S8) and MTX binds to i-motifs. However same could be
validated only after doing experimental studies for synthesized series hence would be reported
subsequently.
4
. Conclusion
Anthracycline class of drugs are most commonly used chemotheuraptic agents. Number of
analogues of this class of compounds are reported but Adriamycin, Daunomycin and
Mitoxantrone are presently in use in cancer hospitals. Mitoxantrone is proposed to be less
cardiotoxic over Adriamycin and Daunomycin. Further 1, 4 substituted diamino alkyl chains on
anthraquinone chromophore are proposed to be important along with 5, 8 substituted –OH
groups (Structure 2) for desired biological activity. Anthraquinone ring facilitate the docking of
the molecule in the DNA double helix via intercalation however 1,4 diaminoalkyl side arm
interacts with DNA and imparts biological activity; hence declared to be pharmacophoric group
(Konopa, 2001). In present research paper we have synthesized series of 1-substituted
anthraquinone compounds. 1-substitued side arm is modified by incorporating sulphonamide and
amide functionalities. Sulphonamide and amide functionalities are proved to show
antiproliferative activities in some cell lines as per literature study. All the synthesized molecules
in series (S1-S8) having 1-substitued anthraquinone ring but molecules differs in structure and
functional groups at the side arm. Comprehensive biological and spectrophotometric studies has
been carried out on synthesized molecules (S1-S8) in comparison to the Mitoxantrone.
Significant cytotoxic activity has been observed over Hela cell lines and one of the molecule S4
is showing better activity over Mitoxantrone. Cell cycle analysis of S3 and S4 molecules
indicates the arrest of cell cycle at G1 and G2 phases. Similarly partial intercalation mode of
binding of all the synthesized molecules (S1-S8) is proposed in comparision to Mitoxantrone on
the basis of spectrophotometric study. Antioxidant study could not conclude any result however
small energy gap with high dipole moment of synthesized molecule (S1-S8) than Mitoxantrone
has been observed using DFT/6-31G (d,p) method. Molecules (S1-S8) are proposed to be
reactive and will facilitate in formation of charge transfer complexes. Further synthesized series
are proposed to be good i-motif inhibitor using docking tools. In present study, we conclude that
1
,4 (OH) position and 5,8 diaminoalkyl residue on anthraquinone ring is not mandatory
1
8

requirement for molecule to be anticancer agent, whereas structure and functionality over side
arm is proposed to be important to mark the desired biological activity. Further same would be
confirmed only after we report synthesis of (2); (3); (1,4); (1,8) and (1,5) substituted
anthraquinone with sulfonamide and amide functionality bearing side arms. Chromophore
modification with 1 and 1, 4 substitution with 1-[1-oxo-3-phenyl-(2-benzosulfonamide)-propyl
amido] – anthracene-9, 10-Dione arm will finally confirm the same, hence, will be reported
subsequently. Further studies on thermal stability of DNA on binding to different ligands S1-S8
are expected to provide further insight into the binding mechanism. We have also carried out
antibacterial and antifungal affects of synthesized series. S1-S8 molecules showed mild activities
for antibacterial and antifungal actions.
Acknowledgements
This work was supported by the Department of Science & Technology, Government of India,
New Delhi, under grant no. SR/WOS-A/CS-1030/2015 in Women Scientist Scheme and CSIR
UGC-JRF fellowship. Authors are Thankful to SAIF lab, Punjab University, Chandigarh for
NMR and ESI-MS experiments. In vitro SRB assay for anti-cancer activity was carried out at
Anti-Cancer Drug screening facility (ACDSF) at ACTREC, Tata Memorial Centre, Navi
Mumbai. Authors acknowledge the cell cycle analysis work at Pharmacology and Toxicology
lab, Food and Nutraceuticals Division, CSIR-IHBT, Palampur. Authors also acknowledge the
support extended by Dr. S.S. Kanwar and his group, Department of Biotechnology, Himachal
Pradesh University, Shimla for MTT assay and antibacterial studies. The facilities provide by
National Institute of Technology, Hamirpur are highly acknowledged. The paper is original
research of authors and also filed as a patent (Indian patent application no. 201711026145).
References
Adachi, C., Zhang, Q., Kuwabara, H., Potscavage, W. J., Huang, S., Hatae Y. & Shibata, T.
(2014). Anthraquinone based intramolecular charge-transfer compounds: Computational
molecular design, thermally activated delayed fluorescence, and highly efficient red
electroluminescence. Journal of American Chemical Society, 136, 18070-18081.
dx.doi.org/10.1021/ja510144h.
1
9

Alderton, P. M., Gross, J. & Green, M. D. (1992). Comparative study of doxorubicin,
mitoxantrone, and epirubicin in combination with ICRF-187 (ADR-529) in a chronic
cardiotoxicity animal model. Cancer Research, 52, 194-201.
Alsughayer, A., Elassar, A-Z. A., Mustafa, S. & Sagheer, F. A. (2011). Synthesis, structure
analysis and antibacterial activity of new potent sulfonamide derivatives. Journal of Biomaterials
and Nanobiotechnology, 2, 144-149. doi:10.4236/jbnb.2011.22018.
Awasthi, P., Dogra, S. & Barthwal, R. (2013). Multispectroscopic methods reveal different
modes of interaction of anticancer drug mitoxantrone with Poly (dG-d C). Poly (dG-dC) and
Poly (dA- dT). Poly (dA- dT). Journal of Photochemistry and Photobiology B: Biology, 127, 78-
8
7. https://doi.org/10.1016/j.jphotobiol.2013.07.023.
Bailly, C., Routier, S., Bernier, J-L. & Waring, M-J. (1996). DNA recognition by two
mitoxantrone analogues: influence of the hydroxyl groups. FEBS Letters, 379, 269-272.
doi/10.1016/0014-5793(95)01528-0.
Balasubramanian, S. & Neidle, S. (2009). G-Quadruplex nucleic acids as therapeutic agents.
Current opinion in chemical biology, 345-353.
Barthwal, R., Tripathi, S., Pradeep, P, Tarikere (2015). 123 NMR study of interaction of
mitoxantrone to G-quadruplex sequences leading to stabilization and inhibition of telomerase
enzyme.
Journal
of
biomolecular
structure
and
Dynamics,
33
doi/org/10.1080/07391102.2015.1032756
Beziane, A., Teerme, T. & Vanelle, P. (2005). An electron transfer approach to the preparation
of highly functionalized anthraquinones. Molecules, 10, 289-294. doi:10.3390/10010289.
Cairns, D., Michalitsi, E., Jenkins, T. C. & Mackay, S. P. (2002). Molecular modelling and
cytotoxicity of substituted anthraquinones as inhibitors of human telomerase. Bioorganic &
Medicinal Chemistry, 10, 803-807. doi: 10.1016/S0968-0896(01)00337-6.
Cheng-Zee, Robert, K.-Y., Cheng, C. C. (1978). Antineoplastic agents. Structure-activity
relationship study of bis-(substituted amino-alkylamino) anthraquinones. Journal of Medicinal
Chemistry, 21, 291-294. doi:10.1021/jm00201a012.
2
0

Chung, Y-L., Pan, C-H., Liou, W-H., Sheu, M-J., Lin, W-H., Chen, T-C., Huang, H-S. & Wu, C-
H. (2014). NSC746364, a G-quadruplex- stabilizing agent, suppreeses cell growth of A549
Human lung cancer cells through activation of the ATR/ Chk1- dependent pathway. Journal of
Pharmacological Sciences, 124, 7-17. doi: 10.1254/jphs.13096FP.
Collier, D. A. & Neidle, S. (1988). Synthesis, Molecular modelling, DNA binding and antitumor
properties of some substituted amidoanthraquinones. Journal of Medicinal Chemistry, 31, 4, 847-
8
57. doi:10.1021/jm00399a028.
Dogra, S., Awasthi, P., Nair, M. & Barthwal, R.(2013). Interaction of anticancer drug
mitoxantrone with DNA hexamer sequence d-(CTCGAG)2 by absorption, fluorescence and
circular dichromism. Journal of Photochemistry and photobiology B: Biology, 123 , 48-54. doi
1
0.1016/j.j photobiol.2013.03.015.
Ella, D. A. A. E., Ghorab, M. M., Heiba, H. I. & Soliman, A. M. (2011). Synthesis of some new
thiazopyrane and thiazolopyranopyrimidine derivatives bearing a sulfonamide moiety for
evaluation as anticancer and radiosensitizing agents. Medicinal Chemistry Research, 21: 2395–
2
407. doi 10.1007/s00044-011-9751-9.
Freccero, M., Percivalle, C., Sissi, C., Greco, M. L., Musetti, C., Mariani, A., Artese, A. Costa,
G., Perrore, M. L. & Alcaro, S. (2014). Aryl ethynyl anthraquinones: a useful platform for
targeting telomeric G-quadruplex structures. Organic and Biomolecular Chemistry, 12, 3744-
3
754. doi:10.1039/c4ob00220b.
Fukuoka, K., Usuda, J., Iwamoto, Y., Fukumoto, H., Nakamura, T., Yoneda, T., Narita, N.,
Saijo, N. & Nishio, K. (2001). Mechanisms of action of the novel sulfonamide anticancer agent
E7070 on cell cycle progression in human non-small cell lung cancer cells. Investigational New
Drugs, 19, 219-227. https://doi.org/10.1023/A:1010608317361.
Guidi, A., Canfarini, F., Giolitti, A., Pasqui, F., Pestellini, V. & Arcamone, F. (1994). Synthesis
of new antitumor anthracyclines: Derivatives bearing a fluorine substitution at position 8 or 10.
Pure and Applied Chemistry, 66, 2319-2322. doi.10.1351/pac199466102319.
2
1

Horn, D. E., Leonard, M. E., Fischl, A. J., Gray, M., Clark, D. W., A- Mahlock, T. & Sader, C.
N. (2000). Synthesis of symmetrically 1, 4- bis [(aminoalkyl) amino]-5, 8- Dimethylanthracene-
9
, 10-diones. ARKIVOC, 1, 876-881.
Huang, H-S., Chiu, H-F., Lu, W-C. & Yuan, C-L. (2005). Synthesis and antitumor activity of 1,
-Diaminoanthraquinone derivatives. Chem. Pharm. Bull., 53, 9, 1136-1139. doi:
8
Kang, K., Fong, W-P. & Tsang, P. W-K. (2010). Novel antifungal activity of purpurin against
Candida species in vitro. Medical Mycology, 48, 904-911. DOI: 10.3109/13693781003739351.
Kim, N. W., Piatyszek, M. A., Prowse, K. R., Harley, C. B., West, M. D., Ho, P. L. C., Coviello,
G. M., Wright, W. E., Weinrich, S. L. & Shay, J. W. (1994). Specific association of human
telomerase activity with immortal cells and cancer. Science 266, 2011-2015.
Khan, S.N., Lal, S.K., Kimar, P. & Khan, A. U. (2010). Effect of Mitoxantrone on proliferation
dynamics and cell- cycle progression. Bioscience Reports, 30, 375-381. doi
1
0.1042/BSR20090119.
Konopa, J. (2001). Antitumor acridines with diaminoalkylo pharmacophoric group. Pure Applied
Chemistry, 73, 1421-1428. doi:10.1351/pac200173091421.
Khan, S.N., Islam, B., Yennamalli, R., Sultan, A., Subbarao, N., Khan. A.U. (2008). Interaction
of mitoxantrone with human serum albumin: Spectroscopic and molecular modelling studies.
European journal of pharmaceutical sciences, 35, 371-382.
Krapcho, A. P., Getahun, Z., Avery, K. L., Vargas, K. J. & Hacker, M. P., (1991). Synthesis and
antitumor evaluations of symmetrically and unsymmetrical substituted 1,4- bis [( aminoalkyl)
amino] anthracene-9,10- diones and 1,4- bis [( aminoalkyl)amino]-5,8-dihydroxyanthracene-9,10
diones. Journal of Medicinal Chemistry, 34, 2373-2380. doi: 10.1021/jm00112a009.
Krugh, T. R. & Reinhardt, C. G. (1975). Evidence for Sequence Preferences in the Intercalative
Binding of Ethidium Bromide to Dinucleoside Monophosphates. Journal of Molecular Biology
9
7, 133-162. doi:10.1016/80022-2836(75)8031-3.
K-Chyla, A., Jedrzejczak, M., Skierski, J., Kania, K. & Jozwiak, Z. (2005). Mechanisms of
induction of apoptosis by anthraquinone anticancer drugs aclarubicin and mitoxantrone in
2
2

comparison with doxorubicin: Relation to drug cytotoxicity and caspase-3 activation. Apoptosis,
0, 1497-1514. DOI: 10.1007/s10495-005-1540-9.
1
Lakowicz, I. R.. Principles of fluorescence spectroscopy. Kluwer Academic, New York, 239-
40.
2
Li, N., Ma, Y., Yang, C., Guo, L. & Yang, X. (2005). Interaction of anticancer drug
mitoxantrone with DNA analyzed by electrochemical and spectroscopic methods. Biophysical
Chemistry, 116, 199-205. doi:10.1016/j.bpc.2005.04.009.
Lu, Y., Wang, G-K, Lv, J., Zhang, G-S. & Liu, Q-F. (2010). Studies on the interaction of an
anthracycline disaccharide with DNA by spectroscopic techniques and molecular modelling.
Jounal of Fluorescence, 21, 409–414. DOI 10.1007/s10895-010-0729-7.
Murdock, K. C., Child, R. G., Fabio, P. F., Angier, R. B. (1979). Antitumor agents. 1. 1, 4- Bis
[
(aminoalkyl) mino]-9, 10- anthracenediones. Journal of Medicinal Chemistry, 22, 9, 1024-1029.
doi: 10.1021/jm00195a002.
Neidle, S. & Parkinson, G. (2002). Telomere maintenance as a target for drug discovery. Nature
Reviews Drug Discovery, 1, 383-393.
Nor, S. M. M., Sukari, M. A. H. M., Azziz, S. S. S. A., Fah, W. C., Alimon, H. & Juhan, S. F.
(2013). Synthesis of new cytotoxic aminoanthraquinone derivatives via nucleophilic substitution
reactions. Molecules, 18, 8046-8062. doi:10.3390/molecules18078046
Pasternack, R. F., Gibbs, E. J., & Villafrancas J. J. (1983). Interactions of Porphyrins with
Nucleic Acids. Biochemistry 22, 5409-5417. doi: 10.1021/bi00292a024.
Phan, A. T. & Mergny, J-L. (2002). Human telomeric DNA: G-quadruplex, i-motif and Watson-
crick double helix. Nucleic acids research 30, 21, 4618-4625. doi:10.1093/nar/gkf597. x
Reszka, K., Hartley, J. A., Kolodziejczyk, P. & Lown, J. W. (1989). Interaction of the
peroxidase- derived metabolite of mitoxantrone with nucleic acids. Evidence for covalent
1
4
binding of C-labeled drug. Biochem Pharmacology, 23, 4253-4260. doi: 10.1016/0006-
952(89) 90523.
2
2
3

Routier, S., Bernier, J-L., Catteau, J-P., Riou, J-F. & Bailly, C. (1998). Synthesis, DNA binding,
topoisomerase III inhibition and cytotoxicity of two guanidine- containing anthracene-9, 10
diones. Anticancer Drug Design, 13, 407-415.
S-Aliabadi, H., Tabarzadi, M. & Zarghi, A. (2004). Synthesis and cytotoxic evaluation of two
novel anthraquinone derivatives. FARMACO, 59, 645-649. Doi: 10.1016/ j.farmac.2004.03.006.
Searle, M. S. & Balkwill, G. D. (2006). DNA Qadruplex-ligand recognition: Structure and
Dynamics in: S. Neidle, S. Balasubramanian Quadruplex nucleic acids, RSC Publishing,
Cambridge, 131-149.
Shukla, B. K., Yadava, U., Roychoudhury, M. (2015). Theoretical explorations on the molecular
structure and IR frequencies of 3-phenyl-1-tosyl-1H-pyrazolo [3, 4-d] pyrimidin-4-amine in view
of
experimental
results.
Journal
of
Molecular
Liquids,
212,
325-330.
http://dx.doi.org/10.1016/j.molliq.2015.09.021
Sissi, C., Moro, S., Zagotto, G., Ellis, M., Krapcho, A. P., Menta, E. & Palumbo, M. (1999).
Binding of bis-substituted 2-aza-antracenedione regioisomers to DNA: effects of the relative
positioning of the side chains. Anticancer Drug Design, 14, 265-274.
Skehn P., Storeng, R., Scudiero, A., Monks, J., McMohan, D., Vistica, D., Jonathan, T. W.,
Bokesch, H., Kenney, S., Boyd, M. R. (1990). New colorimetric cytotoxicity assay for anticancer
drug screening. J. Natl. Cancer Inst., 82, 1107. Doi: 10.1093/jnci/82.13.1107.
Supuran, C. T., Briganti, F., Tilli, S., Chegwidden, W. R. & Scozzafava, A. (2001). Carbonic
anhydrase inhibitors: Sulfonamides as antitumor agents. Bioorganic and Medicinal Chemistry, 9,
7
03-714. Doi: 10.1016/s0968-0896(00)00288-1.
Tan, C., Noronha, R de, Devi, N. S., Jabbar, A. A., YLiu, S. K, , Mooring, S. R., Nicolaou, K.
C., Wang, B., Mair, E. G. V. (2011). Sulfonamides as a new scaffold for hypoxia inducible factor
pathway inhibitors. 21 (18), 5528-5532. https://doi.org/10.1016/j.bmcl.2011.06.099.
Tarasiuk, J., Gobis, K. T., Stefanska, B., Dzieduszycka, M., Priebe, W., Martellli, S. &
Borowski, E. (1998). The role of structural factors of antraquinone compounds and their
quinone- modified analogues in NADH dehydrogenase- catalysed oxygen radical formation.
Anticancer Drug Design, 13, 923-939.
2
4

Tripathi, S., Pradeep, P, Tarikere & Barthwal Ritu. (2016). Molecular Recognition of Parallel
DNA Quadruplex d (TTAGGGT)4 by Mitoxantrone: Binding with 1:2 Stochiometry Leading to
Thermal Stabilization and Telomerase Inhibition. CHEM BIO CHEM communications, 17, 554-
5
60. doi: 10.1002/cbic.201500588 .
Vanicha Vichai & Kanyawim Kirtikara (2006). Sulforhodamine B colorimetric assay for
cytotoxicity screening. Nature Protocols 1, 1112 – 1116. https://doi.org/10.1038/nprot.2006.179.
Vatsal, M., Devi, V. & Awasthi, P. (2018). Structural and theoretical study of of 1-[1-oxo-3-
phenyl-(2-benzosulfonamide)-propyl amido] – anthracene-9, 10-Dione to be i-motif inhibitor.
Journal of Molecular structure, 1157, 230-238. https://doi.org/10.1016/j.molstruc.2017.12.054
Waller, Z. A. E., Sewitz, S. A., Hsu, S-T. D., Balasubramanian, S. (2009). A small molecule that
disrupts G-quadruplex DNA structure and enhances gene expression. Journal of American
chemical society, 131, 12628-12633. doi: 10.1021/ja901892u.
Wei, B-L., Wu, S-H., Chung, M-I., Won, S-J. & Lin, C-N. (2000). Synthesis and cytotoxic effect
of 1, 3-dihydroxy-9, 10-anthraquinones derivatives. European Journal of Medicinal Chemistry,
3
5, 1089-1098. https://doi.org/10.1016/S0223-5234(00)01190-9.
Wright. E.P., Day. H. A. Ibrahim. M.A., Kumar. J., Boswell. L.J.E, Huguin. C, Stevenson.
C.E.M, Pors. K and Waller. Z.A.E., (2016). Mitoxantrone and Analogues Bind and Stabilize i-
Motif Forming DNA Sequences. Nature, doi: 10.1038/srep39456.
Yang, A. S. & Issa, J-P. J. (2003). A Hypomethylating agent. Cancer Biology and Therapy,
2
64-265.
Zagotto, G., Moro, S., Uriate, E., Ferrazzi, E., Palu, G. and Palumbo, M. (1997). Amido analogs
of mitoxantrone: physico-chemical properties, molecular modelling, cellular effects and
antineoplastic potential. Anti-Cancer Drug Design, 12, 99-112.
Zagotto, G., Sissi, C., Gatto, B., Palumbo, M. (2004). Aminoacyl- analogues of mitoxantrone as
novel DNA-damaging cytotoxic agents. ARKIVOC, 204-218. Doi: http://sci-
hub.tw/http://dx.doi.org/10.3998/ark.5550190.0005.519
2
5

Zagotto, G., Supino, R., Favini, E., Moro, S. & Palumbo, M. (2000). New 1, 4- anthracene -9,
1
8
0-dione derivatives as potential anticancer agents. IL FARMACO, 55, 1-5. doi:10.1016/s0014-
27x(99)00091-9.
Zhang, J., Redman, N., Litke, A. P., Zeng, J., Zhan, J., Chan, K. Y., Chang, C-W. T. (2011).
Synthesis and antibacterial activity study of a novel class of cationic anthraquinone analogs.
Biorganic and Medicinal Chemistry, 19, 498-503. doi:10.1016/j.bmc.2010.11.001.
Zhong, W., Yu, J. S., Huang, W. L., Ni, K. Y. & Liang, Y. Q. (2001). Spectroscopic studies of
interaction of chlorobenzylidine with DNA. Biopolymers, 62, 315-323. doi:10.1002/bip.10001.
Zuravka, I., Sosic, A., Gatto, B. & Goottlich, R. (2015). Synthesis and evaluation of a bis-3-
chloropiperidine derivative incorporating an anthraquinone chromophore. Biorganic and
Medicinal Chemistry Letters, 25, 4606-4609. http://dx.doi.org/10.1016/j.bmcl.2015.08.042.
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6

H
N
R1
O
C
OH
OH
O
O
HN
HN
OH
OH
H O
N
n
A
O
HN
C
S
R2
O
N
H
O
R1= CH3
R1= H
(S5, S6, S7 & S8)
S1, S2, S3 & S4)
(S1, S3, S4,S5, S7 & S8)
(S2 & S6)
(
CH2C6H5
&
CH(CH3)2
R =
2
n= 2
Structure 1. Basic structure of synthesized compounds. Structure 2. Structure of Mitoxantrone.
2
7

*
Table 1a. Cytotoxic studies on Hep2C cell lines (Hela Derivative)
Hep-2C (Hela cells) Human cervix carcinoma
IC value (Concentration of an inhibitor where
50
the response (or binding) is reduced by half)
MTX
2.5μg/ml
S-1
9μg/ml
NE
S-2
S-3
9μg/ml
1.008μg/ml
NE
S-4
S-5
S-6
NE
S-7
S-8
N
NE
NE= Not effective.
*
Cervix carcinoma at 24 h, as determine by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide assay.
2
8

Table 1b. Measurement of Potential Cytotoxicity of (S1-S8) series by Sulforhodamine B (SRB)
Assay on MCF-7 (Human breast cancer cell line) and PC-3 (Human prostate cancer cell line).
Compounds
MCF-7 (GI = Concentration of drug PC-3 (GI = Concentration of
50 50
causing 50% inhibition of cell drug causing 50% inhibition
growth) (μg/ml) of cell growth)
S1
S2
S3
S4
S5
S6
S7
S8
MTX
<10
41.2
58.0
NE
>80
NE
-
NE
NE
NE
NE
NE
NE
NE
NE
<10
NE
<10
2
9