Synthesis, spectral characterization, crystal structure, cytotoxicity and apoptosis—Inducing activity of two derivatives of 2-hydroxy-1,4-naphthaquinone

Article abstract of DOI:10.1016/j.pdpdt.2017.01.180

A phenaxazone compound [5H-Benzo[a]phenoxazin-5-one (BP)] along with an aminoquinone[2-[(o-hydroxyphenyl)amino]-1,4-naphthaquinone (HAN)] derivatives were synthesized from lawsone using ultrasound irradiation technique. The structure of the compounds were

Full text of DOI:10.1016/j.pdpdt.2017.01.180

Accepted Manuscript
Title: Synthesis, Spectral Characterization, Crystal structure,
Cytotoxicity and Apoptosis − inducing Activity of two
derivatives of 2-hydroxy-1,4-naphthaquinone
Authors: Kavitha Rani P.R., Annette Fernandez, Shiny P.
Laila, Arunkumar B., Sreelakshmi C.S., Vishnu V.S.
PII:
S1572-1000(16)30165-X
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.pdpdt.2017.01.180
PDPDT 891
To appear in:
Photodiagnosis and Photodynamic Therapy
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Revised date:
Accepted date:
26-8-2016
3-12-2016
19-1-2017
Please cite this article as: P.R.Kavitha Rani, Fernandez Annette, Laila Shiny
P, B.Arunkumar, C.S.Sreelakshmi, V.S.Vishnu.Synthesis, Spectral Characterization,
Crystal structure, Cytotoxicity and Apoptosis − inducing Activity of two
derivatives of 2-hydroxy-1,4-naphthaquinone.Photodiagnosis and Photodynamic
Therapy http://dx.doi.org/10.1016/j.pdpdt.2017.01.180
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Synthesis, Spectral Characterization, Crystal structure, Cytotoxicity
and Apoptosis – inducing Activity of two derivatives of 2-hydroxy-
1,4-naphthaquinone
Kavitha Rani P. Ra, Annette Fernandez b, Shiny P. Lailac, Arunkumar Bb,
Sreelakshmi C.Sd , Vishnu V.S b
a
. Dept. of Chemistry, S.FR College for Women, Sivakasi, 626123, Tamilnadu,
India
b
Dept. of Chemistry, College of Engineering, Trivandrum, 695016, Kerala. India.
c Dept. of Chemistry,University College, Trivandrum, 695014, Kerala. India.
d. Dept. of Chemistry, Mohandas College of Engineering and Technology
Thiruvananthapuram, 695544, Kerala. India
1

Highlights
. Ultrasound assisted synthesis of lawsone derivatives with anticancer properties
. Single crystal X-ray analysis of 5H–Benzo[a]phenoxazin–5–one
. Find the ADMET properties
. In silico molecular docking studies
. Cytotoxic and preliminary apoptosis inducing activity of two derivatives
2

Abstract
A phenaxazone compound [5H–Benzo[a]phenoxazin–5–one (BP)]along with an
aminoquinone[2–[(o–hydroxyphenyl)amino]–1,4–naphthaquinone (HAN)] derivatives were
synthesized from lawsone using ultrasound irradiation technique. The structure of the
compounds were characterized by elemental analysis and various spectral studies. Optoelectronic
properties were studied using Schrodinger material science suit (2015). The compounds exhibit
fluorescence emission in longer wave length it may find applications in photodynamic therapy.
Single crystal X-ray diffraction studies reveals that the compound BP crystallizes in monoclinic
space group. The antioxidant activity of HAN and BP were determined using DPPH radical
scavenging assay and the results indicate that both the compounds have good antioxidant
capacity, HAN having more scavenging activity than BP. Lead molecules were identified using
insilico molecular socking studies as a green chemistry approach. iGEMDOCK, GOLD and
Schrödinger softwares were used for these studies. The docking studies reveal that the structural
modification of the parent compound gave more active compounds making them promising lead
molecules. The lead molecules were subjected to in vitro studies. The cytotoxicity of BP and
HAN was studied using human breast cancer (SKBR3) cell lines. The IC50 value of HAN was
found to be 19.8 µM while BP was found to have cell viability, less than 10% even at 25 µM
concentration. The chemotherapeutic agents kill the cancer cells mainly through apoptosis.HAN
and BP wwere subjected to apoptosis studies. BP was found to more active than HAN. Thus it
can be suggested that the mechanism of cell death may be through apoptosis.
Key words : Ultrasound irradiation, lawsone, orthoaminophenol and in silico molecular
docking studies
1.
Introduction
Naphthoquinone compounds are widely distributed in the environment both as natural
products and as pharmaceutical agents. 1,4–naphthoquinone is an important example of the
quinone family and it is industrially used as a raw material for pharmaceuticals, agrochemicals
and other functional chemicals. There are many clinically important antitumor drugs containing
quinone nucleus. The amino and substituted quinone derivatives are an important class of
molecules having immense applications in biology and pharmacology. Phenoxazines and its
3

derivatives find application as cancer drugs. Actinomycin D is a drug against cancer having
phenoxazine moiety. Another two compounds having phenoxazine moiety,
pyridophenazinediones and pyridazinophenazinedione are known for its antitumour effects [1-2].
These compounds find applications as multidrug modulators in cancer cells. By virtue of their
DNA intercalating ability, they exhibit antitumor activity in leukemia and solid tumors.
Fluorescent phenoxazine derivatives find applications as emitters for electroluminescence
devices, organic semiconductors, photo-sensitizers in photodynamic therapy, promoter for
proliferation, electrochemical, and biosensors [3-4].
Docking is frequently used to predict the binding orientation , affinity and activity of
molecule, with protein targets. For this study the protein kinase CK2 was selected as target
protein. Protein Kinase plays a central role in controlling nearly all cellular functions, with
special reference to signal transduction, gene expression and DNA synthesis and repair. So the
development of efficient and selective inhibitors of protein kinases represents a powerful tool for
drug design particularly in oncology[5]
The aim of the present work deals with the isolation of lawsone, synthesis of its two
amino derivatives, HAN and BP, their characterization by various spectral techniques including
single crystal X-ray diffraction studies, the in silico and in vitro cytotoxic studies of the
compounds and to find out whether these compounds are suitable lead molecules for drug
discovery.
2. Experimental
2.1. Materials and instrumentations
Isolation and purification of lawsone was done as per literature [6-7]. Ortho amino
phenol(Merck), (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) (MTT), DPPH
were purchased and used without purification. TLC analyses were performed on precoated
aluminum plates of silica gel 60 F 254 plates (0.25 mm, Merck).Column chromatography was
performed using silica gel G (mesh 100-200). In vitro cytotoxicity assay was conducted in
SKBR3 breast cancer cell line using MTT assay. DNA–binding dyes, Acridine Orange and
Ethidium Bromide (Sigma, Aldrich) were used for the morphological detection of apoptotic and
necrotic cells. The morphological changes were observed by a fluorescence microscope. Melting
4

points were obtained using a capillary melting point apparatus. IR spectra were recorded on a
1
13
Shimadzu IR Prestige-21 FT-IR spectrometer on KBr pellets. H and C NMR spectra were
measured on a 500 MHz Bruker advanced DPX spectrometer with CDCl3 as solvent. CHN
analyses were carried out on an Elemental Analyzer. UV-Visible spectrum was recorded on
Shimadzu UV-3600 UV-VIS-NIR spectrometer. The fluorescence emissions were measured by a
Horiba FluoroLog Spectrophotometer. Mass spectra were recorded on a Shimadzu Gas
Chromatographic Mass Spectrometer. X-ray diffraction studies were carried using a Bruker AXS
Kappa Apex2 CCD diffractometer, with graphite-mono-chromated Mo Kα (λ=0.7103 Å)
radiation.
2.2 Synthesis of 5H–Benzo[a]phenoxazin–5–one (BP) and 2–[(o–hydroxyphenyl )amino]–1,4 –
naphthaquinone (HAN)
A solution of lawsone 0.174 g (1 mmol) in 25 mL methanol was added to 0.109 g (1
mmol) ortho amino phenol dissolved in 25 mL methanol. The mixture was sonicated for 40 min
at room temperature until it became dark red in color and was monitored using TLC in benzene
after each 10 min for the completion of the reaction. The resulting solution was concentrated on
a rotavapour. The crude precipitate was dried and subjected to column chromatography. Isocratic
elution with pure benzene gave yellow colored crystals of BP. Yield 25 % and m.p 220 °C. The
remaining portion of the above mixture was eluted with methanol: acetone in the (1:3) ratio. The
resulting solution gave dark red colored crystals of HAN, yield 60% and m.p 190 °C. The
scheme of the reaction is shown in Scheme .1.
5

2–[(o–hydroxyphenyl)amino]–1,4–
5H–Benzo[a]phenoxazin–5–one
naphthaquinone (HAN)
(BP)
Scheme 1.
Synthesis of
2–[(o–hydroxyphenyl)amino]–1,4–naphthaquinone (HAN)
5H–Benzo[a]phenoxazin–5–one(BP)
2.3. X – Ray crystallography
The unit cell dimensions and intensity data were recorded at 293 K. The program SAINT
/ XPREP was used for data reduction and APEX2/SAINT for cell refinement [8]. The structure
was solved using SIR92 and refinement was carried out by full matrix least squares of F2 using
SHELXL-97[9-10]. Molecular graphics employed was MERCURY.
2.4. In vitro pharmacology analysis
2.4.1. DPPH radical scavenging.
Free- radical scavenging activity of lawsone and its derivatives were studied using
DPPH (diphenylpicrylhydrazylradical) according to the procedure[11-12].
2.4.2. MTT Assay
In vitro cytotoxicity of the derivatives SKBR3 human breast cancer cell lines was
determined using MTT assay according to the procedure [13].The test compounds with various
6

concentrations (3.125µM, 6.25µM, 12.5 µM, 25 µM, 50 µM) were added to the cells and
incubated for 48h.
2.4.3. Determination of Apoptosis by Acridine Orange (AO) and Ethidium Bromide (EB) double
staining method
DNA–binding dyes, Acridine Orange and Ethidium Bromide were used for the
morphological detection of apoptotic and necrotic cells. AO is taken up by both viable and non
viable cells and emits green fluorescence if intercalated into double stranded nucleic acid. EB is
taken up only by non–viable cells and emits red fluorescence by intercalation into DNA. After
treatment with samples in a concentration of 50 μg/mL for 48 h, the cells were washed by cold
PBS (Phosphate buffered saline) and then stained with a mixture of AO (100 μg/mL) and EB
(100 μg/mL) at room temperature for 10 min. The stained cells were washed twice with 1x PBS
3–
(1x PBS should have a concentration of 10 mMPO4 , 137mMNaCl, and 2.7 mMKCl) and
observed by a fluorescence microscope using blue filter (Olympus CKX41 with an Optika Pro5
camera) [14].
2.5Insilico analysis
In silico molecular docking studies of lawsone and its derivatives were carried out using
three different software iGEMDOCK, GOLD and Schordinger (Maestro) suit. The selected
target protein was protein kinase CK2 and PDB ID was 1M2R. In the Schordinger modelling
software, protein was optimised using the Protein Preparation Wizard. The compounds were
preprocessed by the LigPrep module . Interactions of ligand and amino acid in the active site of
CK2 was visualizes by XP visualizer. ADMET (absorption, distribution, metabolism, and
excretion and toxicity) properties were predicted using Medchem designer software. Medchem
Designer is an advanced molecule design software application that combines an intuitive sketch
interface with fast and accurate ADMET properties prediction.
3.Results and discussion
Spectral studies of the extracted lawsone were carried out and the data were found to be
consistent with the reported data [14]. The structure of lawsone is as shown in (Scheme. 1)
3.1 CHN analysis
7

The elemental analysis data were in good agreement with the stoichiometry of HAN and
BP. The percentage composition of the elements present in the BP, C16H9O2N was found
to be C= 77.30% , H=3.49%, N=5.45 and calculated as C=77.72%, H= 3.67%, N= 5.67% . For
HAN it was found to be C= 72.40% , H=4.25%, N=5.17%, calculated as C=72.38%, H=4.15 %
and N=5.27%.Values for C, H, N also confirmed the stoichiometry of BP and HAN.
3.2. UV-Visible and Fluorescence spectra
From the electronic spectrum of HAN, the band observed at 266 nm was assigned to the
π–π* transition of the naphthaquinone ring and the imine function of the amino phenol moiety.
The π–π* transitions was observed at 350 nm in the electronic spectra of BP. The n–π*
transitions were observed at 436 nm in HAN and 440 nm in BP. Both the derivatives have no
appreciable change in n–π* transitions.
3.3. Fluorescence Emission spectra of HAN and BP
The fluorescence emission spectra of HAN and BP was examined in methanol at room
temperature (298K). The spectra are shown in Fig.1(a) and (b). Upon photo excitation at the
corresponding absorption bands (275 nm for HAN and 398 nm for BP) the derivatives exhibited
intense emission bands at 328 nm in the spectrum of HAN and 523 nm in the spectrum of BP.
The fluorescence behavior in both the cases may be attributed to an intra-ligand (π-π*)
emission[16]. Both the compounds gave emission spectra. HAN and BP exhibited fluorescence
emission at 328 and 523 nm with corresponding stokes shift of 53 and 125 nm respectively. It is
reported that the phenoxazine dyes tend to have large Stokes shifts. This is useful in simplifying
the type of filtering needed to differentiate between excitation and emission wavelengths and
also may enable sensitivity limits to be improved [17-18]. In the case of BP, greater stokes shift
was observed which may be due to energy transfer or excited state reaction [19-20].Fluorescent
compounds with particularly high Stokes shift possess application in fields, where long optical
path are required like sunlight collection, scintillation and various biological applications [1].
Since BP possess favorable absorption properties ie, emission in the photodynamic window, this
dye may find application as a sensitizer for the use in photodynamic therapy (PDT) which finds
scope for further studies.
8

Schrodinger quantum chemistry package(Jaguar)to calculate properties like oxidation –
reduction potential, reorganization energy and HOMO-LUMO energy are given in Table 1. Low
reorganization energy for efficient charge transfer process. The compound HAN and BP have
low reorganization energy than the parent compound. The lowest S1-T1 gap found in BP, the
derivatives HAN and BP excited by light and can be converted to triplet excited state. BP has
highest triplet energy than HAN and which is higher than parent compound. So the species
may lead to the formation of singlet oxygen and radicals that oxidize biomolecules [21].The
compound BP covers larger area in emission spectrum. BP exhibiting the highest oxidation
potential and lowest reduction potential So it is expecting that such type of materials can be used
as photosensitizers
3.4. Infrared Spectra
The IR spectra of the derivatives are given in (Fig. S1 and Fig.S2). The parent
compound, lawsone exhibited a broad spectrum at 3526 cm–1 due to the hydrogen bonded OH
group. This band was found to be absent in HAN indicating that substitution must have occurred
at the hydroxyl group. The –OH stretching frequency of the amino phenol moiety was observed
at 3060 cm–1 in HAN indicating the strong intramolecular hydrogen bonding. A strong band
observed at 3280 cm–1 was attributed to N–H vibration. A broad, medium intense band observed
at 817 cm–1 was due to the out of plane N–H wagging absorption. The 1,4–naphthaquinone
carbonyl absorption bands of lawsone observed at 1680 cm–1 (C8=O) and 1640 cm–1 (C1=O)
were also observed in HAN. In HAN, the carbonyl group was found to be hydrogen bonded with
the neighboring NH group since it exhibited a lower carbonyl frequency of 1612 cm–1. Another
peak at 1544 cm–1 was assigned to the N–H…..O deformation [22]
The hydrogen bonded –OH group absorption observed in lawsone and the hydrogen
bonded –OH group absorption observed in HAN were both absent in the derivative BP. The
absorption due to the –NH vibration at 3280 cm–1 observed in HAN, was also absent in BP. The
absorption band at 1638 cm–1 in BP was assigned to the C(8)=O group. This gave evidence that
coupling has occurred in the C(1) carbonyl group of lawsone and was further confirmed by
crystal structure studies.
3.5.1H NMR spectra of HAN and BP
9

1
1
The H NMR spectrum of the compound, HAN is given in Fig. S3. In the H NMR
spectrum, the singlet observed at 6.4 ppm was assigned to the C(3) proton and another one
singlet at 5.4 ppm to the NH proton with more down field shift due to hydrogen bonding.
The –OH protons appeared as a singlet at down field, 7.7 ppm was due to the presence of
intermolecular hydrogen bonding in HAN. Intra and inter molecular hydrogen bonding were
observed in this compound and was further proved by the crystal structure [22] obtained.
1
The H NMR spectrum of the compound BP, recorded in CDCl3 is given in Fig. S4. A
sharp singlet which integrated as one hydrogen at 6.3 ppm was assigned to the proton attached to
the C(9) of naphthaquinone ring. The singlet absorption peak at 5.4 ppm assigned to the NH
proton observed in HAN was absent in BP. The absorption due to –OH group in lawsone was
found to be absent in the derivative BP which indicated that the condensation has occurred and
which was further confirmed by the crystal structure obtained.
1H–1H COSY spectrum of BP is given in Fig. S5. The expansion of the COSY spectrum
in the aromatic region is a good example for the assignment of aromatic systems. The safe
distinction between H-5 and H-14 was recognized in the COSY spectrum. It was observed that
H-5 coupled with H-4 and H-14 coupled with H-13.
3.613C NMR Spectra of HAN and BP
The 13C NMR Spectrum of HAN with the different resonant peaks with respect to
respective carbon atoms are presented in Fig. S6. The C(4) carbonyl carbon atom resonance was
observed farthest downfield at δ value 183 ppm and C(1) carbonyl carbon atom resonance at
δ value 180 ppm. This gave the evidence of the presence of two carbonyl peaks. In the
naphthaquinone ring, the C(2)carbon atom adjacent to the more electronegative nitrogen atom
(N1) were shifted further down field at δ value 155 ppm. The C–H methane of the
naphthaquinone and phenyl carbons were observed at 135-126 ppm. Whereas it was fairly safe to
assign the signal at 108 ppm to C(11) carbon due to its β-position with respect of the hydroxyl
group.
The 13C NMR spectrum of BP is given in Fig. S7. 13C NMR spectrum of HAN gave two
different quinone carbonyl peaks at 183 and 180 ppm. In BP, only one carbonyl peak was
10

observed at 183 ppm which indicated that the condensation has occurred at the C(1) carbonyl
group which was further confirmed by the crystal structure of BP shown in Fig. 2.
3.7 Mass Spectra of HAN and BP
Mass Spectrum of HAN was obtained by the direct ionization method. The
chromatogram obtained is presented in Fig. S8. A molecular ion peak (M+) ion was observed at
m/z = 265 corresponding to the molecular formula C16H11O3N. The base peak was obtained at
m/z = 236 corresponding to the elimination of –CO group from the molecular ion. From these
spectral studies the tentative structure of the compound was assigned. Its crystal data have been
already deposited in CCDC No. 724554.
The chromatogram of BP is given in Fig. S9. The mass spectra of the compound gave a
molecular ion peak at m/z = 247 which was also the base peak and corresponds to C16H9O2N.
Another peak at m/z = 219 was found to be due to the loss of a –CO group.
3.8. Crystal Structure of BP
The derivative BP crystallizes in monoclinic space group P21/c with Z=4 and unit cell
dimensions a = 13.061(5) (Å), b = 3.859(5) (Å), c = 22.788(5) (Å), V = 1132.3 (a3); d = 1.450 (g
cm–3). Crystallographic data is summarized in Table 2.
Selected bond length and bond angles of BP are given in Table 3. The carbon nitrogen
double bond distance C1=N1 was 1.297Å, where as carbon -nitrogen single bond distance
C(16)–N(1) was 1.389 Å. This was a clear evidence of the presence of C–N as well as C=N in
the compound. C=N distance between C(10)–O(2) was 1.364 Å and C(11)–O(2) distance was
1.377 Å indicating the presence of C–O–C bond [23-24]. The carbon–oxygen double bond
distance C(8)–O(3) was 1.224 Å. This indicated that the compound existed in the keto form. In
addition to this, C–C–C and C–C–O angles were obtained in the range 120.04º–120.17°as
expected for sp2 hybridized atoms.
4. In silico Molecular docking studies
4.1ADMET Properties
11

ADMET properties including PKa, lipophilicity, solubilities, permeability, toxicities and
metabolic profiles, were predicted using Medchem designer. It calculates many physical
properties and some mathematical descriptors like nonpolar and polar surface area, water
solubility, logP etc. Molecular lipophilicity usually quantified as logP and aqueous solubility
usually defined as logS are important molecular characteristic in medicinal chemistry. The logP
and logS coefficients are for the estimation of lipophilicity and solubility of the chemical
compound and determine their pharmacokinetic properties. ADMET results are shown in
Table 4. It was found that the parent lawsone and its derivatives obeyed the Lipinski’s rule and
may further be subjected to docking studies.
4.2. Molecular docking
In silico molecular docking studies of lawsone, HAN and BP were done for predicting
its pharmacokinetic properties. For this study the target selected was protein kinase CK2. Its
structure was retrieved from Protein Data Bank (PDB) and PDB ID is 1M2R [25-27]. Lawsone
and its derivatives were docked into the target protein kinase CK2 using the three softwares
iGEMDOCK, GOLD and Schrödinger software. Many types of drug design approaches are
there. For the present study structure based drug design approach was used, which is a very
powerful method in drug design.
Table 5 represent the various interaction scores obtained for lawsone, HAN and BP using
iGEMDOCK software. Fitness is the total energy of a predicted pose in the binding site. The
scoring function of iGEMDOCK is estimated as sum of these energies like vanderwaals energy,
hydrogen bonding energy and electrostatic energy. Highest negative scores indicate a better
active compound. The standard, 5,8–di–amino–1,4–dihydroxy–anthraquinone belongs to
quinone family was used to compare the docking score had a total energy of -95.6622
Kcal/mole. It was found that HAN had higher activity than the standard and the energy values of
lawsone and BP were comparable with that of the standard.
Table 6 demonstrates the docking scores obtained for lawsone and its derivatives using
GOLD. The interaction data represent the interaction between various amino acid present in the
active site of the protein. The table also provides the hydrogen bonding interaction of the ligand
with the respective amino acid Tyrosine115, Asparagine165, Asparagine117, Lysine64 and
12

Valine 116 present in the active site. Short contacts [vanderwaal’s (internal, external), torsional
strain energy] represents various other interactions between ligand and amino acids in the active
site. Higher scores indicate maximum interaction and a score above 40 is considered as the best
fit[28-30]. The same standard was used to compare GOLD and has a score of 61.5222Kcal/mole.
Lawsone and its derivatives were found to have comparable scores with the standard and
exhibited best fit score values. The derivatives, HAN and BP were found to be more active than
the parent compound, lawsone. Fig. 3 and Fig. 4 represents the various interactions of ligand
and amino acids in the active site of CK2 [31]. This was visualized by discovery studio.
The glide score obtained from Schordinger Mastero software is shown in Table 7.The
ligand binding protein through , electrostatically hydrogen bonding and vander waals
interaction. The compound HAN has the lowest docked binding energy of -2.65 kcal/mol and
forms various interaction with active site residue THR320, PRO322, HIE321, LYS303, LBJ313,
ARG306, GLU311, ARG10 and GLU317 shown in Fig.5. BP has the docked binding energy -
1.60 kcal/mol and interacted with active site TYR138, GLU331, ARG328, ASN332, THR320,
MET319, LEU316, ASP145, TYR146, PRO92 and LYS 142 shown in Fig 6. The score obtained
for standard compound shown in Fig 7. Lawsone and its derivatives have better score than
standard compound. Interactions of ligand and amino acid in the active site of CK2 was
visualizes by XP visualizer.
The comparison of scores using Schordinger Mastero software , iGEMDOCK and
GOLD are given in Table 6 and it was found that HAN gave a better score in all the docking
softwares. Lawsone and its two derivatives were found to have better energy scores and
interactions. Hence from the docking studies it was predicted that these compounds pose to be
lead molecules for drug discovery and can be further subjected to in vitro studies.
5. Pharmacological Studies
5.1 DPPH radical scavenging
Radical-scavenging activity of lawsone and its derivatives were determined using DPPH
radical scavenging assay shown in Fig. 8. The scavenging activity of the compounds HAN and
BP were tested and compared to that of the standard ascorbic acid, which was used as the
positive control. This activity was evaluated by the determination of the IC50 values, From the
13

results it was seen that HAN was more potent against DPPH than BP and therefore exhibited
higher antioxidant properties. The IC50 value of lawsone was found to be 62 µM/mL, for HAN
52 µM/mL and that for BP 97 µM/mL.
5.2(a) In-vitro cytotoxicity studies
The cytotoxicity of the compounds were screened by MTT assay against the human
breast cancer SKBR3 cell lines. The inhibition percentages are shown in Fig. 9. SKBR3 cells
were cultured in the presence of BP and HAN at various concentrations (3-25 µM) for 48 h and
the percentage of cell viability was evaluated by MTT assay. The compounds BP and HAN
exhibited potential cytotoxic behavior against SKBR3cells. Comparing the results, BP was found
to be more active than HAN. From the graph the IC50 value of HAN was found to be 19.8 µM,
while BP was found to have a cell viability less than 10% even at 25 µM concentration which
proves that it has very good anticancer properties. Since BP exhibited very good anticancer
activity, it was further subjected to the preliminary apoptosis inducing activity to confirm it as a
promising candidate for drug discovery.
5.2(b) Determination of Apoptosis by Acridine Orange (AO) and Ethidium Bromide (EB) double
staining method
Induction of tumor cell apoptosis has already been used as an important indicator to
detect the ability of chemotherapeutic drugs to inhibit tumor growth[32-33]. In the present study
we adopted the fluorescent staining method for morphological assessment of cell death by the
compounds HAN and BP. After treatment of HAN and BP with cells in a concentration of 50
μg/mL for 48 h, the morphological changes are given in Fig. 10, it was found that in HAN, 90%
of cells are viable as indicated by green flouresence, some of the cells undergo early apoptosis
having chromatin condensation and was visible as green fragments or patches. Only few cells
show late apoptosis as indicated by the orange red fluorescence. In BP, 95% of the cells
underwent apoptosis which gave orange red fluorescence.The mechanism of cell death may be
due to apoptosis, and further investigations are needed to verify the mechanism of apoptosis.
From the results it was found that they pose to be lead molecules in anticancer drug discovery
Conclusion
14

Two amino derivatives of lawsone, HAN and BP were synthesized using ortho
aminophenol by the ultrasound irradiation technique. The synthesis of the derivative HAN has
been reported earlier by another method. But its pharmacological properties have not been
reported. In the present investigation using the efficient and simple method of ultrasound
irradiation technique, we have not only obtained HAN but also another product BP by the single
reaction. BP is a phenoxazone compound which is proved to be biologically active. Both the
derivatives exhibited fluorescence emission. HAN was found to possess more antioxidant
property than BP.ADMET results reveal that the compounds possess physico-chemical drug absorption
and drug like properties. Docking studies of all the compounds were investigated
usingiGEMDOCK and GOLD. Protein kinase (CK2) was selected as the target protein for
docking studies as CK2 be a promising target for cancer therapy. From these results it was predicted
thatall the derivatives behave as lead compounds for in vitro analysis.Cytotoxic studies were further
conducted in SKBR3 cell lines. Even at 25 µM concentration, BP was found to exhibit a viability
of 10%, thus proving to be highly cytotoxic. Since HAN and BP exhibited anticancer activity,
preliminary apoptosis inducing activity studies were further conducted. In this study also, BP
was found to be more active. Thus it can be suggested that the mechanism of cell death was
through apoptosis. Thus the derivative HAN and BP find scope for further investigation and
poses to be lead molecules for anticancer drug discovery.
Acknowledgements
The authors would like to express deep gratitude to the Department of Chemistry, S.F.R
College for Women, Sivakasi, for providing the laboratory, instrumental and software facilities
under DST fund. We would extend thank to Department of Chemistry, Govt. College for
Women, Thiruvananthapuram, School of Chemistry (IISER), Thiruvananthapuram, College of
Engineering Trivandrum and RGCB Thruvananthapuram .
Supplementary data
Crystallographic data (excluding structure factors) for the structure reported in this paper
have been deposited with the Cambridge Crystallographic Data Centre as supplementary
publication. CCDC No. 1011433. These data can be obtained free of charge via
15

http://www.ccdc.cam.ac.uk/conts/retrieving.html or from CCDC, 12 Union Road,
CambridgeCB2 1EZ, UK, E–mail: deposit@ccdc.cam.ac.uk
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List of Tables
Table 1
Optoelectronic Properties Lawsone and its derivatives
LW
1.56
-0.92 0.74
0.64
1.57
0.56
6.69
-1.69 1.07
-5.84
-3.35 1.07
0.37
17

BP
2.19
1.68
-0.69 0.43
-0.98 0.24
0.44
0.36
1.98
1.62
0.44
0.42
8.46
7.35
-1.55 0.91
-1.47 0.79
-6.47
-5.96
-3.58 0.91
-3.29 0.79
0.18
0.12
HAN
Table 2
Crystal data of BP
Empirical formula
C21H17N3O3
Formula weight
Temperature/K
Crystal system
Space group
a (Å)
247.24
293
monoclinic
P21/c.
13.061 (5)
3.859 (5)
22.788 (5)
90 .00
b (Å)
c (Å)
α
96.65

90.00
1132.3
1.450
4

Volume (A3)
D (g cm–3)
Z
µ(mm–1)
F000
0.097
512
h,k,l
15,4,26
Wave length (A°)
No of Reflections
Final R indices
R indices (all data)
wR2
0.71069
0.0267
R1=0.0443
R1=0.0671
0.1602
R(reflections)
Goodness of fit
0.0991(1876)
1.127
Table 3
Selected bond lengths (Å ) of BP
18

Bond lengths
C1–N1
1.297
1.389
1.466
1.464
1.364
1.377
1.338
1.395
1.207
C2–C7
1.396
1.390
1.379
1.382
1.393
1.493
1.376
1.378
1.382
C16–N1
C1–C2
C2–C3
C3–C4
C1–C10
C10–O2
C11–O2
C10–C9
C16–C11
O1–C8
C4–C5
C6–C7
C7–C8
C11–C12
C12–C13
C13–C14
Bond angles (°)
N1–C1–C10
N1–C1–C2
C10–C1–C2
C3–C2–C7
C7–C2–C1
C4–C3–C2
O1–C8–C9
120.81
120.27
116.92
119.32
120.32
120.52
121.38
O1–C8–C7
120.64
122.44
117.17
123.17
121.17
118.90
119.17
C9–C8–C9
O2–C8–C7
C9–C10–C1
N1–C16–C11
N1–C16–C15
C10–O2–C11
Symmetry equivalent positions as xyz
x, y, z : -x, y+1/2, -z+1/2 ; -x, -y, -z ; x, -y-1/2, z-1/2
Table 4
ADMET results
19

Para
Standard
Lawsone
HAN
BP
Limiting Value
meters
S+logP
S+logD
MlogP
MWt
S+logP<5
S+logD<4.1
MlogP<4.15
MWt<500
HBDH<5
1.462
0.896
1.437
-0.405
1.225
3.242
3.225
2.334
265.27
2.000
4.000
66.400
0.000
3.035
3.035
0.344
1.680
270.246
6.000
174.157
1.000
247.225
0.000
HBDH
MNO
MNO<10
6.000
3.000
3.000
TPSA
TPSA =140Å
Rule of 5
126.640
1.000
54.370
0.000
43.100
0.000
Rule of 5
Table 5
iGEMDOCK Docking score (protein 1M2R)
Compound
Vander
Waals
H-bond
energy
Electrostatic Total
energy
Energy
energy
(kcal/mol)
(kcal/mol)
(kcal/mol)
(kal/mol)
Standard
Lawsone
BP
-84.5593
-11.1029
-21.4998
-12
0
0
0
0
-95.6622
-85.584
-64.0842
-71.6381
-90.7962
-83.6381
-103.824
HAN
-13.0274
Table 6
GOLD Docking Score (Protein 1M2R)
Compound
Gold
score
H bond
Short contacts
Standard
61.5222
Lysine 64
Asparagine117
Asparate 165
Leu168
Asparate 160
20

Lawsone
BP
46.2983
49.5545
54.1711
Valine 116
Tyrosine115
Lysine64
Nil
Tyrosine115
Asparagine165
Tyrosine115
HAN
Lysine64
Table 7
Schordinger mastero docking scores
Ligand
Glide Dock Vander Hydro Electrost
Amino acid interacting to
ligand
Score Score
-2.07 -2.07
waals
interact
ion
gen
bond
atic
interacti
on
Lawsone
-1.05
-0.77
-0.35
-0.35
-0.22
THR320,PRO322,HIE321,LYS
303,LEU313,GLU317
THR320,
ALA318,LEU313,GLU317,LYS30
3, ASP299,PRO322, HIE321
Standard -1.6
-1.6
-0.95
0
BP
-1.46 -1.46
-2.65 -2.65
-1.54
-0.81
-0.06
-0.71
TYR138,GLU331,
ARG328,ASN332,THR320,ME
T319,LEU316,ASP145,TYR14
6,PRO92 , LYS 142.
HAN
-1
THR320,PRO322,HIE321,LYS
21

303,LBJ313, ARG306,
GLU311,ARG10, GLU317
List of Figure
Fig. 1. Fluorescence Emission spectra of (a) HAN and (b) BP
Fig. 2.Molecular structure of BP
Fig. 3. Interactions of standard with various amino acids using GOLD software
Fig. 4. Interactions of HAN with various amino acids using GOLD software
Fig 5 Interactions of HAN with various amino acids using Schordinger software
Fig.6. Interactions of BP with various amino acids using Schordinger software
Fig 7 Interactions of STD with various amino acids using Schordinger software
Fig. 8 DPPH radical scavenging activity of HAN and BP
Fig. 9. Plot of the % cell inhibition of BP and HAN in SKBR3 cell lines
Fig. 10. AO/EB-stained SKBR3 cell lines
22

Fig. 1. Fluorescence Emission spectra of HAN and BP
5500000
5000000
4500000
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
2000000
1800000
1600000
1400000
1200000
1000000
800000
600000
400000
200000
0
350
400
450
500
550
600
650
700
750
250
300
350
400
450
500
550
Wave length(nm)
Wave length(nm)
(b) BP
Fluorescence Emission spectra of (a) HAN
23

Fig. 2.Molecular structure of BP
24

Fig. 3. Interactions of standard with various amino acids using GOLD software
25

Fig. 4. Interactions of HAN with various amino acids using GOLD software
Fig 5 Interactions of BP with various amino acids using Schordinger software
26

Fig 6 Interactions of HAN with various amino acids using Schordinger software
Fig 7 Interactions of STD with various amino acids using Schordinger software
90
HAN
BP
80
Ascorbicacid
Lawsone
70
60
50
40
30
20
10
0
20
40
60
80
100
Concentration(Micromolar)
27

Fig. 8 DPPH radical scavenging activity of HAN and BP
100
90
BP
HAN
80
70
60
50
40
30
20
10
0
5
10
15
20
25
Concentration (Micromolar)
Fig. 9. Plot of the % cell inhibition of BP and HAN in SKBR3 cell lines
Fig. 10 AO/EB-stained SKBR3 cell lines
28