Synthesis of 1-(Indol-2-yl)-phenoxazine hybrids from quinacetophenone precursors and their biological evaluation as DNA intercalating agents

Article abstract of DOI:10.1016/j.molstruc.2020.128311

An operationally simple reaction between quinacetophenone and aniline derivatives in the presence of triethylamine at room temperature afforded 1-(indol-2-yl)-phenoxazine hybrids in good yields. This unique transformation proceeds via sequential aza-Micha

Full text of DOI:10.1016/j.molstruc.2020.128311

Journal Pre-proof
Synthesis of 1-(Indol-2-yl)-phenoxazine hybrids from quinacetophenone precursors
and their biological evaluation as DNA intercalating agents
Saiprasad N. Nunewar, Naveen Kotla, Jaya Lakshmi Uppu, Apoorva Dixit, Venkatesh
Pooladanda, Chandraiah Godugu, Neelima D. Tangellamudi
PII:
S0022-2860(20)30636-0
DOI:
https://doi.org/10.1016/j.molstruc.2020.128311
MOLSTR 128311
Reference:
To appear in: Journal of Molecular Structure
Received Date: 20 November 2019
Revised Date: 14 April 2020
Accepted Date: 20 April 2020
Please cite this article as: S.N. Nunewar, N. Kotla, J. Lakshmi Uppu, A. Dixit, V. Pooladanda, C.
Godugu, N.D. Tangellamudi, Synthesis of 1-(Indol-2-yl)-phenoxazine hybrids from quinacetophenone
precursors and their biological evaluation as DNA intercalating agents, Journal of Molecular Structure
(2020), doi: https://doi.org/10.1016/j.molstruc.2020.128311.
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Synthesis of 1-(Indol-2-yl)-Phenoxazine Hybrids from Quinacetophenone Precursors
and their Biological Evaluation as DNA Intercalating Agents
Saiprasad N. Nunewar: Mechanistic studies, Viscosity studies-
Naveen Kotla, Apoorva Dixit: Synthesis and characterization.
Jaya Lakshmi Uppu: Viscosity studies, Nanodrop method.
Venkatesh Pooladanda, Chandraiah Godugu: MTT assay
Neelima D. Tangellamudi: Concept, Writing, Reviewing and Editing

Synthesis of 1-(Indol-2-yl)-Phenoxazine Hybrids from Quinacetophenone Precursors
and their Biological Evaluation as DNA Intercalating Agents
Saiprasad N. Nunewar,a Naveen Kotla,aJaya Lakshmi Uppu,b Apoorva Dixit,a Venkatesh
Pooladanda,b Chandraiah Godugu,bNeelima D. Tangellamudi*, a
a Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research,
Hyderabad 500037.
bDepartment of Regulatory Toxicology, National Institute of Pharmaceutical Education and Research,
Hyderabad 500037.
OH
O
MnO2, TEA
G
+
G
HO
HO
N
H
CHCl3, rt, 6h
NH2
O
CH3
N
II
I
G
1-(Indol-2-yl) Phenoxazines
40-62% 15 examples
G= H, 4-Cl, 4-F, 4-CH3, 2,3-di-CH3, 2,4-di-CH3, 2,5-di-CH3, 4-iPr 2-MeO,
4-OMe, 3,4-di-OMe,3,4,5-tri-OMe
Short reaction time
Ability to intercalate DNA
New frameworks
Anti-cancer activity

Synthesis of 1-(Indol-2-yl)-Phenoxazine Hybrids from Quinacetophenone Precursors
and their Biological Evaluation as DNA Intercalating Agents
Saiprasad N. Nunewar,a Naveen Kotla,aJaya Lakshmi Uppu,b Apoorva
Dixit,aVenkateshPooladanda,b Chandraiah Godugu,bNeelima D. Tangellamudi*, a
a Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research,
Hyderabad 500037.
bDepartment of Regulatory Toxicology, National Institute of Pharmaceutical Education and Research,
Hyderabad 500037.
Abstract
An operationally simple reaction between quinacetophenone and aniline derivatives in the
presence of triethylamine at room temperature afforded 1-(indol-2-yl)-phenoxazine hybrids in
good yields. This unique transformation proceeds via sequential aza-Michael attack and
imine formation to yield 1-(indol-2-yl)-phenoxazine hybrids. The most plausible reaction
mechanism was established based on the mechanistic studies carried out with Mass
spectrometry.The synthesized molecules were subjected to in-vitroanti-proliferative
evaluation on various cancer cell lines by MTT assay where compound 8was found to display
signifiacnt IC50 value of 3.71ꢀ±ꢀ0.57ꢀµM on A-549 cell line. The ability of compound 8 to
intercalate DNA was also confirmed by DNA Nanodrop method and viscosity experimental
studies.
Keywords:Quinacetophenone, Aza-Michael attack, Phenoxazine hybrids, DNA intercalation.
1

Introduction
Quinacetophenone (2,5-dihydroxyacetophenone) is an easily accessible precursor for the
syntheses of different pharmacologically active compounds.1 Quinacetophenone is an
important scaffoldfrom the viewpoints of both synthesis and chemical reactivity that is
inherent in the hydroxyl, methyl ketone, carbonyl and aromatic moieties. Thus
quinacetophenone has been used as a precursor to afford privileged motifs of various kinds
viz., chalcones, chromones, flavonoids, coumarins, aurones, phenazines and azoles.2
In presence of various oxidants, quinacetophenone is oxidized to give 2-acetyl-[1,4]-
benzoquinone A,3a,b which is most prominently, attacked by nucleophiles selectively at C-6
providing 6-substituted quinacetophenone, driven by the resonance stabilization associated
with the intermediate I as shown in Figure.1.4 The 1-acetyl group is also known for its high
reactivity which could be attributed to proton donation to the carbonyl group from the
adjacent 2-hydroxy group and resonance of the carbonyl group with the aromatic ring .5,6
Figure 1. 2-acetyl 1,4- benzoquinone (A); Intermediate (I) and formation of 6-substituted quinacetophenone (Q)
by rearomatization
We have earlier reported the synthesis of benzoxazines7a and benzoxazoles7bfrom 2,5-
dihydroxy benzaldehyde by a novel one pot synthesis using manganese dioxide an oxidant.
Further, we have recently reported a methodology for a facile synthesis of phenoxazines from
2

quinacetophenone precursor and anilines in the presence of an oxidizing agent and absence of
any additive.7cBenzoxazines/phenoxazines have been studied intensively as important
heterocyclic system for the synthesis of herbicides, fungicides and therapeutically usable
drugs.8a,b Several benzoxazine derivatives are currently in the development phase as potential
new drugs.11cIndole is a prominent constituent of flower perfumes, pharmacologically active
alkaloids, therapeutics, drug candidates and hormones.9a Some naturally occurring indole
alkaloids including vincristine, vinblastine and vindesine for anti-tumor activity have gained
FDA approval.9b,10 In view of the potential pharmacological effects of indoles and
phenoxazines and recognizing the enhanced synergistic effects that the pharmacophore
hybrids offer,11,12,13we presumed that hybrids containing indole and phenoxazine in a single
heteroaromatic framework might offer potential anti-proliferative effect. Further, our goal
was to evaluate their ability as DNA intercalators owing to their typical structural features
DNA intercalators are molecules capable of fitting between nucleic acid base pairs in a
14
reversible action. The intercalation process
involves the transfer of the intercalating
molecule from an aqueous environment to the hydrophobic space between two adjacent DNA
base pairs. DNA undergoes conformational changes involving an increase in the vertical
separation in order to accommodate the ligand between the base pairs. Once the ligand gets
sandwiched between the DNA base pairs, the stability of the complex is optimized by a
number of non-covalent interactions, ionic interactions and hydrogen bonding.15This
intercalation prevents DNA replication, leading to possible cell death and anti-cancer action
in rapidly growing cancer cells.
The characteristic features of the synthesized compounds like fused ring structure of
phenoxazine chromophore, presence of two basic groups that can be protonated easily and the
relatively planar geometry of the compounds are the typical features of DNA intercalating
agents. These features impart DNA intercalating ability to these chemical structures, as
3

cationic species are more efficient DNA intercalators because they interact better with the
negatively charged DNA sugar–phosphate backbone under physiological conditions.15 The
planar geometry allows the DNA to accommodate the molecule between the base pairs. Thus
we embarked on a journey to synthesize these 1-(indol-2-yl)-phenoxazine hybrids from an
operationally simple reaction between quinacetophenone and aniline derivatives and further
evaluate their ability to intercalate DNA.
Results and discussion
Optimization conditions
As a sequel to the synthesis of phenoxazines,7c we wished to arrive at phenoxazine-indole
hybrids by extending the same methodology by addition of an additive. In this context, we
reasoned that triethylamine might activate the carbonyl for imine formation and a probable
cyclization to indoles to afford our much desired hybrids.
Thus triethylamine was added as an additive to investigate its effect on activating the
carbonyl group to validate our reasoning. As expected, a mere addition of 0.5 equiv. of
triethylamine to the reaction mixture containing quinacetophenone, manganese dioxide and
aniline in toluene, led to the formation of 1-(indol-2-yl)-phenoxazine hybrid. (Scheme. 1)
Scheme 1:Synthesis of phenoxazines and 1-(indol-2-yl)-phenoxazine from quinacetophenone
4

Table 1. Optimization of reaction conditionsa
S.No Oxidant Additive Solvent Temp. Yieldb
1
2
MnO2
MnO2
MnO2
MnO2
MnO2
Ag2O
TEA
TEA
TEA
TEA
TEAc
TEA
-
Toluene
r.t
22%
Toluene Reflux 12%
3
Dioxane
DCE
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
27%
45%
62%
30%
30%d
15%d
13%
09%
19%
10%d
4
5
CHCl3
CHCl3
CHCl3
CH2Cl2
CH2Cl2
DCE
6
7
MnO2
PIDA
8
-
9
PIDA
TEA
TEA
TEA
-
10
11
12
PIDA
Pb(OAc)2
Ag2O
CH2Cl2
CHCl3
aReaction condition: Quinacetophenone (l, 1.0 mmol), Aniline (ll, 2.0 mmol), Oxidant (10.0 mmol), solvent (10
mL), b: The yields were 11% when I and II were taken in 1:1 ratio which increased to 22% when taken in 1:2
ratios, c: 0.5 equivafforded optimum yields, d: Simple phenoxazine was obtained 7c.
Our optimization studies of the reaction started by using I and II in 1:1 ratioas the starting
materials in presence of manganese dioxide as oxidant in toluene, in the presence of 1
equivalent of triethylamine, wherein we obtained 1-(indol-2-yl)-phenoxazine hybrid, 1 in
5

11% yields (Table 1, entry 1). The yields have increased to 22% when I and II were takenin
1:2 ratios. Thus we continued using I and II in 1:2 ratios and 0.5 equiv. of triethylamine for
further optimization. Increase in the amount of triethylamine had no positive effect on the
yields or reaction times. Encouraged by the formation of 1-(indol-2-yl)-phenoxazine hybrids,
we have tried other oxidants such as silver (I) oxide, phenyl iodine diacetate (PIDA) and lead
acetate in different solvents like toluene, dioxane, dichloroethane and chloroform to improve
the yields. Reaction using silver (I) oxide in presence of triethylamine in chloroform showed
slight improvement in yields (Table 1, entry 6). In the absence of triethylamine, PIDA, silver
(I) oxide and manganese dioxide afforded simple phenoxazine in 30% yields (Table 1,
entries 7, 8 and 12).7c When we performed reaction with PIDA, the product 1 was obtained
in 13% and 9% respectively in dichloromethane and dichloroethane respectively (Table 1,
entries 9 and 10). Lead acetate afforded the product 1 in 19% yields (Table 1, entry 11).
While dioxane showed no improvement in yield (Table 1, entry 3), dichloroethane showed
improvement in yields to 45% (Table 1, entry 4). Higher temperatures led to drastic decline
in the yields (Table 1, entry 2). Among all, manganese dioxide and 1 equivalent of
triethylamine in chloroform as solvent offered the best yields of 1 in 62 % (Table 1, entry
5). The yields remained the same when the amount of triethylamine was reduced to 0.5
equiv. The yields of 1 were dropped drastically when the amount of triethylamine was
decreased any further.
The versatility/limitations of this method were evaluated by examining substrate scope by
reacting a varied set of substituted anilines with quinacetophenone (Table 2, isolated yields
listed) using the conditions outlined above. The results show that R-group substitution can
have a significant effect on product yield. For instance, electron-donating methoxy and
methyl substituents at 2, 3, 4 positions with respect to the aniline (Table 2, entries 4-12) are
well tolerated by this reaction. The enhancement of nucleophilicity of carbonyl by electron
6

donating character of the methoxy/ methyl group facilitating the imine formation could be a
plausible explanation for such OMe and methyl group tolerance in o, m and p-positions. It
was reported earlier that trimethoxyphenyl moiety is one of the important structural units
present in several known natural antimitotic agents like combretastatin A-4, colchicine and
podophyllotoxin. The trimethoxyphenyl moiety apparently binds at the colchicine site of
tubulin.16We believed that incorporation of such a crucial structural feature of lead anticancer
compounds into our 1-(2′-indolyl)-phenoxazines may result in a potent anticancer compound.
Thus 12 (entry 12) was synthesized with trimethoxy susbstituents which was obtained in
similar yields.
It is also interesting to note that when G is a halogen (Cl or F), (entries 2-3) the yield drops
substantially (40%; the inductively withdrawing character of Cl and F may have had the
opposite effect of the -OMe group). However, presence of Br has not yielded any product
(entry 13, 14). Not surprisingly, when G is an electron withdrawing group, the reaction did
not afford any product. Thus, anilines with substituents like nitro, cyano and ester afforded no
products (entries 15-22). If nucleophilicity of nitrogen is the criterion, then aryl amines like
adenine and 2-amino pyridine (due to electronegativity of nitrogen) must also either not react
or afford product in low yields. Thus, we have explored the reaction of quinacetophenone
with adenine and 2-amino pyridine to validate our assumption. As expected, no product
formation was seen from 1a (not shown in Table 1).
Table 2. One step synthesis of 1-(indol-2-yl)-phenoxazine hybrids from quinacetophenone
and substituted anilines
7

Entry
-G
Product
Yield
45
1
H
1
40
2
4-Cl
2
40
3
4-F
3
4
5
4
4-CH3
52
5
2,3-di-CH3
35
8

6
2,4-di-CH3
52
6
7
2,5-di-CH3
40
7
8
4-iPr
42
50
45
8
9
2-OMe
9
10
4-OMe
10
9

11
3,4-di-OMe
60
11
12
3,4,5-tri-OMe
62
12
13
14
2-Br
4-Br
48
52
13
14
15
16
17
18
19
20
21
22
4-CH3;3-NO2
2-CH3;4-NO2
2-NO2
NR
NR
NR
NR
NR
NR
NR
NR
-
-
-
-
-
-
-
-
3-NO2
4-NO2
3-CF3
4-COOMe
2-CN
NR: no reaction
10

Plausible mechanism
To study the mechanism of the reaction we have made ESI-MS spectrometry as techniques of
our choice, in order to establish the most plausible mechanism. NMR spectroscopy alone was
useful only in validating the products formed.Figure 2 (A-E) shows the mass spectra of the
reaction at different intervals. We have captured reaction intermediates by spectroscopic
techniques and established the most probable reaction pathway.
In presence of manganese dioxide, quinacetophenone is oxidized to give 2-acetyl-[1,4]-
benzoquinone A,3a, 3bwhich is most prominently, attacked by nucleophiles selectively at C-6
providing 6-substituted quinacetophenone, Thus, 1 min following the addition of aniline, the
presence of a prominent peak at m/z 244. 0977 amu corresponding to [B-I +H]+ in Figure 2A
provided key information on the composition of intermediate B-I, thus allowing the
intermediate to be associated with the formation of the aza-Michel addition product for the
eventual formation of the desired product.3b
Figure 1A.ESI-MS of the reaction mixture, 1 min after the addition of aniline to quinacetophenone. The signals
due to species [B-l +H] + [P + H] + are encircled.
11

Figure 2B.ESI-MS of the reaction mixture, 1.5 h after the addition of aniline to quinacetophenone. The signals
due to species [P +H] + are encircled.
Figure 2C. ESI-MS of the reaction mixture, 5h after the addition of aniline to quinacetophenone. The signals
due to [B-l +H] + and [P + H] +are encircled.
12

Figure 2D.ESI-MS of the reaction mixture, 5h after the addition of aniline to quinacetophenone. The signals
due to [D+H] + are encircled.
Figure 2E. ESI-MS of the reaction mixture 5h after the addition of aniline to quinacetophenone. The signals due
to [PI +H2O + H] + encircled.
The peak at m/z 244.0977 amu [B-l +H] + was found to coexist with the second molecular ion
peak at m/z 242.0820 amu corresponding to [P+ H] + which is indicative of rapid cyclization
of aza-Michael adduct to phenoxazine P in accordance with the mechanism proposed in
13

Scheme 1(Figure 2A) Further, ESI-MS of sample after 1.5 h showing a tall peak at m/z
242.0821 confirmed the formation of P(Phenoxazine) in the reaction mixture (Figure 2B)
The sample taken after 5 h showed the less intense peaks corresponding to [B-l + H] + and [P
+ Na] + indicating a decreasing concentration of the intermediates (Figure 3C). The ESI-MS
of the reaction mixture after 5 h also showed the rising of peak at m/z 317.1284 amu
+
corresponding to [D + H] indicating the initiation of imine formation (Figure 2D). The
+
strong peak at m/z 333.1247 amu corresponding to [PI + H2O +H] after 5h is indicative of
the formation of PI (Figure 2E). There are peaks detected by ESI-MS, which may be species
that may have formed during the ionization process and may be slightly different from the
true species present in the reaction.
The most plausible mechanism delineated for the reaction to account for the formation
of product is shown in Scheme 2. The initial event may be considered as the nucleophilic
attack of amine in an aza-Michael fashion on 1-acetyl benzoquinone A, to generate an
intermediate B-I. Intermediate B-I undergoes subsequent electro cyclization to form C and
undergoes eventual rearomatization to form the initial phenoxazine P. P in the presence of
triethylamine reacts with a second molecule of aniline to form a phenolic Schiff base D. D
on oxidation in the presence of MnO2 forms a transient enamine E, which furtherundergoes
1,5-H shift to form a cyclization precursor F. F on annulation forms an exocyclic imine G
which finally rearomatizes to the final product PI(Phenoxzine-Indole).
14

H
O
N
OH
MnO2
O
O
O
HO
O
N
O
H
H
H O
O
HO
O
H2N
A
B-I
C
Exact Mass: 243.0895
O
O
O
Et3N
MnO2
N
H
HO
N
H
N
H O
O
NH2
H
O
N
HN
H
Exact Mass: 241.0739
P
D
E
Exact Mass: 316.1212
Exact Mass: 316.1212
O
O
O
N
H O
N
O
N
HO
H
MnO2
H
H
1,5
- H
shift
HN
N
N
Et3N
H
Exact Mass: 316.1212
Exact Mass: 314.1055
Exact Mass: 314.1055
F
G
PI
Scheme 1. Plausible mechanism
Biological activity
The newly synthesized indolyl phenoxazine derivatives 1-12 were screened for their
cytotoxicity against various human cancer cell lines A549 (human lung cancer), MG-63
(human bone cancer), BT-474 (human brest cancer), HepG2 (human hepatic cancer), HCT-
116 (human colon cancer), along with normal lung epithelial tissue cells (L-132) purchased
from American Type Culture Collection (ATCC) USA, by utilizing 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) assay.16 The IC50 (μM) values from the in
vitro cytotoxic evaluation of the synthesized derivatives 1-12as well as standard drugs are
reflected in Table 3.Remarkably, the tested derivatives showed good to moderate cytotoxicity
on the selected cancer cell lines. Preliminary results indicated that among all the
derivatives 6,7and8 showed significant antiproliferative activities against the tested cancer
15

cell linespointing towards hydrophobicity which describes the tendency towards participation
in the London dispersion forceswhich are quite important for hydrophobicsubstances
tointeract within themselves and other neighbouring moieties. Compound 8 possessing 4-iso-
propyl substituent was most active with an IC50 value of 3.71ꢀ±ꢀ0.57ꢀµM, followed by
compound
6(4.43ꢀ±ꢀ0.64ꢀµM)with
3,4-dimethyl
substituent
and
compound
7(6.08ꢀ±ꢀ0.78ꢀµM)with 2,5-dimethyl substituent against A-549. Compound 7was also found
to be fairly active against HCT-116 (9.19ꢀ±ꢀ0.19ꢀµM).compound 2, 3 and 9 with the F, Cl and
2-methoxy respectively displayed weak antiproliferative activities with the IC50 values >
30.0ꢀµM. Since halogen substituents increase the lipohilicity, the molecule might probably
assume a bigger size.Compound1, 4, 5 which areeither unsubstitutedwith hydrophobic
substituents on 2-, 3-, and 4- positions, and10, 11with methoxy substitutionswere found to be
inactive.Thus, steric bulk at4-, and 5- positions of phenoxazine seem crucial for activity. (not
shown)
Table 3. In vitro anticancer activity of compounds 1-12.
Compounds
A549b
MG-63c
>30
>30
19.19 ± 0.17
BT-474d
HepG2e
>30
>30
HCT-116f
>30
>30
12.04 ± 1.78 24.11 ± 0.94
19.52 ± 1.09 20.17 ± 0.08 10.23 ± 0.32
L132g
>30
>30
2
>30
>30
3
>30
>30
8
15.94 ± 1.42
>30
>30
3.71 ± 0.57
11.08 ± 1.02
>30
12
14.25. ± 1.14
25.55 ± 1.02
21.04 ± 0.95
19.15 ± 0.97
0.78 ± 1.55
9
28.56 ± 1.04 25.33 ± 0.52 18.19 ± 1.01 15.36 ± 1.20
16.15 ± 1.06 10.29 ± 0.12
25.48 ± 0.94 13.55 ± 0.18 18.55 ± 0.44 13.96 ± 0.51
2.81 ± 1.17 2.44 ± 0.17 0.68 ± 0.11 0.95 ± 0.84
7
6.08 ± 0.78
4.43 ± 0.64
1.08 ± 0.28
7.13 ± 0.78
9.19 ± 0.19
6
Doxorubicini
[a] 50% Inhibitory concentration after 72ꢀh of drug treatment, [b] human lung cancer, [c] human bone
cancer, [d] human brest cancer, [e] human hepatic cancer, [f] human colon cancer, [g]human lung epithelial
16

cells, [i] Reference compound. All the values are expressed as Meanꢀ±ꢀSEM in which each treatment was
performed in triplicate wells.
DNA-Nanodrop method
DNA intercalation is determined by nanodrop spectrophotometric analysis which measures
the absorbance and calculates the concentration of nucleic acids (260 nm). The blue and red
shifts, hypo and hyperchromic effects, are spectral properties of DNA-drug interaction, which
are closely related with the double helix structure [17a]. The possibility of interaction of
ligands to DNA can be investigated according to changes in the absorption spectra before and
[17b]
after the reaction. In general, an intercalation induces the hyperchromicity at 260ꢀnm.
Such a rise in the absorbance on intercalation is observed owing to the possible enhancement
of axial length of the DNA. Hypochromicity at the maximum absorption of DNA indicates
the compaction of DNA due to the electrostatic interaction whereas binding of certain groove
binders cause little or no change in the absorbance.18b, 18c
.
In our present experiment, the CT-DNA concentration was fixed at 50 × 10−6 M, in TAE
buffer and incubated it with test compound (5µM), Ethidium Bromide (1 µM) for ten
minutes. TAE buffer was used as blank and absorbance was determined by using Nano
DropTM 2000/2000c Spectrophotometer (Thermo fisher scientific, USA). We have observed
that in comparison to the control DNA, Ethidium Bromide, a known intercalator and
compound 8 have shown an increase in the intensity of absorbance at 260nm, indicating their
binding ability to bind to DNA thereby validating the possibility of DNA intercalation.
17

Figure 2. DNA intercalation studies A) Spectrophotometric analysis by nanodrop spectrophotometer for
determination of absorbance: EtBr and Compound 8 have enhanced DNA absorptivity compared to control. B)
Relative viscosity measurements to determine the DNA intercalation: Hoechst-33258 and EtBr used as reference
standards.
DNA intercalation using viscosity measurements
We have performed viscosity measurements to track the changes of DNA viscosity in the
presence of compound 8 in order to validate the internal binding to DNA. When a ligand
binds externally either in the major grove or minor grove, a slight bending of DNA helix
leads to decrease in relative length of DNA as observed by a decrease in its viscosity.
However, when the ligand intercalates between the DNA bases, it results in local unwinding
and lengthening of DNA leading to an increase in viscosity. Here, we have used EtBr, an
intercalator as a positive control along with Hoechst-33258, a known DNA minor groove
binder and compound 8 in our viscosity measurements to compare the changes in viscosity on
their reaction with DNA. In the viscosity experiments we have conducted, although the
viscosity of compound 8 was not as high as that of Ethidium bromide (EtBr), the DNA
showed higher viscosity as compared to Hoechst 33258. Viscosity measurements were
conducted using a Lovis 2000 M/ME Rolling-ball viscometer (Anton Paar GmbH, Graz,
Austria), based on the falling ball principle. CT-DNA concentration was fixed at 50 × 10−6
M, in 100 mMTris-HCl (pH 7.4). Compound 8was added at various concentrations to CT-
DNA solution. A calibrated 1.59 mm glass capillary containing a steel ball was filled with the
sample and viscosity was measured by measuring the ball falling time at angles in the range
from 20o to 70o. Ethidium bromide, Hoechst-33258 were used as controls. Data was
18

represented graphically as (ŋ/ŋ0)1/3 vs. the ratio of the concentration of the hybrid to CT-
DNA, where ŋ is the viscosity of CT-DNA in the presence of the derivative and ŋ0 is the
viscosity of CT-DNA solution.
Conclusion
In conclusion, we have demonstrated the utility of the versatile, easily accessible building
block quinacetophenone, for the synthesis of 1-(indol-2-yl)-phenoxazines via C-N formation
as a key step. ESI-MS was used as a tool to identify the intermediates in order to establish the
mechanism. The method is tolerant of electron donating groups.In vitro cytotoxic evaluation
for these derivatives were screened against the seleted panel of human cancer cell lines, from
preliminary screeing data it was clear thatcompound 8 displyaed signifiacnt IC50 value of
3.71ꢀ±ꢀ0.57ꢀµM on A-549 cell line. DNA nanodrop method and viscosity experiments have
ben conducted to study the changes in absorbances and viscosity of DNA to confirm the
DNA intercalating ability of compound 8.
Experimental section
General Methods: All reactions were carried out under an inert atmosphere with dry solvents
unless otherwise stated. Reactions were monitored by thin layer chromatography (TLC) on
silica gel plates (60 F254), using UV light detection. Visualization of the spots on TLC plates
was achieved either by UV light or by staining the plates in 2, 4-Dinitro phenyl hydrazine/
Ninhydrin stains and charring on a hot plate. Flash chromatography was performed on silica
1
13
gel (200-400 mesh) using distilled hexane, ethyl acetate, dichloromethane. H NMR and C
NMR spectra were recorded in CDCl3 solution by using Bruker 500 MHz NMR spectrometer.
Chemical shifts are reported as δ values relative to internal CDCl3 δ 7.26 or TMS δ 0.0,
1
13
1
DMSO-d6 δ 2.5 for H NMR and CDCl3 δ 77.0, DMSO-d6 δ 39.52 for C NMR. H NMR
data were recorded as follows: chemical shift [multiplicity, coupling constant(s) J (Hz),
relative integral] where multiplicity is defined as s (singlet), d (doublet), t (triplet), q
19

(quartet), dd (doublet of doublet), m (multiplet), bs (broad singlet). FT-IR spectra were
recorded on PerkinElmer spectrometer. High-resolution mass spectra (HR-MS) [ESI] + were
obtained using Agilent Q-TOF mass spectrometer 6540 series instrument
General procedure for the synthesis of 1-(indol-2-yl)-phenoxazine derivative. (1-14):
A suspension of quinacetophenone I (0.152 g, 1 mmol) and MnO2 (0.860g, 10 mmol) is
cooled in an ice bath to 0 °C under constant stirring. Anilines II (0.182 mL, 2.0 mmol) in
anhydrous chloroform and triethylamine (0.068 mL, 0.5 mmol) were added and stirred at rt
for 6h. The mixture was filtered through celite. Removal of the solvent under reduced
pressure followed by column chromatography (5% EtOAc/Hexane) yielded pure 1-(indol-2-
yl)-phenoxazines (PI) in moderate to good yields.
1-(3H-indol-2-yl)-10H-phenoxazin-2-ol (1)
Orange solid, Yield (45%), m. p. = 165-167 °C, Rf 0.5 (30:70 Ethyl acetate: Hexane);1H
NMR (500 MHz, CDCl3): δ 8.19 (s, 1H), 7.43–7.35 (m, 4H), 7.30–7.21 (m, 4H), 7.13 (d, J =
7.5 Hz, 2H), 6.04 (s, 1H), 2.65 (s, 2H). 13C NMR (125 MHz, CDCl3): δ 178.58, 177.88,
146.43, 139.22, 137.06, 129.71, 129.05, 127.05, 126.17, 124.28, 122.79, 107.80, 99.14,
32.61. IR (neat, cm-1): 3291, 3046, 2804, 1649, 1626, 1615, 1509, 1233, 1156. HRMS m/z
calculated for C20H14N2O3 [M + H2O + H] +: 333.1239 found: 333.1238.
7-chloro-1-(5-chloro-3H-indol-2-yl)-10H-phenoxazin-2-ol (2)
1
Orange solid, Yield (40%), m. p. = 203-205 °C, Rf 0.4 (30:70 Ethyl acetate: Hexane); H
NMR (500 MHz, CDCl3): δ 8.12 (s, 1H), 7.38 (d, J = 8.7 Hz, 2H), 7.32 (d, J = 8.65 Hz, 2H),
7.17 (d, J = 8.7 Hz, 2H), 7.04 (d, J = 8.61 Hz, 2H), 5.89 (s, 1H), 2.59 (s, 2H). 13C NMR (125
MHz, CDCl3): δ 178.42, 177.72, 162.35, 161.66, 160.38, 159.69, 146.89, 134.74, 132.84,
126.04, 125.97, 125.07, 125.01, 119.08, 166.62, 166.09, 115.90, 107.38, 32.63. IR (neat, cm-
1): 3289, 3076, 2954, 2849, 2917, 1652, 1615, 1593, 1229, 1170, 753. HRMS m/z calculated
for C20H12Cl2N2O3 [M + H2O + Na] +:423.0279 found: 423.0269.
20

7-fluoro-1-(5-fluoro-3H-indol-2-yl)-10H-phenoxazin-2-ol (3)
o
Orange coloured solid, Yield (40%), m. p = 200-202 C, Rf 0.5 (30:70 Ethyl acetate:
Hexane); 1H NMR (500 MHz, CDCl3): δ 8.08 (s, 1H), 7.26–7.03 (m, 8H), 5.86 (s, 1H), 2.64
13
(s, 2H). C NMR (125 MHz, CDCl3): δ 178.42, 177.72, 162.35, 161.36 (d, J = 247.23 Hz),
160.67 (d, J = 247.67 Hz), 158.48, 146.88, 135.12, 132.87, 129.27, 126.01 (d, J = 8.37 Hz),
125.05 (d, J = 8.26 Hz), 122.05, 119.63, 116.80 (d, J = 23.16 Hz), 116.01 (d, J = 23.16 Hz),
107.76, 98.96, 32.66. IR (neat, cm-1): 3304, 3081, 2923, 2852, 1651, 1615, 1289, 1185, 734.
HRMS m/z calculated for C20H12F2N2O3 [M + H2O + H] +:369.1051 found: 369.1051.
7-methyl-1-(5-methyl-3H-indol-2-yl)-10H-phenoxazin-2-ol (4)
o
Orange solid, Yield (52%), m. p. = 198-203 C, Rf 0.5, (30:70 Ethyl acetate: Hexane);1H
NMR (500 MHz, CDCl3): δ 8.16 (s, 1H), 7.21 (d, J = 10 Hz, 2H), 7.16–7.11 (m, 4H), 7.01 (d,
J = 10 Hz, 2H), 5.98 (s, 1H), 2.65 (s, 2H), 2.35 (s, 6H). 13C NMR (125 MHz, CDCl3): δ
178.45, 177.83, 146.70, 136.95, 136.20, 134.40, 130.24, 129.67, 123.97, 122.76, 32.62,
21.11, 20.99. IR (neat, cm-1): 3316, 2922, 2852, 1743, 1645, 1621, 1287, 1191. HRMS m/z
calculated for C22H18N2O3 [M + H2O + H] +: 361.1552 found: 361.1552.
1-(6,7-dimethyl-3H-indol-2-yl)-8,9-dimethyl-10H- phenoxazin-2-ol (5)
Orange solid, Yield (35%), m. p. = 169-173 oC, Rf0.5 (30:70 Ethyl acetate: Hexane);1H NMR
(500 MHz, CDCl3): δ 8.00 (s, 1H), 7.15–7.04 (m, 5H), 6.85 (d, J = 7.5 Hz, 1H), 5.50 (s, 1H),
2.72 (s, 2H), 2.31 (s, 6H), 2.16 (s, 6H). 13C NMR (125 MHz, CDCl3): δ 178.14, 177.84,
148.18, 138.69, 137.94, 138.02, 135.01, 130.94, 132.08, 128.94, 128.86, 126.35, 125.82,
122.80, 122.44, 107.37, 99.14, 32.60, 20.41, 14.41, 13.98. IR (neat, cm-1): 3314, 2919, 1737,
1651, 1614, 1515, 1386, 1217. HRMS m/z calculated for C24H22N2O3 [M + H2O + H]
+:389.1865 found: 389.1862.
1-(5,7-dimethyl-3H-indol-2-yl)-7,9-dimethyl-10H- phenoxazin -2-ol (6)
21

Orange solid, Yield (52%), m. p. = 202-207oC, Rf 0.5, (30:70 Ethyl acetate: Hexane);1H
NMR (500 MHz, CDCl3): δ 7.89 (s, 1H), 7.03 (d, J = 8.5 Hz, 2H), 6.97 (d, J = 8.0 Hz, 2H),
6.89 (d, J = 8.0 Hz, 1H), 6.80 (d, J = 7.5 Hz, 1H), 5.51 (s, 1H), 2.64 (s, 2H), 2.24 (d, J = 4.5
Hz, 6H), 2.15 (d, J = 8.0 Hz, 6H). 13C NMR (125 MHz, CDCl3): δ 178.15, 177.81, 147.85,
137.10, 132.85, 132.50, 132.03, 131.46, 127.63, 127.15, 124.45, 99.99, 32.62, 21.03, 20.95,
17.93, 17.61. IR (neat, cm-1): 3313, 2922, 2652, 1731, 1560, 1515. HRMS m/z calculated for
C24H22N2O3 [M + H2O + H] +: 389.1865 found: 389.1865.
1-(4,7-dimethyl-3H-indol-2-yl)-6,9-dimethyl-10H-phenoxazin -2-ol (7)
Orange solid, Yield (40%), m. p. = 188-193oC, Rf 0.3 (30:70 Ethyl acetate: Hexane);1H NMR
(500 MHz, CDCl3): δ 8.00 (s, 1H), 7.16 (d, J = 10.0, 7.5 Hz, 1H), 7.12 (d, J = 15 Hz, 1H),
7.05 (s, 1H), 7.00 (d, J = 7.5 Hz, 2H), 6.81 (s, 1H), 5.66 (s, 1H), 2.70 (s, 2H), 2.32 (d, J = 15
Hz, 6H), 2.23 (d, 13.5 Hz, 6H). 13C NMR (125 MHz, CDCl3): δ 178.27, 177.83, 147.56,
138.39, 137.00, 136.96, 136.28, 135.03, 131.21, 130.57, 129.75, 129.26, 128.14, 127.91,
125.10, 124.86, 107.32, 99.96, 32.56, 20.93, 20.85, 17.57, 17.25. IR (neat, cm-1): 3254,
3218, 2922, 2853, 1695, 1615, 1471, 1238, 1121. HRMS m/z calculated for C24H22N2O3 [M +
H2O + H] +: 389.1865 found: 389.1867.
7-isopropyl-1-(5-isopropyl-3H-indol-2-yl)-10H-phenoxazin-2-ol (8)
1
Orange solid Yield (42%), m. p. = 148-151°C, Rf 0.4, (30:70 Ethyl acetate: Hexane); H
NMR (500 MHz, CDCl3): δ 8.11 (s, 1H), 7.18 (d, J = 4.0 Hz, 2H), 7.17 (d, J = 8.5 Hz, 2H),
7.09 (d, J = 8.5 Hz, 2H), 6.98 (d, J = 8.5 Hz, 2H), 5.94 (s, 1H), 2.87-2.81 (m, 2H), 2.58 (s,
2H), 1.18 (dd, J = 2.0, 2.0 Hz, 12H). 13C NMR (125 MHz, CDCl3): δ 178.49, 177.82, 147.81,
147.17, 146.68, 136.82, 134.66, 127.62, 127.03, 124.06, 122.79, 107.70, 99.15, 33.72, 32.60,
23.87. IR (neat, cm-1): 3218, 2960, 2868, 1739, 1693, 1610, 1635, 1576, 1555, 1417, 1261,
1149. HRMS m/z calculated for C26H26N2O3 [M + H2O + H] +: 417.2178 found: 417.2175.
9-methoxy-1-(7-methoxy-3H-indol-2-yl)-10H-phenoxazin-2-ol (9)
22

Orange solid, Yield (50%), m. p. = 150-155oC, Rf 0.5,(30:70 Ethyl acetate: Hexane);1H NMR
(500 MHz, CDCl3): δ 8.53 (s, 1H), 7.30 (d, J = 8.75 Hz 1H), 7.27 (m, 2H), 7.16 (d, J = 8.35
Hz 1H), 7.10-7.02 (m, 2H), 6.93-6.83 (m, 4H), 6.06 (s, 1H), 3.85 (s, 3H), 3.74 (s, 3H), 2.59
(s, 2H). 13C NMR (125 MHz, CDCl3): δ178.31, 177.72, 158.49, 158.02, 156.36, 131.96,
129.66, 125.33, 125.04, 124.68, 119.34, 119.05, 115.52, 114.94, 114.29, 113.64, 107.42,
98.89, 55.52, 55.44, 32.64. IR (neat, cm-1): 3318, 2924, 2853, 1689, 1639, 1590, 1462, 1211.
HRMS m/z calculated for C22H18N2O5 [M + H2O + H] +: 393.1450 found: 393.1452.
7-methoxy-1-(5-methoxy-3H-indol-2-yl)-10H-phenoxazin-2-ol (10)
Orange solid, Yield (45%), m.p 172-176°C, Rf 0.4(30:70 Ethyl acetate: Hexane);1H NMR
(500 MHz, DMSO-d6): δ 8.05 (s, 1H), 7.09 (d, J = 8.5 Hz, 2H), 6.98 (d, J = 8.0 Hz, 2H), 6.86
(d, J = 9.0 Hz, 2H), 6.80 (d, J = 8.5Hz, 2H), 5.81 (s, 1H), 3.74 (d, J = 6.5 Hz, 6H), 2.62 (s,
2H). 13C NMR (125 MHz, DMSO-d6): δ 178.28, 178.11, 152.68, 147.00, 128.37, 128.07,
127.62, 126.29, 125.72, 124.20, 121.32, 120.78, 112.64, 112.21, 97.78, 56.32, 56.12, 32.51.
IR (neat, cm-1): 3217, 2923, 2840, 1688. HRMS m/z calculated for C22H18N2O5 [M + H2O +
H] +: 393.1450 found: 393.1448.
1-(5,6-dimethoxy-3H-indol-2-yl)-7,8-dimethoxy-10H-phenoxazin -2-ol (11)
Orange coloured solid, Yield (60%), m. p. = 192-195oC, Rf 0.5 (30:70 Ethyl acetate:
Hexane);1H NMR (500 MHz, DMSO-d6): δ 9.38 (s, 1H), 7.01 (d, J = 9.0 Hz, 2H), 6.90 (d, J
= 8.5 Hz, 2H), 6.81 (s, 1H), 6.70 (d, J = 7.5 Hz, 1H), 5.73 (s, 1H), 3.77 – 3.71 (m, 12H), 2.26
(s, 2H). 13C NMR (125 MHz, CDCl3): δ 178.63, 178.47, 149.60, 149.06, 148.49, 147.73,
147.48, 132.60, 130.95, 116.87, 112.55, 111.91, 109.41, 109.23, 109.16, 96.27, 79.64, 56.23,
56.15, 56.10, 56.02, 32.43. IR (neat, cm-1): 3292, 2927, 2837, 1647, 1605, 1594, 1557, 1513,
+
1295, 1262, 1162. HRMS m/z calculated for C24H22N2O7 [M + H2O + H] : 453.1662 found:
453.1665.
6,7,8-trimethoxy-1-(4,5,6-trimethoxy-3H-indol-2-yl)-10H-phenoxazin-2-ol (12)
23

o
1
Orange solid, Yield (62%), m. p. = 125-130 C, Rf 0.5 (30:70 Ethyl acetate: Hexane); H
NMR (500 MHz, CDCl3): δ 8.12 (s, 1H), 6.46 (s, 2H), 6.35 (s, 2H), 6.02 (s, 1H), 3.85-3.82
(m, 18H), 3.82 (s, 6H), 2.64 (s, 2H). 13C NMR (125 MHz, CDCl3): δ178.94, 178.57, 153.72,
153.68, 153.27, 148.04, 136.32, 136.10, 135.92, 135.41, 133.79, 124.33, 123.10, 119.76,
109.79, 109.37, 108.72, 102.76, 102.44, 102.35, 96.96, 6062, 60.57, 56.57, 56.44, 32.36. IR
(neat, cm-1): 3258, 2924, 2852, 1586, 1452, 1416, 1228, 1182, 1123, 1042. HRMS m/z
calculated for C26H26N2O9 [M + H2O + H] +:513.1873 found: 513.1871.
1-(9-bromo-3-((2-bromophenyl)amino)-2-hydroxy-10H-phenoxazin-1-yl)ethan-1-one (13)
1
Brown Solid; Yield- 48%, m. p. = 202-202 °C, Rf 0.3 (30:70 Ethyl acetate: Hexane); H
NMR (500 MHz, DMSO- d6): δ 12.34 (s, 1H), 9.39 (s, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.68 (d,
J = 8.0 Hz, 1H), 7.51 (t, J = 8.0 Hz, 1H), 7.46 (d, J = 6.5 Hz, 1H), 7.37 (t, J = 7.6 Hz, 1H),
13
7.32 (d, J = 7.0 Hz, 2H), 7.23 (t, J = 7.6 Hz, 1H), 5.19 (s, 1H), 2.44 (s, 3H), C NMR (125
MHz, DMSO- d6): δ 200.27, 178.31, 178.02, 148.45, 138.84, 136.37, 133.95, 133.08,
129.49, 129.05, 128.65, 128.39, 127.95, 120.71, 119.56, 108.86, 98.03, 32.59. IR (neat, cm-1):
3277.84, 3003.17,2 779.41, 1737.20, 1356.93, 1288.47, 1027.99, 759.24, 661.20. HRMS m/z
calculated for C20H14Br2N2O3 [M + H] +: 490.9429 found 490.9431.
1-(7-bromo-3-((4-bromophenyl)amino)-2-hydroxy-10H-phenoxazin-1-yl)ethan-1-one (14)
1
Red Solid; Yield- 52%, m. p. = 215-217 °C, Rf 0.3 (30:70 Ethyl acetate: Hexane); H NMR
(500 MHz, DMSO- d6): δ 11.07 (s, 1H), 9.60 (s, 1H), 7.61 (s, 1H), 7.47 - 7.38 (m, 5H), 7.31
(t, J = 8.0 Hz, 1H), 7.19 (d, J = 8.8 Hz, 1H), 5.86 (s, 1H), 2.32 (s, 3H), 13C NMR (125 MHz,
DMSO- d6): δ 199.81, 179.56, 179.04, 147.46, 139.92, 131.69, 131.08, 129.31, 128.87,
127.12, 126.8, 123.67, 123.0, 122.3, 121.74, 110.26, 97.69, 32.30. IR (neat, cm-1): 3300.03,
3000.28, 2950.35, 1737.20, 1653.44, 1310.23, 1215.30, 1000.25, 820.30, 650.80. HRMS m/z
calculated for C20H14Br2N2O3 [M + H] +: 490.9429 found 490.9425.
24

Pharmacology
Cell culture
Human cancer lines such as A549 (human lung cancer), MG-63 (human bone cancer), BT-
474 (human brest cancer), HepG2 (human hepatic cancer), HCT-116 (human colon cancer),
along with human lung epithelial tissue cells (L-132) were procured from National Centre for
cell science (NCCS) Pune India. All cells were maintained with appropriate DMEM and
RPMI 1640 media (Sigma Aldrich) cells were also supplemented with 10% Fetal bovine
serum stabilized with 1% antibiotic-anti mycotic solution (Sigma Aldrich) in CO2 incubator
o
at 37 C. when cells reached up to 80-90% confluency, they were sub-cultured by using
0.25% trypsin/1mM EDTA for further passage. Compounds were dissolved in DMSO to
prepare the stock solution of 10 mM. Further dilutions were made accordingly with
respective to media to get required concentration.
Evaluation of in vitro cytotoxic assay
Cytotoxicity of synthesized compounds was evaluated by MTTassay. Briefly, cells were
seeded in 96-well plates at a density of 4000cells per well in 100 µL of complete medium and
allowed to growovernight for attachment onto the wells. Then the cells were treated with
various concentrations of the compounds for a period of 72h. After the treatment, 100 µL of
MTT (0.5 mg/mL) was added and incubated at 37 °C for 4h. Then MTT reagent was
aspirated and the formazan crystals formed were dissolved by the addition of 200 µL of
DMSO for 20 mins at 37 °C. The quantity of formazan product wasmeasured by using a
spectrophotometric microtiter plate reader(Spectra max, M4 molecular devices, USA) at 570
nm wavelength.Initially, cytotoxicity effects of the synthetic derivatives were screened
by MTT assay at 30 µM concentration. Among these, the compoundwhich showed IC50 value
< 30 µM was used for the dose dependentstudies at various concentrations ranging from 0.78
µM to 30 µM inserial dilutions and the percentage of cytotoxicity was calculated.
25

DNA Nanodrop method
DNA intercalation is determined by nanodrop spectrophotometric analysis. The
intercalatingagents decrease the absorption and increase the wavelengths. In our experiment,
weincubated the 50 μM of calf thymus DNA (Sigma-Aldrich, USA) with compound 8,
Ethidium bromide (EtBr) Hoechst 32258 for 10 min at 1 μM concentration. Later, the TAE
buffer used as blank and absorbance and concentrations were determined by
NanoDrop™ 2000/2000c Spectrophotometer (Thermo fisher scientific, USA).
DNA binding using viscosity measurements
Relative studies were determined by Ostwald viscometer. The titrations were conducted for
most active compound 8, EtBr and Hoechst-33258 at 1.0 μM while and they are added to CT-
DNA solution(50 μM) and exposed to viscometer. Here, the DNA solution was prepared in
100 mMTrisHCl (pH 7.0). Data were represented as (ŋ/ŋo)1/3 versus the ratio of the
concentration of thecompound 8, EtBr and Hoechst-33258 to CT-DNA, where ŋ is the
viscosity of CTDNA in the presence of intercalating agents and ŋo is the viscosity of CT-
DNA alone.
Acknowledgements
The authors are thankful to NIPER Hyderabad for facilities and Mr. B. Prashanth, NIPER
Hyderabad for ESI-MS studies. The authors are also thankful to Department of Science and
Technology for the financial support (DST-File No: SB/S1/OC-55/2013).
Author Contributions
The manuscript was written through contributions of all authors. All authors have given
approval to the final version of the manuscript.
26