Synthesis and biological evaluation of 6H-1-benzopyrano[4,3-b] quinolin-6-one derivatives as inhibitors of colon cancer cell growth

Article abstract of DOI:10.3329/bjp.v10i3.23645

A convenient synthesis of 6H-1-benzopyrano[4,3-b]quinolin-6-one derivatives was reported using 4-chloro-2-oxo-2H-chromene-3-carbaldehyde with different aromatic amines using silica sulfuric acid. The compounds were tested for their anticancer activity against colon (HCT-116 and S1-MI-80), prostate (PC3 and DU-145), breast (MCF-7 and MDAMB-231) cancer cells. These compounds showed more selectivity and potent cytotoxic activity against colon cancer cells. 3c was tested against five other colon cancer cell lines (HT-29, HCT-15, LS-180, LS-174, and LoVo), which had similar cytotoxicity and selectivity. 3c did not induce PXR-regulated ABCB1 or ABCG2 transporters. In fact, 3c induced cytotoxicity in HEK293 cells over expressing ABCB1 or ABCG2 to the same extent as in normal HEK293 cells. It was cytotoxic approximately 3- and 5-fold to resistant colon carcinoma S1-MI-80 cells. 3c also produced concentration-dependent changes in HCT-116 colon cancer cells, in mitochondrial membrane potential, leading to apoptosis, and submicromolar concentrations caused chromosomal DNA fragmentation.

Full text of DOI:10.3329/bjp.v10i3.23645

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Synthesis and biological evaluation of 6H-1-benzopyrano[4,3-b]
quinolin-6-one derivatives as inhibitors of colon cancer cell growth
Tie-Ling Li1, Hai-Feng Guo2, Fu-Juan Li3, Zhi-Guo Sun2 and Heng-Chun Zhang2
2
1Department of Pathology, MuDanJiang Medical University, Mudanjiang 157 011, China; Department of General
Surgery, The affiliated Red Hospital of Mudanjiang Medical University, Mudanjiang 157 011, China; 3Department of
Pathogenic Biology and Immunology Laboratory，MuDanJiang Medical University, Mudanjiang 157 011, China.
Article Info
Abstract
Received:
Accepted:
Available Online:
12 June 2015
2 July 2015
2 August 2015
A convenient synthesis of 6H-1-benzopyrano[4,3-b]quinolin-6-one derivatives
was reported using 4-chloro-2-oxo-2H-chromene-3-carbaldehyde with differ-
ent aromatic amines using silica sulfuric acid. The compounds were tested for
their anticancer activity against colon (HCT-116 and S1-MI-80), prostate (PC3
and DU-145), breast (MCF-7 and MDAMB-231) cancer cells. These com-
pounds showed more selectivity and potent cytotoxic activity against colon
cancer cells. 3c was tested against five other colon cancer cell lines (HT-29,
HCT-15, LS-180, LS-174, and LoVo), which had similar cytotoxicity and
selectivity. 3c did not induce PXR-regulated ABCB1 or ABCG2 transporters.
In fact, 3c induced cytotoxicity in HEK293 cells over expressing ABCB1 or
ABCG2 to the same extent as in normal HEK293 cells. It was cytotoxic
approximately 3- and 5-fold to resistant colon carcinoma S1-MI-80 cells. 3c
also produced concentration-dependent changes in HCT-116 colon cancer
cells, in mitochondrial membrane potential, leading to apoptosis, and sub-
micromolar concentrations caused chromosomal DNA fragmentation.
DOI: 10.3329/bjp.v10i3.23645
Cite this article:
Li TL, Guo HF, Li FJ, Sun ZG, Zhang
HC. Synthesis and biological evalua-
tion of 6H-1-benzopyrano[4,3-b]
quinolin-6-one derivatives as inhibi-
tors of colon cancer cell growth.
Bangladesh J Pharmacol. 2015; 10: 660
-71.
et al., 2007; Hegab et al., 2007 ) or 1-benzopyrano[3,4
-f]quinoline (Tabakovic et al., 1983) have also been
identified for the promising bioactive molecules.
Introduction
Coumarin is
a fragrant organic chemical com-
pound in the benzopyrone chemical class isolated
from different plant sources (Geissman 1962; Har-
borne, 1988a; Harborne 1988b) and has various
pharmacological activities. For example, the couma-
rin nucleus is present in a number of potential drug
molecules such as nonpeptidic HIV protease inhibi-
tors, (Thaisrivongs et al., 1996) topoisomerase II
(Rappa et al., 2000) and tyrosine kinase inhibitors
(Yang 1999).
The combination of quinoline and coumarin in a
single molecule, for example, 6H-1-benzopyrano[4,3-
b]quinolin-6-one (Tabakovic et al., 1987; Mulakayala
et al., 2012) is acknowledged as a separate class of
heterocycle. It can be used as a model for the detec-
tion of bioactive molecules is not common. By know-
ing the cytotoxicities of coumarins and chromeno
[4,3-b]quinoline derivatives (Ajdini et al., 1984), we
hypothesized that design of small molecules based
on 6H-1-benzopyrano[4,3-b]quinolin-6-one might
show anticancer properties.
Alternatively quinolines and their derivatives are
known to have several applications in drugs and
pharmaceuticals. A combination of benzopyran or
chromen with a quinoline moiety in a single mole-
cule, for example, 6H-chromeno[4,3-b]quinoline (Vu
Herein we report the synthesis of a number of 6H-1-
benzopyrano[4,3-b]quinolin-6-one derivatives along

Bangladesh J Pharmacol 2015; 10: 660-671
with the in vitro anticancer properties of the com-
661
pounds synthesized.
Materials and Methods
Chemistry
Chemicals and reagents were purchased either from
Sigma or Merck, and all reagents were of analytical
reagent grade. Thin-layer chromatography (TLC)
was performed on Merck silica gel 60 F254 plates and
visualized under UV light. 1H NMR spectra were
recorded with Varian Mercury Plus 400 MHz
instrument. 13C NMR spectra were recorded with a
Varian Gemini 100 MHz instrument. All the
chemical shifts are reported in µ (ppm) using TMS
as an internal standard. Multiplicity is indicated by
white solid; mp 234–236ºC; 1H NMR (400 MHz,
DMSO-d6):  9.1 (s, 1H), 8.85 (dd, J = 7.5 and 1.3 Hz,
1H), 7.56–7.62 (m, 3H), 7.38–7.45 (m,2H), 7.24 (d, J =
5.7 Hz, 1H), 4.15 (s, 3H); 13C NMR (100 MHz,CDCl3):
 160.6, 153.2, 152.3, 150.0, 136.1, 129.7, 129.6, 127.7,
127.1, 126.5, 125.8, 124.3, 122.0, 110.1, 114.8, 112.2,
56.1; MS (ES mass):m/z 278 (M+1, 10%); HRMS:
calcd for C17H12NO3: 278.0815, found278.0817.
9,10-Difluoro-6H-chromeno[4,3-b]quinolin-6-one
(3c)
one or more of the following abbreviations:
s
(singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), br (broad); the coupling constants (J)
correspond to the order of the multiplicity assign-
ment. Mass spectra were recorded with a PE Sciex
model API 3000 instrument. All the reactions were
carried out under nitrogen atmosphere.
General procedure for the preparation of 6H-chromeno
[4,3- b]quinolin-6-one (3a–j)
white solid; mp 252–255ºC; 1H NMR (400 MHz,
CDCl3): µ 9.23 (s, 1H), 8.84 (dd, J = 8.1 and 1.5 Hz,
1H), 7.83–7.87 (m, 1H), 7.63–7.67 (m, 1H), 7.41–7.56
(m, 3H); 13C NMR (100 MHz, CDCl3): µ 160.9, 152.8,
150.1, 140.2,140.1, 132.8, 125.3, 125.2, 119.2, 117.5,
115.9, 115.7, 114.5, 114.4, 114.3, 114.2; MS(ES mass):
m/z 284 (M+1, 100%). HRMS: calcd for
C16H8F2NO2:284.0465, found: 284.04653.
A solution of 4-chloro-2-oxo-2H-chromene-3-carb-
aldehyde (1 mmol) (Bairagi et al., 2009) and aroma-
tic amine (1.5 mmol) in ethanol (5 mL) was added to
amberlyst 15 (10 mol%). The reaction was refluxed
for 2 hours and after completion of the reaction the
reaction mixture was filtered to remove the catalyst.
The filtrate was concentrated and the residue was
purified by flash chromatography (n-hexane/ethyl-
acetate 3:1) to afford the desired product.
10,11-Difluoro-6H-chromeno[4,3-b]quinolin-6-one
(3d)
6H-Chromeno[4,3-b]quinolin-6-one (3a)
white solid; mp 224–227ºC; 1H NMR (400 MHz,
DMSO-d6): µ 9.34 (s, 1H), 8.65 (dd, J = 7.5 and 1.5
Hz, 1H), 8.30 (d, J = 8.1 Hz, 1H), 8.21 (d, J = 8.7 Hz,
1H), 8.03–8.05 (m, 1H), 7.64–7.74 (m, 2H), 7.45–7.49
(m, 2H); 13C NMR (100 MHz, DMSO-d6): µ 160.7,
152.7, 150.4, 149.4, 141.2, 134.1, 133.0, 130.2, 129.0,
128.0, 127.3, 125.2, 125.0, 119.6, 117.6, 116.4; MS (ES
mass): m/z 248 (M+1, 100%); HRMS: calcd for
C16H10NO2: 248.0702, found 248.0701.
white solid; mp 257–260ºC; 1H NMR (400 MHz,
DMSO-d6): µ 9.46 (s, 1H), 8.62 (dd, J = 7.5 and 1.3
Hz, 1H), 8.24–8.28 (m, 1H), 7.84–7.91 (m, 1H), 7.75–
7.70 (m, 1H), 7.41–7.50 (m, 2H); 13C NMR (100 MHz,
CDCl3): µ 160.7, 152.8, 150.5, 141.0, 133.1, 125.6,
125.4, 125.3 (2C), 125.2, 125.1, 124.8, 119.0, 118.7,
118.5, 117.3; MS (ES mass): m/z 284 (M+1, 100%);
HRMS: calcd for C16H7F2NO2: 284.0486, found
284.0478.
11-Methoxy-6H-chromeno[4,3-b]quinolin-6-one (3b)
10-Bromo-6H-chromeno[4,3-b]quinolin-6-one (3e)

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Bangladesh J Pharmacol 2015; 10: 660-671
white solid; mp 229–231ºC; 1H NMR (400 MHz,
CDCl3): µ 9.2 (s, 1H), 8.77 (dd, J = 9.9 and 1.5 Hz,
1H), 8.13 (d, J = 9.1 Hz, 1H), 7.54–7.60 (m, 2H), 7.38–
7.44 (m, 2H), 7.22 (d, J = 2.7 Hz, 1H), 3.98 (s, 3H); 13C
NMR (100 MHz, TFA): µ 159.5, 153.7, 151.5, 147.5,
142.1, 138.8, 134.6, 134.5, 127.1, 124.7, 124.1, 122.0,
121.7, 118.8, 105.8, 105.5, 58.5; MS (ES mass): m/z
278.1 (M+1, 10%); HRMS: calcd for C17H12NO3:
278.0815, found 278.0813.
White solid; mp 245–247ºC; 1H NMR (400 MHz,
CDCl3): µ: 9.21 (s, 1H), 8.74 (dd, J = 8 and 1.5 Hz,
1H), 8.44 (s, 1H), 7.88 (d, J = 8.7 Hz, 1H), 7.72 (dd, J =
8.7 and 1.5 Hz, 1H), 7.61–7.65 (m, 1H),7.38–7.47 (m,
2H); 13C NMR (100 MHz, DMSO-d6): µ 161.1, 152.9,
150.5, 141.2, 132.9, 132.7, 131.9, 131.8, 131.3, 130.4,
125.9, 125.5, 125.2, 117.5, 115.9, 110.1; MS (ES
mass):m/z 327.1 (M+2, 100%); HRMS: calcd for
C16H9BrNO2: 327.2545,found 327.2541.
9-Methyl-6H-chromeno[4,3-b]quinolin-6-one (3i)
10-Fluoro-6H-chromeno[4,3-b]quinolin-6-one (3f)
white solid; mp 234–236ºC; 1H NMR (400 MHz,
CDCl3): µ 9.11 (s, 1H), 8.76 (dd, J = 8.1 and 1.5 Hz,
1H), 8.13 (d, J = 8.3 Hz, 1H), 7.73–7.75 (m, 2H), 7.55–
7.57 (m, 1H), 7.37–7.44 (m, 2H), 2.58 (s, 3H); 13C
NMR (100 MHz, DMSO-d6): µ 160.8, 152.7, 150.7,
149.5, 145.0, 140.8, 132.88, 130.2, 129.8, 128.1, 125.7,
125.2, 124.5, 119.7, 117.6, 115.5, 22.2; MS (ES mass):
m/z 262;2 (M+1, 100%); HRMS: calcd, for
C17H12NO2: 262.0869, found 262.0867.
white solid; mp 234–237ºC; 1H NMR (400 MHz,
CDCl3): µ 9.20 (s, 1H), 8.76 (dd, J = 8.1 and 1.5 Hz,
1H), 8.01–8.05 (m, 1H), 7.85 (dd, J = 9.8 and 2.3
Hz,1H), 7.58–7.65 (m, 1H), 7.38–7.48 (m, 3H);
13CNMR (100 MHz,DMSO-d6): µ 160.6, 153.1, 151.5,
150.6, 141.6, 133.5, 125.5, 125.2, 124.9, 119.5, 118.9,
118.6, 117.8, 116.1, 112.8, 112.6; MS (ES mass): m/z
266.2 (M+1, 10%); HRMS: calcd for C16H9FNO2:
266.2021, found 266.2018.
9-Chloro-6H-chromeno[4,3-b]quinolin-6-one (3j)
9-Fluoro-6H-chromeno[4,3-b]quinolin-6-one (3g)
white solid; mp 224–226ºC; 1H NMR (400 MHz,
CDCl3): µ 9.18 (s, 1H), 8.78 (dd, J = 8.1 and 1.5 Hz,
1H), 8.26–8.27 (m, 1H), 7.58–7.73 (m, 3H), 7.41–7.46
(m, 2H); 13C NMR (100 MHz, DMSO-d6): µ 160.9,
152.8, 150.3, 149.4, 141.3, 134.1, 133.0, 130.2, 129.1,
127.8, 127.4, 125.2, 125.1, 119.6, 117.6, 116.3; m/z
266.4 (M+1, 100%); HRMS: calcd for C16H9FNO2:
266.3032, found 266.3028.
white solid; mp 242–244ºC; 1H NMR (400 MHz,
CDCl3): µ 9.0 (s, 1H), 8.75 (dd, J = 7.5 and 1.5 Hz,
1H), 8.17–8.21 (d, J = 9.3 Hz 1H), 8.1 (s, 1H), 7.86
(dd, J = 9.1 and 2.5 Hz, 1H), 7.58–7.63 (m, 1H), 7.38–
7.45 (m, 2H); 13C NMR (100 MHz, CDCl3): µ 160.8,
152.4, 149.6, 149.1, 139.8, 134.1,133.2, 132.5, 131.1,
127.6, 127.5, 125.1, 125.0, 119.1, 117.3, 116.3; MS (ES
mass): m/z 282.2 (M+1, 100%); HRMS: calcd for
C16H9ClNO2, 282.0244, found 282.0242.
9-Methoxy-6H-chromeno[4,3-b]quinolin-6-one (3h)

Bangladesh J Pharmacol 2015; 10: 660-671
10-Chloro-6H-chromeno[4,3-b]quinolin-6-one (3k)
663
maintained in medium with 80 µM mitoxantrone
(Miri et al., 2011; Robey et al., 2001). The assay
media for PXR transactivation assays included
phenol red-free DMEM (Lonza) supplemented with
5% charcoal/dextran-treated FBS (HyClone) and the
other additives.
Cl
N
O
PXR transactivation assay
O
HepG2 cells were transfected with pcDNA3-hPXR
and CYP3A4-luc plasmids using FuGENE 6 (Pro-
mega). After 24 hours of transfection in growth
media, 10,000 cells were plated into wells of 96-well
culture plates (PerkinElmer), and treated with
DMSO, RIF, or 3c for an additional 24 hours. At 48
hours of transfection, a luciferase assay was perfor-
med to measure luminescence using the Neolite
Reporter Gene Assay System (PerkinElmer) and a
FLUOstar Optima microplate reader (BMG Labtech).
Normalized CYP3A4 promoter activity was express-
ed as fold induction over the DMSO control. Cell
viability was measured in parallel by CellTiter-Glo
luminescent assays (Promega), which determine the
number of metabolically active cells by quantifying
the ATP present. Luminescence was measured with
a FLUOstar Optima plate reader (BMG Labtech).
1
White solid; m.p. 243 -245ºC; H NMR (400 MHz,
CDCl3): δ 9.05 (s, 1H), 8.70 (d, J = 6.7 Hz, 1H), 8.12
(d, J = 9.1 Hz, 1H), 7.93 (d, J = 1.8 Hz, 1H), 7.82 (dd, J
= 8.9 Hz and 2.1, 1H), 7.55 – 7.60 (m, 1H), 7.34 – 7.42
(m, 1H); 13C NMR (100 MHz, CDCl3); δ 161.0, 152.8,
150.6, 149.5, 140.0, 134.3, 133.6, 132.7, 131.2, 127.8,
127.7, 125.3, 125.1, 119.4, 117.5, 116.5; MS (ES mass):
m/z 282.2 (M+1, 100%); HRMS calcd for
C16H8O2NCl, 281.0242 found 281.0246
9,10-Dimethoxy-6H-chromeno[4,3-b]quinolin-6-one
(3l)
MeO
MeO
N
O
Cell cytotoxicity as determined by MTT assays and
morphological analysis
O
1
Yellow solid; mp 291-294 oC, H NMR (400 MHz,
CDCl3): δ 8.94 (s, 1H), 8.66 (d, J = 7.7 Hz, 1H), 7.53 (t,
J = 7.5 Hz, 1H), 7.47 (s, 1H) 7.37 (dd, J = 7.8 and 14.3
Hz, 2H), 7.11 (s, 1H), 4.10 (s, 3H), 4.03 (s, 3H); 13C
NMR (100 MHz, CDCl3); δ 161.7, 156.1, 152.5, 150.8,
149.3, 148.1, 137.8, 131.7, 124.8, 124.7, 123.6, 120.1,
117.4, 114.2, 107.6, 105.6, 56.6, 56.4; MS (ES mass):
m/z 307.2 (M+1, 100%); HRMS calcd for C18H13O4N,
307.0840found 307.0834.
The MTT assay (Carmichael et al., 1987) was used to
determine cytotoxicity of the compounds to the follow-
ing cells: HCT-116, HCT-15, HT-29, LS-180, LS-174,
Lovo, S1, DU-145, PC-3, MDA-MB-231 and MCF-7.
Briefly, the cells were harvested with trypsin and
suspended at a final concentration of 5 X 103 cells/well.
Cells were seeded (180 µL/well) into 96-well multi-
plates. Different concentrations of 6H-1-benzopyrano
[4,3-b]quinolin-6-one derivatives (20 µL/well) were
added. After 72 hours of incubation, 20 µL of MTT
solution (4 mg/mL) was added in each well, and the
plates were incubated further 4 hours, allowing the
viable cells to convert the yellow-colored MTT into dark
-blue formazan crystals. Subsequently, the medium was
removed, and DMSO (100 µL) was added in each well
to dissolve the formazan crystals. The absorbance was
determined at 570 nm with an OPSYS microplate
Reader (DYNEX Technologies, Inc., Chantilly, VA,
USA). The means ± SD concentrations were calculated
from at least three experiments performed in triplicate.
The IC50 values were calculated from survival curves
using the Bliss method. At 68 hours, cells with or
without treatment were photographed by use of an
inverted microscope (Olympus, BX53F) with fluores-
cent lamps and digital cameras. The data were acquired
and analyzed by CellSens software.
Biology
DMEM media, 0.25% trypsin, propidium iodide,
paclitaxel, buffers, and reagents were purchased
from VWR (VWR International Inc., Suwanee, GA).
MitoTracker Red and Alexa Fluor 488 annexin V kits
for flow cytometry were purchased from Life
Sciences (Molecular Probes Inc., Invitrogen, Eugene,
OR). DMSO and RIF were purchased from Sigma.
Cell culture
Colon carcinoma cell lines [HCT-116, HCT-15, HT-
29, Lovo, LS-180, LS-174, S1 (a clone of LS174T
cells)] and prostatic cancer cell lines [DU-145 and PC
-3] and breast carcinoma cells [MDA-MB-231 and
MCF-7] were grown as adherent monolayers in
flasks with Dulbecco’s Modified Eagle Medium
(DMEM) supplemented with 10% fetal bovine serum
(FBS) in a humidified incubator containing 5% CO2
at 37ºC. A G482 mutant ABCG2-overexpressing,
drug-resistant colon cancer cell line, S1-M1-80, was
Mitochondrial membrane potential and DNA
fragmentation analysis
Mitochondrial membrane potential and apoptosis

664
Bangladesh J Pharmacol 2015; 10: 660-671
were measured for colon cancer HCT-116 cells using
MitoTracker Red and Alexa Fluor 488 annexin V kits
for flow cytometry (Molecular) Probes Inc.,
Invitrogen, Eugene, OR). Briefly, apoptosis was
induced by exposing HCT-116 cells (seeded in 6 well
-plates) with or without 3C at 1 or 10 µM concentra-
tions for 4 hours. Equal amounts of cells were
harvested, and, to each mL of cells, 4 µL of 10 µM
working solution of MitoTracker Red was added.
Cells were stained at 37ºC for 30 min in an atmos-
phere of 5% CO2. The cells were washed with PBS
and suspended in 100 µL of 1X annexin-binding
buffer, to which 5 µL of Alexa Fluor 488 annexin V
was added. Cells were incubated for an additional
15 min, after which 400 µL of 1X annexin-binding
buffer was added. The stained cells were counted by
flow cytometry, measuring the fluorescence at 530
nm and 585 nm (BD FACSCalibur Flow Cytometer
using FlowJo FACS data analysis software).
were used for checking the compatibility of this
method. All the amines including aromatic and
substituted aromatic amines were well tolerated
with the current methodology to yield the required
products in good to excellent yields. Thus, the
structures of the synthesized compounds are shown
in Table I.
Biological evaluation
Growth curves, cell cytotoxicity, and morphological
analysis for 6H-1-benzopyrano [4,3-b]quinolin-6-
one derivative derivatives (3a–3l)
6H-1-benzopyrano[4,3-b]quinolin-6-one derivatives
(3a–3l) were evaluated for cytotoxic effects on
threetypes of cancer cells, that is, colon (HCT-116
and S1), prostate (PC3 and DU-145), breast (MCF-7
and MDAMB-231) cell lines. The 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay was used. All compounds
were tested at concentrations ranging from 0.1 µM to
100 µM. The 50% minimum inhibitory concentra-
tions (IC50) are summarized in Table I.
A characteristic feature of apoptosis is induction of
oligonucleosomal DNA fragmentation by cytotoxic
compounds, which activate nucleases that degrade
the higher-order chromatin structure of DNA into
mono- and oligonucleosomal DNA fragments (Hen-
kels and Turchi, 1999; Wu et al., 2005). To establish
the mode of action of 3C in colon cancer cytotoxi-
city, a DNA fragmentation assay was performed.
Apoptotic degradation of DNA was analyzed by
agarose gel electrophoresis. Briefly, HCT-116 cells
were cultured in the presence of 3c (2.5 µM) for 6
hours. Genomic DNA was extracted from the cells
by use of Promega Wizard Genomic DNA purifica-
tion kits (Promega Corporation, Madison, WI) and
resolved on 1% agarose gels at 40V for 4 hours. DNA
was visualized by ethidium bromide staining and
photographed.
6H-1-benzopyrano [4,3-b]quinolin-6-one derivatives
(3a–3l) were exhibited variable degrees of growth
inhibitory activity towards the three human cancer
cell lines (Table I). Compound 3d, showed only a
modest inhibitory activity (IC50 >40 µM) on the
tested cells. Other compounds, with the exception of
3d, showed moderately more or less cytotoxic
activity relative to 3c.
Compound 3c with a 9,10-difluoro substitution, was
most effective against breast (MDAMB-231) cancer
cells, with IC50 values ranging between 44 and 50
µM. A similar cytotoxic profile against colon cancer
(HCT-116 and S1) and breast cancer cells was
observed for 3f, which has a 10-fluoro substitution.
Among the eight compounds studied, 3C, with a
difluoro substitution on the 9 and 10 position of the
6H-1-benzopyrano [4,3-b]quinolin-6-one ring, show-
ed selective and potent growth inhibition against
colon cancer cells. The IC50 values of 3c against HCT
-116 and S1 cells were 0.6 lM and 0.8 lM,
respectively. Prostate cancer cells (PC-3 and DU-145)
were also inhibited by 3c, with an IC50 values
ranging from 0.8 lM to 1.2 lM. Further, 3c showed
moderate growth inhibitory activity (IC50 >10 lM)
against breast (MCF-7 and MDAMB-231) cancer
cells. Overall, these results established that the 9,10-
difluoro substitution is significant to the anticancer
activity of 6H-1-benzopyrano [4,3-b]quinolin-6-one
and that compound 3c was a promising candidate
for further studies with colon cancer cells.
Results and Discussion
Chemistry
To find the convenient procedure we investigated
the cylclization of 4-chloro-2-oxo-2H-chromene-3-
carbaldehyde (1) with aniline (2) using Amberlyst-15
resin as catalyst in ethanol under reflux (Scheme-1)
for 2 hours which yielded 6H-1-benzopyrano[4,3-b]
quinolin-6-one 3a in 91% (Table I). Several amines
3c exhibits potent and selective cytotoxic activity on
various colon cancer cells results of compound 3c on
colon carcinoma cells encouraged us to expand our
Scheme I: Synthetic scheme of 6H-1-benzopyrano[4,3-b]
quinolin-6-one derivatives from 4-chloro-2-oxo-2H-
chromene-3-carbaldehyde

Bangladesh J Pharmacol 2015; 10: 660-671
665
Table I
Synthesis of a novel series of substituted 5-(aminomethylene)thiazolidine-2,4-dionesa
Yield (%)b
92
Entry
1
Amine (2)
Product
3a
3b
3c
2
3
93
81
3d
3e
3f
4
5
6
7
78
84
87
82
3g
3h
3i
8
9
85
92

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Bangladesh J Pharmacol 2015; 10: 660-671
Table I
Synthesis of a novel series of substituted 5-(aminomethylene)thiazolidine-2,4-dionesa (Cont.)
Yield (%)b
96
Entry
10
Amine (2)
Product
3j
3k
3l
11
12
96
97
aAll the reactions were performed using 4-chloro-2-oxo-2H-chromene-3-carbaldehyde 1 (1.0 mmol), aniline 2 (1.1 mmol) and amberlyst-15 (15
mol%), ethanol (10 vol.), reflux, 2-3 hours; b Isolated yields
Table II
Activity of 6H-1-benzopyrano [4,3-b]quinolin-6-one derivatives against various cell lines
Compound
Colon
Prostate
Breast
MDAMB-231
HCT-116
S1
PC3
DU-145
MCF-7
3a
48.6 ± 8.6
54.2 ± 9.8
65.2 ± 13.2
71.3 ± 11.2
69.4 ± 12.4
71.9 ± 12.9
76.5 ± 16.4
86.2 ± 14.3
78.5 ± 14.2
82.1 ± 12.3
3b
56.2 ± 11.3
61.2 ± 10.6
3c
3d
3e
1.6 ± 0.1
4.6 ± 0.4
1.7 ± 0.1
3.8 ± 0.3
0.9 ± 0.2
5.2 ± 0.5
1.6 ± 0.6
6.4 ± 0.4
7.6 ± 0.8
11.2 ± 1.1
74.6 ± 12.6
6.6 ± 0.4
13.2 ± 1.6
72.6 ± NA
82.5 ± 14.3
86.5 ± 15.6
93.2 ± 17.6
91.4 ± 16.3
3f
3g
3h
3i
12.1 ± 3.2
13.5 ± 2.9
91.6 ± 12.4
86.3 ± 14.3
12.9 ± 2.8
14.6 ± NA
93.5 ± 4.2
84.3 ± 15.2
15.3 ± 5.3
21.6 ± 4.3
16.2 ± 4.8
25.3 ± 4.3
18.3 ± 5.3
17.6 ± 4.3
93.2 ± 14.2
72.3 ± NA
17.6 ± NA
18.3 ± 5.3
91.3 ± 7.5
75.8 ± 12.9
120.3 ± 18.3
95.6 ± 15.6
114.6 ± 17.2
92.3 ± 14.3
3j
89.5 ± NA
85.4 ± 14.2
95.8 ± 18.2
83.4 ± 7.4
44.6 ± NA
87.3 ± 11.2
94.5 ± 14.6
106.7 ± 19.4
58.4 ± 6.9
110.5 ± 18.6
62.4 ± 4.6
3k
111.4 ± 18.3
3l
91.6 ± 12.7
72.8 ± 11.6
57.3 ± 15.8
53.7 ± 17.8
78.6 ± 16.2
71.6 ± 12.4
Cell survival assay was determined by the MTT assay. IC50 values are represented as means ± SD of three independent experiments performed in
triplicate. A mean IC50 value of 100 mM was the cut off. NA, not assessed

Bangladesh J Pharmacol 2015; 10: 660-671
667
studies to different types of colon cancer cells (HCT-15,
HT-29, Lovo, LS-180, and LS-174). All these cancer cells
were sensitive to 3c at concentrations ranging from µ0.6
to 1.0 µM (Table II, Figure 1A and B). Notably, 3c was
10–15 fold more selective for inhibition of colon cancer
cells relative to HEK293 cells (Figure 1A and B); 10–30
Figure 1: Cytotoxic effects of 3c
A) Survival of colon cancer cells S1, HCT-116, HCT-15, HT-29, Lovo, and LS-180 compared to that of human embryonic kidney cells, EK293/
pcDNA3.1; B) IC50 values of 3c for colon cancer cells S1, HCT-116, HCT-15, HT-29, Lovo, and LS-180 relative to human embryonic kidney cells
(HEK293/pcDNA3.1). Cell survival was determined by the MTT assay. IC50 values are represented as means ± SD of three independent experiments
performed in triplicate. Statistically, *p<0.05; **p<0.01, colon cancer cells versus the HEK293/pcDNA3.1; C) Morphological analysis of the cytotoxic
effects of 3c (0, 0.5, 2 and 10 M) on colon cancer cells, HCT-116 and HCT-15; prostate cancer cells, DU-145; and human embryonic kidney cells HEK-
293, exposed for 68 hours, was made by microscopy at 20x. The cells were photographed for each triplicate treatment with an inverted microscope
(Olympus, BX53F) with fluorescent lamps and digital cameras. A representative figure is shown for each treatment. The data were acquired and
analyzed with Cell Sens software

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Bangladesh J Pharmacol 2015; 10: 660-671
116, HCT-15, and HEK293 cells with 0, 0.5, 2, or 10 µM
Table II
3c, morphological changes, complementing the cytoto-
xicity data, were evident in all the cells (Figure 1c
shows representative images). In sum, the cytotoxic
effect of 3c on colon cancer cells is particularly notable
because of its selectivity and its IC50 values of <1 µM.
IC50 values for IND-2 inhibition of various colon
cancer cell lines
Colon cancer cells
IC50 ± SD (nM) 3c
HCT-15
670.6 ± 48.9
634.5 ± 120.1
3C does not activate pregnane X receptor (PXR), is
not a substrate of ABCB1 or ABCG2 transporters,
and is cytotoxic to chemoresistant colon cancer cells
HCT-116
HT-29
LS-180
LS-174
S1
790.8 ± 103.5
1023.5 ± 230.6
874.6 ± 89.7
798.3 ± 137.4
993.8 ± 142.6
{
PXR is a ligand-dependent nuclear receptor involved in
regulating the expression of ATP-binding cassette
(ABC) transporters, including ABCB1 (a.k.a. P-
glycoprotein) and ABCG2 (a.k.a. breast cancer esistance
protein, BCRP) (Pondugula et al., 2009, Pondugula and
Mani, 2013). Activation of PXR by xenobiotics leads to
chemoresistance, concern in drug development and
clinical therapy. The possible activation of PXR by3C
was examined with HepG2 cell-based reporter gene
assays. Although rifampicin (RIF), an agonist of human
Lovo
fold more selective for other cancer cells. (breast,
prostate) (Table I); After 68 hours of treatment of HCT-
Figure 2: 3c does not induce human PXR transactivation of CYP3A4 promoter activity
A) CYP3A4 prompter activity was determined in HepG2 cells after transient cotransfection with pGL3-CYP3A4-luc reporter and pcDNA3-hPXR
plasmids for 24 hours, followed by treatment with DMSO, RIF, or 3c for an additional 24 hours. The induction of CYP3A4 promoter activity was
normalized as fold increase over the DMSO control. Data represent means ± SD from three different experiments. Statistical significance (*p<0.05)
was determined with Student’s t test by comparing the effects of RIF or 3c with DMSO; B) Effect of 3c on HepG2 cell viability. During the PXR trans-
activation studies, viability of HepG2 cells was determined simultaneously in parallel experiments with the Cell Titer-Glo luminescent cell viability
assay kit and the data are expressed as luminescence units. Data represent the means ± SD of triplicate determinations. Statistical significance
(*p<0.05) was determined with Student’s t test by comparing the effects of RIF or 3c with DMSO; C) Effect of 3c on the survival of cells overexpress-
ing ABCB1 or ABCG2.32 Effects of 3c, topotecan, and SN-38 on S1 and S1-M1-80 cells. MTT cytotoxicity assays were used to measure survival of
HEK293/pcDNA3.1, HEK293/R2, HEK293/ABCB1, S1, and S1-M1-80 cells. Data points represent the means ± SD of triplicate determinations. Rep-
resentative results from three independent experiments, each performed in triplicate, are shown

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669
Figure 3: Effects of 3c on mitochondrial membrane potential and DNA fragmentation
A) HCT-116 cells in complete medium were expose to 3c (0, 1, or 10 mM) for 4 hours. Cells were then treated with the reagents of the MitoTracker
Red and Alexa Fluor 488 annexin V kits for flow cytometry. Representative results from two independent experiments, each performed in triplicate,
are shown; B) 3c induced DNA damage in HCT-116 cells. Chromosomal DNA was extracted from HCT-116 cells and resolved on a 1% agarose gel at
40 V for 6 hours. Lane 1: ‘M’, Marker; lanes 2 and 3: untreated HCT cells, 500 ng and 1 lg, respectively; lanes 4 and 5: HCT cells incubated with 3c (2.5
mM) for 6 hours

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Bangladesh J Pharmacol 2015; 10: 660-671
PXR, induced PXR transactivation of CYP3A4 promoter
activity, 3c at 1 or 10 µM did not affect PXR
transactivation (Figure 2A), showing that 3c does not
activate PXR at concentrations cytotoxic to colon cancer
cells and indicating that 3c does not induce PXR-
mediated drug resistance in cancer cells. Similar to RIF,
3c at 10 µM was marginally cytotoxic, although 3c at 1
µM was non-cytotoxic (Figure 2B), showing that the
lack of effect of 3c on PXR was not due to cytotoxicity.
released from mitochondria initiate apoptotic cell
death. In the process of apoptosis, phospha-
tidylserine (PS), otherwise located on the cyto-
plasmic surface, is displaced from the inner to the
outer leaflet of the plasma membrane. The
extracellular exposed PS has high affinity for
binding to the human vascular anticoagulant,
annexin V, a 35–36 kDa, Ca2+-dependent, phospho-
lipid-binding protein. This principle is used to
determine the binding of annexin V, labeled with a
fluorophore or biotin, to exposed PS on the outer
leaflet and thereby to identify apoptotic cells.
(Koopman et al., 1994) Live cells, 63.11% of
untreated HCT-116 cells with intact mitochondrial
membranes, are seen in quadrant I of Figure 3A. Of
these cells, 27.6% had a loss in mitochondrial
membrane potential and had started the process of
apoptosis. Treatment with 3c, even at 1 µM, for 4
Multi-drug resistance (MDR) factors, such as ABCB1
and ABCG2 transporters, present limitations for the
development of anticancer agents. (Perin et al., 2011)
These transporters are expressed in a variety of solid
and hematological malignancies, where they are
involved in the efflux of various therapeutic drugs,
leading to MDR. Ideally, drugs that are not
substrates for these transporters are preferred as
anticancer agents. 3c was screened against ABCB1-
and ABCG2-over-expressing cells, and its effects
were compared with positive controls paclitaxel and
mitoxantrone, respectively. There was no substantial
resistance to 3c in ABCB1- or ABCG2-overexpress-
ing cells, as all the cells were sensitive at similar
concentrations of 3c (Figure 2C), indicating that 3c is
not a substrate for ABCB1 and ABCG2 transporters.
These results support our PXR analysis (Figure 2A).
Additionally, the cytotoxic potential of 3c was
hours produced
a significant loss (p<0.01) of
membrane potential in 68.46% of the cells, as seen in
quadrant II of Figure 3A. At 10 µM, 3c treatment for
4 h showed that almost all the cells (98.36%) had no
or low mitochondrial membrane potential and thus
were primed to undergo apoptosis (quadrant II of
Figure 3A). These data show that 3c produces
concentration-dependent decreases in mitochondrial
potential of HCT-116 colon cancer cells, leading to
apoptosis.
evaluated for
a
G482 mutant ABCG2-over-
expressing, drug-resistant colon cancer cell line, S1-
M1-80. S1-M1-80 cells showed significant resistance
(p<0.001) to SN-38, an active metabolite of irino-
tecan and topotecan (Figure 2D). 3c, however,
inhibited the resistant S1-M1-80 cells 3–5 fold more
potently (p<0.001) than SN-38 and topotecan (Figure
2D). This could be because: (a) 3c did not interact
with ABCG2 transporters, was not effluxed by
resistant S1-M1-80 cells, and maintained its cytotoxic
concentration, or (b) 3c blocked the ABCG2
transporters and retained its cytotoxic potential in
resistant S1-M1-80 cells. If the second reason is true,
3c may be beneficial in combination chemotherapy
against colorectal and other tumors with agents that
are substrates of ABCG2 transporters. However,
further mechanistic experiments including ATPase,
photo-affinity labeling assays, and combination
chemotherapy are necessary to confirm the
interactions of 3c with ABCB1 and ABCG2
transporters.
The extent of 3c-induced DNA damage in HCT-116
colon cancer cells was assessed by chromosomal
DNA fragmentation.Treatment with 2.5 µM 3c for 6
h led to fragmentation of chromosomal DNA (Figure
3B). In contrast, chromosomal DNA of untreated
cells was intact. These results indicate that 3c
induces DNA breakage at multiple positions across
chromosomal DNA, leading to apoptosis.
Conclusion
The present study describes the synthesis of 6H-1-
benzopyrano[4,3 -b]quinolin-6-one
deriva -
tives derivatives (3a–3l). The synthesized analogues
3a-3l was evaluated their anti-cancer activity. 9,10-
difluoro-6H-chromeno[4,3-b]quinolin-6-one 3c was
shown potent inhibition against colon cancer cells.
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Author Info
Hai-Feng Guo (Principal contact) e-mail: guohaifeng07@gmail.com;