Benzo[α]phenoxazines and benzo[α]phenothiazine from vitamin K3: Synthesis, molecular structures, DFT studies and cytotoxic activity

Article abstract of DOI:10.1039/c5ra08496b

Synthesis and characterization of fluorescent benzo[α]phenoxazines viz., M-1B (10-chloro-6-methyl-7a,11a-dihydro-5H-benzo[α]phenoxazin-5-one), M-2B 6,10-dimethyl-7a,11a-dihydro-5H-benzo[α]phenoxazin-5-one), M-3B (6-methyl-7a,11a-dihydro-5H-benzo[α]phenoxazin-5-one) and benzo[α]phenthiazine, M-4B (6-methyl-5H-benzo[α]phenothiazin-5-one) were carried out. ¹H and ¹³C chemical shifts were assigned from the 2DgHSQCAD NMR experiments. Compound M-1B crystallizes in the orthorhombic space group P2₁2₁2₁, while M-2B and M-4B crystallize in the monoclinic space group P2₁/c. The crystal network of M-1B showed slipped π-π stacking and Cl...Cl interactions, while M-2B facilitated ladder like π-π stacked polymeric chains. The C...S contacts were observed in the crystal environment of M-4B. All these structures possess C-H...O interactions. Electronic structure and charge distribution in terms of molecular electrostatic potential and frontier orbital analyses based on the MO6-2X based density functional theory further showed that monomer and dimer structures are in keeping with the single crystal X-ray data and provide insights for the growth of the crystal network. Antiproliferative activity of M-1B-M-4B was determined from the MTT assay against a human breast adenocarcinoma cell line (MCF-7), human carcinoma cell line (HeLa) and normal skin cell line. All these compounds showed significant cytotoxic activity against MCF-7 and HeLa by inducing apoptosis and thus can be viewed as potential candidates for antitumor therapy. Compounds M-2B and M-4B were further found to be topoisomerase II inhibitors.

Full text of DOI:10.1039/c5ra08496b

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PAPER
Benzo[a]phenoxazines and benzo[a]phenothiazine
from vitamin K3: synthesis, molecular structures,
DFT studies and cytotoxic activity†
Cite this: RSC Adv., 2015, 5, 57917
Dattatray Chadar,a Soniya S. Rao,a Ayesha Khan,a Shridhar P. Gejji,a
a
b
a
*
Kiesar Sideeq Bhat, Thomas Weyhermuller and Sunita Salunke-Gawali
¨
Synthesis and characterization of fluorescent benzo[a]phenoxazines viz., M-1B (10-chloro-6-methyl-
7a,11a-dihydro-5H-benzo[a]phenoxazin-5-one), M-2B 6,10-dimethyl-7a,11a-dihydro-5H-benzo[a]-
phenoxazin-5-one), M-3B (6-methyl-7a,11a-dihydro-5H-benzo[a]phenoxazin-5-one) and benzo[a]-
phenthiazine, M-4B (6-methyl-5H-benzo[a]phenothiazin-5-one) were carried out. 1H and 13C chemical
shifts were assigned from the 2DgHSQCAD NMR experiments. Compound M-1B crystallizes in the
orthorhombic space group P212121, while M-2B and M-4B crystallize in the monoclinic space group P21/c.
The crystal network of M-1B showed slipped p–p stacking and Cl/Cl interactions, while M-2B
facilitated ladder like p–p stacked polymeric chains. The C/S contacts were observed in the crystal
environment of M-4B. All these structures possess C–H/O interactions. Electronic structure and charge
distribution in terms of molecular electrostatic potential and frontier orbital analyses based on the MO6-
2X based density functional theory further showed that monomer and dimer structures are in keeping
with the single crystal X-ray data and provide insights for the growth of the crystal network.
Antiproliferative activity of M-1B–M-4B was determined from the MTT assay against a human breast
adenocarcinoma cell line (MCF-7), human carcinoma cell line (HeLa) and normal skin cell line. All these
compounds showed significant cytotoxic activity against MCF-7 and HeLa by inducing apoptosis and
thus can be viewed as potential candidates for antitumor therapy. Compounds M-2B and M-4B were
further found to be topoisomerase II inhibitors.
Received 8th May 2015
Accepted 26th June 2015
DOI: 10.1039/c5ra08496b
www.rsc.org/advances
inammatory,12 anti-tuberculosis13 and multidrug resistance
activities14 prevent human amyloid disorders15 and protect
neuronal cells from death by oxidative stress.16,17 Furthermore, it
Introduction
Benzo[a]phenoxazines are uorescent molecules with a wide
range of pharmacological applications.1 Phenoxazine derivatives
viz., benzo[a]phenoxzinium salts, show absorption maxima
higher than 600 nm and the strong red uorescence thereof
facilitates their use as long wavelength uorophores.2,3 Preferred
for biological applications4–9 these encompass labelling of small
synthetic molecules as well as large biomolecules including
proteins, antibodies or nucleotides in DNA studies. Phenox-
azines possessing antiproliferative,10 antitumor,11 anti-
was shown that the use of phenothiazine derivatives belonging to
neuroleptic drugs,18 reduce tumor-antigen expression19 and
cytotoxicity against myelogeneous leukemia cell lines.20 The
underlying mechanism for antiproliferative activity of phenox-
azine derivatives stem from DNA intercalative binding facilitated
via noncovalent interactions or p–p stacking21,22 which render
stability to these planar polycycles. Alternatively phenoxazine
systems engender free radical intermediates leading to oxidative
stress.23 Some of these phenoxazine derivatives displayed
apoptotic activity against different cell lines, both in caspase
dependent and independent manner.24,25 Fluorescent heterocycle
of phenoxazines are valuable for staining nucleic acids in solu-
tions, electrophoretic gels, matrices, blotting experiments and
assays employing intact, live cells.26 Furthermore, modications
of phenoxazine moiety yield compounds those directly bind to
proteins through hydrophobic interactions which subsequently
are utilized in monitoring protein conformation alterations or for
therapeutic purposes.
aDepartment of Chemistry, Savitribai Phule Pune University, Pune 411007, India.
E-mail: sunitas@chem.unipune.ac.in; Fax: +91 2025693981; Tel: +91 2025601397
ext. 531
b
¨
¨
MPI fur Chemische Energiekonversion, Stistr. 34-36, 45470 Mulheim an der Ruhr,
Germany
† Electronic supplementary information (ESI) available: Characterization of M-1B
to M-4B by FT-IR (Fig. S1–S5 and S22), GC-MS (Fig. S6–S9), DSC (Fig. S10–S13) and
1H,13
C
and 2DgHSQCAD NMR (Fig. S14–S21). Crystallography gures
Fig. S23–S27, cytotoxicity assay Fig. S28–S30. Table S1 (FT-IR data) and
crystallographic Table S2–S12. CCDC 1039204–1039206. For ESI and
crystallographic data in CIF or other electronic format see DOI:
10.1039/c5ra08496b
Topoisomerase are the enzymes those control the DNA
topology, a requisite for division or proliferation of cells. These
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enzymes are important target for cancer chemotherapy and
assist numerous DNA transactions through a complex series of
DNA strand breakage and rejoining reactions. Topoisomerases
resolves the entanglement problems generated during DNA
replication, repair and transcription processes. Depending on
the DNA breaks there are two types of topoisomerases; Topo I,
which break single strand DNA, while Topo II generate double
strand DNA breaks.27,28 Topo II enzymes plays key role in DNA
metabolism and chromosome structure29 and it is the primary
cytotoxic target for the potent anticancer drugs, which include
anthracyclines, acridines and epipodo phyllotoxins.30 Depend-
ing on the mode of action the topoisomerase II inhibitors are
classied as (i) topoisomerase II poisons, which involves in
breaking–rejoining reaction of the enzyme that involves trap-
ping of covalent reaction intermediates, usually referred as the
cleaved complexes, or (ii) catalytic inhibitors inhibiting the
activity of topoisomerase II prior to the formation of the cleaved
complex.31 Heteroatom planar polycyclic compounds are used
in cancer chemotherapy as topoisomerase inhibitor/poison.32–39
DNA intercalation has been believed to be a prefer mode of
action for such compounds.
Scheme 1 General reaction scheme for synthesis of M-1B to M-4B.
In present endeavour, benzo[a]phenoxazines were synthe-
sized by reacting vitamin K3 (menadione, 2-methyl-1,4-
naphthoquinone) with 2-aminophenols (M-1B to M-3B) and
Fig. 1 UV-visible and fluorescence spectra of M-1B to M-4B in DMSO.
Fig. 2 1H and 13C NMR chemical shifts of M-1B to M-4B.
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2-aminothiophenol (M-4B) derivatives. Molecular structures of the density functional based calculations. The antiproliferative
M-1B, M-3B and M-4B have been studied by single crystal X-ray activity against human carcinoma HeLa cell line, drug uptake
diffraction studies, which revealed their p–p stacking abilities. and topoisomerase II inhibitor activity of these compounds
The structural investigations were combined with those from have further been investigated.
Fig. 3 ORTEP plots of M-1B, M-2B and M-4B.
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nm (of parent MQ) showed shi ꢃ25 to 30 nm (ꢃ355 nm). Two
another bands observed in UV region ꢃ260 nm to 290 nm were
assigned to p–p* transitions. The n–p* transition was observed
ꢃ450 nm (M-1B to M-3B) which shis to ꢃ480 nm in M-4B. A
uorescence emission band was observed between 450–700 nm.
M-2B showed maximum uorescent intensity than other
compounds despite using the same concentrations in recording
of the spectra.
Results and discussion
Synthesis
Benzo[a]phenoxazine derivatives were synthesized by conden-
sation of vitamin K3 (1) with 4-R-2-aminophenols (R ¼ Cl ¼ M-
1B, CH3 ¼ M-2B, H ¼ M-3B) reuxed in methanol, while M-4B
ꢀ
was obtained at room temperature (26 C) by reacting vitamin
K3 and 2-aminothiophenol (Scheme 1). 6-Methyl-5H-benzo[a]
phenoxazine-5-one and 6-methyl-5H-benzo[a]phenothiazin-5-
one (M-3B and M-4B respectively in present investigation)
were previously synthesized by reactions of alkyl radicals on
corresponding 5H-benzo[a]phenoxazine-5-one40 and 5H-benzo
[a]phenothiazin-5-one41 respectively.
1H and 13C chemical shi were assigned to all compounds
and are presented in Fig. 2.
Single crystal X-ray diffraction studies of M-1B, M-2B and M-4B
M-1B crystallizes in orthorhombic space group P212121, while
M-2B and M-4B crystallize in monoclinic space group P21/c.
ORTEP plots are presented in Fig. 3, and the crystallographic
1
FT-IR, UV-visible, uorescence, H and 13C NMR studies
The nC]O frequency in all compounds was assigned at 1629 ꢁ 1 data is summarized in Table 1. All the crystallized compounds
cmꢂ1, while nC]N stretching frequency42–45 was assigned at 1585 showed planar polycycles. The carbonyl distance C(2)–O(1) was
ꢁ 10 cmꢂ1 in the FT-IR spectra except in M-1B it was observed at observed as ꢃ1.23 A in M-1B and M-2B,10,42–45 while it was longer
˚
1574 cmꢂ1 (Fig. S22 and Table S1 in ESI†). The para-naph- in M-4B (1.246 A). The bond distance of C(9)–N(10) was found to
˚
thoquinone (p-NQ) vibration10 of quinonoid ring (ꢃ1260 cmꢂ1
)
be ꢃ1.30 A similar to the imino functional moiety (C]N) of the
˚
was absent in all compounds however a peak due to nC–O was naphthoquinoneoximes.46 The bond distances in all these
observed ꢃ1240 cmꢂ1 in M-1B, M-3B and M-4B and ꢃ1226 cmꢂ1 crystalline systems usually by and large, match with those for
in M-2B. vC–Cl stretching frequency was assigned to 736 cmꢂ1 in quinonoid bond distances in ring B (Scheme 1).
M-1B.
Each M-1B molecule was in vicinity to eight neighbouring
There were mainly two bands observed in UV-Visible spectra molecules (Fig. S23†) via C–H/O, C–H/Cl, Cl/Cl (Table 2)
of parent vitamin K3 in DMSO at 266 nm and 330 nm and four and p–p stacking interactions. Slipped p–p stacking were
bands to all compounds (Fig. 1). In M-1B to M-3B the band ꢃ330 observed in neighbouring molecules, carbon C(8)/C(11)
Table 1 Crystallographic data of M-1B, M-2B and M-4B
Identication code
M-1B
M-2B
M-4B
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
C
17H10ClNO2
C18H13NO2
275.29
C17H11NOS
277.33
295.71
100(2) K
0.71073 A
Orthorhombic
P212121
100(2) K
1.54178 A
100(2) K
0.71073 A
˚
˚
˚
Monoclinic
P21/c
Monoclinic
P21/c
˚
˚
˚
˚
˚
˚
Unit cell dimensions
a ¼ 4.7569(9) A, b ¼ 9.660(2) A,
a ¼ 7.1715(6) A, b ¼ 21.816(2) A,
a ¼ 15.172(3) A, b ¼ 3.8385(8) A,
˚
˚
˚
˚ ˚
c ¼ 28.117(5) A
b ¼ 112.283(4) A, c ¼ 8.9610(9) A
b ¼ 107.902(9) A, c ¼ 22.105(4) A
3
˚
3
˚
3
˚
Volume
1292.0(4) A
1297.3(2) A
1225.0(4) A
Z
4
4
4
Density (calculated)
Absorption coefficient
F(000)
Crystal size
Theta range for data collection
Index ranges
1.520 mg mꢂ3
1.410 mg mꢂ3
1.504 mg mꢂ3
0.299 mmꢂ1
608
0.742 mmꢂ1
576
0.257 mmꢂ1
576
0.264 ꢄ 0.037 ꢄ 0.012 mm3
3.028 to 30.210ꢀ
ꢂ6 # h # 6, ꢂ13 # k # 13,
ꢂ39 # l # 39
35 695
0.32 ꢄ 0.12 ꢄ 0.08 mm3
4.053 to 67.383ꢀ
ꢂ7 # h # 8, ꢂ26 # k # 25,
ꢂ10 # l # 10
0.40 ꢄ 0.02 ꢄ 0.02 mm3
2.724 to 28.999ꢀ
ꢂ20 # h # 20, ꢂ5 # k # 5,
ꢂ30 # l # 30
15 404
Reections collected
30 049
Independent reections
Completeness to theta ¼ 25.242ꢀ
Absorption correction
3840 [R(int) ¼ 0.0956]
2298 [R(int) ¼ 0.0581]
3243 [R(int) ¼ 0.0734]
99.6%
98.0%
99.6%
Gaussian
Gaussian
Gaussian
Max. and min. transmission
Renement method
0.99706 and 0.95811
Full-matrix least-squares on F2
3840/0/191
0.94601 and 0.84701
Full-matrix least-squares on F2
2298/0/193
0.99571 and 0.94895
Full-matrix least-squares on F2
3243/0/182
Data/restraints/parameters
Goodness-of-t on F2
1.040
1.059
1.116
Final R indices [I > 2sigma(I)]
R indices (all data)
Extinction coefficient
R1 ¼ 0.0397, wR2 ¼ 0.0813
R1 ¼ 0.0545, wR2 ¼ 0.0870
n/a
0.314 and ꢂ0.378 e A
R1 ¼ 0.0543, wR2 ¼ 0.1482
R1 ¼ 0.0660, wR2 ¼ 0.1611
0.0032(8)
0.295 and ꢂ0.297 e A
R1 ¼ 0.0657, wR2 ¼ 0.1519
R1 ¼ 0.0898, wR2 ¼ 0.1659
n/a
0.720 and ꢂ0.611 e A
ꢂ3
ꢂ3
ꢂ3
˚
˚
˚
Largest diff. peak and hole
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˚
˚
(3.365(3)A, 1 + x, y, z) and C(15)/C(18) (3.367(3)A, 1 + x, y, z) slipped p–p stacking interactions (Fig. S24a†). A zig-zag chain of
were involved in the same. When viewed down a axis polymeric Cl/Cl contacts were further observed; the :Cl/Cl/Cl being
chains of M-1B molecules extend via C–H/O and Cl/Cl nearly 90ꢀ as shown in Fig. S24b.†
˚
(3.358(3)A, 1/2 ꢂ x, 1/2 ꢂ y, ꢂz) interactions (Fig. 4). A fasci-
M-2B molecule was in vicinity to four neighbouring mole-
nating architecture with herring borne like structure for M-1B cules via C–H/O and p–p stacking interactions (Fig. S25†).
molecules can be noticed down c-axis (Fig. 5) consequent to M-2B molecules with head to tail orientation lead to a dimer via
Table 2 Hydrogen bond geometries for M-1B, M-2B and M-4B
˚
˚
˚
Com.
M-1B
M-2B
M-4B
Sr. no.
D–H/A
D–H (A)
H/A (A)
D/A (A)
:D–H/A (ꢀ)
1
2
3
4
5
6
C(5)–H(5)/Cl(20)a
C(15)–H(15)/O(1)b
C(20)–H(20C)/O(1)c
C(4)–H(4)/O(1)d
0.950(2)
0.951(1)
0.951(2)
0.951(2)
0.951(3)
0.980(3)
2.797 (1)
2.399(2)
2.686(2)
2.695(1)
2.623(2)
2.830(3)
3.743(3)
3.285(3)
3.481(3)
3.356(3)
3.382(4)
3.685(4)
177.0(1)
154.8(1)
138.5(1)
127.2(1)
137.1(2)
146.1(2)
C(13)–H(13)/O(1)e
C(21)–H(21C)/C(19)f
a
b
ꢂ2 + x, 1 + y, z. 3 ꢂ x, 1/2 + y, 1/2 ꢂ z. c 1 ꢂ x, 1 ꢂ y, 1 ꢂ z. d x, 1/2 ꢂ y, ꢂ1/2 + z. e x, 2.5 ꢂ y, 1/2 + z. f x, 1 + y, z.
Fig. 4 Polymeric chains of M-1B via C–H/O and Cl/Cl interactions.
Fig. 5 Molecular packing in M-1B down the c-axis.
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C–H/O interaction of C(19)–CH3 and carbonyl oxygen (C(2)–
Crystal structures showed the p–p stacking in addition to
O(1)). The dimers also reveal p–p stacking interactions of hydrogen bonding interactions in M-1B, M-2B and M-4B.
˚
C(2)/C(12) (3.216(2)A, 1 ꢂ x, 1 ꢂ y, 1 ꢂ z), C(4)/C(14)
˚
˚
(3.392(3)A, 1 ꢂ x, 1 ꢂ y, 1 ꢂ z), C(8)/C(14) (3.386(3)A, ꢂx, 1 ꢂ y,
DFT investigations
˚
1 ꢂ z) and C(8)/C(16) (3.331(2)A, 1 ꢂ x, 1 ꢂ y, 1 ꢂ z) (Fig. 6).
Optimized structures of M-1B, M-2B, M-3B and M-4B from the
M06-2x/6-31+G(d,p) density functional theory are shown in
Fig. 9. Selected bond distances (in A) are given along with the
Moreover, p–p stacking interactions from C(8)/C(14) extend-
ing as ladder structure of dimers, can further be noticed when
viewed down c-axis of dimers. Neighbouring ladders of dimers
were joined via C–H/O interactions. The “roller coaster” chain
of dimers can be viewed down the c-axis (Fig. 7).
˚
net atomic charges obtained from Hirscheld partitioning
scheme in parenthesis. The structural parameters thus
obtained agree fairly well with those from X-ray crystal data. To
gain molecular insights for charge distribution governing dimer
formation highest occupied molecular orbital HOMO were
derived. An isosurface of ꢂ12.5 kcal molꢂ1 are portrayed in
Fig. 10 in the M-1B to M-4B systems. It is evident that the
electron-rich regions are largely localized on aromatic moieties.
Remarkably enough, Fig. 10 points to diminutive charge local-
ization around the phenolic ring, which partly has been
attributed to ꢂI effect from chlorine center. It may as well be
inferred that p–p stacking contribute signicantly along with
hydrogen bonding interactions in the extended crystal network
of M-1B, M-2B and M-4B.
M-4B possess four neighbours surrounding the central
molecules connected via C–H/O interaction and C/S inter-
˚
action (Fig. S26†). The slipped C(18)/S(17) (3.458(3)A, x,1 + y, z)
interactions are supported by C–H/p interactions (C(19)/
˚
˚
H(21C) ¼ 2.830 A, C(19)/C(21) ¼ 3.685(4)A, :C(21)–H(21C)/
C(19) ¼ 146.1ꢀ) (Fig. S26†). M-4B possess buttery like molecular
packing when viewed down the c-axis (Fig. S27†), where the
plane of molecules makes an angle of nearly 45ꢀ to one another.
Slipped C/S stacked polymeric chains further can be viewed
down a-axis wherein the C–H/O interactions are present
(Fig. 8).
Fig. 6 p–p stacked ladders in M-2B.
Fig. 7 “Roller coaster” dimer chain in M-2B down the c-axis.
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Fig. 8 C/S stacked polymeric chains of M-4B facilitated via C–H/O interactions.
Fig. 9 M06-2x/6-31+G(d,p) optimized structures of M-1B, M-2B, M-3B and M-4B. Bond distances and net atomic charges (in parentheses) are
given.
To understand interplay between stacking interactions and diffraction experiments. SCRF-PCM calculations further
hydrogen bonding, we further delve into dimer formation demonstrated that the solvent (DMSO) has no profound effect
accompanying M-1B to M-4B which has been shown in Fig. 11. on the electronic structure.
The p–p stacking separations in the dimers range from 3.1–4.7
˚
A. Besides slipped p–p stacking interactions contributing to M-
1B can be noticed from Fig. 11. The present theoretical calcu-
lations thus support inferences from the single crystal X-ray
Antiproliferative activity
The in vitro cytotoxicity of compounds M-1B to M-4B against
MCF-7 (breast) and HeLa (cervical) cancer cell lines and normal
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skin cell cancer cell lines has been tested by MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. All
the compounds did not show any toxicity to normal skin cell;
however, exhibit signicant cytotoxicity in concentration depen-
dent manner against MCF-7 and HeLa cells (Fig. S28–S30 in
ESI†). The percent cell viability of MCF-7, HeLa and normal skin
cell lines in the presence of complex M-1B to M-4B was measured
in the concentration range of 5 mM to 25 mM, the compounds
tested were found to be active at lower concentrations.
The IC50 values against MCF-7 and HeLa cell lines are pre-
sented in Table 3, which is comparable to cis-platin47 having
IC50 of 16.7 ꢁ 2.5 mM aer 48 hours. Interestingly the M-2B is
highly toxic against both MCF-7 and HeLa cell lines than the
other compounds which possibly can be attributed to its
stronger p–p stacking ability. Compounds M-1B to M-4B
exhibits signicant cytotoxicity against both cell lines viz. HeLa
and MCF-7 by inducing apoptosis and can be envisaged as
potential candidates for antitumor therapy. Cis-platin exhibits
cytotoxic effect by covalent binding to DNA forming cis-platin-
DNA adduct, which obstruct with DNA transcription,
Table 3 Antiproliferative data (IC50) of M-1B to M-4B
Compound
MCF-7 (mM)
HeLa (mM)
M-1B
M-2B
M-3B
M-4B
21.52 ꢁ 0.23a
3.86 ꢁ 0.33
8.32 ꢁ 0.25
3.52 ꢁ 0.21
25.35 ꢁ 0.13
4.35 ꢁ 0.08
12.62 ꢁ 0.12
13.75 ꢁ 0.13
Fig. 10 Frontier orbitals (isosurfaces of ꢂ12.5 kcal molꢂ1 in HOMO and
LUMO).
a
Each value reported here is
a
mean value of
3
independent
experiments obtained aer 48 hours.
Fig. 11 M06-2x/6-31+G(d,p) optimized structures of M-1B, M-2B, M-3B and M-4B dimers.
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Fig. 12 Fluorescence microscopy images of HeLa cells incubated (48 h) with 15 mM of compounds M-1B to M-4B.
replication, and eventually leading cell death. It may thus be
conjectured that the anticancer activities of M-1B to M-4B arise
from partial intercalation or groove binding; the specic
chemical structure and the nature of these compounds, on the
other hand contribute signicantly to enhanced cytotoxicity.
Cellular uptake
HeLa cells were treated with 25 mM of the respective compounds
and aer 24 h the cells were xed and the images were captured
Fig. 13 Lane 1: kDNA with no topoisomerase II; lane 2, decatenated
kDNA control with no topoisomerase II in the assay buffer. Lane 3;
Topo II + M-1B; lane 4; Topo II + M-3B; lane 5; Topo II + M-2B; lane 6;
Topo II + M-4B. ORI, loading well origin; NOC, nicked, open circular
decatenated kDNA; CC, covalently closed circular decatenated kDNA.
The assay buffer contained 1.0 unit of topoisomerase II and 100 ng of
kDNA.
using a uorescence microscope. The cellular localization of
compounds is monitored by uorescence microscopy as these
compounds emit green light on excitation with visible light.
These compounds stain the nucleus of the cells (Fig. 12) and the
mode of cell death was found to be apoptosis.
Fig. 13 depicts the gel decatenation assay analyzed for the
compounds M-1B to M-4B. A wide network of many interlocked
circular DNA forms the kDNA. Topoisomerase II, decatenates
the kDNA which yields 2.5 kilobase relaxed decatenated kDNA
monomers. Since smaller in size the monomers tend to run
much faster on the gel.48 Topo-II act on the intact kDNA
substrate to produce covalently closed circular decatenated
kDNA (CC in Fig. 13). While its action on the nicked kDNA
substrate formed during the isolation of kDNA produces
nicked, open circular decatenated kDNA (NOC in Fig. 13).
Noteworthy enough, compounds M-2B and M-4B are able to
inhibit the Topo-II catalyzed decatenation of kDNA.
Conclusions
Benzo[a]phenoxazines (M-1B to M-3B) and benzo[a]phenthia-
zine (M-4B) have been synthesized and characterized in this
investigation. The precursor vitamin K3 was used in synthesis
for the very rst time with various derivatives of amino phenol
and thioaminophenol. All synthesized planar polycycles are
uorescent. Molecular structures of M-1B, M-2B and M-4B
revealed p–p stacking interactions as well as C–H/O, C–H/Cl
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and Cl/Cl non-covalent interactions. MO6-2X based density reactions were monitored by thin layer chromatography with
functional theory predicted electronic structures of M-1B to M- 2% methanol in toluene (methanol : toluene (2 : 8)) as a mobile
4B monomers as well as dimers agree well with the single crystal phase. Orange band was separated for all three compounds as a
X-ray experiments wherein the p–p interactions are evident. major product which was dried in air followed by vacuum. The
The in vitro cytotoxicity of all compounds evaluated against X-ray quality crystals for M-1B and M-2B were obtained aer
MCF-7 (breast), HeLa (cervical) and normal skin cancer cell recrystallization in toluene. In M-4B a dark orange coloured
lines. All the compounds exhibits signicant cytotoxicity precipitate was obtained within 5 minutes of mixing the reac-
against the MCF-7 and HeLa cell line by inducing apoptosis and tants at room temperature 26 ꢀC, which showed single spot on
further can be regarded as potential candidates for antitumor TLC. This precipitate further ltered and dried in vacuum. An X-
therapy. Moreover, and more important, compounds M-1B to ray quality crystal of M-4B was obtained aer recrystallization of
M-4B showed very low cytotoxicity vs. normal skin cell lines. the powder product in toluene.
Cellular uptake studies on HeLa cell line of all compounds stain
the nucleus of the cells and apoptosis causing the cell death.
DNA catenation assay further suggested M-2B and M-4B are
Characterization
potential topoisomerase II inhibitors.
10-Chloro-6-methyl-7a,11a-dihydro-5H-benzo[a]phenoxazin-
5-one; M-1B. Dark orange solid, yield: 0.492 g (29%), mp 222.58
ꢀC. Anal. data calc. for C17H10ClNO2: C, 69.05; H, 3.41; N, 4.14;
found: C, 69.19; H, 3.78; N, 4.56. FT-IR (KBr, nmax/cmꢂ1): 3063,
3030, 2953, 2914, 2852, 1630, 1574, 1523, 1462, 1413, 1381,
1348, 1306, 1259, 1240, 1151, 1095, 1082, 962, 922, 871, 825,
783, 736, 682, 642, 588, 538, 497, 437. 1H NMR (500 MHz, CDCl3,
d/ppm): 2.221 (s, 3H), 7.264 (d, 1H, J ¼ 8.00 Hz), 7.794 (d, 1H, J ¼
8.00 Hz), 7.239 (s, 1H), 7.756 (2, 2H, J ¼ 8.00 Hz), 8.239 (d, 1H,
7.25 Hz), 8.646 (d, 1H, J ¼ 8.00 Hz). 13C NMR (500 MHz, CDCl3,
d/ppm): C(1) ¼ 124.85, C(2) ¼ 132.11, C(3) ¼ 132.21, C(4) ¼
126.32, C(4A) ¼ 131.99, C(5) 183.71, C(6) ¼ 116.99, C(6A) 143.41,
C(7A) ¼ 133.50, C(8) ¼ 117.04, C(9) ¼ 130.84, C(10) ¼ 129.93,
C(11) ¼ 129.16, C(11A) ¼ 147.43, C(12A) ¼ 148.52, C(12B) ¼
130.80, C(13) ¼ 8.37. UV-Vis; (lmax/nm, DMSO): 450, 355, 297.
GC-MS (EI) m/z: 295 (M+ + H).
6,10-Dimethyl-7a,11a-dihydro-5H-benzo[a]phenoxazin-5-one;
M-2B. Dark orange solid, yield: 0.603 g (40%), mp 193.24 ꢀC.
Anal. data calc. for C18H13NO2: C, 78.53; H, 4.76; N, 5.09; found:
C, 78.53; H, 5.11, N, 5.44. FT-IR (KBr, nmax/cmꢂ1): 3066, 3030,
2916, 2739, 1630, 1585, 1532, 1458, 1373, 1338, 1309, 1280, 1267,
1134, 1093, 1039, 966, 871, 783, 700, 642, 588, 536, 464, 434. 1H
NMR (500 MHz, CDCl3, d/ppm); 2.230 (s, 3H), 7.205 (d, 1H, J ¼
7.50 Hz), 7.261 (d, 1H, J ¼ 8.25 Hz), 7.589 (s, 1H), 7.729 (m, 2H, J
¼ 8.25 Hz), 8.311 (d, 1H, J ¼ 8.00 Hz), 8.769 (d, 1H, J ¼ 8.00 Hz).
13C NMR (500 MHz, CDCl3, d/ppm): C(1) ¼ 124.70, C(2) ¼ 131.74,
C(3) ¼ 131.85, C(4) ¼ 126.30, C(4A) ¼ 132.59, C(5)183.81, C(6) ¼
116.14, C(6A) ¼ 142.86, C(7A) ¼ 135.00, C(8) ¼ 115.60, C(9) ¼
131.19, C(10) ¼ 132.59, C(11) ¼ 129.83, C(11A) ¼ 141.36, C(12A)
¼ 148.08, C(12B) ¼ 132.07, C(13) ¼ 8.36, C(130) ¼ 21.06. UV-Vis;
(lmax/nm, DMSO): 445, 355, 293. GC-MS (EI) m/z: 279 (M+ + H).
Experimental section
General materials and methods
The materials used viz. vitamin K3 (2-methyl-1,4-naphthoquinone),
2-aminophenol, 2-aminothiophenol were purchased from Sigma-
Aldrich, 2-amino-4-methylphenol and 2-amino-4-chlorophenol
were obtained from Across chemicals. The solvents used such as
toluene, methanol are of analytical grade were purchased from
Merck Chemicals. Solvents were distilled by standard methods49
and dried wherever necessary.
The FT-IR spectra of all the compounds were recorded
between 4000–400 cmꢂ1 as KBr pellets on SHIMADZU FT 8400
spectrometer (Fig. S1–S5 in ESI†). Mass of all compounds were
determined by GC-MS 2010-eV (Make SHIMADZU) (Fig. S6–
Fig. S9 in ESI†). Melting points of all compounds were deter-
mined using melting point apparatus (Make-METTLER) and
were corrected using DSC (Differential Scanning Calorimetry)
(Make-TA Q2000) (Fig. S10–S13 in ESI†). UV-Visible spectra of
compounds were recorded on SHIMADZU UV 1650 in DMSO
between 200 to 800 nm. The uorescence spectra of the
compounds were recorded on JASCO spectrouorometer FP-
1
8300. H,13CNMR and 2DgHSQCAD of compounds was recor-
ded in CDCl3 on Varian mercury 500 MHz NMR instrument,
TMS (tetramethylsilane) was used as the internal reference
(Fig. S14–S21 in ESI†). Elemental analysis was performed on
Elementar Vario EL III.
Synthesis
Synthesis of M-1B, M-2B, M-3B, M-4B. Vitamin K3, 1 g (5.8
6-Methyl-7a,11a-dihydro-5H-benzo[a]phenoxazin-5-one; M-3B.
ꢀ
mM) was dissolved in 25 mL of dry methanol, the solution was Dark orange solid, yield: 0.58 g (39%), mp 179.45 C. Anal. data
stirred for 20 min. The solids of 2-amino-4-chlorophenol (0.834 calc. for C17H11NO2: C, 78.15; H, 4.24; N, 5.36; found: C, 77.84; H,
g, 5.8 mM) for M-1B, 2-amino-4-methyl-phenol (0.850 g, 5.8 4.24, N, 4.93. FT-IR (KBr, nmax/cmꢂ1): 3061, 2995, 2914, 2850, 1936,
mM) for M-2B, 2-aminophenol (0.872 g, 5.8 mM) for M-3B, 2- 1836, 1628, 1591, 1523, 1452, 1379, 1352, 1313, 1230, 1184, 1149,
1
aminothiophenol (0.621 mL, 5.8 mM) for M-4B were dissolved 1089, 1028, 962, 893, 856, 763, 684, 642, 586, 538, 474, 436. H
in 15 mL of dry methanol independently; each solution was NMR (500 MHz, CDCl3, d/ppm): 2.239 (3H, s), 7.270 (d, 1H, J ¼
added drop wise to the solution of vitamin K3 with continuous 8.00 Hz), 7.795 (d, 1H, J ¼ 7.50 Hz), 7.324 (t, 1H, J ¼ 7.25 Hz), 7.457
magnetic stirring. The color of solution turns dark red to brown. (t, 1H, J ¼ 7.75 Hz), 7.773 (m, 2H, J ¼ 8.00 Hz), 8.307 (d, 1H, J ¼
The reaction mixtures of M-1B to M-3B were stirred at room 7.00 Hz), 8.935, (d, 1H, J ¼ 7.50 Hz). 13C NMR (500 MHz, CDCl3, d/
temperature (26 ꢀC) for 24 hours and are further reuxed for 60, ppm): C(1) ¼ 124.71, C(2) ¼ 131.79, C(3) ¼ 131.90, C(4) ¼ 126.21,
32 and 28 hours respectively for M-1B, M-2B and M-3B. The C(4A) ¼ 132.03, C(5) ¼ 183.82, C(6) ¼ 116.39, C(6A) ¼ 144.85,
57926 | RSC Adv., 2015, 5, 57917–57929
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C(7A) ¼ 132.84, C(8) ¼ 115.98, C(9) ¼ 125.11, C(10) ¼ 131.21, C(11)
¼ 129.84, C(11A) ¼ 147.47, C(12A) ¼ 147.86, C(12B) ¼ 131.10,
C(13) ¼ 8.24. UV-Vis; (lmax/nm, DMSO): 443, 356, 296. GC-MS (EI)
m/z: 261 (M+ + H).
Cytotoxicity studies
Maintenance of cancer cell line. MCF-7, HeLa and normal
skin cell lines were obtained from National Centre for Cell
Sciences Repository, Savitribai Phule Pune University, Pune,
India. The cells were maintained in DMEM media with 10% FBS
and 0.1% antibiotic solution at 37 ꢀC at 5% CO2 in the steri-cycle
CO2 incubator with HEPA Class 100 lters, Thermo Electron
Corporation.
Preparation of sample for cell line testing. The compounds
were dissolved in 1% DMSO to obtain a solution of 1 mM
concentration each. These samples were then lter sterilized
using a 0.22 mM lter using syringe lter.
Testing of compounds on cell line. The cells were trypsinized
using TPVG solution. 1 mL of 1 ꢄ 105 cells per mL of medium
and dilutions of concentration 5, 10, 15 and 20 mM was added in
96 well plates (Tarsons) and kept in the CO2 incubator for 24, 48,
72 and 96 hours. All experiments were carried out in laminar
ow hoods, Laminar Flow Ultraclean Air Unit, Microlt, India.
The cells were visualized using an Inverted Microscope,
Olympus.
6-Methyl-5H-benzo[a]phenothiazin-5-one;
M-4B.
Dark
orange crystal, yield: 1.30 g (80%), mp 178.06 ꢀC. Anal. data calc.
for C17H11NOS: C, 73.62; H, 4.00; N, 5.05; S, 11.16; found: C,
73.24; H, 4.22, N, 5.44. FT-IR (KBr, nmax/cmꢂ1): 3055, 2982, 2899,
1628, 1593, 1541, 1439, 1310, 1244, 1184, 1095, 1031, 966, 891,
756, 688, 617, 569, 518, 449. 1H NMR (500 MHz, CDCl3, d/ppm):
2.210 (s, 3H), 7.271 (d, 1H, J ¼ 7.75 Hz), 7.405 (t, 1H, J ¼ 7.50
Hz), 7.465 (t, 1H, 7.75 Hz), 7.914 (d, 1H, J ¼ 8.00 Hz), 7.727 (m,
2H, J ¼ 7.50 Hz), 8.316 (d, 1H, 7.50 Hz), 8.853 (d, 1H, J ¼ 7.50
Hz). 13C NMR (500 MHz, CDCl3, d/ppm): C(1) ¼ 125.23, C(2) ¼
131.37, C(3) ¼ 131.61, C(4) ¼ 126.27, C(4A) ¼ 133.88, C(5) ¼
179.62, C(6) ¼ 124.00, C(6A) ¼ 134.63, C(7A) ¼ 133.88, C(8) ¼
125.73, C(9) ¼ 127.78, C(10) ¼ 129.70, C(11) ¼ 133.21, C(11A) ¼
138.54, C(12A) ¼ 144.99, C(12B) ¼ 132.30, C(13) ¼ 13.27. UV-Vis;
(lmax/nm, DMSO): 258, 314, 380, 483. GC-MS (EI) m/z: 277
(M+ + H).
MTT assay. Solution of 5 mg mLꢂ1 MTT was dissolved in PBS
and lter sterilized using syringe lter. Aer incubation for the
stipulated time 20 mL of MTT solution was added to 200 mL of
cell content solution. The plate was incubated for 2 hours in the
CO2 incubator. Aer incubation the media was removed. 200 mL
of DMSO was added to each well to dissolve the crystals. The
plate was kept into the 37 ꢀC incubator for 5 min. Reading was
taken on Plate reader, Thermo Electron Corporation and
absorbance was measured at 540 and 620 nm.
Cellular uptake. For preparation for uorescent images
(HeLa cell line preinoculated with compounds, in 24 well plate),
100 mL of cell content was pipetted out and centrifuged at 5000
rpm for 10 minutes. Cells were briey washed with PBS twice.
The pellet was resuspended in 50 mL of PBS. Ten microliters of
the suspended cells was placed on a clean glass slide and a cover
slip was imposed in such a way that no air bubbles were
obtained. Images were taken in a Carl Zeiss Axio Scope A1
uorescence microscope with lter set no. 9 and excitation at
450–490 nm.
X-ray crystallography
An orange single crystal of M-1B, M-2B and M-4B were coated
with peruoropolyether, picked up with nylon loops and
mounted in the nitrogen cold stream of the Bruker APEX-II
diffractometer. Graphite monochromated Mo-Ka radiation (l
˚
¼ 0.71073 A) from a Mo-target rotating-anode X-ray source was
used. Final cell constants were obtained from least squares ts
of several thousand strong reections. Intensity data were cor-
rected for absorption using intensities of redundant reections
with the program SADABS.50 The structure was solved readily by
direct methods and subsequent difference Fourier techniques.
The Siemens ShelXTL51 soware package was used for solution
and artwork of the structures, ShelXL97 (ref. 52) was used for
the renement. All non-hydrogen atoms were anisotropically
rened and hydrogen atoms were placed at calculated positions
and rened as riding atoms with isotropic displacement
parameters. The crystallographic data is presented in Table 1.
DNA decatenation assay. Reagents for the assay were
purchased from TopoGEN (catalogue number 1001-1, Topo-
GEN, Inc., Port Orange, FL). Topoisomerase II was extracted
according to the manufacturer's instructions from nuclei of
HeLa cells (NCCS repository, India) and contained both iso-
forms as assessed by Western blotting. Calculation of specic
topoisomerase II decatenation activity was based on complete
decatenation of a given amount of catenated input DNA (100 ng
kDNA) by a dened amount of nuclear extract containing the
alpha and beta isoforms or of a dened amount of puried
enzyme in a particular amount of time. The topoisomerase II
activity from nuclei decatenated 0.07 ng catenated kDNA minꢂ1
ngꢂ1 extract. Puried topoisomerase II alpha (see above) deca-
tenated 0.13 ng catenated kDNA minꢂ1 ngꢂ1. Vehicle (DMSO) or
drug substance at concentrations to yield the desired end
concentrations were added and 20 mL of the samples were
preincubated for 5 minutes at 37 ꢀC and 400 rpm in an
Computational details
Structures of M-1B, M-2B, M-3B and M-4B were optimized
within the framework of dispersion corrected M06-2x based
density functional theory employing the Gaussian-09
program.53 The internally stored 6-31G basis set with diffuse
functions being added on all the heavy atoms (designated as 6-
31+G(d,p) basis) have been used for these optimizations.54,55
Stationary point structures thus obtained were conrmed to be
local minima on the potential energy surface through vibra-
tional frequency calculations (all the normal vibration
frequencies were turned out to be real). Frontier orbital were
subsequently analyzed for these local minima structures at the
M06-2x/6-31+G(d,p) level of theory. To derive molecular insights
accompanying extension of crystal networks in M-1B–M-4B
their dimeric structures were optimized subsequently.
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Eppendorf thermomixer. Reactions were started by adding ATP 15 T. Klabunde, H. M. Petrassi, V. B. Oza, P. Raman, J. W. Kelly
(to 450 mM nal concentration) to each sample and incubation and J. C. Sachhettini, Nat. Struct. Biol., 2000, 7, 312–321.
was continued for 20 minutes. Reactions were terminated by 16 B. Moosmann, T. Skutella, K. Beyer and C. Behl, Biol. Chem.,
placing the samples on ice and adding 4 mL stop/gel loading 2001, 382, 1601–1612.
buffer. 20 mL of each sample were separated by 1% agarose gel 17 C. Y. Soto, N. Andreu, I. Gilbert and M. Luquin, J. Clin.
electrophoresis. Gels were analyzed under UV trans- Microbiol., 2002, 40, 3021–3024.
illuminator and decatenated kDNA products were quantied 18 B. Morak-Młodawska, M. Jelen and K. Pluta, Pol. Merkuriusz
a
´
using Alpha Ease FC (Fluor Chem 8900) image analysis soware
Lek., 2009, 26, 671–675.
version 3.2.3 (Alpha Innotech, San Leandro, CA).
19 J. Molnar, R. Pusztal, A. Hever, S. Nagy and N. Motohashi,
Anticancer Res., 1995, 15, 2013–2016.
20 N. Motohashi, H. Sakagami, K. Kamata and Y. Yamamoto,
Anticancer Res., 1991, 11, 1933–1937.
Acknowledgements
SSG is grateful the Department of Biotechnology, India for the 21 A. Bolognese, G. Correale, M. Manfra, A. Lavechhia,
nancial support. DC thanks the University Grants Commis-
sion, New Delhi, India for the Junior Research Fellowship. SSR
acknowledges the nancial support from the Savitribai Phule
O. Mazzoni, E. Novellino, V. Barone, A. Pani,
E. Tramontano, P. L. Colla, C. Murgioni, I. Serra, G. Setzu
and R. Loddo, J. Med. Chem., 2001, 45, 5205–5216.
Pune University, Pune, India for the award of research fellow- 22 A. Bolognese, G. Correale, M. Manfra, A. Lavecchia,
ship through the potential excellence scheme. Authors
E. Novellino and S. Pepe, J. Med. Chem., 2006, 49, 5110–5118.
acknowledge Institute of Bioinformatics & Biotechnology (IBB), 23 A. Alberti, A. Bolognese, M. Guerra, A. Lavecchia,
Savitribai Phule Pune University for Animal Tissue Culture
facility.
D. Macciantelli, M. Marcaccio, E. Lovellino and
F. Paolucci, Biochemistry, 2003, 42, 11924–11931.
24 A. Abe, M. Yamane and A. Tomoda, Anti-Cancer Drugs, 2001,
12, 377–382.
25 K. Shirato, K. Imaizumi, A. Abe and A. Tomoda, Biol. Pharm.
Bull., 2007, 30(2), 331–336.
References
1 J. Jose and K. Burgess, Tetrahedron, 2006, 62, 11021–11037.
2 B. R. Raju, A. Daniela, G. Firmino, A. L. S. Costa, 26 B. S. B. Salomi, C. K. Mitra and L. Garton, Synth. Met., 2005,
P. J. G. Coutinho and M. S. T. Gonçalves, Tetrahedron,
2013, 69, 2451–3246.
155, 426–429.
27 J. C. Wang, Annu. Rev. Biochem., 1996, 65, 635–692.
3 L. Yuan, W. Lin, K. Zheng, L. He and W. Huang, Chem. Soc. 28 J. C. Wang, Annu. Rev. Biochem., 1985, 54, 665–697.
Rev., 2013, 42, 622–661.
4 A. D. G. Firmino and M. S. T. Gonçalves, Tetrahedron Lett.,
2012, 53, 4946–4950.
29 J. M. Berger, S. J. Gamblin and J. C. Wang, Nature, 1996, 379,
225–232.
30 L. F. Liu, Annu. Rev. Biochem., 1989, 58, 351–375.
5 J. Xu, S. Sun, Q. Li, Y. Yue, Y. Li and S. Shao, Analyst, 2015, 31 A. Y. Chen and L. F. Liu, Annu. Rev. Pharmacol. Toxicol., 1994,
140, 574–581.
34, 191–218.
6 K. Tanabe, Z. Zhang, T. Ito, H. Hatta and S.-I. Nishimoto, 32 S.-T. Zhuo, C.-Y. Li, M.-H. Hu, S.-B. Chen, P.-F. Yao,
Org. Biomol. Chem., 2007, 5, 3745–3757.
7 M. Liu, M. Hu, Q. Jiang, Z. Lu, Y. Huang, Y. Tan and Q. Jiang,
RSC Adv., 2015, 5, 15778–15783.
8 R. Sun, W. Liu, Y.-J. Xu, J.-M. Lu, J.-F. Ge and M. Ihara, Chem.
Commun., 2013, 49, 10709–10711.
9 S. S. Bag, S. Ghorai, S. Jana and C. Mukherjee, RSC Adv.,
2013, 3, 5374–5377.
S.-L. Huang, T.-M. Ou, J.-H. Tan, L.-K. An, D. Li, L.-Q. Gu
and Z.-S. Huang, Org. Biomol. Chem., 2013, 11, 3989–4005.
33 J.-J. Liu, J. Sun, Y.-B. Fang, Y.-A. Yang, R.-H. Jiao and
H.-L. Zhu, Org. Biomol. Chem., 2014, 12, 998–1008.
34 M.-Y. Kim, W. Duan, M. Gleason-Gunman and L. H. Hurley,
J. Med. Chem., 2003, 46, 571–583.
35 F. M. Deane, E. C. O'Sullivan, A. R. Maguire, J. Gilbert,
J. A. Sakoff, A. McCluskey and F. O. McCarthy, Org. Biomol.
Chem., 2013, 11, 1334–1344.
10 L. Kathawate, P. V. Joshi, T. K. Dash, S. Pal, M. Nikalje,
¨
T. Weyhermuller, V. G. Puranik, V. B. Konkimalla and
S. Salunke-Gawali, J. Mol. Struct., 2014, 1075, 397–405.
11 K. Hara, M. Okamoto, T. Aki, H. Yagita, H. Tanaka,
36 J. G. Atwell, W. G. Rewcastle, C. B. Baguley and W. A. Denny,
J. Med. Chem., 1987, 30, 664–669.
Y. Mizukami, H. Nakamura, A. Tomoda, N. Hamasaki and 37 M.-Y. Kim, W. Duan, M. Gleason-Guzman and L. H. Hurley,
D. Kang, Mol. Cancer Ther., 2005, 4, 1121–1127. J. Med. Chem., 2003, 46, 571–583.
12 B. Blank and L. L. Baxter, J. Med. Chem., 1968, 11, 807–811. 38 M. Duca, P. B. Arimondo, S. Leonce, A. Pierre, B. Pfeiffer,
´
´
13 F. Paula Carneiro, M. D. Carmo, F. R. Pinto, S. T. Coelho,
B. C. Cavalcanti, C. Pessoa, C. A. D. Simone, I. K. C. Nunes,
C. Monneret and D. Dauzonne, Org. Biomol. Chem., 2005,
3, 1074–1080.
N. M. D. Oliveira, R. T. D. Almeida, A. V. Pinto, 39 N. Vicker, L. Burgess, I. S. Chuckowree, R. Dodd, A. J. Folkes,
K. C. D. G. Moura, K. D. Moura, P. A. D. Silva and
E. N. D. Silva Jr, Eur. J. Med. Chem., 2011, 46, 4521–4529.
14 O. Wesolowaska, J. Molnar, G. Westman, K. Samuelsson,
M. Kawase, I. Ocsovszki, N. Motohashi and K. Michalak, In
Vivo, 2006, 20, 109–114.
D. J. Hardick, T. C. Hancox, W. Miller, J. Milton, S. Sohal,
S. Wang, S. P. Wren, P. A. Charlton, W. Dangereld,
C. Liddle, P. Mistry, A. J. Stewart and W. A. Denny, J. Med.
Chem., 2002, 45, 721–739.
40 Y. Ueno, Monatsh. Chem., 1982, 113, 641–643.
57928 | RSC Adv., 2015, 5, 57917–57929
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
41 J. Koshitani and Y. Ueno, J. Prakt. Chem., 1983, 325, 165–167. 52 G. M. Sheldrick, SHELX-97 Program for crystal structure
¨
42 R. Patil, D. Chadar, D. Chaudhari, J. Peter, M. Nikalje,
solution and renement, University of Gottingen, Germany,
¨
T. Weyhermuller and S. Salunke-Gawali, J. Mol. Struct.,
1997.
2014, 1075, 345–351.
43 S. Pal, M. Jadhav, T. Weyhermuller, Y. Patil, M. Nethaji,
53 M. J. Frisch G. W. Trucks H. B. Schlegel G. E. Scuseria
M. A. Robb J. R. Cheeseman J. A. Montgomery Jr,
T. Vreven, K. N. Kudin, J. C. Burant, J. M. Millam,
S. S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi,
G. Scalmani, N. Rega, G. A. Petersson, H. Nakatsuji,
M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa,
M. Ishida, T. Akajima, Y. Honda, O. Kitao, H. Nakai,
M. Klene, X. Li, J. E. Knox, H. P. Hratchian, J. B. Cross,
V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts,
R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi,
C. Pomelli, J. W. Ochterski, P. Y. Ayala, K. Morokuma,
G. A. Voth, P. Salvador, J. J. Dannenberg, V. G. Zakrzewski,
S. Dapprich, A. D. Daniels, M. C. Strain, O. Farkas,
D. K. Malick, A. D. Rabuck, K. Raghavachari,
J. B. Foresman, J. V. Ortiz, Q. Cui, A. G. Baboul, S. Clifford,
J. Cioslowski, B. B. Stefanov, G. Liu, A. Liashenko,
P. Piskorz, I. Komaromi, R. L. Martin, D. J. Fox, T. Keith,
M. A. Al-Laham, C. Y. Peng, A. Nanayakkara,
M. Challacombe, P. M. W. Gill, B. Johnson, W. Chen,
M. W. Wong, C. Gonzalez and J. A. Pople, Gaussian03,
revision Gaussian, Inc., Pittsburgh, PA, 2003.
¨
U. Kasabe, L. Kathawate, V. B. Konkimalla and S. Salunke-
Gawali, J. Mol. Struct., 2013, 1049, 355–361.
44 O. Pawar, A. Patekar, A. Khan, L. Kathawate, S. Haram,
G. Markad, V. Puranik and S. Salunke-Gawali, J. Mol.
Struct., 2014, 105, 68–74.
45 S. Salunke-Gawali, O. Pawar, M. Nikalje, R. Patil,
¨
T. Weyhermuller, V. G. Puranik and V. B. Konkimalla, J.
Mol. Struct., 2014, 1056–1057, 97–103.
46 D. R. Thube, A. Todkary, S. Y. Rane, K. Joshi, S. P. Gejji,
S. A. Salunke, F. Varret and J. Marrot, J. Mol. Struct.:
THEOCHEM, 2003, 622, 211–219.
47 C. Tan, S. Wu, S. Lai, M. Wang, Y. Chen, L. Zhou, Y. Zhu,
W. Lian, W. Peng, L. Ji and A. Xu, Dalton Trans., 2011, 40,
8611–8621.
48 B. B. Hasinoff, A. M. Creighton, H. Kozlowska, P. Thampatty,
P. William and J. C. Yalowich, Mol. Pharmaceutics, 1997, 52,
839–845.
49 D. D. Perrin and W. L. Armarego, Purication of Laboratory
Chemicals, Pergamon Press, London, 1988, p. 260.
50 G. M. Sheldrick, SADABS, Bruker–Siemens Area Detector 54 Y. Zhao and D. G. Truhlar, Acc. Chem. Res., 2008, 41, 157–167.
Absorption and Other Correction, Version 2008/1, University 55 Y. Zhao and D. G. Truhlar, J. Chem. Theory Comput., 2005, 1,
¨
of Gottingen, Germany, 2006.
415–432.
51 ShelXTL 6.14, Bruker AXS Inc., Madison, WI, USA2003.
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