An unorthodox metal-free synthesis of dihydro-6: H-quinoline-5-ones in ethanol/water using a non-nucleophilic base and their cytotoxic studies on human cancer cell line

Article abstract of DOI:10.1039/c9nj06346c

A DBU-catalysed metal-free domino reaction strategy has been developed for the facile synthesis of dihydro-6H-quinoline-5-ones. This protocol employs a very expedient route to the synthesis of pyridine frameworks using β-chloro-α,β-unsaturated aldehydes, 1,3-diketones, and ammonium acetate in ethanol : water (1 : 1) solvent under eco-friendly conditions. Diverse types of acyclic and cyclic β-chloro-α,β-unsaturated aldehydes were used to obtain a variety of dihydro-6H-quinoline-5-ones. The mechanism of the domino reaction was established by isolating the intermediate compound, which was subjected to the next step of the reaction to obtain the target product. The structure of the intermediate was established from spectral and single crystal XRD studies. Most of the synthesized dihydro-6H-quinoline-5-one derivatives were found to be cytotoxic to the HeLa cell lines, showing profound cytotoxicity in MTT assays. The DNA fragmentation assay showed no fragmented DNA in the treated sets, which indicated that the compounds did not induce apoptosis of the HeLa cells. In most of the cases, autophagic cell death was evident from fluorescence microscopy studies, though necrosis was also observed in some cases.

Full text of DOI:10.1039/c9nj06346c

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An unorthodox metal-free synthesis of dihydro-
6H-quinoline-5-ones in ethanol/water using a
non-nucleophilic base and their cytotoxic studies
on human cancer cell line†
Cite this:DOI: 10.1039/c9nj06346c
a
c
Arijit Kundu,
Subhabrata Paul,d Indira Majumdere and Rita Kundue
Bhaswati Bhattacharyya,b Kaliprasanna Dhara,
*
A DBU-catalysed metal-free domino reaction strategy has been developed for the facile synthesis of
dihydro-6H-quinoline-5-ones. This protocol employs a very expedient route to the synthesis of pyridine
frameworks using b-chloro-a,b-unsaturated aldehydes, 1,3-diketones, and ammonium acetate in
ethanol : water (1 : 1) solvent under eco-friendly conditions. Diverse types of acyclic and cyclic b-chloro-
a,b-unsaturated aldehydes were used to obtain
a variety of dihydro-6H-quinoline-5-ones. The
mechanism of the domino reaction was established by isolating the intermediate compound, which was
subjected to the next step of the reaction to obtain the target product. The structure of the
intermediate was established from spectral and single crystal XRD studies. Most of the synthesized
dihydro-6H-quinoline-5-one derivatives were found to be cytotoxic to the HeLa cell lines, showing
profound cytotoxicity in MTT assays. The DNA fragmentation assay showed no fragmented DNA in the
treated sets, which indicated that the compounds did not induce apoptosis of the HeLa cells. In most of
the cases, autophagic cell death was evident from fluorescence microscopy studies, though necrosis
was also observed in some cases.
Received 23rd December 2019,
Accepted 17th February 2020
DOI: 10.1039/c9nj06346c
rsc.li/njc
Introduction
In the recent eon of research, the discovery of small molecules
with the efficiency to work in complex biological processes to
prevent diseases has been one of the major challenges facing
researchers.1 This trend has clearly indicated a paradigm shift
of the spotlight from natural product chemistry to combinatorial
chemistry.2 Among numerous nitrogen heterocycles, quinoline is
a scaffold of crucial importance with respect to biomedical use.
Several quinoline derivatives, isolated from natural resources
or prepared synthetically, show a wide variety of biological
activities.3 One such type of important quinoline moiety is
dihydroquinoline, which exhibits interesting biological and
pharmaceutical activities including antitubercular (1), anti-HIV (2),
anticancer (3), apical sodium-dependent bile acid transporter (ASBT)
a Department of Chemistry, Maulana Azad College, Kolkata-700013, India
b Department of Chemistry, Government Girls General Degree College, Ekbalpur,
Kolkata-700023, India
Fig. 1 Some naturally occurring quinoline derivatives.
c Department of Chemistry, University College of Science and Technology,
University of Calcutta, Kolkata-700009, India. E-mail: chemkpd@gmail.com
d Department of Botany, Presidency University, Kolkata-700019, India
e Department of Botany, University of Calcutta, Kolkata-700019, India
† Electronic supplementary information (ESI) available. CCDC 1542885 and
1542887. For ESI and crystallographic data in CIF or other electronic format
see DOI: 10.1039/c9nj06346c
inhibition (4), (Fig. 1) etc. Compounds, streptonigrin (5), ascidiathia-
zones and lavendamycin, (Fig. 1) showed antibiotic, anticancer, and
antiproliferative activities, respectively.3
Numerous skeletal analogues incorporating these crucial
moieties have been prepared using synthetic approaches to
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accomplish a vast and diverse chemical library.4 Development
of a green technique requires fulfillment of certain criteria such
as, use of nontoxic reagents and absence of additives, ambient
reaction conditions, atom economy and procedural simplicity.
Coupled with this concern, one-pot multicomponent reactions
are equally significant as they give rise to complex structures by
simultaneous formation of two or more bonds in time-saving
procedures.5 A number of synthetic strategies have been utilized
for the preparation of dihydro-6H-quinoline-5-one skeletons
either by the reaction of b-enaminones with b-dicarbonyls in
the presence of CeCl3/NaI in 2-propanol,6 DBU in butanol,7
ammonium acetate using microwave,8 K5CoW12O40Á3H2O9 etc.
or by the reaction between a ketone, 1,3-diketone, and an aldehyde
in the presence of a copper incorporated nanorod like mesoporous
silica catalyst using molecular O210 or MCM-41 supported HPF6.11
One crucial feature of multicomponent reactions is cost efficiency.
Besides many other factors, cost efficiency depends on the number
of starting materials and reagents used or steps required for the
formation of the product. In this regard, development of reaction
protocols involving inexpensive starting materials or minimal
number of steps to reach the target molecule is more acceptable.
A literature review on the synthetic methodologies for 7,8-
dihydroquinolin-5-ones and similar derivatives reveal that most
of the synthetic techniques engage either more than one step
with costly metal catalysts or a three to four component-strategy.
The majority of groups have reported the preparation of dihydro-6H-
quinoline-5-one derivatives using a two-step procedure entailing
b-enaminone intermediates, which could be obtained from the
reaction between dimethylformamide, dimethyl acetals and aryl
methyl ketones.6–9 Mukhopadhyay et al.10,11 demonstrated a four-
component-reaction strategy for the preparation of dihydro-6H-
quinoline-5-one derivatives (Scheme 1).
To enrich this library, we focused on the synthesis of different
dihydroquinoline derivatives. Here, the synthesis of dihydro-6H-
quinoline-5-one derivatives was developed by taking the advan-
tages of the skeletal motif of b-chloro-a,b-unsaturated aldehydes.
Goroi et al.12 had reported the synthesis of dihydro-6H-quinolin-5-
ones from similar motifs of b-bromo-a,b-unsaturated aldehydes by
using a three-component methodology between 1,3-diketones, and
ammonium acetate without any additional catalyst or metal salt at
120 1C, where the methodology involves the elimination of a more
labile bromide group. Moreover, flexibility of the methodology
with an acyclic analog of b-bromo-a,b-unsaturated aldehydes was
not reported. Additionally proof of mechanistic pathway was not
established in the approach. In the pursuit of an easier and more
green technique at a lower temperature we have adopted a metal-
free protocol in an ethanol/water solvent starting from different
Scheme 1 A comparative study of previous and present work.
apoptosis, dysregulate cell cycles and inhibit angiogenesis (Afzal
et al., 2015).13 Koprulu and coworkers (2019)14 reported the
synthesis of methoxy and hydroxy substituted quinoline com-
pounds and their antiproliferative properties against human
adenocarcinoma (HT29), human cervical cancer cell line-HeLa
and rat glioblastoma (C6). In the present study, the anticancer
activity of the synthesized dihydro-6H-quinoline-5-one derivatives
on cervical cancer cells in vitro (HeLa, HPV 18 positive cell line) was
explored. As the compounds have a planar structure, they tend to
intercalate within the DNA bases, causing DNA damage and
inhibiting replicative processes (Okten et al., 2017),15 leading to
cell death. To evaluate the mode of cell death, DNA fragmentation
assays were undertaken and the presence of autophagic vacuoles
was studied.
Results and discussion
b-chloro-a,b-unsaturated aldehydes as a source of carbonyl In order to find out the suitable reaction conditions associated
functionalities. To the best of our knowledge this is the first with the synthesis of dihydro-6H-quinoline-5-one, we optimized
report of the formation of dihydro-6H-quinoline-5-ones from the reaction by carrying out extensive screening tests utilizing a
b-chloro-a,b-unsaturated aldehydes as a source of carbonyl representative reaction between 3-chloro-2-methyl-3-phenyl-
functionality.
acrylaldehyde (1h) (1 mmol) and dimedone (2a) (2.0 mmol) in
Quinolines and their derivatives are known for their diverse the presence of NH4OAc (5 mmol) and different bases by varying
(antimicrobial, anticancer, anti-HIV, anti-inflammatory, anti- solvents to obtain 3h.
depressant etc.) biological activities and have been studied for
All the results are summarized in Table 1. Initially the
centuries. Anticancer drugs with quinoline moieties can induce reaction was carried out by using potassium carbonate as the
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Table 1 Optimization study using 1h and aldehyde 2a
S. no.
Basea
Solventb
Time (h)
Temp. (1C)
Yieldc,d (%)
1
2
3
4
5
6
7
8
K2CO3
Water
Water
Ethanol/water (1 : 1)
Water
Ethanol/water (1 : 1)
water
Ethanol/water (1 : 1)
Ethanol/water (1 : 1)
Ethanol/water (1 : 1)
Ethanol/water (1 : 1)
Ethanol/water (1 : 1)
Ethanol/water (1 : 1)
Ethanol/water (1 : 1)
Ethanol/water (1 : 1)
Ethanol/water (1 : 1)
Ethanol/water (1 : 1)
Ethanol/water (1 : 1)
Ethanol/water (1 : 1)
12
8
8
8
8
6
6
6
5
5
4.5
4
3
1.5
2
1
2.5
2
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
80
10
10
20
25
45
55
75
60
75
80
85
85
85
90
85
96
90
92
Triethyl amine
Triethyl amine
Morpholine
Morpholine
Pyridine
Pyridine
Piperidine
a-Picoline
b-Picoline
g-Picoline
Lutidine
Bipyridine
DABCO
DBU
DABCO
DABCO
DABCO
9
10
11
12
13
14
15
16
17
18
60
70
a
b
c
All the reactions were carried out with 3 mmol base. 10 ml of solvent was used in each case. All the reactions were carried out with 3-chloro-2-
d
methyl-3-phenyl-acrylaldehyde (1.0 mmol) and dimedone (2.0 mmol). Yield of isolated product.
base in water, but it resulted in low yield of the product even herein is a mixture of ethanol and water and the base DABCO is
after a long reaction time at 100 1C (Table 1, entry 1). In the completely benign for the environment.
pursuit of more fruitful reaction conditions, we performed
Besides cost efficiency, the methodology involved procedural
the same reaction in water and ethanol–water mixture in the simplicity, easy work-up and minimization of chemical impu-
presence of triethyl amine as the base (Table 1, entries 2 and 3) rities. The structures of the products were well characterized by
and it was observed that there was a slight increase in the yield using spectral (IR, 1H and 13C NMR) and elemental analysis
of the product in the ethanol–water (1 : 1) solvent after a long data. The structural pattern of the products was fully established by
reaction time. Consequently, we studied the reaction in water X-ray crystallographic analysis of compound 6a (CCDC 1542885)†
and ethanol–water (1 : 1) in the presence of morpholine and (Fig. 2). We herein synthesized twenty-three compounds and of
pyridine as the bases (Table 1, entries 4–7), where a marginal them, seven are new additions to the literature.
enhancement in the product yield was noticed when pyridine
A plausible mechanistic pathway was proposed to explain
was used as the base in an ethanol–water (1 : 1) solvent. the base-catalyzed formation of dihydro-6H-quinoline-5-ones
Henceforth screening tests were done in ethanol–water (1 : 1) from b-chloro-a,b-unsaturated aldehydes (Fig. 3). The b-chloro-
solvent to obtain the best reaction conditions by studying with a,b-unsaturated aldehyde initially went through a Knoevenagel
different non-nucleophilic bases and various reaction times condensation with the 1,3-diketone in the presence of a non-
(Table 1, entries 8–15). Ultimately the optimum conditions were nucleophilic base. Subsequently the presence of a vinylic chlorine
realized by using DABCO as the base at 80 1C, which afforded atom facilitated the incorporation of the second molecule of 1,3-
96% yield of the desired product in ethanol–water (1 : 1) solvent diketone to produce the xanthene-dione intermediate (8). The
(Table 1, entry 16). A study to observe the effect of temperature in situ product 8 immediately reacted with ammonia, generated
on the yield of the product was also performed (Table 1, from ammonium acetate under the reaction conditions, to form
entries 17 and 18), which indicated that any further alteration the b-enaminone through ring opening. This b-enaminone
of the reaction temperature decreased the yield of the product. ultimately formed the dihydro-6H-quinoline-5-one through internal
To show the scope of this protocol, we utilized the new methodology rearrangement involving N–C bond formation and C–C bond
using different 1,3-dicarbonyl systems with various acyclic b-chloro- cleavage losing one molecule of the 1,3-diketone.
a,b-unsaturated aldehydes (Table 2) and cyclic b-chloro-
a,b-unsaturated aldehydes (Table 3). In all the cases, the reac- mentioned reaction pathway for the formation of dihydro-6H-
tion proceeded efficiently with good to excellent yields. quinoline-5-one derivatives, had been isolated in the following way.
The xanthene-dione intermediate (8), proposed in the above-
The reactions were simultaneously carried out at the 1 mmol Initially, the reaction between/with (Z)-3-chloro-3-phenylacrylal-
scale and no change in the yield of the product was observed dehyde (1a) and dimedone (2a) was carried out in the presence
when scaled up to the 10 mmol scale. From the point of view of of base DABCO in water at room temperature for ten minutes.
green chemistry, it was interesting to note that the solvent used The intermediate 8 was immediately precipitated, which was
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Table 2 Base catalysed dihydro-6H-quinoline-5-one derivatives from acyclic b-chloro-a,b-unsaturated aldehydes
a
% yield was calculated from the isolated product.
Table 3 Base catalysed dihydro-6H-quinoline-5-one derivatives from cyclic b-chloro-a,b-unsaturated aldehydes
a
% yield was calculated from the isolated product.
filtered and further reacted in the presence of ammonium procedure is shown in Fig. 4. The structure of the xanthene-dione
acetate in ethanol–water (1 : 1) solvent at 80 1C when 3a was intermediate (8) was confirmed by spectroscopic and X-ray
obtained after 1.2 hours. A schematic diagram of the aforesaid crystallographic studies (CCDC 1542887)† (Fig. 5).
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Isolation of intermediate 8 confirmed the proposed mechanistic
pathway as shown in the scheme shown in Fig. 3. The role of vinylic
chlorine present in the b-chloro-a,b-unsaturated aldehyde for the
formation of 8 was thus established.
Being a class of substituted pyridine scaffolds, dihydro-6H-
quinoline-5-one moieties show high potential towards biological
systems. In the present study, we have evaluated the cytotoxic
potential of the synthesized dihydro-6H-quinoline-5-one scaffolds
using MTT cell viability assays on a cervical cancer cell line
(HeLa) followed by a DNA fragmentation assay. Morphology of
acidic vesicles (vacuoles and lysosomes) and the presence of
autophagosomes were observed by fluorescence microscopy after
staining with a vital metachromatic dye acridine orange (AO).
Biological study
Fig. 2 X-ray crystallographic structure of compound 6a.
Cytotoxicity assay. Many of the synthesized quinoline com-
pounds bearing different functional groups exhibit antimeta-
static effects. The nature and position of the substituent on the
Fig. 3 Plausible mechanistic pathway for the synthesis of dihydro-6H-quinoline-5-one derivatives.
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Fig. 4 Schematic diagram of the formation of dihydro-6H-quinoline-5-one derivative through xanthene-dione intermediate.
We thereby undertook a detailed study with the compounds
containing methoxyl groups only. From the MTT assay (Table 4),
it was evident that less cytotoxicity was observed in HeLa cells
treated with 3f, 7c, and 6b (IC50 120 mm, 100 mm, and 90 mm
respectively), whereas cells treated with 4f, 6c, and 7b showed
significant cytotoxicity. Among these compounds, it was found
that compound 6c is the most cytotoxic when compared with
the others (IC50 B 0.95 mM) (Fig. 6).
DNA fragmentation assay. DNA fragmentation is a hallmark
of apoptotic cell death. However, in the treated cells no sign of
laddering of DNA (fragments with 180 kb or its multiples) was
observed (Fig. 7). Intact genomic DNA was found in all the
treated sets, which indicate that apoptosis might not be the
mechanism for inducing cell death in the treated ones.
Fluorescence microscopy with acridine orange dye. From the
fluorescence microscopy study, it was observed that in the sets
treated with compounds 3f, 4f, 6b and 7b formation of acidic
vacuoles (autophagosomes) was evident. Increased amounts of
Fig. 5 X-ray crystallographic structure of compound 8.
quinoline framework show variety in their efficacy in terms of diffused red specks can be easily observed indicating that
anticancer activity, thus generating structure–activity relation- the cell death pathway was mediated by autophagy, which
ship studies.16 In our case, the presence of methoxyl groups at was induced after 24 hours of chemical treatments, whereas a
different positions with respect to quinoline nitrogen has shown necrotic cellular morphology was observed in the sets treated
relatively higher cytotoxicity than that of other substituents. with compounds 6c and 7c (Fig. 8).
Table 4 Results obtained from an MTT assay of HeLa cells with different dihydro-6H-quinoline-5-one derivatives
Dose
Compound
% cell viability
0.05 mM
0.1 mM
0.5 mM
0.75 mM
1 mM
25 mM
50 mM
100 mM
250 mM
3f
Average
SD
Average
SD
Average
SD
Average
SD
92
0.65
99.33
1.25
90.33
2.51
89.67
0.73
92
1.15
90.67
0.23
97.66
3.1
87
0.5
83.67
0.72
95
1.15
65.66
0.65
97.26
4.2
81.77
0.05
88.33
0.45
47
0.43
90.23
3.5
75.33
1.6
88
0.75
35.33
1.3
82.03
3.7
71.67
1.63
72.33
2.08
20.33
0.05
48.44
0.8
69.33
1.05
45.67
1.13
15
1.5
31.05
1.8
3.67
0.4
—
—
—
—
—
—
6b
95.33
0.65
93.33
0.6
101.17
4.6
6c
4f
104.69
3.5
7b
Average
SD
Average
SD
101.7
7.8
102.54
8.14
99.61
1.2
100.2
1.25
100.2
1.5
97.07
2.98
98.44
0.41
95.31
0.8
90.43
2.12
26.75
10.44
81.84
0.11
22.07
0.265
67.77
2.6
19.94
1.01
51.37
2.62
—
—
—
—
7c
101.37
1.82
Doxorubicin
IC50
1 mM17
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in 5 ml water at room temperature for 10 min. The compound
3,3,6,6-tetramethyl-9-(2-oxo-2-phenylethyl)-3,4,5,6,7,9-hexahydro-
1H-xanthene-1,8(2H)-dione was precipitated as a white solid,
which was isolated by filtration and washed with water. The
isolated xanthene dione was further reacted in the presence of
5 mmol ammonium acetate in 10 ml ethanol/water (1: 1) solvent
at 80 1C for 1.2 hour and the expected 7,7-dimethyl-2-phenyl-7,8-
dihydroquinolin-5(6H)-one was obtained.
General procedure for in vitro evaluation of anticancer activity
Maintenance of cell line. Human cervical cancer cell line,
HeLa, a HPV 18 positive cell line was obtained from NCCS,
Pune. Cells were maintained in EMEM (Eagle’s minimum essential
medium), supplemented with 10% FBS, non-essential amino acid
and glutamic acid, in a humidified CO2 (5%) at 37 1C.
Fig. 6 Bar graph showing IC50 doses of the compounds in HeLa cells.
Cytotoxicity assay. Cytotoxicity of the chemicals towards
HeLa cells was investigated using standard MTT reduction
assays.18 Positively charged tetrazolium dye MTT can penetrate
viable cell membranes easily. After reduction by viable cell
mitochondrial dehydrogenases and oxidoreductases, the yellow
colored MTT dye turns to purple colored crystals. These water
insoluble formazan crystals can be solubilized and quantified
spectrophotometrically to determine the viability percentage.
1 Â 103 cells were seeded in a 96-well plate and were incubated
overnight at 37 1C. Cells were then treated with the synthesized
compounds in a concentration range of 0.05 mM to 100 mM for
24 hours. Then 100 mg of MTT (Sigma) (5 mg MTT per ml PBS)
was added to each well, mixed and then incubated in a 5% CO2
incubator for another 3–4 hours. Then the media was removed
from the wells and the formazan crystals were dissolved with
80 ml of DMSO (Merck). Absorption spectra were recorded using
an ELISA microplate reader (BioRad) at 595 nm. The extent of
cytotoxicity was determined using the following formula:
Fig. 7 HeLa cells grown in T25 flask were treated with IC50 doses of
corresponding treatments for 24 hours. Lane 1: DNA marker (1.5 kb), lane 2:
control set, lane 3: compound 3f, lane 4: compound 6b, lane 5: compound 6c,
lane 6: compound 4f, lane 7: compound 7b, and lane 8: compound 7c.
Experimental
% inhibition = [(absorbance of control set
Procedure for the synthesis of dihydro-6H-quinoline-5-one derivatives
À absorbance of treated set)/absorbance of control set] Â 100
2 mmol of a 1,3-diketone compound and 1.2 mmol of b-chloro-
a,b-unsaturated aldehydes were stirred in the presence of
5 mmol ammonium acetate in 10 ml ethanol/water (1: 1) solvent
using DABCO as a base at 80 1C for 1–1.6 hours. The reaction was
monitored by TLC. After completion of the reaction the whole
mixture was extracted with ethyl acetate and the ethyl acetate part
was washed with brine three times and evaporated under
vacuum. The reaction mixture obtained was subjected to column
chromatographic separation using different proportions of a
petroleum ether:ethyl acetate mixture. The product was obtained
from a 20% ethyl acetate petroleum ether mixture. The products
were well characterized by IR, 1H NMR, 13C NMR, elemental
analysis and an X-ray crystallographic study.
From the absorption spectral data, cell viabilities (in percentage)
in treated and control sets were calculated along with standard
deviations and bar graphs were made using the standard software
(Microsoft Excel).
DNA fragmentation
Apoptotic cell death can be established by DNA fragmentation
assays. DNA ladder assays have previously shown fragmented
DNA of 180 base pairs and its multiples (which is an apoptotic
phenomenon) in Hep-2 cells treated with only AMK OX-12, a
1,3,4-oxadiazole derivative. To assess the cell cytotoxic potential
of the synthesized chemicals on the HeLa cell line, cells were
seeded in surface-treated 60 mm culture dishes for 24 hours.
Treatments were added according to the IC50 doses from MTT
cell viability assays and incubated for 24 hours. Total DNA was
isolated according to Herrmann and his coworkers (1994) after
slight modification.19 1 Â 106 HeLa cells were harvested using a
Synthesis of 3,3,6,6-tetramethyl-9-(2-oxo-2-phenylethyl)-3,4,5,6,7,9-
hexahydro-1H-xanthene-1,8(2H)-dione (8) (proposed intermediate)
and its conversion to the 7,7-dimethyl-2-phenyl-7,8-dihydroquinolin-
5(6H)-one (3a)
A mixture of 3-chloro-3-phenylacrylaldehyde (1a) (1 mmol) and policeman (cell scrapper) and washed with chilled PBS. Then the
dimedone (2a) (2 mmol) was stirred in the presence of DABCO cells were dissolved in Tris–EDTA (TE) buffer. Equal amounts of
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Fig. 8 Fluorescence microscopic study with acridine orange; fluorescence (right) and phase contrast (left) images of treated HeLa cells. Cells were
treated with IC50 doses of the respective compounds.
2Â lysis buffer (200 mM NaCl, 20 mM Tris, 50 mM EDTA, 1% SDS Cells were stained with 10 mg mlÀ1 AO and visualized under a
and 2% NP-40) was added, tapped and incubated in ice for fluorescence microscope (Leica).
30 minutes. Then a phenol–chloroform–isoamyl alcohol (25 : 24 : 1)
mixture was used to eliminate fats and proteins. DNA was collected
from the aqueous phase and precipitated with isopropanol and
Conclusion
sodium acetate after two rounds of centrifugation. The collected
pellet was redissolved in TE buffer and incubated with RNaseA
to remove RNA. DNA was finally precipitated with isopropanol
and sodium acetate. DNA pellets were washed in 70% ethanol
and air dried to evaporate alcohol fully. Then the pellets were
dissolved in TE buffer and run on a 1.5% agarose gel and
visualized under a UV documentation system.
A green, atom economic metal-free methodology has been
developed using a non-nucleophilic base for the syntheses
of dihydro-6H-quinoline-5-one derivatives from b-chloro-a,b-
unsaturated aldehydes with good to excellent yield of the
products. Molecular structures of two representative molecules
were investigated by means of X-ray diffraction analysis.
A mechanistic pathway was established by isolating the xanthene-
dione intermediate, which was further subjected to the second step
of the domino reaction to yield the corresponding dihydro-6H-
quinoline-5-one derivative. The biological study revealed that
dihydro-6H-quinoline-5-one derivatives were cytotoxic to HeLa cells
and among them, the compound 6c showed profound cytotoxicity.
As the DNA fragmentation assay showed no fragmented DNA in the
treated sets, it could be concluded that the dihydro-6H-quinoline-5-
one compounds did not induce apoptosis to the HeLa cells. In most
of the cases autophagic cell death was evident from fluorescence
microscopy studies, though necrosis was also observed in a
few cases.
Fluorescence microscopic study of acidic cellular
compartments with acridine orange dye
Acridine orange (AO) is a metachromatic dye. Cell cytoplasm
and nucleus are stained as green and the acidic compartments of a
cell viz., lysosome and vacuoles are stained orange. Formation of
these kinds of acidic compartments in a higher degree represents
an increased degree of autophagosome formation, a hallmark of
autophagic cell death. In most of the autophagy pathway, the
lysosome after fusion with an autophagosome forms an auto-
phagolysosome that can engulf other cellular components
leading to completion of the autophagic cell death cascade.
Some key proteins are involved for conducting this pathway.
Beclin-1, Atg group of proteins are the positive regulatory
upstream proteins and LC3B proteins are the key proteins,
which act as downstream regulators leading to the formation of
an autophagosome.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
In this experiment 1 Â 104 HeLa cells were seeded on poly-lysine
coated 60 mm Petri dishes. On addition of treatments with their The authors gratefully acknowledge the Department of Chemistry,
respective IC50 doses, cells were incubated overnight at 37 1C. University of Calcutta for Crystallographic and NMR studies and
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the Department of Botany, University of Calcutta for the biological
studies. The authors also thank Maulana Azad College, Kolkata for
use of the departmental facilities.
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