Anti-cancer potential of (1,2-dihydronaphtho[2,1-b]furan-2-yl)methanone derivatives

Article abstract of DOI:10.1016/j.bmcl.2020.127476

A series of 1,2-dihydronaphtho[2,1-b]furan derivatives were synthesized by cyclizing 1-(aryl/alkyl(arylthio)methyl)-naphthalen-2-ol and pyridinium bromides in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in very good yield. The synthesized compounds were evaluated for their anti-proliferative potential against human triple negative MDA-MB-468 and MCF-7 breast cancer cells and non-cancerous WI-38 cells (lung fibroblast cell) using MTT experiments. Among 21 synthesized compounds, three compounds (3a, 3b and 3 s) showed promising anti-cancer potential and compound 3b was found to have best anti-proliferative activities based on the results of several biochemical and microscopic experiments.

Full text of DOI:10.1016/j.bmcl.2020.127476

Journal Pre-proofs
Anti-cancer potential of (1,2-dihydronaphtho[2,1-b]furan-2-yl)methanone de‐
rivatives
Kobirul Islam, Kunal Pal, Utsab Debnath, R. Sidick Basha, Abu Taleb Khan,
Kuladip Jana, Anup Kumar Misra
PII:
S0960-894X(20)30587-4
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127476
BMCL 127476
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
30 January 2020
2 August 2020
5 August 2020
Please cite this article as: Islam, K., Pal, K., Debnath, U., Sidick Basha, R., Taleb Khan, A., Jana, K., Kumar
Misra, A., Anti-cancer potential of (1,2-dihydronaphtho[2,1-b]furan-2-yl)methanone derivatives, Bioorganic &
Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127476
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Graphical Abstract
Anti-cancer potential of (1,2-
dihydronaphtho[2,1-b]furan-2-yl)methanone
derivatives
Leave this area blank for abstract info.
Kobirul Islam,a,b Kunal Pal,a Utsab Debnath,a R. Sidick Basha,b Abu Taleb Khan,b Kuladip Janaa* and Anup
Kumar Misraa *
2
1
R
R
S
Me
One-Pot
[
]
Compound 3b
O
1
H
R
IC = 15 ± 3.1 against MDA-MB-468
O
50
N
3
3
H
R
DBU
R
17± 2.65 against MCF-7
19± 2.1 against MDA-MB-231
21±1.9 against 4T1
+
Br
2
R
CH CN, reflux
3
O
O
H
3
58 ± 1.7 against WI-38
1
R
= Ar , Heter oaryl, alkyl
R
= Ar, Heteroaryl, Alkyl, H
= H, Br
3a-u
21 examples,
2
R
up to 86% yield

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Anti-cancer potential of (1,2-dihydronaphtho[2,1-b]furan-2-yl)methanone
derivatives
Kobirul Islam,a,b,c Kunal Pal,a,c Utsab Debnath,a R. Sidick Basha,b Abu Taleb Khan,b Kuladip Janaa* and
Anup Kumar Misraa*
a Bose Institute, Division of Molecular Medicine, P-1/12, C.I.T. Scheme VII M, Kolkata 700054, India; E-mail:
akmisra69@gmail.com; kuladip.jana@gmail.com
b Department of Chemistry, Indian Institute of Technology Guwahati, Guwahati 781 039, India.
c
Contributed equally.
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A series of 1,2-dihydronaphtho[2,1-b]furan derivatives were synthesized by cyclizing 1-
(aryl/alkyl(arylthio)methyl)-naphthalen-2-ol and pyridinium bromides in the presence of 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) in very good yield. The synthesized compounds were
evaluated for their anti-proliferative potential against human triple negative MDA-MB-468 and
MCF-7 breast cancer cells and non-cancerous WI-38 cells (lung fibroblast cell) using MTT
experiments. Among 21 synthesized compounds, three compounds (3a, 3b and 3s) showed
promising anti-cancer potential and compound 3b was found to have best anti-proliferative
activities based on the results of several biochemical and microscopic experiments.
Keywords:
dihydronaphtho[2,1-b]furan
DBU
anti-proliferative
anti-cancer
2009 Elsevier Ltd. All rights reserved.
apoptosis
Cancer is most lethal disease which is characterized by
abnormal proliferation of cells, which invades other parts of the
body through metastasis and affects the normal functioning of vital
organs of the body.1 Various therapeutic strategies such as
chemotherapy, immunotherapy, radiation therapy, targeted
therapy, gene therapy have been explored for the treating of the
disease.2 Despite of several advanced treatment strategies,
metastasis and cancer relapsed with resistance remain a significant
challenge in cancer treatment. Recently, the reports show that
more or less 8.2 million people around the globe are suffering from
the irreversible metastatic condition of malignant tumors with drug
resistance.3 Among several organ specific cancer complications,
breast cancer is frequently found in women throughout the world.4
Because of its metastatic nature, breast cancer usually transfers to
distant organs such as the bone, liver, lung and brain. Early
diagnosis of the disease can lead to a good prognosis and a high
survival rate. Although several anticancer therapeutics are
available in clinics to arrest breast cancer, they are suffering from
undesirable side-effects and toxicities.5-7 Several new molecular
signaling and pathways in oncogenic transformation, tumor
progression and metastasis have been identified utilizing several
bio-chemical techniques like protein-protein interaction, protein-
DNA/RNA interactions.8,9 Therefore, targeting the pathways in
oncogenic transformation, tumor progression and metastasis
through chemically synthesized small molecules may be effective
to develop chemoprevention of cancer.
A wide range of biologically active natural products contain
2,3-dihydrobenzofuran as the active framework for their biological
activities.10 Several compounds having 2,3-dihydrobenzofuran
scaffold within were reported as antibacterial,11 antitumor,12
antifungal,13 and insecticidal agents14 (Figure 1).
O
OH
O
HO
O
O
(+)-Conocarpan
( )-Liliflol-B
OH
OH
OMe
O
O
OH
O
Annullatin A
Callislignan A
OH
O
HO
OMe
OH
OH
O
OMe
OMe
(2R,3S)-3,4'-di-O-methylcedrusin
O
Megapodiol
Figure 1: Biologically active compounds having 1,2-benzofuran
scaffold.

experimentation, it was observed that best yield of (1,2-
dihydronaphtho[2,1-b]furan-2-yl)(p-tolyl)methanone (3b) can be
obtained by refluxing the reaction mixture in CH3CN using DBU
(1.0 equiv.) as the cyclizing agent (Table 1; entry 4). Use of other
bases as cyclizing agents did not give satisfactory yield of the
product. The substrate scope of the reaction condition was
excellent since a series of (1,2-dihydronaphtho[2,1-b]furan-2-
yl)methanone derivatives (3a-u) were synthesized using a variety
However, reports on the synthesis of 1,2-dihydronaphthofuran
derivatives15 and their biological activity are rather scarce. In spite
of their versatile pharmaceutical potential, only a few reports on
their enzyme inhibitory activities16-18 are available (Figure 2).
O
O
HO
N
NH2
O
N
H
Br
O
O
of
1-(alkyl/arylthio)methyl)-naphthalen-2-ol
(1a-o)
and
O
pyridinium bromides (2a-f) following similar reaction condition as
mentioned in Table 2. It is noteworthy that the reaction condition
is highly stereoselective and use of substituted 1-
(alkyl/arylthio)methyl)-naphthalen-2-ol furnished exclusively
single diasteriomeric products (3g-u) without formation of the
other isomer. The starting materials napthalen-2-ol sulfides (1a-o)
were prepared following reported reaction conditions.20 All
synthesized compounds were purified using flash chromatography
and obtained with high purity. Most of the compounds tested for
biological potential were in the range of 96-98% purity and
characterized by their NMR and mass spectral analysis.
Melatonin
receptor
5-Lipoxygenase
inhibitor

-Chymotrypsin
inhibitor
Figure 2: 1,2-Dihydronaphthofuran derivatives as enzyme
inhibitors.
It was decided to synthesize a library of 2,3-dihydronaphthofuran
derivatives to evaluate their anti-proliferative potential against
different breast cancer cells towards the development of novel
anti-cancer agents. As an extension of the recent report19 on the
synthesis of unsymmetrical ethers by the cleavage of C-S bond in
napthalen-2-ol sulphide, it was envisaged that combination of C-S
bond cleavage in 1-(aryl/alkyl(arylthio)methyl)-naphthalen-2-ol
along with tandem C-C and C-O bond cyclization might lead to
the synthesis of 2-acyl-1,2-dihydronaphtho [2,1-b]furan
derivatives (Scheme 1).
Table 1: Optimization of reaction condition for the synthesis of
(1,2-dihydronaphtho[2,1-b]furan-2-yl)(p-tolyl)methanone (3b).
O
S Tol
Cyclizing
agent
O
OH
O
N
O
R1
R3
R1 S Tol
+
Br
Solvent
Cyclizing agent
CH3CN, Reflux
O
OH
+
O
1a
2b
3b
N
R3
R2
Sl.
No.
1
2
3
Base
Solvent
Temp Time Yield
R2
Br
2-7h
(h)
5
5
8
(%)
ND
ND
60
(C)
r t
reflux
Reflux
Reflux
R1, R2, R3 = H, alkyl and aryl
--
--
CH3CN
CH3CN
CH3CN
CH3CN
Scheme 1: Cleavage of C-S bond along with tandem C-C and C-
O bond cyclization for the synthesis of 1,2-dihydronaphtho[2,1-
b]furan derivatives.
DBU (0.5 equiv)
DBU (1.0 equiv)
4
5
80
5
DBU (1.0 equiv.) THF
Reflux 10
50
6
7
8
9
DBU (1.0 equiv)
DBU (1.0 equiv)
DBU (1.0 equiv)
DABCO (1.0
equiv)
Et3N (1.0 equiv)
iPr2NH (1.0
equiv)
DMF
EtOH
80
80
8
10
72
35
20
50
In
a
set of initial experiments, cyclization of 1-(p-
(1a) with 1-(2-oxo-2-p-
tolylthiomethyl)naphthalen-2-ol
Toluene Reflux 10
CH3CN
tolylethyl)pyridinium bromide (2b) was attempted using a variety
of organic bases such as DBU, DABCO, Et3N, (iPr)2NH, K2CO3
in different organic solvents such as EtOH, CH3CN, DMF, THF,
toluene at variable reaction temperature. The reactions were
continued till the formation of the desired product in maximum
yield. The temperature and time of the reactions were changed to
obtain maximum yield of the product. After a set of
Reflux 10
10
11
CH3CN
CH3CN
Reflux 10
Reflux 10
45
55
12
K2CO3 (1.0
equiv)
CH3CN
Reflux
8
40
Table 2: Synthesis of (aryl)(1-(aryl/alkyl-1,2-dihydronaphtho-[2,1-b]furan-2-yl) methanone (3a-u).
O
R1 S Tol
R1
R3
O
R3
OH
+
DBU
CH3CN, Reflux
O
N
R2
Br
R2
2-7h
3a-u
2a-g
1a-o
R2
Sl.
No.
R1
R3
Me
Product
Time
(h)
0.75
Yield
(%)
68
1
2
H
H
O
Me
O
3a
O
H
H
4-MeC6H4
3
80
Me
O
3b

O
3
4
H
H
H
H
4-BrC6H4
5
5
85
86
Br
O
3c
O
4-FC6H4
F
O
3d
O
5
6
7
8
H
H
H
Br
H
2-furyl
C6H5
C6H5
C6H5
6
5
5
3
75
71
85
70
O
O
O
3e
O
Br
3f
Me
Pr
O
Me
O
3g
H
O
O
3h
9
Cyclohexyl
H
H
C6H5
5
5
69
70
O
O
3i
10
C6H5
C6H5
O
O
3j
11
12
4-MeC6H4
H
H
C6H5
2
4
75
79
Me
O
O
3k
4-MeOC6H4
C6H5
MeO
O
O
3l

13
3,4,54-MeOC6H2
H
C6H5
4
72
OMe
MeO
MeO
O
O
3m
14
15
16
17
4-HOC6H4
4-ClC6H4
4-BrC6H4
4-CN-C6H4
H
H
H
H
C6H5
C6H5
C6H5
C6H5
4
4
3
6
71
81
79
85
HO
O
O
3n
3o
3p
Cl
O
O
Br
O
O
NC
O
O
3q
3r
3s
18
19
20
21
2-naphthyl
H
Br
Br
H
C6H5
7
3
63
78
75
41
O
O
C6H5
C6H5
O
O
Br
C6H5
2-furyl
4
O
O
O
Br
3t
2-naphthyl
4-CN-C6H4
12
O
CN
O
3u

compounds, Compounds 3b, 3c and 3s showed significantly
higher efficacy in blocking proliferation of MDA-MB-468 cells
with LD50 values of (15 ± 3.1, 18 ± 1.2, 18 ± 2.4 µM respectively).
Similar anti-proliferative efficacy of Compounds 3b, 3c and 3s
was also found in the case of MCF-7 cells with LD50 values of (17
±2.65, 21 ± 3.9 and 19 ±2.2 µM respectively). Subsequently, when
the selectivity index (SI) compounds were calculated by
comparing the cytotoxic LD50 value of the compound in normal
non-cancerous lung fibroblast cells (WI-38) versus cancer cells
(MDA-MB-468 and MCF-7) it was observed that compound 3b
possessed higher SI (SI = 3.87±0.058 and 3.11 ±0.062) as
compared to compound 3c (SI = 3.11±0.062 and 2.66 ±0.075) and
compound 3s (SI = 2.89±0.076 and 2.74±0.027) (Figure 3).
Therefore, amongst the 21 screened compounds, compound 3b
showed significant anti-cancer activity based on its highest
selectivity index.
Biology
Compound 3b showed selective cytotoxicity towards the
cancer cells
Primarily, 21 synthesized compounds along with Etoposide, a well
known anticancer agent, were assessed for their ability to block
proliferation of human triple negative MDA-MB-468 [(ER-, PR-
and HER2-) breast cancer cells], MCF-7 [human (ER+, PR+ and
HER2-) breast cancer cells] and non-cancerous WI-38 cells (lung
fibroblast cell) using MTT experiments.21 For this purpose, cells
were treated with varying concentrations of the compounds (0-50
µM) and cell proliferation was measured. The anti-proliferative
activities of the tested compounds in terms of LD50 were presented
in Table 3. From the MTT assay it was found that three
Table 3: The anti-proliferative activities of the synthesized compounds and standard anticancer agent expressed in terms of LD50 (µM).
Sl.
No.
Compound
MDA-MB-468 MCF-7
WI-38
S.I. ( WI-38/
MDA-MB 468)
S.I. (WI-38/
MCF-7)
1
3a
3b
3c
3d
3e
3f
22 ± 1.7
15 ± 3.1
18± 1.2
26± 2.5
17± 2.65
21± 3.9
29± 2.9
47± 2.8
58 ± 1.7
56± 0.59
54± 2.37
51 ± 1.7
73± 1.23
64 ± 1.7
79 ± 2.13
74 ± 2.9
89 ± 2.8
72 ± 0.8
83± 1.4
2.13± 0.056
3.87± 0.058
3.11± 0.062
2.00± 0.056
1.5± 0.026
2.28± 0.036
1.88± 0.051
1.14± 0.048
1.6± 0.036
1.24± 0.026
1.14± 0.032
0.70± 0.023
1.45± 0.036
1.42± 0.054
1.1± 0.087
1.22± 0.032
1.35± 0.045
2.56± 0.086
2.89± 0.076
1.8± 0.056
2
3.41± 0.054
2.66± 0.075
1.86± 0.045
1.38± 0.066
1.35± 0.067
1.68± 0.084
1.07± 0.044
1.76± 0.064
1.30± 0.071
1.01± 0.036
0.98± 0.028
1.49± 0.068
1.49± 0.059
1.05± 0.076
1.37± 0.068
1.41± 0.043
2.10± 0.032
2.74± 0.027
3
4
27 ± 2.5
34 ±1.9
5
37± 1.16
54 ± 2.17
38 ± 2.36
74 ± 1.27
42 ± 1.14
68 ± 1.8
71 ± 1.6
84 ± 2.9
86 ± 1.9
77 ± 2.1
9 1± 2.4
56 ± 3.2
51 ± 2.2
28 ± 2.6
19± 2.2
6
32±1.87
34 ± 1.9
69 ± 1.87
46± 3.4
72 ± 1.7
63± 2.3
118 ± 1.8
88 ± 3.1
81 ± 2.4
87± 2.5
63 ± 3.2
53 ± 2.3
23± 2.3
18 ± 2.4
7
3g
3h
3i
8
9
10
11
12
13
14
15
16
17
18
19
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
128± 0.93
115 ± 1.9
96 ± 3.27
77 ± 0.59
72 ± 0.7
59 ± 0.8
52 ± 0.68

20
21
22
3t
68± 2.8
72± 2.8
57± 3.9
92± 0.88
88 ± 1.4
1.35± 0.051
1.63± 0.076
1.06± 0.046
1.28± 0.018
1.54± 0.027
0.65± 0.066
3u
54 ± 1.8
Etoposide
27.88± 3.62
45.31± 2.41 29.63 ± 2.19
in Figure 4. From the AnnexinV-FITC/PI staining on the cancer
cells (MDA MB-468 and MCF-7) and non-cancer cell lines (WI-
38), it was observed that there was no substantial apoptosis took
place in case of the non-cancer cells treated with compound 3b
while in case of the cancer cells the percentage of apoptotic cell is
significantly high. Therefore, it was decided to identify the
mechanism of the induction of apoptosis in cancer cells by
compound 3b employing a series of biochemical and microscopic
analysis.
In order to evaluate the cytotoxic effects of the active compounds
(3b, 3c and 3s) against other triple negative breast cancer cell lines,
a comparative MTT assay was also performed using the active
compounds against MDA-MB-231 (Human) and 4T1 (Murine)
cells. Compounds 3b, 3c and 3s showed similar trends in
cytotoxicity against other cell lines such as MDA-MB 231, 4T1
and as presented in Table 4.
Table 4: The anti-proliferative activities of compounds 3b, 3c and 3s
against MDA-MB-231 (Human), 4T1 (Murine) and WI-38 cells
expressed in terms of LD50 (µM).
Compd. MDA- 4T1
WI-
38
SI(WI-38/ SI(WI-38/
MDA-MB 4T1)
231)
MB
231
3b
3c
3s
19±2.1 21±1.9 58 ± 3.05±0.037 2.76±0.051
1.7
23±1.6 25±2.4 56±
0.59
2.43±0.058 2.24±0.068
22±1.8 24±1.7 52 ± 2.36±0.054 2.16±0.048
0.68
Figure 4: (a) Dot plot of Annexin V-FITC/PI for evaluation of
apoptosis in MDA-MB 468, MCF-7 and WI-38 cells treated with
the compound 3b; (b) Quantification of apoptotic cells for
evaluation of apoptosis treated with compound 3b at doses 10 µM,
20 µM and 30 µM.
Figure 3: (a) Assessment of cytotoxicity of compound 3b on triple
negative breast cancer cell lines, MDA-MB 468, MDA-MB-231,
4T1, MCF-7 and non-cancerous cell line WI-38 by MTT assay
which shows dose dependent decrease in cell survivability in cells
treated with compound 3b, (b) LD50 values of compound 3b in
MDA-MB 468, MDA-MB-231, 4T1, MCF-7 and WI38 cells.
Treatment of compound 3b promotes ROS generation,
mitochondrial permeability transition (MPT), DNA damage
and apoptosis in cancer cells.
Generation of reactive oxygen species (ROS) is a critical
phenomenon linked with several anti-proliferative processes.23
The ROS production in cancer cells in response to the treatment
with compound 3b has been assessed using the DCFDA method.24
Briefly, cells were treated with varying doses (0, 10, 20 and 30
µM) of compound 3b for 12 h followed by 30 min of DCFDA (100
µM) staining and microscopy. The photomicrographs
demonstrated concentration dependent increase in the intensity of
the green colour in response to the treatment with compound 3b
suggesting significant induction of ROS generation in MDA-MB-
468 and MCF-7 cells as shown in Figure 5. Pre-treatment with 10
mM of cellular ROS scavenger, N-acetyl cysteine (NAC)
In order to study the mechanism of action of the cytotoxicity
against the cancer cells of the most promising compound 3b based
on the selectivity index (SI= 3.87±0.058 and 3.41 ±0.054), the
induction of apoptosis was measured using Annexin V FITC/PI
staining.22 After treatment of MDA-MB-468 and MCF-7 cells with
different concentrations of compound 3b (0, 10, 20 and 30 μM)
for 24 h, the flow cytometry analysis showed significant induction
of apoptosis in a concentration dependent manner in the cancer
cells in comparison with the non-cancer cells (WI-38). Quite
evidently, compound 3b was able to induce nearly 69% apoptosis
in MDA-MB-468 and 61% in MCF-7 cells at 30 μM dose as shown

significantly reverses the ROS levels in the MDA-MB 468 and
MCF-7 cells treated with the 30 µM of Compound 3b as seen in
Figure 5a. The WI-38 cells also exhibits no such significant
enhancement in ROS production when they were treated with the
doses of 10, 20 and 30µM as shown in Figure 1 in supporting
information. Therefore, it can be confirmed that intracellular ROS
was generated in presence of the compound 3b selectively in the
cancer cells.
The mitochondrial ROS production was further assessed by
staining the cells with MitoSOX™ Red reagent.27 The high red
fluorescence due to the presence of oxidized products indicates the
induction of mitochondrial ROS generation.28 In the present study
an enhanced high red fluorescence was observed in case of cancer
cells treated with different concentrations of compound 3b (10
µM, 20 µM and 30 µM) (Figure 7). Hence it can be concluded that
compound 3b mediated cancer cell apoptosis took place due to
increased mitochondrial ROS production.
Figure 5: Determination of cellular ROS with DCFDA method.
(a) Fluorescence microscopic image of treated cells and (b)
Spectrophotometric fluorescence intensity measurement which
indicates the enhancement of ROS in cells treated with the
compound 3b at doses 10 µM, 20 µM and 30 µM. NAC is used as
a ROS inhibitor. The data is the average of three experiments ±SD.
* represents P value < 0.05, ** represents P value < 0.01.
Figure 7: (a) Mitochondrial ROS determination by Mitosox™
Red on MDA-MB-468 and MCF-7 cells which clearly indicates
the generation of mitochondrial ROS in cells treated with
compound 3b at doses 10 µM, 20 µM and 30µM; (b)
Spectroscopic fluorescence intensity of the induced mitochondrial
ROS in MDA-MB- 468 and MCF-7 cells. The data is the average
of three experiments ± SD. * represents P value < 0.05, **
represents P value < 0.01.
Since ROS plays critical role in changing the mitochondrial
permeability transition (MPT),25 the effect of compound 3b on
mitochondrial damage in MDA-MB-468 and MCF-7 cells was
investigated by JC-1 staining.26 A drastic alteration of the redox
status of cellular mitochondria in response to the treatment of
compound 3b was observed in a concentration and time dependent
manner (Figure 6). However, the WI-38 cells treated with
compound 3b at the same doses did not exhibit any significant
transition in mitochondrial membrane potential as seen in Figure
2 in supporting information. The shift of fluorescence from red to
green or a decrease in the red/green ratio indicated the increase in
the mitochondrial permeability in response to compound 3b only
in cancer cells. These results altogether implies that compound 3b
mediated generation of ROS may have decisive role in the
mitochondrial permeability transition in cancer cells.
Figure 8: Comet assay of MDA-MB-468 cells following treatment
with compound 3b at the doses of 10 μM, 20 μM and 30 μM.
In order to accumulate more evidences to support cancer cell
apoptosis mediated by the treatment of compound 3b, damage of
DNA in MDA-MB-468 cancer cells in response to the treatment
with compound 3b was assessed using Comet Assay.29 Comet
assay (also known as Single Cell Gel Electrophoresis Assay) is a
facile method for measuring DNA strand breaks in eukaryotic
cells.30 The term “comet” refers to the pattern of DNA migration
through the electrophoresis gel, which often resembles a comet. It
was found that the fluorescence was confined mostly to the
nucleus in control cells because of the undamaged DNA, whereas
negatively charged DNA fragments were released from the
nucleus of MDA-MB-468 cells treated with compound 3b due to
DNA damage and migrate toward the anode leading to a prolonged
tail formation. It was observed that tail length upon treatment with
Figure 6: Mitochondrial membrane potential measurement by JC1
on (a) MDA-MB-468 and (b) MCF-7, which clearly indicates the
changes in the mitochondrial membrane potential transition in
cancer cells treated with the compound 3b at doses 10 µM, 20 µM
and 30 µM.

compound 3b at 20 µM and 30 µM concentrations for 24 h were
32 ± 3 nm and 63 ± 2 nm respectively. This increase of tail length
is an indication of DNA damage in cancer cells in comparison to
the untreated cells which had no or negligible tail length (Figure
8).
growth.31 In order to establish the induction of apoptosis,
expression of different pro-apoptotic and anti-apoptotic proteins
were carried out using western blot analysis.32 The data showed
increase in the expression of pro-apoptotic protein Bax, whereas
the expression of anti-apoptotic protein Bcl-2 and pBad was
decreased significantly in MDA-MB-468 cells following
treatment with compound 3b. The expression of p53 in MDA-MB-
468 cells was also enhanced upon treatment with the compound
3b. Moreover, upon treatment with compound 3b showed
cleavage mediated activation of caspase-3 and thereby suggesting
caspase mediated induction of apoptosis.33 Altogether these results
suggested that there was a significant change occurred in Bax/Bcl-
2 ratio and the expression of p53, pBad, caspase-3 in MDA-MB-
468 cancer cells due to the treatment with compound 3b, which
play critical roles in apoptosis (Figure 9). It was observed that a
general caspase-3 inhibitor Z-VAD-FMK was able to diminish the
expression of the cleaved caspase-3 which confirmed the cell death
was caspase dependent. However the WI38 cells that were treated
with a dose of 10, 20 and 30 µM of compound 3b did not show
any significant enhanced expression of cleaved caspase-3 protein
(as shown in Figure-3 in supporting information), which
suggested that compound 3b was selectively effective against the
cancer cells only.
Molecular docking study of three selective compounds
In order to elucidate the structure activity relationship (SAR)
between the biological activity of compound 3b and its binding
affinity towards the active domain of caspase-3 enzyme, molecular
docking experiment was carried out.34,35 It is clear that hydrogen
bonding interactions with the binding pocket of caspase-3 enzyme
play crucial roles for its caspase-3 inhibitory activity (Figure 10a).
Nevertheless, Gibbs free energy (ΔG) indicates that π-π stacking
and CH-π hydrophobic interactions with surrounding amino acids
(i.e. Tyr 204, Arg 207, Trp 206, Gly 122 and Thr 166) of the
binding site are also crucial to improve the biological activity
(Table 5, Figure 10). The 3D docked conformation of compound
3b showed that naphthafuran scaffold is important to increase their
common - as well as CH- stacking hydrophobic interactions.
Also, the tetrahydrofuran group of naphthafuran scaffold provide
a strong H-bond interaction with Cys 163 (compound 3b, ΔG =
8.15 kcal/mol, IC50: 15±3.10 for MDA-MB-468 and 17±2.65 for
MCF-7). In addition, a carbonyl (C=O) linker in between
napthafuran and 4-methyl phenyl group showed another H-bond
interaction with Tyr 204 which was used to bind the molecule
more tightly with the caspase-3 enzyme.36 From 2D view, it can be
concluded that naphthalene is a very important part of
naphthafuran as it binds with Arg 207 closely which may induces
its better potential activity against the enzyme (Figure 10b).
Furthermore, Figure 10C revealed that compound 3b is nicely
superimposed with the indole-2,3-dione group of the co-crystal
ligand. These superimposed structures give significant
information to compare their anti-cancer activity. Therefore, it can
be concluded that (a) targeting of Cys 163, Thr 166 and Arg 207
amino acids of caspase-3 enzyme’s binding pocket are vital for
anti cancer activity of compound 3b and (b) naphthofuran moiety
acts a central role to develop the potency of the molecule.34,37,38
Figure 9: (a) Western blot analysis of apoptotic proteins like Bcl2,
Bax, cleaved caspase-3, p53, pBad suggesting up-regulation of
apoptic pathway upon treatment of the MDA-MB 468 cells with
compound 3b at doses 10 µM, 20 µM and 30 µM; (b)
Quantification of the apoptotic protein expression; (c) Western
blot of MDA-MB 468 cells exposed to Caspase inhibitor Z-FAD-
FMK and then estimating its cleaved caspase-3 expression with
Compound 3b at doses 10µM, 20 µM and 30 µM; (d)
Quantification data of cleaved caspase-3.
It is well established that generation of ROS and change in MPT
are linked with apoptosis which is a process that controls cellular

References and notes
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In summary, a series of (1,2-dihydronaphtho[2,1-b]furan-2-
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for their anti-proliferative activities against cancer cells (MDA-
MB-468 and MCF-7) and normal cells (WI-38; human lung
fibroblast). Three compounds (3b, 3c and 3s) showed significant
cytotoxic effects on MDA-MB-468 and MCF-7 cancer cell lines
as well as other triple negative cancer cells such as MDA-MB-231
and 4T1 cells. Based on the selectivity index, compound 3b was
considered as most promising anticancer agent. Further
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Acknowledgements
K.I. and U.D. thank SERB, India for NPDF fellowships
[PDF/2017/001211 (KI) and PDF/2017/001782 (UD)].
Conflicts of interest
There are no conflicts to declare.

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of synthesized compounds.
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Anti-cancer potential of (1,2-dihydronaphtho[2,1-b]furan-2-yl)methanone
derivatives
Kobirul Islam,a,b,c Kunal Pal,a,c Utsab Debnath,a R. Sidick Basha,b Abu Taleb Khan,b Kuladip Janaa* and
Anup Kumar Misraa*
a Bose Institute, Division of Molecular Medicine, P-1/12, C.I.T. Scheme VII M, Kolkata 700054, India; E-mail:
akmisra69@gmail.com; kuladip.jana@gmail.com
b Department of Chemistry, Indian Institute of Technology Guwahati, Guwahati 781 039, India.
c Contributed equally.
Research highlights:
 1,2-dihydronaptho[2,1-b]furan derivatives were synthesized.
 One-pot condition has been used to prepare compounds in high yield.
 Synthetic compounds were evaluated for their potential as anticancer agents.
 Three compounds showed promising cytotoxicity against human breast cancer.
 Biochemical and microscopic experiments were carried out using the best compound.