Combined palladium/eosin Y-catalysed direct synthesis of anticancer biarylquinolinooxazocino-quinoxaline-1-ones under visible light in one-pot sequence: a revisited proof of concept

Article abstract of DOI:10.1007/s00706-020-02641-2

Abstract: A novel, efficient, and green catalytic system under visible light irradiation has been introduced for the synthesis of anticancer biarylquinolooxazocino-quinoxaline-1-ones in one-pot sequence. The developed synthetic approach will be successfully utilized for the synthesis of various bioactive biarylquinolone in near future. Also, the cytotoxicity profile of these scaffolds has unveiled their superb cytoselectivity in HeLa and MCF-7 cell line compared to cisplatin. Graphic abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00706-020-02641-2

Monatshefte für Chemie - Chemical Monthly
https://doi.org/10.1007/s00706-020-02641-2
ORIGINAL PAPER
Combined palladium/eosin Y‑catalysed direct synthesis of anticancer
biarylquinolinooxazocino‑quinoxaline‑1‑ones under visible light
in one‑pot sequence: a revisited proof of concept
Nilmadhab Roy1 · Priyankar Paira1
Received: 11 March 2020 / Accepted: 8 June 2020
© Springer-Verlag GmbH Austria, part of Springer Nature 2020
Abstract
A novel, efcient, and green catalytic system under visible light irradiation has been introduced for the synthesis of antican-
cer biarylquinolooxazocino-quinoxaline-1-ones in one-pot sequence. The developed synthetic approach will be successfully
utilized for the synthesis of various bioactive biarylquinolone in near future. Also, the cytotoxicity profle of these scafolds
has unveiled their superb cytoselectivity in HeLa and MCF-7 cell line compared to cisplatin.
Graphic abstract
Keywords Anticancer drug · Biarylquinolinooxazocino-quinoxaline-1-ones · Green technology · Eosin Y · Cellular
imaging
Introduction
naturally obtained biologically active compounds possessed
heterocyclic ring as fundamental ingredients in their skele-
ton. Therefore, these compounds and their modifed products
were then started to be utilized as famous pharmacophore
for preparation of drugs. In this situation, the more signif-
cant N-heteroaromatic compounds snatched the attention of
researchers for being utilized as drugs. It has been seen that
among the numerous N-heterocyclic compounds, quinolones
and quinoxalines are two valuable nuclei as they conform
the framework of several antibiotics like ofoxacin [1–3],
norfoxacin [4], A-62176 [5], echinomycin, leromycin, and
actinomycin [6–8]. Two nitrogen atoms at the para position
of quinoxaline ring are very much responsible for exhibit-
ing wide range of physicochemical and biological activi-
ties [9, 10]. Hence, quinoxaline is a versatile heterocyclic
moiety comprising of huge therapeutic potential [11]. It
showcases several biological activities such as anticancer
[12–18], antitubercular [19, 20], antiamoebic [21], antimi-
crobial [22–27], anti-infammatory [28], antimalarial [29],
The unrestricted pervasion of cancer throughout the world
is now a matter of great trepidation to the human being and,
therefore, the researchers have engrossed themselves in deep
research in view to fnd out the anticancer drug after contem-
plating the perilous impact of cancer. In this circumstance,
when stepping along the way of long research, an abundance
of natural products on the lap of Mother Nature drew the
attention of researchers and they observed that most of the
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00706-020-02641-2) contains
supplementary material, which is available to authorized users.
* Priyankar Paira
priyankar.paira@vit.ac.in
1
Department of Chemistry, School of Advanced Sciences,
Vellore Institute of Technology, Vellore, Tamilnadu 632014,
India
Vol.:(0123456789)
1 3

N. Roy, P. Paira
antiviral [30], antibiotic (echinomycin, leromycin, actino-
mycin) [31, 32], antidepressant [33], antichagasic [34, 35],
and kinase inhibitory activity [36]. In addition to this, qui-
noxaline nucleus is also associated with the application in
dyes [37], organic semiconductors [38], dihydroannulenes
[39], cavitands [40], pesticides [41], corrosion inhibitors
[42], and an efcient electroluminescent material. Like-
wise, quinolone derivatives are also prevalent in a variety of
pharmacologically active synthetic and natural compounds
and quinolones can display the antiseptic, antipyretic, and
antiperiodic properties and are useful as antimalarials. The
discovery of chloroquine, the most famous drug containing
this scafold, resulted in control and treatment of malaria for
decades. Also, the quinolone drugs are capable of inhibiting
lymphocyte apoptosis [43] and can be used as the skeleton
of DNA intercalating agents [44] as well as very much harsh
for a number of transplantable tumors. The DNA intercala-
tive power can be conferred to these drugs only by giving
the planarity to them and hence the presence of planar lin-
early fused tri-cyclic system can aford them the efciency
of DNA intercalation which can be vindicated by the fusion
of tetra-, penta-, and hexacyclic compounds having one or
two heteroatoms with quinoline ring in a linear fashion and
these type of moieties are found in natural products. So,
they are being extensively used in synthetic compounds of
biological interest to exhibit antitumor and anticancer prop-
erties. The key strategy of designing an anticancer agent is
to damage DNA by diferent binding modes such as elec-
trostatic binding, covalent binding, hydrophobic binding
to the minor groove and intercalation. Therefore, to induce
an intercalative property to the scafolds, a planar geom-
etry is very essential. Furthermore, the synthesis of biaryl
derivatives is a prime aim in current drug discovery as the
presence of biaryl motifs is very much important in variety
of pharmaceuticals, herbicides, and natural products [45,
46], especially as the biarylquinolones are known to pos-
sess neuroprotective properties [47]. On the other hand,
biaryl rings are responsible for increasing hydrophobicity
of the drugs making them easily permeable to cell mem-
brane. The substitution at the biaryl rings can gear up the
delocalisation of π-electrons due to extended conjugation
and thereby makes the drug more fuorescent enabling them
cellular imaging capability. Due to these diverse biological
properties of quinoxaline, biarylquinoline, and quinolones,
there was an ardent zeal to devise a simple and efcient
method for the construction of these scafolds. In previously
reported article, some novel methodology for synthesis of
these compounds was developed, but in this recent work, we
have emphasised on the resynthesis of these molecules fol-
lowing environmentally friendly green technology in a one-
pot sequence as well as to evaluate their anticancer activity
using human breast carcinoma cell line (MCF-7), human
epitheloid cervix carcinoma (HeLa) cell line.
It is widely acknowledged that there is a growing need
for more environmentally acceptable processes to control
environment pollution. Therefore, green chemistry has
encouraged the development of cleaner chemical processes
and new technologies [48–51]. Green chemistry addresses
the environmental impact of both chemical products and
the processes by which they are produced. In our previous
report, we highlighted some novel techniques for the syn-
thesis of biarylquinolone analogues [52, 53]. However, there
is a growing need for more environmentally benign method
to isolate the biarylquinolones in one-pot sequence. In this
context, visible light was considered as supreme reaction
promoter for organic transformations, since it is non-toxic,
inexpensive, abundant, and renewable as well as generates
no waste. Alternatively, visible light-mediated photoredox
transformations have been fruitfully established by several
research groups [54–63]. Nowadays, visible light-mediated
several oxidative cross-coupling reactions have also been
developed for the construction of C‒C bonds and C‒het-
eroatom bonds [64]. In comparison to iridium, ruthenium,
or copper complexes, specially eosin Y, eosin B, fuores-
cein, and rose bengal (organic dyes), have been proven to
be efcient catalysts in visible light-induced organic trans-
formations due to its atom economy and overall sustainable
nature [65–75]. Among all these photocatalysts, eosin Y has
appeared to the forefront of the organic photo catalytic reac-
tions as a most powerful entity to activate small molecules
by converting visible light into chemical energy and triggers
the single-electron transfer (SET) with organic reactants and
thereby generates reactive intermediates [76]. Therefore, the
approach of photoredox reaction does not demand any radi-
cal initiator or any oxidants and reductants and even can be
accomplished under mild condition using metal free photo
catalyst like eosin Y which is very much efective to pro-
vide the economically and ecologically acceptable synthetic
route. As a consequence, our key objective of the present
work is to resynthesize our previously reported compounds
applying green methodology under visible light irradiation
in presence of eosin Y in one-pot sequence and to bring
these scafolds into light as good anticancer agents.
Result and discussion
Synthesis and characterization
The typical optimization of this reaction was accomplished
by selecting 5,7-dibromo-8-hydroxyquinoline (1), 2,3-bis-
(bromomethyl)quinoxaline (2), and phenyl boronic acid
(3a) as the model substrates together with the application of
various combined catalyst and solvents under inert atmos-
phere. At the outset, 5,7-dibromo-8-hydroxyquinoline (1)
was treated with 1.5 equivalents of 2,3-bis-(bromomethyl)
1 3

Combined palladium/eosin Y-catalysed direct synthesis of anticancer…
quinoxaline (2) and 2.1 equivalents of phenyl boronic acid
(3a) in presence of 1.0 mol% combined catalytic mixture of
Pd(0) and eosin Y in ethanol under continuous irradiation of
visible light for 24 h (Table 1, entry 1). The progress of the
reaction was monitored by TLC. After 24 h of irradiation,
the reaction mixture was evaporated through rotary evapo-
rator followed by addition of distilled water to the crude
mixture. Then the mixture was extracted with ethyl acetate
and the undissolved impurities were removed out by fltra-
tion. After that, the pure ethyl acetate extract was kept for
crystallization and the crystalline 4,6-diphenyl-8,15-dihy-
dro-1H-quinolino[8′,1′:2,3,4][1,4]oxazocino[6,7-b]quinox-
alin-1-one (4a) was obtained with moderate yield (65%). To
observe the betterment of the yield of this reaction, a num-
ber of other combined catalytic systems like Pd(0)/eosin B,
Pd(0)/rhodamine B, Pd(0)/rose bengal, Pd(0)/acridine red,
Pd(0)/fuorescein (entries 2–6) in ethanol were used for the
study. However, we could not observe any improvement of
the yield (<50%) which helped us to apprehend the efectiv-
ity of Pd(0)/eosin Y combined catalytic mixture for the syn-
thesis of 4,6-biaryl-8,15-dihydro-1H-quinolino[8′,1′:2,3,4]-
[1,4]oxazocino[6,7-b]quinoxalin-1-one. Furthermore, sol-
vents including CH3CN, H2O, THF, DCE (dichloroeth-
ane), and EtOH/H2O, were also tested using Pd(0)/eosin
Y as combined catalytic system at room temperature, and
we found that EtOH/H2O (1:2 v/v) was superior to others
(Table 1, entries 8–14). It is also noteworthy that no biaryl-
8,15-dihydro-1H-quinolino[8′,1′:2,3,4][1,4] oxazocino[6,7-
b]quinoxalin-1-one was found to be formed in absence of
light.
After optimizing the reaction condition, we intrigued
our attention to investigate the scope of the substrates for
the visible light-initiated direct 4,6-biaryl-8,15-dihydro-
1H-quinolino[8′,1′:2,3,4][1,4]oxazocino[6,7-b]-quinoxalin-
1-ones 4a–4i formation in one-pot sequence. As shown in
Table 2, a variety of aryl boronic acid, bearing electron-
donating groups (R = OMe, Me) on the aryl ring, reacted
with dibromoquinolone, afording 4,6-di-(p-tolyl)-8,15-di-
hydro-1H-quinolino[8′,1′:2,3,4][1,4]- oxazocino[6,7-b‑z]-
quinoxalin-1-one (4d) and 4,6-bis(4-methoxyphenyl)-
8 , 1 5 - d i h y d r o - 1 H - q u i n o l i n o [ 8 ′ , 1 ′ : 2 , 3 , 4 ] -
[1,4]oxazocino[6,7-b]quinoxalin-1-one (4e) with high
Table 1 Optimization of the reaction conditions
O
N
Combined catalyst (1.0 mol%)
N
Br
N
Br
Br
N
N
TBHP, Solvent
Ph
O
+
OH
OH
Br
N
Ph
B
3a
Ph
OH
11 W LED, rt, 24 h
1
2
4a
Entry
Combined catalysta
Solvent
Yield/%b
1
2
3
4
5
6
7
8
Pd(0)/eosin Y
Pd(0)/eosin B
Pd(0)/rhodamine B
Pd(0)/rose bengal
Pd(0)/acridine red
Pd(0)/fuorescein
Pd(0)/eosin Y
Pd(0)/eosin Y
Pd(0)/eosin Y
Pd(0)/eosin Y
Pd(0)/eosin Y
Pd(0)/eosin Y
Pd(0)/eosin Y
Pd(0)/eosin Y
Pd(0)/eosin Y
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
CH3CN
i-PrOH
CH2Cl2
THF
65
42
45
30
35
25
55
60
40
50
60
55
70
92c
NId
9
10
11
12
13
14
15
H2O
DMF
EtOH/H2O (1:1)
EtOH/H2Oc (1:2)
EtOH/H2O (1:2)
aReaction conditions: under a nitrogen atmosphere, 1 (0.33 mmol), 2 (0.50 mmol), catalyst (1.0 mol %), TBHP (3.0 equiv.), solvent 10 cm3,
room temperature, 24 h
bIsolated yield. c11 W green LED. dIn the dark. NI not isolated, TBHP tert-butyl hydroperoxide solution in decane. cScope of the chemistry has
been extended (4a–4i) using this solvent systems
1 3

N. Roy, P. Paira
Table 2 Scope of biarylquinolonoquinoxalines
O
N
Pd(0) / eosin Y (1.0 mol%)
TBHP, EtOH/H2O (1:2)
N
Br
N
Br
Br
N
N
Ar
O
+
OH
OH
Br
N
3
Ar
B
Ar
OH
11 W LED, rt, 24 h
1
2
4a-4i
Compound
Ar
Yield/%
M.p./°C
Lit. m.p./°C
4a
4b
4c
4d
4e
4f
4g
4h
4i
C6H5
92
88
86
95
96
90
90
92
90
224
220 [52]
4-F-C6H4
4-Cl-C6H4
4-Me-C6H4
4-MeO-C6H4
2-Furanyl
2-Naphthalenyl
2-Pyridinyl
197–198
190–192
188–190
196–198
180–182
200–202
147–149
200–202
195–196 [52]
192–194 [52]
190–192 [52]
198–200 [52]
182–184 [52]
202–204 [52]
150–152 [52]
202–204 [52]
1-Naphthalenyl
yields (95–96%), whereas arylboronic acid with diferent
electron-withdrawing group such as fuoro and chloro pro-
duced their corresponding biarylquinolones i.e. 4,6-bis(4-
fuorophenyl)-8,15-dihydro-1H-quinolino[8′,1′:2,3,4][1,4]-
oxazocino[6,7-b]quinoxalin-1-one (4b) and 4,6-bis(4-
chlorophenyl)-8,15-dihydro-1H-quinolino[8′,1′:2,3,4][1,4]-
oxazocino[6,7-b]quinoxalin-1-one (4c) with slightly low
yields (86–88%). Additionally, heteroarylboronic acids 3f–3i
took part for the formation of 4,6-di(furan-2-yl)-8,15-di-
hydro-1H-quinolino[8′,1′:2,3,4][1,4]oxazocino[6,7-b]-
quinoxalin-1-one (4f), 4,6-di(naphthalen-2-yl)-8,15-di-
hydro-1H-quinolino[8′,1′:2,3,4][1,4]oxazocino-
[6,7-b]-quinoxalin-1-one (4 g), 4,6-di(pyridin-2-yl)-8,15-di-
hydro-1H-quinolino[8′,1′:2,3,4][1,4]oxazocino[6,7-b]-
quinoxalin-1-one (4 h), and4,6-di(naphthalen-1-yl)-8,15-di-
hydro-1H-quinolino[8′,1′:2,3,4][1,4]oxazocino[6,7-b]-
quinoxalin-1-one (4i) with a high yield (90–92%). The for-
mation of the biarylquinolones 4a-4i was confrmed by Rf,
electron spray ionization mass spectroscopy (ESI–MS), and
literature melting point [52].
intermediate [A] took part in the reaction with quinoxaline
dibromide to give fused quinolinium salts [B] under expo-
sure to visible light. Hydroxyl anion generated from TBHP
attacked to the C-2 position of quinolinium salts [B] fol-
lowed by single-electron transfer (SET) with eosin Y·+ to
form intermediate [C]. Subsequently, the tautomerization
of intermediate [C] aforded the desired quinolones. Then,
Pd(0) catalyst was utilized for the preparation of biarylqui-
nolones from dibromoquinolones following the well-known
oxidative addition and reductive elimination pathway.
To demonstrate this concept, we treated the compound
1 with dibromo alkanes 5a, 5b in presence of eosin Y cata-
lyst, TBHP under visible light (11 W LED). Eventually, we
isolated the desired 9,11-dibromo-3,4-dihydro-2H,6H-[1,4]-
oxazepino[2,3,4-ij]quinolin-6-one (6a) and 10,12-dibromo-
2,3,4,5-tetrahydro-7H-[1,4]oxazocino[2,3,4-ij]quinolin-
7-one (6b) with high yield (Scheme 2). The formation of
the product was confrmed by Rf and literature melting point
[77].
Solubility and lipophilicity study
Proposed mechanistic pathway
Both hydrophilicity and lipophilicity studies were conducted
to know the tumour-inhibiting potential of these biarylqu-
nolones. These complexes were exhibited signifcant solubil-
ity in DMSO, MeOH, EtOH, and CH3CN and moderate sol-
ubility in H2O. These compounds were soluble in the range
of 5–10 mg per cm3 of DMSO-10% DMEM medium (1%
DMSO in DMEM, 1:99 v/v, comparable to cell media) at
25 °C. The lipophilicity of these complexes was performed
by n-octanol/water partition coefcient (log Po/w, where
We have also proposed a general mechanistic way based
on the result obtained in previous section (Scheme 1). At
the beginning, eosin Y was converted to the excited eosin
Y* under the visible light irradiation. A single electron
was transferred from eosin Y* to TBHP which aforded a
tert-butoxyl radical and a hydroxyl anion. Then dibromo-
quinolinol reacted with tert-butoxyl radical to furnish the
corresponding oxygen radical intermediate [A]. Afterwards,
1 3

Combined palladium/eosin Y-catalysed direct synthesis of anticancer…
Scheme 1
Br
+
t-BuOO
Br
N
–
HO
t-BuOOH
OH
–
HO
t-BuOOH
Br
Br
Br
N
N
+
N
*
Eosin Y
Eosin Y
N
N
Br
N
Br
O
Br
Light
O
SET
[A]
[B]
Br
Eosin Y
[B]
[C]
Light
H
H
H
O
N
O
N
N
N
+
+
N
N
Br
O
Br
O
[D]
[C]
Br
N
Br
O
N
N
1
Br
O
Ar-Ar
Ar-Br
Pd(0)
I
Br
1
Ar-Pd(II)-Ar
Ar-Pd(II)-Br
IV
H BO
3
3
–
HO
Ar-Pd(II)-OH
1
Ar B(OH)
2
III
Scheme 2
O
Br
Eosin Y (1.0 mol%)
TBHP, EtOH/H2O (1:2)
N
n
a
b
, n = 3
Br
O
(CH2)nBr2
, n = 4
5a, 5b
(
)
Br
N
Br
6a, 6b
OH
11 W LED, rt, 24 h
1
1 3

N. Roy, P. Paira
Table 3 Preliminary MTT cytotoxicity screening of synthesized qui-
nolones 4a–4i and positive control (cisplatin and doxorubicin) at 48 h
of drug exposure
Po/w = the octanol/water partition coefcient) study using
the shake fask method (Table S1) [49]. The experimen-
tal log Po/w values of these compounds are obtained in the
range 0.7–1.25. Compound 4b displayed the highest log Po/w
(1.25 0.10) due to the presence of lipophilic fuorine atom.
In case of compound 4c, comparable log Po/w (1.18 0.12)
was observed. Compound 4 g and 4i also showed signifcant
lipophilicity (1.10–1.12) due to presence of hydrophobic
naphthalene ring. The lowest log Po/w value was observed in
the compounds 4 h due to presence of hydrophilic pyridine
ring which could be protonated easilyin aqueous medium.
Compound
Cell lines
IC50 /µM[a] SF[b]
HeLa
MCF-7
HEK-293 HeLa MCF-7
4a
9.7 0.9
6.2 0.8
6.8 0.9
8.6 1.8
8.4 0.9
10.3 1.4 11.8 1.2 >100
7.2 0.9 8.2 1.1 >100
12.8 1.3 13.6 1.1 >100
10.8 0.5 >100
>10.3 >9.2
>16.1 >12.8
>14.7 >12.3
>11.6 >11.2
>11.9 >11.3
4b
7.8 0.8
8.1 0.6
8.9 1.1
8.8 0.6
>100
>100
>100
>100
4c
4d
4e
Anticancer study
4f
>9.7
>13.8 >12.2
>7.8 >7.2
>13.3 >8.6
>8.4
4g
To bring out the biomedical applications of those com-
pounds, cytotoxicity study of all synthesized biarylquinolo-
nes 4a–4i was carried out via typical 3-(4,5-dimethylthi-
azol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
protocol beside a panel of cancer cell lines i.e. human breast
carcinoma cell line (MCF-7), human epitheloid cervix carci-
noma (HeLa), cell line and one normal cell line i.e. Human
embryonic kidney cells (HEK-293) in triplicates. The cells
were treated with synthesized quinolones along with cispl-
atin as the standard positive control with variable concen-
trations (0–300 μM) for 48 h. All the compounds exhibited
higher potency (IC50 =6–12 µM) and selectivity (7–16 fold)
in HeLa and MCF-7 cells with respect to normal HEK-293
cell line compare to cisplatin (Table 3). Among all these
scafolds, compound 4b and 4c showed the best potency and
selectivity in cancer cell than normal cell. For example, IC50
values of compound 4b and 4c were observed as6.2 0.8 µM
(p<0.05) and 6.8 0.9 µM (p<0.05) in HeLa cell, respec-
tively. Similarly, in MCF-7 IC50, values were found as
7.8 0.8 µM (p<0.05) and 8.1 0.6 µM (p<0.05) for the
compound 4b and 4c, respectively (Table 3). If we look the
cytotoxicity results of doxorubicin, except compound 4 h,
all other compounds exhibited much lower toxicity against
HeLa cells compared to this positive control. However, all
the synthesized scafolds were less potent than doxorubicin
against MCF-7 cell line. Surprisingly, in selectivity concern,
all these scafolds were much more efective than both the
positive controls (Fig. 1).
4h
4i
7.5 1.2
–
18.4 0.9 12 0.4
11.6 1.1 >100
DMSO
Cisplatin
–
–
–
–
2.2
7.8
38.2 1.8 2.1
10.9 1.2 1.01
Doxorubicin 10.8 1.1 1.4 0.7
aIs the 50% growth inhibition of the tested cell line relative to control
experiments. It was calculated by non-linear sigmoidal curve ftting
in dose–response model using Origin 8.5. Each value represents the
mean SD (standard deviation) of three independent experiments
bSF (selectivity factor)=ratio of IC50 for HEK-293 and IC50 for all
the cancer cell lines. HEK-293 fbroblasts are generally selected as
the model for healthy cells in the evaluation of chemotherapeutic
drug selectivity. Statistical signifcance is indicated as p<0.05 based
on Student’s t test using Origin 8.5 software (the statistical signif-
cance (p) of the data is<0.05 or better)
fuorescence of compound 4b inside the live cells were
monitored under fuorescence microscope (Fig. 2) which
clearly indicated the strong cellular uptake of this compound
within 4 h incubation with HeLa cell line. Co-localization
of the complex with DAPI displayed that the intracellular
distribution of these complexes was mainly in the nucleus,
rather than in other organelles (Fig. 2).
Structure activity relation (SAR) study
The result of anticancer screening assisted us to derive the
SAR of the hypothetically designed biarylquinolone com-
pounds. The structure–activity relationship (SAR) studies
revealed that the introduction of most electronegative and
lipophilic fuorine atom at the phenyl ring (4b) exhibited
the best cytotoxicity profle (IC50 = 6–7 µM) against Hela
and MCF-7 cell lines. Similar result was also obtained in
case of compound 4c having –Cl atom in their phenyl sub-
stitution. However, cytotoxicity in both the cell lines was
reduced a bit (compound 4d and 4e; IC50 = 8–9 µM) after
introduction of electron-donating methyl and methoxy
groups at the phenyl ring in compound 4d and 4e. At the
same time, signifcant cytotoxic profles (IC50 = 7–11 µM)
Cellular imaging study
To get insight into the imaging quality of our compounds,
cellular imaging experiment was performed using HeLa cell
line. HeLa cells were incubated with fresh Dulbecco’s Modi-
fcation Eagle’s Medium (DMEM, with 1% pen/strep and
10% FBS) containing 50 µM of compound 4b (in DMSO/
culture medium, 1/99 v/v) for 4 h at 37 °C, and then washed
gradually with PBS, the images were capturedby Olympus
fuorescence microscope under 405 nm excitation.Green
1 3

Combined palladium/eosin Y-catalysed direct synthesis of anticancer…
were also observed in compound 4 g and 4i having hydro-
phobic naphthalene ring at the ffth and seventh position of
quinolone. However, the presence of polar furan and pyri-
dine substitution compelled to reduce the potency of com-
pound 4f and 4 h in both the cell lines (IC50 = 10–13 µM).
On the basis of cytotoxicity profles, it can be assumed
that—(i) lipophilic fuorine or chlorine substitution is
important for cellular accumulation; (ii) hydrophobic
naphthalene ring also helps for cellular permeability; (iii)
quinolone ring with electronegative substituents is the
key protagonist for anticancer potency; and (iv) planar
quinoxaline ring essential for targeting the nuclear DNA
which was vindicated by the cellular co-localization study
(Fig. 3).
4a
(a)
(b)
4b
100
4a
4b
4c
4d
4e
4f
4g
4h
100
80
60
40
20
4c
4d
4e
4f
4g
4h
80
60
40
20
4i
cisplatin
Doxorubicin
4i
cisplatin
Doxorubicin
0.4
0.8
1.2
1.6
2.0
2.4
0.4
0.8
1.2
1.6
2.0
2.4
Log (concentration /μM)
Log (concentration /μM)
Fig. 1 Comparison of cytotoxicity for complexes 4a–4i, cisplatin, doxorubicin against a HeLa cell and b MCF-7 cell
Fig. 2 Co-localization images of compound 4b (50 µM) in live HeLa cells after 4 h of incubation with nucleus dye DAPI. Excitation wavelength
is 405 nm. Scale bar 20 µm
Fig. 3 SAR study of
4,6-biaryl-8,15-dihydro-
1H-quinolino[8′,1′:2,3,4][1,4]-
oxazocino[6,7-b]quinoxalin-
1-ones 4a–4i
1 3

N. Roy, P. Paira
for crystallization and crystalline biarylquinolonoquinoxa-
lines 4a–4i were obtained with high yield (~ 95%).
Conclusions
Coming to the end, it is a time to give out the essence of
the whole work where we focused our attention on the
anticancer activity of previously reported biarylquinoli-
nooxazocino-quinoxaline-1-one molecule by resynthesiz-
ing this molecule with the help of environmentally benign
green methodology. In a nutshell, here we have developed
an efcient Pd(0)/eosin Y-mixed catalyzed visible light-
mediated direct synthesis of biarylquinolnes from dibro-
moquinolinol. The scope of this chemistry is well tolerated
with the use of various aryl and heteroarylboronic acids
to obtain corresponding biarylquinolones in high yields.
It can be depicted that this method is highly advantageous
in light of commercially available starting materials and
catalysts, use of environmentally benign solvents, ease of
isolation of the products, convenient reaction condition
and outstanding tolerance of functional groups without
addition of bases, ligands or additives. In connection with
this, these compounds displayed an astounding cytoselec-
tivity in two cancer celllines viz human breast carcinoma
cell line (MCF-7) and in human epitheloid cervix carci-
noma (HeLa) cell line than normal kidney cell, HEK-293
(human embryonic kidney cells). Therefore, it can be
anticipated that the development of biarylquinolinooxa-
zocino-quinoline-1-ones compounds will be highly useful
in cancer therapy by means of their anticancer efciency
obeying the necessities of green and sustainable chemistry
and our developed synthetic approach will also delineate
an appropriate route for synthesis of various other antican-
cer biarylquinolones to be appeared as anticancer candi-
dates in forthcoming days.
General procedure for the synthesis of fused
quinolones
100 mg of 5,7-dibromo-8-hydroxyquinoline (1, 0.33 mmol)
was taken in 10 cm3 of ethanol/water mixture (1:2 v/v) in
a 100 cm3 RB fask under inert atmosphere, 4 equivalent
of dibromoalkane 5a or 5b, 1.0 mol% of combined cata-
lyst eosin Y and 3 equivalent of TBHP was added, and the
mixture was stirred under constant irradiation from 11 W
LED for 24 h. After completion of the reaction, the reaction
mixtures were evaporated to dryness and distilled water was
added to it. The mixture was extracted with ethyl acetate and
the undissolved impurities were removed by fltration. Then
the pure ethyl acetate extract was kept for crystallization and
crystalline fused quinolones 6a, 6b were obtained with high
yield (~90%).
In vitro cytotoxic activities (MTT assay) [78]
In vitro cytotoxicity was determined using the standard MTT
assay protocol. The MTT proliferation assay is based on the
reduction of the yellow MTT tetrazolium salt (3-(4,5-dimeth-
ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) by mito-
chondrial dehydrogenases to form a blue MTT formazan in
viable cells. Synthesized quinolones 4a–4i were practiced
aforementioned to the experiment by dissolving in 0.1%
DMSO and then serial dilution with medium. Two diferent
types of cancer cell lines i.e. human breast carcinoma cell
line (MCF-7), human epitheloid cervix carcinoma (HeLa),
and one normal human embryonic kidney cells (HEK-293)
were used in the assay. Approximately, 1×104 cells per well
for all the cell lines were cultured in 100 mm3 of a growth
medium in 96-well plates and incubated at 37 °C under a
5% CO2 atmosphere. The cells were then treated with difer-
ent concentrations of the complexes (1–200 µM) in the vol-
ume of 100 µM/well. Cisplatin is used as standard positive
control drug. Cells in the control wells accepted the same
volume of medium containing 0.1% DMSO. After 24 h, the
medium was discarded and cell cultures were incubated with
100 mm3 MTT reagent (1 mg/cm3) for 5 h at 37 °C. Then
the suspension was placed on microvibrator for 10 min and
subsequently the absorbance was recorded by the ELISA
reader at λ=570 nm. The experiment was also performed
in triplicate. The data were expressed as the growth inhibi-
tion percentage calculated according to the equation: % Cell
viability = [ODsample − ODblank/ODcontrol—ODblank] × 100,
where ODsample is the measured absorbance in wells contain-
ing samples, ODcontrol is the absorbance measured for cells
with a medium and a vehicle and ODblank is the absorbance
Experimental
General procedure for the synthesis
of biarylquinolonoquinoxalines
100 mg of 5,7-dibromo-8-hydroxyquinoline (1, 0.33 mmol)
was taken in 10 cm3 of ethanol/water mixture (1:2 v/v) in
a 100 cm3 RB fask under inert atmosphere, 155 mg of
quinoxaline dibromide (2, 0.50 mmol), 1.0 mol% of com-
bined catalyst Pd(PPh3)4/eosin Y, 3 equivalent of TBHP
and 2.5 equivalent of aryl boronic acid 3a–3i was added,
and the mixture was stirred under constant irradiation from
11 W LED for 24 h. After completion of the reaction, the
reaction mixtures were evaporated to dryness and distilled
water was added to it. The mixture was extracted with
ethyl acetate and the undissolved impurities were removed
by fltration. Then the pure ethyl acetate extract was kept
1 3

Combined palladium/eosin Y-catalysed direct synthesis of anticancer…
Gosmann G, Jarry C, Déléris G, Sonnet P, Grellier P (2011) Eur
J Med Chem 46:2310
measured for blank well (no cells). Dose–response curve
was ftted in Origin 8.5 software and IC50 was calculated.
30. Tanis SP, Strohbach JW, Parker TT, Moon MW, Thaisrivongs
S, Perrault WR, Hopkins TA, Knechtel ML, Oien NL, Wieber
JL, Stephanski KJ, Wathen MW (2010) Bioorg Med Chem Lett
20:1994
Acknowledgements The authors gratefully acknowledge VIT for pro-
viding ‘VIT SEED GRANT’, Vellore for the support through Seed
Grant for Research. We acknowledge DST, New Delhi, India for DST-
FIST project.
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