Natural product-inspired rational design, synthesis and biological evaluation of 2,3-dihydropyrano[2,3-f]chromen-4(8H)-one based hybrids as potential mitochondrial apoptosis inducers

Article abstract of DOI:10.1016/j.ejmech.2016.06.044

Synthesis of novel pyranochromanone amide hybrids, by combining pyranochromanone pharmacophore and privileged scaffolds such as 2-amino-1,3,4-thiadiaole/2-aminothiazole/aminopyridine/aminonaphthalene and anti-cancer evaluation of a series led us to discover a series of new chemical entities (NCEs) showing broad spectrum of anti-cancer activity against three different human cancer cell lines (MCF-7, A549 and HeLa), at IC₅₀values ranging from 14.3 to 97.8?μM. Among them, some compounds such as 15b, 15d, 20a and 20b displayed excellent activity against breast cancer cell line MCF-7. Detailed biological studies such as AO/EB dual staining, Hoechst 33342 staining, FACS analysis of mitochondrial membrane potential (Δψ_m) using JC-1 dye and DNA fragmentation confirmed the apoptosis induced by the hybrids. Gene expression studies by Real time RT-PCR has shown that these compounds are efficient regulator of anti-apoptotic gene Bcl-2. Western blot analysis also revealed that these compounds persuade apoptosis through intrinsic pathway by up-regulating the pro-apoptotic protein Bax and down-regulating the anti-apoptotic protein Bcl-2. Molecular docking studies reveal that compounds 15b and 20b binds efficiently with Bcl-2 promoter G-quadruplex.

Full text of DOI:10.1016/j.ejmech.2016.06.044

European Journal of Medicinal Chemistry 122 (2016) 302e318
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Natural product-inspired rational design, synthesis and biological
evaluation of 2,3-dihydropyrano[2,3-f]chromen-4(8H)-one based
hybrids as potential mitochondrial apoptosis inducers
,
Palaniappan Sakthivel a, Andivelu Ilangovan a *, Mahabir Prasad Kaushik b
a School of Chemistry, Bharathidasan University, Tiruchirappalli, 620 024, Tamil Nadu, India
b Process Technology Development Division, Defence R&D Establishment, Jhansi Road, Gwalior, 474002, Madhya Pradesh, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of novel pyranochromanone amide hybrids, by combining pyranochromanone pharmacophore
and privileged scaffolds such as 2-amino-1,3,4-thiadiaole/2-aminothiazole/aminopyridine/amino-
naphthalene and anti-cancer evaluation of a series led us to discover a series of new chemical entities
(NCEs) showing broad spectrum of anti-cancer activity against three different human cancer cell lines
Received 1 March 2016
Received in revised form
30 May 2016
Accepted 24 June 2016
Available online 27 June 2016
(MCF-7, A549 and HeLa), at IC50 values ranging from 14.3 to 97.8 mM. Among them, some compounds
such as 15b, 15d, 20a and 20b displayed excellent activity against breast cancer cell line MCF-7. Detailed
biological studies such as AO/EB dual staining, Hoechst 33342 staining, FACS analysis of mitochondrial
membrane potential (Djm) using JC-1 dye and DNA fragmentation confirmed the apoptosis induced by
the hybrids. Gene expression studies by Real time RT-PCR has shown that these compounds are efficient
regulator of anti-apoptotic gene Bcl-2. Western blot analysis also revealed that these compounds
persuade apoptosis through intrinsic pathway by up-regulating the pro-apoptotic protein Bax and down-
regulating the anti-apoptotic protein Bcl-2. Molecular docking studies reveal that compounds 15b and
20b binds efficiently with Bcl-2 promoter G-quadruplex.
Keywords:
Hybrid molecule
Deguelin
(þ)-Calanolide A
1,3,4-Thiadiazole
Aminopyridine
Mitochondrial apoptosis
G-Quadruplex DNA
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
cancer agents have intensified.
Inhibition of Bcl-2 (B-cell CLL/lymphoma 2), an oncoprotein
Cancer, a major life threatening disease having a high mortality
rate arises out of uncontrolled cell growth in living systems [1,2].
According to WHO estimates more than 20% of population world-
wide are affected with cancer, among which 13.1 million deaths is
projected to occur before 2030. There are 200 different types of
cancer named upon the organ or type of cell from which it origi-
nates. In particular, breast cancer, a type of cancer originating from
breast tissue is the most common among women all over the world.
According to Globocan (WHO), in the year 2015, an estimated
75,957 women died in India due to breast cancer, more than any
other country in the world. Keeping this in mind, the researchers
are keen to eradicate cancer by developing novel therapeutics.
Although, chemotherapeutic agents active against a few types of
cancer have been developed, cure against cancer remains as a major
challenge. Over the last few decades, the race to find new anti-
functioning against cell apoptosis [3,4], can decrease cellular pro-
liferation and enhance the efficacy of chemotherapy. The G-rich
strand of the DNA found in the 39-bp GC-rich P1 promotor region of
Bcl-2 can form G-quadruplex structures which can be stabilized by
G-quadruplex interactive agents [5,6], which ultimately leads to
down-regulation of Bcl-2 [7e9]. Thus, the Bcl-2 G-quadruplex has
emerged as an attractive target for developing potential anticancer
agents.
The number and quantity of natural products available for
chemical biology and medicinal chemistry research are limited.
Hybridization is an evolving structural modification tool to design
new chemical entities (NCEs). This elegant idea of integrating
skeletons of different natural products or drugs, in part or full,
makes it possible to get NCEs in millions of combinations [10,11]. In
this background, biology oriented synthesis (BIOS) of natural
product-inspired scaffolds and their hybrids shows promising re-
sults in finding new lead structures [12e14] which may show
improved bioactivity and less toxicity compared to the parent
compound. A number of potent hybrid anticancer molecules have
* Corresponding author.
E-mail address: ilangovanbdu@yahoo.com (A. Ilangovan).
http://dx.doi.org/10.1016/j.ejmech.2016.06.044
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

P. Sakthivel et al. / European Journal of Medicinal Chemistry 122 (2016) 302e318
303
been developed recently by combining different pharmacophores
[15].
molecule [34]. 1,3,4-Thiadiazole, a notable PS found among com-
pounds achieving broad-spectrum anticancer activity, by targeting
Bcl-2, Bcl-XL and Bax proteins, has good hydrogen bond forming
ability and/or chelation with certain metal ions due to presence of
two nitrogens [35e37]. Similarly, thiazole [38e40] and pyridine
[41,42] ring incorporated compounds shows excellent anti-cancer
activity in addition to diverse biological activities. Furthermore,
naphthalene skeleton is an important building block for a large
number of clinical drugs [43,44] and resveratrol analogues which
are most effective compounds in causing apoptosis in human breast
cancer cell line.
Thus, in an effort to design promising anti-cancer agents, and in
continuation of our interest on study of calanolide analogs [29], we
synthesized new pyranochromanone amides 15aef, 20aeb, 21 and
22 as novel hybrid molecules by combining the pyranochromanone
pharmacophore, found in degulin as well as 12-oxocalanoide and
2,3-Dihydropyrano[2,3-f]chromen-4(8H)-one (which will be
subsequently called as pyranochromanone) is a novel structural
framework found in natural products and bio-active molecules
[16e18]. (ꢀ)-Deguelin (i, Fig. 1) is a natural product [19,20] pos-
sessing pyranochromanone moiety in its basic scaffold exhibits
cytotoxicity against a wide range of human cancer cell lines
including MCF-7 [20e23]. However, potential side effects of
deguelin over certain dose is a substantial obstacle to its clinical use
[24]. Therefore structural modification of deguelin (i) was carried
out either by reducing the double bond in ring C (ii) [24] or by
altering ring D [25] to develop analogues with improved activity.
(þ)-Calanolide A (iii, Fig. 1), was the first natural product
identified to show anti-HIV-1 activity and later found to show anti-
tubercular activity [26e28]. However, its further development as a
drug did not progress beyond phase II/III clinical trials due to the
fact that it has poor stability in biological pH and poor bio-
availability [28]. In order to overcome these drawbacks, several
structural modification studies were carried out. Among which,
aza-calanolide prepared us by modifying coumarin ring into qui-
nazolinone ring (ring B) [29], dihydrocalanolide obtained by
reduction of double bond in ring C [30], and 12-oxo-calanolide (iv)
obtained by modification of hydroxyl group in ring D [28,31e33]
were found to be useful. Thus, pyranochromanone ring system
found in both deguelin (i), 12-oxo-calanolide (iv) was found to be
an important pharmacophore to achieve biological activity.
On the other hand, privileged structures or privileged scaffolds
(PS) are structures that are able to provide high-affinity ligands for
more than one type of receptor to increase the drug likeness of the
PSs
2-amino-1,3,4-thiadiaole/2-aminothiazole/aminopyridine/
naphthalene found among important anti-cancer active com-
pounds. All the ten compounds were screened against three
different types of cancer cell lines, and the hit candidate molecules
were further examined to establish mechanism of action. The re-
sults are given below.
2. Results and discussion
2.1. Chemistry
The target pyranochromanone amide was visualized as a penta-
substituted aromatic core (ring A) fused on either side with chro-
manone (ring D), chromene (ring C) and connected with PS through
Fig. 1. Designing 2,3-Dihydropyrano[2,3-f]chromen-4(8H)-one-based hybrids to achieve potential anti-cancer activity.

304
P. Sakthivel et al. / European Journal of Medicinal Chemistry 122 (2016) 302e318
covalent amide linkage. Successive introduction of these three
parts on commercially available 2,4-dihydroxybenzoic acid (1,
resorcylic acid) forms the basic strategy of our synthetic endeavour
(Scheme 1).
at 60e70 ꢁC (Scheme 3).
b
-
The synthesis of target molecule pyranochromanone-linked
1,3,4-thiadiazole hybrids (15aef) in good yield, was carried out
by coupling pyranochromanone acid 11 with a series of 2-amino-
1,3,4-thiadiazoles 14aef using the coupling agent EDCI$HCl/HOBt
and DIPEA in DMF at room temperature (Scheme 4). The formation
of coupled products 140aef was confirmed by analysing its 1H NMR
spectra where the amide NeH peak appeared at 11.2e11.4 ppm. The
expected molecular weight of these hybrids 140a (462.1476), 140b
(476.1653), 140c (552.1773), 140d (507.1336), 140e (463.1466) and
140f (468.1046) was confirmed by characteristic [MþH]þ ion peak
observed in the HRMS spectra.
The synthesis was initiated with an aim to introduce dime-
thylchromanone ring 7 (ring D) on ester 2 which in turn was pre-
pared from its acid 1 (Scheme 2). Attempted Friedel-Crafts C-
acylation of 2 using tigloyl chloride ((E)-2-methylbut-2-enoyl
chloride, 4) in the presence of different Lewis acids such as AlCl3,
TiCl4, ZnCl2, Yb(OTf)3 and SnCl4 only lead to the formation of un-
desired 4-O-tigloylated product. To circumvent this, 4-OH group of
compound 2 was selectively methylated to get ester 3, which was
treated with tigloyl chloride (4) in the presence of AlCl3 in PhNO2/
CS2 solvent at room temperature to get acylated product 5 in 70%
yield and compound 6 in 10% yield (Scheme 2). In the 1H NMR
spectrum of compound 5, two singlets corresponding to two AreH
Similarly, pyranochromanone acid 11 was coupled with 3-
aminopyridine (16), 4-aminopyridine (17), 2-aminothiazole (18)
and 1-naphthylamine (19) using EDCI$HCl/HOBt and DIPEA in DMF
at room temperature to get a pyranochromanone e pyridine/thia-
zole/naphthalene hybrids such as 20aeb, 21 and 22 in moderate to
good yield (Scheme 5). The formation of coupled products 20aeb,
21 and 22 was confirmed by 1H NMR analysis where amide proton
appeared in the range 9.0e11.0 ppm. The expected molecular
weight of these hybrids 154a (379.1656), 154b (379.1656), 155
(385.1219) and 156 (428.1839) was confirmed by characteristic
[MþH]þ ion peak observed in the HRMS spectra.
were observed at
d 6.47 and 7.67 which confirmed regioselective
acylation of compound 3. Further, compound 5 was demethylated
using AlCl3 in DCM at room temperature to get compound 6 in 90%
yield which was confirmed by the presence of a peak at d 12.62 ppm
due to intramolecular hydrogen bonded eOH. Subsequently, com-
pound 6 was subjected to intramolecular oxa-Michael addition
using triethylamine in CHCl3 at room temperature to give com-
pound tc-7 in 90% yield as a cis and trans diastereomeric mixture in
1:1 ratio.
Attempted coupling of pyranochromanone acid 11 with
2-amino-1,3,4-oxadiazole,
2-chloro-3-aminopyridine
and
Next, the construction of chromene segment (ring C) was car-
ried out on tc-7. The reaction of tc-7 with 3-chloro-3-methyl-1-
butyne (8) in acetone in the presence of K2CO3, KI and CuI at
reflux afforded a separable mixture of trans and cis propargylic
ether tc-9 in 80% yield and the product formation was confirmed by
1H NMR analysis particularly using acetylenic proton appearing at
2-aminopyridine in the presence of various coupling agents such as
EDCI/HOBt, BOP, HBTU, DCC, T3P® or ClCOOEt failed. Having ach-
ieved synthesis of all the target molecules, study of its anti-cancer
activity and mechanistic insights was carried out.
2.2. Evaluation of biological activity
d
2.68 ppm. Subsequently, when propargylic ether tc-9 was treated
with N,N0-dimethylaniline at 190e195 ꢁC for 5 h an inseparable
mixture of trans and cis pyranochromanone ester tc-10 was pro-
duced in 95% yield. While our attempts to separate these isomers
by column chromatography were not successful, one of the isomer
t-10 crystallized out, but not completely, in hexane/EtOAc (6:4). 1H
NMR spectrum of t-10 showed two doublets, corresponding to the
H-9 and H-10 in ring C, appeared separately at 6.60 (d, J ¼ 10 Hz)
2.2.1. MTT assay
All the synthesized pyranochromanone based hybrid molecules
15aef, 20aeb, 21 and 22 were screened for in vitro cytotoxic ac-
tivities against three different human cancer cell lines namely
breast cancer cell line (MCF-7), lung carcinoma cell line (A549) and
cervical cancer cell line (HeLa) by 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) method while (ꢀ)-deguelin
[20] and anti-cancer drug levamisole [49] were taken as the
reference. The results are summarized in Table 1. Gratifyingly, these
hybrids have shown considerable cytotoxicity against tested cancer
and 5.63 (d, J ¼ 10.4 Hz) and H-2 appeared at
d 4.29e4.22 ppm (For
numbering see Scheme 1). The structure of t-10 was also confirmed
by single crystal X-ray analysis (Fig. 2) [45]. The chromene ring
formation results from a thermally induced sequence of aryl-
Claisen rearrangement followed by 1,5-hydrogen shift and elec-
trocyclic ring closure [46].
Finally ester tc-10 was hydrolysed using aq.NaOH in methanol at
room temperature to afford pyranochromanone acid 11 in 90%
yield. Even if we started with pure distereomer t-10, isomerisation
took place during the hydrolysis so that we could not get pure
diastereomeric product 11. The overall yield in achieving the syn-
cell lines with IC50 values ranging from 14.3 to 97.8 mM.
It is inferred from the study that, these hybrids showed higher
cytotoxic activity in the MCF-7 breast cancer cell line as compared
to the other cancer cell lines. Compound 11 having no PS showed
lowest cytotoxicity against all the tested cancer cell lines as
compared to hybrids containing PS. Further, hybrids containing
methyl (15b) and nitro (15d) group substituted 2-amino-1,3,4-
thiadiazole as PS exhibited excellent cytotoxic activity as
compared to other thiadiazole derivatives (15a/c/e/f). Also, hybrids
20a and 20b containing 3-aminopyridine and 4-aminopyridine as
PS displayed good cytotoxic activity. Unfortunately, compounds 21
thesis of compound 11 from b-resorcylic acid (1) is 33.89%. Further,
2-amino-1,3,4-thiadiazoles 14a-f were prepared [47] by reaction of
nitrile 12 [48] with thiosemicarbazide 13 in trifluroaceticacid (TFA)
Scheme 1. Retrosynthetic analysis of target NCEs.

P. Sakthivel et al. / European Journal of Medicinal Chemistry 122 (2016) 302e318
305
Scheme 2. Synthesis of pyranochromanone acid 11: Reagents and conditions: (i) CH3OH/Con.H2SO4, reflux, 24 h, 90%; (ii) CH3I (1.0 equiv), K2CO3, acetone, reflux, 3 h, 92%; (iii) 4,
AlCl3, PhNO2/CS2, r.t., 3 h, 70%; (iv) AlCl3, DCM, r.t., 10 h, 95%; (v) Et3N, CHCl3, r.t., 10 h, 90%; (vi) 8, K2CO3, KI, CuI, acetone, reflux, 8 h, 80%; (vii) N,N-Dimethylaniline, 190e195 ꢁC, N2
atm., 5 h, 95%; (viii) Aq.NaOH, CH3OH, r.t., 5 h, 90%.
Based on the results obtained from the MTT assay, the hybrids
15b, 15d, 20a and 20b showing higher cytotoxicity was selected for
further mechanistic studies such as AO/EB dual staining, Hoechst
nuclear staining, DNA fragmentation and Western blot analysis of
Bcl-2 and Bax proteins expression using MCF-7 cells. In addition,
the most potent hybrids 15b and 20b were also investigated for its
potential to induce mitochondrial dysfunction via JC-1 staining and
molecular docking studies using the NMR structure of the Bcl-2
promoter G-quadruplex.
2.2.2. Acridine orange/ethidium bromide (AO/EB) dual staining
Dual staining techniques is used to differentiate the live and
dead cells under fluorescent microscope [50]. Acridine orange, a
cationic fluorescent dye used in the study stains both live and dead
cells emitting green fluorescence. Similarly, ethidium bromide
which emits orange fluorescence because of DNA intercalation was
used to stain DNA of cells whose membrane integrity is lost. Under
the present study, MCF-7 cells were treated separately with hybrid
molecules 15b, 15d, 20a and 20b at their corresponding IC50 con-
centration during 24 and 48 h time intervals. Followed by this it
was treated with AO/EB and visualized under fluorescence micro-
Fig. 2. X-ray structure of t-10.
scope (Fig. 3A). The change in colour and morphology of cells
and 22 possessing 2-aminothiazole and naphthalene moieties have
shown less cytotoxic activity in almost all cancer cell lines tested.
Thus it is obvious that, the cytotoxicity of these hybrids depends on
the nature of the PS attached.
treated with hybrid molecules compared to untreated cells
revealed apoptotic cell death. While yellow and orange colouration
observed after 24 h treatment indicates early apoptotic stage,
further deepening of colour to orange after 48 h indicated late
Scheme 3. Preparation of 2-amino-1,3,4-thiadiazoles.

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P. Sakthivel et al. / European Journal of Medicinal Chemistry 122 (2016) 302e318
Scheme 4. Synthesis of pyranochromanone e 1,3,4-thiadiazole hybrids.
Scheme 5. Synthesis of pyranochromanone e pyridine/thiazole/naphthalene hybrids.
apoptotic phase. Thus all these morphological changes indicated
occurrence of time-dependent cell death upon treatment with
hybrid molecules at IC50 value. The treated MCF-7 cells showed
higher incidence of apoptosis than necrosis. Also, the hybrids
causes more cells to take to preferentially one particular mode of
cell death during 48 h treatment than 24 h treatment (Fig. 3B).
nuclear counterstain which emits blue fluorescence when bound to
adenine-thymine (AT) rich regions of DNA. This staining was per-
formed to visualize the change in the nuclear morphology [51].
Taking this advantage, a time-dependent study was carried out at
IC50 concentration of the hybrid molecules 15b, 15d, 20a and 20b
(Fig. 4). After a period of 24 h, due to chromatin condensation,
fragmented nuclei, and the presence of apoptotic bodies, relative
fluorescence of the treated cells increased significantly compared to
untreated control cells (Fig. 4A). The occurrence of cytoskeleton
breakdown along with plasma membrane blebbing was
2.2.3. Hoechst 33342 staining
Important characteristics of apoptosis are chromatin conden-
sation and fragmented nuclei. Hoechst 33342 is cell permeating

P. Sakthivel et al. / European Journal of Medicinal Chemistry 122 (2016) 302e318
307
Table 1
DNA appears as ladder. However, in the case of healthy and normal
cells, this endonuclease is inhibited by inhibitor of caspase acti-
vated DNase (ICAD) enzyme. In the present study, MCF-7 cells were
treated for 24 h with hybrids 15b, 15d, 20a and 20b at IC50 con-
centration (Fig. 7). After 24 h, all these tested compounds induced
DNA fragmentation and shown to have characteristic ladder
pattern inferring apoptosis. Meanwhile, no laddering was observed
in the untreated cells.
In vitro cytotoxic effect of 2,3-dihydropyrano[2,3-f]chromen-4(8H)-one based
hybrids.
Sl. No
Compound
IC50 [m
M]a
MCF-7b
A549c
HeLad
1.
2.
3.
4.
5.
6.
7.
8.
11
15a
15b
15c
15d
15e
15f
20a
88.7 0.96
37.8 0.10
14.3 0.11
70.0 0.22
31.2 2.88
57.9 1.57
78.2 2.32
28.7 2.14
26.2 1.63
75.0 1.39
48.1 0.99
10.59 1.38
>100
116.0 1.89
32.4 1.59
20.2 2.28
79.3 2.02
42.3 1.93
75.0 1.62
74.5 2.12
26.9 0.78
39.3 1.85
64.4 1.17
58.1 1.91
e
102.0 0.62
40.2 1.81
25.2 2.02
97.8 2.15
42.9 2.14
74.3 1.28
92.8 2.28
41.2 2.02
28.6 2.14
84.0 0.94
41.7 2.07
e
2.2.6. Real time RT-PCR analysis
It is well known that, Bcl-2 was the first anti-death gene
discovered and play a key role to determine whether or not unleash
the caspase proteolytic cascade [55]. Moreover, several experi-
mental therapies target Bcl-2 family mRNA/proteins in the devel-
opment of new anticancer drugs [56]. Keeping this in mind, mRNA
expression of Bcl-2 gene was carried out on MCF-7 cells in a time
dependent manner using compounds such as 15b and 20b at their
IC50 concentration as shown in Fig. 8. Based on the result, it was
confirmed that, the down-regulation of Bcl-2 gene expression on
treatment with hybrids 15b and 20b. On treatment with hybrid
15b at 12, 24 and 48 h, Bcl-2 was down regulated to 82.7%, 46.0%
and 39.7% of the level of control respectively. Similarly, with hybrid
20b at 12, 24 and 48 h, Bcl-2 was down regulated to 92.2%, 64.0%
and 57.0% of the level of control respectively.
9.
20b
21
22
10.
11.
12.
13.
(ꢀ)-Deguelin (i)
Levamisole
>100
96
a
Concentration required to inhibit 50% cell growth and each value represents
mean S.D. from three different experiments performed in triplicates.
b
MCF-7: breast adenocarcinoma cell line.
A549: lung adenocarcinoma epithelial cell line.
HeLa: cervical cancer cell line.
c
d
significantly large after 48 h of incubation. Also, the number of
abnormal cells is found to increase with incubation time revealing
that all the hybrids bring about cytological changes in a time
dependent manner (Fig. 4B).
2.2.7. Western blot analysis of Bcl-2 and Bax proteins
A number of chemotherapeutic drugs have direct or indirect
implications on the inhibition of apoptosis related proteins. In the
present study, the protein expression pattern of Bcl-2 and Bax was
studied in MCF-7 breast cancer cell line. It is well known that, the
members of the Bcl-2 family both proapoptotic (Bax, Bak, Bad, Bcl-
Xs, Bid, Bik Bim and Hrk) proteins and antiapoptotic (Bcl-2, Bcl-XL,
Bcl-W, Bfl-2 and Mcl-1) proteins play a pivotal role in the regulation
of the mitochondrial apoptotic pathway [3,4,57]. The apoptotic
regulation depends mainly on the internal imbalance of proteins
such as Bcl-2 and Bax. After 24 h of treatment of MCF-7 cells with
hybrids, 15b, 15d, 20a and 20b, at their IC50 concentration, cell ly-
sates were collected and their expression level of Bcl-2 and Bax was
determined by Western Blot analysis. It was observed that, anti-
apoptotic protein Bcl2 was down-regulated as shown in Fig. 9.
Simultaneously, the level of pro-apoptotic protein Bax was upre-
gulated, indicating to persuade apoptosis by intrinsic pathway. In
response to the apoptotic stimuli, cytochrome-c releases and acti-
vates the downstream caspases.
2.2.4. Mitochondria membrane potential (Djm
)
Mitochondria plays a crucial role in the induction and control of
apoptosis through the release of proapoptotic factors such as cy-
tochrome c and other apoptosis-inducing factors. Dissipation of the
mitochondrial electrochemical potential gradient is known as an
early event in apoptosis [52]. In the present study, alterations in
mitochondrial membrane potential (MMP or DJm) was measured
through the examination of 15b and 20b treated MCF-7 cells under
inverted fluorescence microscope after staining with JC-1 cationic
dye (Fig. 5). JC-1 aggregates in the mitochondrial matrix emits red
fluorescence for intact mitochondria with normal DJm, whereas at
depolarized abnormal DJm, JC-1 forms monomers and emits
green fluorescence. Interestingly, after the treatment of MCF-7 cells
with 15b and 20b, at IC50 concentration, decrease in the red fluo-
rescence and increase in intensity of green fluorescence was
noticed. This clearly indicated the depolarisation of cytoplasm.
In general, the effect of compound on mitochondrial membrane
potential (DJm) was studied by JC-1 staining followed by flow
cytometry analysis. Intact mitochondrial membranes produce red
fluorescence due to the accumulation of JC-1 in the mitochondria.
But JC-1 cannot accumulate in the mitochondria once the collapse
of DJm, where it will persist as a monomer in the cytosol and
fluoresce green light [53]. Therefore, marked changes in MMP
during apoptosis is indicated by an increase of green/red fluores-
cence ratio. Taking into account, the effect of hybrids 15b and 20b
on DJm was examined by FACS analysis using JC-1 dye in MCF-
7 cells at their IC50 concentration for 48 h (Fig. 6). Based on the
result, it was observed that, for hybrids 15b and 20b, the percentage
of cells with DJm loss increases and a significant population shifts
to the UR quadrant or FL1 channel. This illustrates that the induc-
tion of apoptosis by hybrids 15b and 20b is associated with the
mitochondrial or intrinsic pathway.
2.2.8. In silico molecular docking
In silico Molecular docking study was an excellent tool to
investigate the nature of drug and DNA interaction. Thus, in our
study the molecular docking was performed for selected com-
pounds like 15b and 20b to further authenticate the bcl-2/Bax
mediated apoptosis as described in the experimental results of
western blot analysis. NMR solution structure of Bcl-2 promoter G-
quadruplex (PDB code: 2F8U) was successfully docked with the
hybrids by using the Autodock4 program. The capability of a drug
molecule to stabilize G-quadruplex structure mainly depends on
the binding affinity and specificity of the molecule. Our results
shows that 15b and 20b possess excellent selectivity towards Bcl-2
promoter G-quadruplex. It was well established that the Bcl-2
promoter G-quadruplex is well determined with various parallel/
antiparallel structure. Furthermore, 15b and 20b also binds effi-
ciently into groove in loop of the Bcl-2 promoter quadruplex [8].
The surface electrostatic potential representations of Bcl2 mid-M-
ligand complex structures are shown in Fig. 10.
2.2.5. DNA fragmentation
Apoptosis is characterized with an event of biochemical level
change as a result of DNA fragmentation [54]. More precisely,
during cell death DNA is cleaved by an endonuclease as a result
In agreement with the empirical data, the calculated binding
affinities (Table 2) indicate that compounds 15b and 20b binds

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P. Sakthivel et al. / European Journal of Medicinal Chemistry 122 (2016) 302e318
Fig. 3. (A) AO/EB dual staining of MCF-7 human breast cancer cells at 24 and 48 h of incubations by the treatment of 15b, 15d, 20a and 20b. (B). AO/EB fluorescent study of 15b, 15d,
20a and 20b induced apoptosis of MCF-7 cells. The graph shows manual count of apoptotic and necrotic cells in percentage.
more strongly with the Bcl-2 promoter G-quadruplex. This result
helps to understand the nature of drug-quadruplex DNA interac-
tion in the drug design and discovery, as well as in the mechanistic
study by introducing a 15b and 20b molecule into the binding site
of the specific DNA target.
Based on the results obtained from western blot analysis and
docking studies, the induction of apoptosis by these new hybrid
molecules is associated with Bcl-2 down-regulation [7]

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309
Fig. 4. (A) Hoechst 33342 staining of MCF-7 human breast cancer cells at 24 and 48 h of incubation by the treatment of 15b, 15d, 20a and 20b. (B). Hoechst 33342 staining study of
15b, 15d, 20a and 20b induced apoptosis of MCF-7 cells. The graph shows manual count of apoptotic cells in percentage.
2.2.9. Hemocompatibility assay
3. Conclusions
To be a potential an anti-cancer agent, it is important for an
organic molecule to be non-toxic to the normal human cells. In
order to check toxicity, hemocompatibility of all the hybrids 15b,
15d, 20a and 20b were examined by measuring the damage to
human RBCs. The mechanisms of direct hemolytic activity for
different toxic agents were found to be non-specific. According to
the International Organization for Standardization/Technical
Report 7406, the admissible level of hemolysis of biological mate-
rials is 5% [59]. As shown in Fig. 11, compared to positive control
(Triton X-100), all the four hybrids exhibited very low hemoglobin
release.
We have designed, by following hybrid organic molecule
approach, pyranochromanone amides as a new class of anti-cancer
agents. Efficient synthetic method was developed for the synthesis
of ten different hybrids in high overall yield. All the hybrids were
tested for anticancer activity against three different human cancer
cell lines such as MCF-7 (breast), A549 (lung) and HeLa (cervix) by
following in vitro cell viability studies (MTT assay). The results
shows that all the tested hybrids exhibits good cytotoxicity effect
on all the three tested cell lines, amongst which the hybrids 15b,
15d, 20a and 20b have shown stupendous cellular toxicity against

310
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Fig. 5. Mitochondrial membrane potency of hybrids 15b and 20b at their IC50 concentration using MCF-7 cells.
Fig. 6. Flow cytometric analysis (FACS) of the mitochondrial membrane potential using the JC-1 dye in MCF-7 cells treated with hybrids 15b and 20b at their corresponding IC50
concentration for 48 h.
Fig. 8. RT-qPCR analysis of mRNA expression of Bcl-2 in MCF-7 cells. MCF-7 cells were
treated with IC50 concentration of compounds 15b and 20b for 0, 12, 24 and 48 h. The
mRNA expression was determined by quantitative real-time PCR and the expressional
Fig. 7. DNA fragmentation analysis of potential hybrids at IC50 concentration in MCF-7
breast cancer cells at 24 h. Lane M: marker (100 bp), lane C: control, lane 1: 20a, lane 2:
20b, lane 3: 15b, lane 4: 15d.
levels were normalized to the level of GAPDH mRNA in each samples. P < 0.05, sta-
tistically significant differences from the control group (Error bars were given as STDEV
of triplicate measurements).

P. Sakthivel et al. / European Journal of Medicinal Chemistry 122 (2016) 302e318
311
synthesized hybrids 15b, 15d, 20a and 20b represent a new struc-
tural lead/hit, which is less complex than their corresponding
natural product but showing closely similar activity, can be
potentially developed into a cost-effective, commercially viable
chemotherapeutic drug to save breast cancer patient’s life. It is very
important to investigate preclinical insights, molecular signaling
pathways including anti-metastasis and angiogenesis and these
studies are in progress. In future, the synthesized hybrids have to be
assessed against different types of cancer cells.
4. Experimental protocols
Fig. 9. Effect of hybrids 15b, 15d, 20a and 20b on Bcl-2 and Bax protein expression
detected using western blot analysis. GAPDH (Glyceraldehyde 3-phosphate dehydro-
genase) was used as a loading control. Lane C: Control, Lane 1: 20a, Lane 2: 20b, Lane
3: 15b, Lane 4: 15d.
4.1. Chemistry
4.1.1. General remarks
Melting points were determined by the open capillary tube
method using a Toshniwal melting point apparatus and are un-
corrected. The 1H and 13C NMR spectra were measured on a Bruker
Avance 400 (400 MHz) NMR spectrometer. Chemical shifts are re-
breast carcinoma cells (MCF-7). Further, it was observed that in
order to achieve better cytotoxicity, it is important to have PS in
addition to pyranone chromanone backbone. Among the different
PSs, 1,3,4-thiadiazole derivatives showed higher cytotoxicity.
Further, by staining and microscopic examination of the cells
treated with hybrid molecules, it was observed that the morpho-
logical change, hence cell death occurs in time-dependent manner.
Interestingly, at half maximal inhibitory concentration (IC50), the
hybrids triggers mitochondrial-driven apoptosis via activation up
and down regulation of Bax/Bcl-2 depicts the improved anticancer
activity of hybrids. On the other hand, in silico docking experiments
have shown that the hybrids interact and bind efficiently with the
crystal structure of Bcl-2 promoter G-quadruplex. All the four hy-
brids exhibited negligible red hemoglobin release, implying that it
is negligibly toxic or safe to normal cells. Interestingly, one of the
ported in ppm (
d) relative to internal standard tetramethylsilane
(TMS, 0.00 ppm). Data are reported as follows: chemical shift
d
(multiplicity [singlet (s), doublet (d), doublet of doublet (dd), triplet
(t), quartet (q), multiplet (m), broad resonance (br)], coupling
constants [Hz], integration). All the NMR spectra were acquired at
ambient temperature. ESI-MS was recorded on Agilent 1100 LC/
MSD (70 ev) spectrometer. High resolution mass spectra (HRMS)
were recorded on a Waters Q-Tof micro mass spectrometer.
Elemental analyses were performed on a CHN analyser. X-ray
crystallographic data were collected on a Bruker SMART APEX-II
CCD diffractometer. Thin layer chromatography (TLC) was per-
formed on Merck pre-coated silica gel 60F plates and visualized by
exposure to UV light. ACME silica gel (100e200 mesh) was used for
column chromatography. All commercially available reagents were
used without purification and were purchased from standard
chemical supplier.
hybrids 15b showed IC50 (14.3
mM) much closer to the natural
product degulin (10.59 M). Overall, the results suggests that the
m
Fig. 10. Interaction of the hybrids 15b and 20b with Bcl-2 promotor G-Quadruplex DNA. Pictures were generated by using PyMOL.
Table 2
Binding affinities (kcal/mol) of the Bcl2mid-M G-quadruplex structure.
Compound
Binding affinitya
DG (kcal/mol)
RMSD UB (upper bound)
RMSD LB (lower bound)
15b
20b
ꢀ7.9
ꢀ7.1
11.872
7.199
7.991
2.975
a
The minimum value for the docking score was calculated using the AutoDock Vina program [58].

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get
a homogeneous mixture. Tigloyl chloride (4, 11.9 mL,
108.79 mmol) in nitrobenzene (25 mL) was added dropwise in
30 min at room temperature and stirred for additional 3 h. The
reaction mixture was quenched into the crushed ice and 1 N HCl.
The semi precipitated product was taken into EtOAc, and the
aqueous solution was extracted with the same solvent (400 mL x 3).
The combined EtOAc solutions were washed with brine, dried over
Na2SO4 and concentrated in vacuo (use high vacuum pump to
remove the nitrobenzene). The residue was purified by silica gel
column chromatography (hexane-EtOAc ¼ 8:2) to afford the title
compound 5 (18.27 g, 70%) as white solid. M.p.124e126 ꢁC; 1H NMR
(400 MHz, CDCl3):
(m,1H), 3.90 (s, 3H), 3.80 (s, 3H),1.91 (s, 3H),1.84 (dd, J ¼ 7.2, 0.8 Hz,
3H); 13C NMR (100 MHz, CDCl3):
196.6, 170.1, 164.7, 163.0, 142.6,
d 11.13 (s, 1H), 7.67 (s, 1H), 6.47 (s, 1H), 6.41e6.35
d
139.1, 131.1, 121.9, 104.5, 99.4, 55.9, 52.1, 14.9, 11.1 ppm; HRMS (ESI):
[MþH]þ Calcd for C14H17O5, 265.1076; found 265.1066.
4.1.5. (E)-Methyl 2,4-dihydroxy-5-(2-methylbut-2-enoyl)benzoate
(6)
To a stirred solution of 5 (17.0 g, 64.39 mmol) in DCM (250 mL)
was added AlCl3 (42.82 g, 321.95 mmol) at room temperature and
then allowed to stir for a period of 10 h. The reaction mixture was
quenched into the crushed ice and 1 N HCl. The aqueous solution
was extracted with DCM (250 mL x 2). The combined DCM layer
was washed with brine, dried over Na2SO4 and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (hexane-EtOAc ¼ 9:1) to afford the title com-
pound 6 (15.29 g, 95%) as colorless crystal. M.p. 90e93 ꢁC; 1H NMR
(400 MHz, CDCl3):
d 12.62 (s, 1H), 11.33 (s, 1H), 8.23 (s, 1H), 6.48 (s,
Fig. 11. In vitro hemocompatibility assay of hybrids 15b, 15d, 20a and 20b exhibits very
less hemolytic activity. No (0%) lysis was noticed in the negative control (NC-PBS
buffer) whereas the positive control (PC- 1% Triton X-100) shows 100% lysis.
1H), 6.15e6.09 (m, 1H), 3.93 (s, 3H), 1.97 (t, J ¼ 0.8 Hz, 3H), 1.91 (dd,
J ¼ 7.0, 1.0 Hz, 3H); 13C NMR (100 MHz, CDCl3):
d 202.2, 169.8, 169.1,
167.2, 137.1, 136.5, 135.8, 112.7, 104.8, 104.6, 52.4, 14.4, 13.3 ppm;
HRMS (ESI): [MþH]þ Calcd for C13H15O5, 251.0919; found 251.0916.
4.1.2. Methyl 2,4-dihydroxybenzoate (2) [60]
4.1.6. (2R,3R)-Methyl 7-hydroxy-2,3-dimethyl-4-oxochroman-6-
carboxylate (trans-7) and (2R,3S)-Methyl 7-hydroxy-2,3-dimethyl-
4-oxochroman-6-carboxylate (cis-7)
To a stirred solution of 2,4-dihydroxybenzoicacid (1, 10.00 g,
64.93 mmol) in methanol (100 mL) was added catalytic amount of
Con.H2SO4 (0.35 mL, 6.49 mmol) and stirred at reflux for 24 h. After
completion of the reaction methanol was evaporated and quenched
with ice water and basified into pH 8 using aq.Na2CO3 solution.
Then precipitated solid was filtered off, washed with ice water and
then dried. The title compound 2 (9.80 g, 90%) was obtained as pale
brown solid. This is pure enough for further step. M.p. 118e120 ꢁC.
To a stirred solution of 6 (15.0 g, 60.0 mmol) in CHCl3 (250 mL)
was added triethylamine (18.18 g, 180 mmol) and then allowed to
stir at room temperature for 24 h. The reaction mixture was
quenched into ice water and then neutralised by 1 N HCl. The
aqueous solution was extracted with CHCl3 (2 ꢂ 150 mL). The
combined CHCl3 layer was washed with brine, dried over Na2SO4
and concentrated under reduced pressure. The crude diastereo-
meric mixture was purified through a silica gel column chroma-
tography (hexaneeEtOAc ¼ 9: 1). First eluted was compound trans-
7 (6.9 g, 46%), obtained as a white solid and second eluted was cis-7
(6.9 g, 46%), obtained as a white solid. The overall yield of tc-7 was
92% (13.8 g). Compound trans-7: M.p. 166e168 ꢁC; 1H NMR
4.1.3. Methyl 2-hydroxy-4-methoxybenzoate (3) [61]
To a stirred solution of methyl 2,4-dihydroxybenzoate (2, 20.0 g,
119.05 mmol) in acetone was added K2CO3 (18.07 g, 130.9 mmol)
followed by dropwise addition of MeI (8.2 mL, 130.9 mmol) at room
temperature. After the completion of addition allowed to stir at
reflux for 3 h. The reaction mixture was evaporated and quenched
into the water and extracted by EtOAc (3 ꢂ 100 mL). The combined
EtOAc layer was washed with water followed by brine to give the
crude which was purified by silica gel column chromatography
(hexane-EtOAc ¼ 9:1) to afford the title compound 3 (19.93 g, 92%)
(400 MHz, CDCl3): d 11.28 (s, 1H), 8.47 (s,1H), 6.45 (s, 1H), 4.31e4.24
(m, 1H), 3.94 (s, 3H), 2.58e2.49 (m, 1H), 1.52 (d, J ¼ 6.4 Hz, 3H), 1.21
(d, J ¼ 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3):
d 192.7, 170.1, 166.9,
166.1, 131.8, 113.8, 107.7, 104.1, 79.6, 52.4, 46.5, 19.7, 10.3 ppm; MS
(ESI): m/z 251 [Mþ1]þ. Anal. Calcd for C13H14O5: C, 62.39; H, 5.64.
Found: C, 62.55; H, 5.70. Compound cis-7: M.p. 105e107 ꢁC; 1H
as colorless solid. M.p. 50e52 ꢁC; 1H NMR (400 MHz, CDCl3):
d 10.97
(s, 1H), 7.73 (d, J ¼ 8.4 Hz, 1H), 6.45e6.42 (m, 2H), 3.91 (s, 3H), 3.82
NMR (400 MHz, CDCl3):
d 11.27 (s, 1H), 8.48 (s, 1H), 6.46 (s, 1H),
(s, 3H); 13C NMR (100 MHz, CDCl3):
107.5, 105.4, 100.7, 55.4, 51.9 ppm.
d 170.42, 165.6, 163.8, 131.2,
4.69e4.63 (m, 1H), 3.93 (s, 3H), 2.69e2.63 (m, 1H), 1.39 (d,
J ¼ 6.4 Hz, 3H), 1.14 (d, J ¼ 7.6 Hz, 3H); 13C NMR (100 MHz, CDCl3):
d
194.3, 170.1, 167.0, 165.8, 131.9, 113.3, 107.8, 104.2, 77.2, 52.4, 45.0,
4.1.4. (E)-Methyl 2-hydroxy-4-methoxy-5-(2-methylbut-2-enoyl)
benzoate (5)
16.1, 9.2 ppm; HRMS (ESI): [MþH]þ Calcd for C13H15O5, 251.0919;
found 251.0919.
To a stirred solution of methyl 2-hydroxy-4-methoxybenzoate
(3, 18.0 g, 98.90 mmol) in CS2 (250 mL) was added AlCl3 (39.46 g,
296.70 mmol) and then nitrobenzene (75 mL) was added dropwise
in 30 min and stirred for additional 15 min at room temperature to
4.1.7. 3-Chloro-3-methylbut-1-yne (8)
To neat 2-methyl-3-butyn-2-ol (5.0 g, 59.52 mmol) at 0 ꢁC was
added CuI (1.13 g, 5.95 mmol) followed by dropwise addition of 37%

P. Sakthivel et al. / European Journal of Medicinal Chemistry 122 (2016) 302e318
313
HCl (29.3 mL, 297.6 mmol). The solution was then brought to 25 ꢁC
and stirred for 30 min. The upper layer was separated and dried
over Na2SO4 to afford pure 3-chloro-3-methyl-1-butyne (8, 3.0 g,
J ¼ 10 Hz, 1H), 5.63 (d, J ¼ 10.4 Hz, 1H), 4.29e4.22 (m, 1H), 3.83 (s,
3H), 2.54e2.46 (m, 1H), 1.52 (d, J ¼ 6.4 Hz, 3H), 1.49 (s, 3H), 1.46 (s,
3H), 1.19 (d, J ¼ 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3):
d 192.7,
50%) as a colorless liquid. 1H NMR (400 MHz, CDCl3):
d
2.67 (s, 1H),
165.0, 159.0, 158.9, 131.4, 129.1, 115.6, 113.6, 113.3, 109.9, 79.8, 78.2,
1.86 (s, 6H); 13C NMR (100 MHz, CDCl3):
d
86.5, 71.9, 56.9, 34.5 ppm.
51.7, 46.4, 28.2, 27.9, 19.7, 10.2 ppm; HRMS (ESI): [MþNa]þ Calcd for
Spectral data matched with literature report [62].
C18H20NaO5, 339.1208; found 339.1207.
4.1.8. (2R,3R)-methyl 2,3-dimethyl-7-(2-methylbut-3-yn-2-yloxy)-
4-oxochroman-6-carboxylate (trans-9) and (2R,3S)-methyl 2,3-
dimethyl-7-(2-methylbut-3-yn-2-yloxy)-4-oxochroman-6-
carboxylate (cis-9)
To a solution of tc-7 (12.0 g, 48.0 mmol) in dry acetone (150 mL)
at 25 ꢁC was added under K2CO3 (8.61 g, 62.4 mmol), KI (8.76 g,
52.8 mmol), CuI (91.4 mg, 0.48 mmol) and 3-chloro-3-methyl-1-
butyne (8, 13.5 mL, 120.0 mmol) under N2 atm. The reaction
mixture was then brought to reflux for 8 h. The reaction mixture
was evaporated and quenched into the water and extracted by
EtOAc (3 ꢂ 200 mL). The combined EtOAc layer was layer was
washed with water and brine followed by concentrated under
reduced pressure. The crude diastereomeric mixture was purified
4.1.10. 2,3,8,8-Tetramethyl-4-oxo-2,3,4,8-tetrahydropyrano[2,3-f]
chromene-6-carboxylic acid (11)
To a stirred solution of compound tc-10 (11.0 g, 34.81 mmol) in
methanol (200 mL) was added 10% aq. NaOH (1.53 g, 38.29 mmol)
at room temperature and then allowed to stir at the same tem-
perature for 5 h. The progress of the reaction was monitored by TLC
(hexane - EtOAc ¼ 7:3). The reaction mixture was evaporated and
quenched into the ice water and then extracted by EtOAc
(2 ꢂ 50 mL). EtOAc layer was separated out and then aqueous layer
was acidified by slow addition of dil. HCl till pH-5. The precipitated
solid was filtered, washed well with ice cold water and then dried.
The product 11 (271 mg, 90%) obtained as an inseparable diaste-
reomeric mixture (trans/cis ¼ 65:35) as a white solid which is pure
enough for further step. M.p. 159e163 ꢁC; 1H NMR (400 MHz,
through
a
silica
gel
column
chromatography
(hexaneeEtOAc ¼ 9:1). First eluted was compound trans-9 (6.2 g,
41%), obtained as a white solid and second eluted was cis-9 (6.2 g,
41%), obtained as a pale yellow solid. The overall yield of tc-9 was
82% (12.4 g). Compound trans-9: M.p. 125e127 ꢁC; 1H NMR
CDCl3):
d
10.49 (br, 1H), 8.62, 8.61 (2s, 1H), 6.69 (dd, J ¼ 10.4, 0.8 Hz,
1H), 5.73 (d, J ¼ 10.4 Hz, 1H), 4.75e4.70 (m, 1Hminor), 4.35e4.28 (m,
1Hmajor), 2.74e2.68 (m, 1Hminor), 2.60e2.52 (m, 1Hmajor), 1.60e1.58
(m, 6H), 1.56 (d, J ¼ 6.0 Hz, 3Hmajor), 1.42 (d, J ¼ 6.8 Hz, 3Hminor), 1.22
(d, J ¼ 6.8 Hz, 3Hmajor), 1.15 (d, J ¼ 7.6 Hz, 3Hminor); 13C NMR
(400 MHz, CDCl3): d 8.38 (s,1H), 7.17 (s, 1H), 4.32e4.24 (m, 1H), 3.83
(s, 3H), 2.68 (s, 1H), 2.58e2.50 (m, 1H), 1.75 (d, J ¼ 5.2 Hz, 6H), 1.53
(100 MHz, CDCl3): d 193.7, 192.1, 164.5, 159.6, 159.3, 156.6, 156.5,
(d, J ¼ 6.4 Hz, 3H), 1.21 (d, J ¼ 7.2 Hz, 3H); 13C NMR (100 MHz,
133.3, 133.2, 128.7, 115.7, 115.2, 114.7, 111.5, 111.4, 109.9, 109.8, 81.1,
80.1, 77.8, 46.4, 44.8, 28.2, 28.2, 28.1, 27.9, 19.7, 16.1, 10.1, 9.1 ppm,
four signals are superimposed; HRMS (ESI): [MþH]þ Calcd for
CDCl3):
d 192.7, 165.4, 164.1, 161.4, 132.1, 117.5, 114.2, 106.8, 84.7,
79.7, 75.3, 73.7, 51.8, 46.4, 29.4, 29.1, 19.7, 10.2, ppm; HRMS (ESI):
[MþH]þ Calcd for C18H21O5, 317.1389; found 317.1385. Compound
C17H19O5, 303.1232; found 303.1223.
cis-9: M.p. 84e86 ꢁC; 1H NMR (400 MHz, CDCl3):
d 8.34 (s, 1H), 7.15
(s, 1H), 4.67e4.61 (m, 1H), 3.80 (s, 3H), 2.68 (s, 1H), 2.64e2.57 (m,
4.1.11. Typical experimental procedure for synthesis of 2-amino-5-
substituted-1,3,4- thiadiazole(14aef) [47]
1H), 1.72 (s, 6H), 1.38 (d, J ¼ 6.8 Hz, 3H), 1.11 (d, J ¼ 7.2 Hz, 3H); 13
C
NMR (100 MHz, CDCl3):
d
194.4, 165.4, 163.8, 161.5, 132.1, 117.5,
To a stirred mixture of nitrile (1.0 mmol) and thiosemicarbazide
(1.1 mmol) was added trifluoroacetic acid (4 mL) and then the
mixture was refluxed for 6 h under stirring. After completion of
reaction cooled to room temperature and diluted with aqueous
ammonia. The precipitated solid was filtered off, washed with hot
water and dried in air. The product 14a-f were used in the next step
without further purification. Spectral data of compounds such as 5-
phenyl-1,3,4-thiadiazol-2-amine (14a) [63], 5-(p-tolyl)-1,3,4-
thiadiazol-2-amine (14b) [63], 5-(3,4,5-trimethoxyphenyl)-1,3,4-
thiadiazol-2-amine (14c) [64], 5-(4-nitrophenyl)-1,3,4-thiadiazol-
2-amine (14d) [63], 5-(pyridin-4-yl)-1,3,4-thiadiazol-2-amine
(14e) [63] and 5-(thiophen-2-yl)-1,3,4-thiadiazol-2-amine (14f)
[63] were agreement with literature report.
113.6,106.8, 84.7, 77.3, 75.4, 73.7, 51.7, 44.9, 29.3, 29.2,16.2, 9.1 ppm;
MS (ESI): m/z 317 [Mþ1]þ. Anal. Calcd for C18H20O5: C, 68.34; H,
6.37. Found: C, 68.51; H, 6.44.
4.1.9. Methyl 2,3,8,8-tetramethyl-4-oxo-2,3,4,8-tetrahydropyrano
[2,3-f]chromene-6-carboxylate (tc-10)
A solution of tc-9 (12.0 g, 37.97 mmol) in dry N,N-dimethyla-
niline (150 mL) was stirred at 190e195 ꢁC for 5 h under a nitrogen
atmosphere. After this time, the reaction mixture was cooled to
room temperature and acidified with dil. HCl. The mixture was
extracted with dichloromethane (3 ꢂ 200 mL), the organic phase
was dried with anhydrous Na2SO4, and the solvent was evaporated
under reduced pressure. The crude diastereomeric mixture was
purified through
a
silica gel column chromatography
4.1.12. 2,3,8,8-Tetramethyl-4-oxo-N-(5-phenyl-1,3,4-thiadiazol-2-
yl)-2,3,4,8-tetrahydropyrano[2,3-f]chromene-6-carboxamide (15a)
To a stirred solution of the acid 11 (400 mg, 1.32 mmol), DIPEA
(375 mg, 2.90 mmol), HOBt (196 mg, 1.45 mmol) in DMF (5 mL) was
added EDCI$HCl (278 mg, 1.45 mmol) at 0 ꢁC. After 5 min, 5-phenyl-
1,3,4-thiadiazol-2-amine (14a, 257 mg, 1.45 mmol) was added to
the ice-cold coupling solution, and the reaction was stirred for
12 h at room temperature. The mixture was poured into the ice
water and then precipitated solid was filtered and washed with ice
water. The crude solid was purified through a silica gel column
chromatography (chloroform-methanol ¼ 9:1) to give title com-
pound 15a (501 mg, 82%, dr: trans/cis ¼ 85:15)) as a white solid.
(hexaneeEtOAc ¼ 9:1) to give an inseparable diastereomeric
mixture (trans/cis ¼ 6:4) tc-10 (11.4 g, 95%) as a white solid. Single
crystals suitable for X-ray studies were grown from a solution of
trans-10 in hexaneeethyl acetate (6:4). Compound tc-10: M.p.
150e153 ꢁC; 1H NMR (400 MHz, CDCl3):
d 8.30, 8.29 (2s, 1H), 6.61
(dd, J ¼ 6.0, 1.2 Hz, 1H), 5.64 (d, J ¼ 10 Hz, 1H), 4.69e4.63 (m,
1Hmajor), 4.30e4.22 (m, 1Hminor), 3.84, 3.83 (2s, 3H), 2.65e2.59 (m,
1Hmajor), 2.55e2.47 (m, 1Hminor), 1.53 (d, J ¼ 6.4 Hz, 3Hminor),
1.50e1.47 (m, 6H), 1.40 (d, J ¼ 6.8 Hz, 3Hmajor), 1.20 (d, J ¼ 7.2 Hz,
3Hminor), 1.13 (d, J ¼ 7.2 Hz, 3Hmajor); 13C NMR (100 MHz, CDCl3):
d
194.3, 192.6, 165.0, 164.9, 159.1, 159.0, 158.9, 158.7, 131.4, 131.3,
129.2, 129.1, 115.6, 115.5, 113.7, 113.6, 113.3, 112.7, 110.0, 109.9, 79.8,
78.3, 78.2, 77.4, 51.7, 46.4, 44.9, 28.2, 28.1, 28.0, 27.9, 19.7, 16.2, 10.2,
9.2 ppm, one signal is superimposed; HRMS (ESI): [MþH]þ Calcd
for C18H21O5, 317.1389; found 339.1385. Compound t-10: M.p.
M.p. 248e252 ꢁC; 1H NMR (400 MHz, CDCl3):
d 11.26 (s, 1H), 8.75,
8.74 (2s, 1H), 7.99e7.96 (m, 2H), 7.49e7.47 (m, 3H), 6.71 (d,
J ¼ 10.0 Hz, 1H), 5.76 (d, J ¼ 10.4 Hz, 1H), 4.78e4.72 (m, 1Hminor),
4.38e4.30 (m, 1Hmajor), 2.77e2.70 (m, 1Hminor), 2.62e2.54 (m,
1Hmajor), 1.71e1.67 (m, 6H), 1.58 (d, J ¼ 6.0 Hz, 3Hmajor), 1.44 (d,
162e166 ꢁC; 1H NMR (400 MHz, CDCl3):
d 8.29 (s, 1H), 6.60 (d,

314
P. Sakthivel et al. / European Journal of Medicinal Chemistry 122 (2016) 302e318
J ¼ 6.8 Hz, 3Hminor), 1.24 (d, J ¼ 7.2 Hz, 3Hmajor), 1.17 (d, J ¼ 7.2 Hz,
3Hminor); 13C NMR (100 MHz, CDCl3):
193.6, 192.1, 163.8, 161.2,
1H), 8.75 (s, 1H), 8.35 (d, J ¼ 8.8 Hz, 2H), 8.16 (d, J ¼ 8.8 Hz, 2H), 6.73
(d, J ¼ 10.0 Hz, 1H), 5.78 (d, J ¼ 10.4 Hz, 1H), 4.78e4.75 (m, 1Hminor),
4.39e4.32 (m, 1Hmajor), 2.76e2.74 (m, 1Hminor), 2.64e2.56 (m,
1Hmajor), 1.72e1.68 (m, 6H), 1.59 (d, J ¼ 6.8 Hz, 3Hmajor), 1.45 (d,
J ¼ 6.4 Hz, 3Hminor), 1.25 (d, J ¼ 7.2 Hz, 3Hmajor), 1.18 (d, J ¼ 7.2 Hz,
d
159.7, 159.3, 158.1, 156.5, 156.4, 132.4, 132.3, 130.6, 130.5, 129.1,
128.8, 127.2, 115.7, 115.2, 114.7, 114.0, 112.5, 112.4, 110.1, 109.9, 80.9,
80.1, 77.9, 77.2, 46.4, 44.9, 31.9, 29.7, 28.4, 28.3, 28.1, 22.6, 16.1, 14.1,
10.1, 9.2 ppm, eight signals are superimposed; HRMS (ESI): [MþH]þ
Calcd for C25H24N3O4S, 462.1488; found 462.1476.
3Hminor); 13C NMR (100 MHz, CDCl3) for trans-15d isomer:
d 192.1,
161.4, 161.2, 159.9, 159.3, 156.4, 148.8, 136.5, 132.5, 128.9, 127.9,
124.4, 115.8, 115.3, 112.2, 110.1, 81.1, 80.3, 77.2, 46.5, 29.7, 28.4, 28.2,
19.7, 10.1 ppm; HRMS (ESI): [MþH]þ Calcd for C25H23N4O6S,
507.1338; found 507.1336.
In the same manner, compound 15b-f, 20a-b, 21 and 22 were
synthesized.
4.1.13. 2,3,8,8-Tetramethyl-4-oxo-N-(5-(p-tolyl)-1,3,4-thiadiazol-2-
yl)-2,3,4,8-tetrahydropyrano[2,3-f]chromene-6-carboxamide (15b)
The reaction was carried out similar to 15a using acid 11
(400 mg, 1.32 mmol), DIPEA (375 mg, 2.90 mmol), HOBt (196 mg,
1.45 mmol), EDCI$HCl (278 mg, 1.45 mmol) and 5-(p-tolyl)-1,3,4-
thiadiazol-2-amine (14b, 277 mg, 1.45 mmol) in DMF (5 mL) for
10 h. The crude solid was purified through a silica gel column
chromatography (chloroform-methanol ¼ 9:1) to give title com-
pound 15b (484 mg, 77%, dr: trans/cis ¼ 90:10) as a pale yellow
solid. M.p. 249e252 ꢁC; 1H NMR (400 MHz, CDCl3): for tc-15b:
4.1.16. 2,3,8,8-Tetramethyl-4-oxo-N-(5-(pyridin-4-yl)-1,3,4-
thiadiazol-2-yl)-2,3,4,8-tetrahydropyrano [2,3-f]chromene-6-
carboxamide (15e)
The reaction was carried out similar to 15a using acid 11
(400 mg, 1.32 mmol), DIPEA (375 mg, 2.90 mmol), HOBt (196 mg,
1.45 mmol), EDCI$HCl (278 mg, 1.45 mmol) and 5-(pyridin-4-yl)-
1,3,4-thiadiazol-2-amine (14e, 258 mg, 1.45 mmol) in DMF (5 mL)
for 12 h. The crude solid was purified through a silica gel column
chromatography (chloroform-methanol ¼ 9:1) to give title com-
pound 15e (440 mg, 72%, dr: trans/cis ¼ 85:15) as a yellow solid.
d
11.23 (s, 1H), 8.74 (s, 1H), 7.86 (d, J ¼ 8.0 Hz, 2H), 7.29e7.26 (m,
2H), 6.71 (d, J ¼ 10.4 Hz, 1H), 5.76 (d, J ¼ 10.0 Hz, 1H), 4.78e4.72 (m,
1Hminor), 4.37e4.33 (m, 1Hmajor), 2.76e2.70 (m, 1Hminor), 2.62e2.54
(m, 1Hmajor), 2.41 (s, 3H), 1.70e1.66 (m, 6H), 1.57 (d, J ¼ 6.4 Hz,
3Hmajor),1.44 (d, J ¼ 6.8 Hz, 3Hminor), 1.24 (d, J ¼ 6.8 Hz, 3Hmajor),1.17
(d, J ¼ 7.6 Hz, 3Hminor); 13C NMR (100 MHz, CDCl3) for trans e15b:
M.p. 244e248 ꢁC; 1H NMR (400 MHz, CDCl3):
d 11.37 (s, 1H),
8.76e8.74 (m, 3H), 7.85 (d, J ¼ 6.4 Hz, 2H), 6.72 (d, J ¼ 10.4 Hz, 1H),
5.78 (d, J ¼ 10.0 Hz, 1H), 4.78e4.74 (m, 1Hminor), 4.39e4.31 (m,
1Hmajor), 2.76e2.73 (m, 1Hminor), 2.63e2.55 (m, 1Hmajor), 1.72e1.68
(m, 6H), 1.59 (d, J ¼ 6.4 Hz, 3Hmajor), 1.45 (d, J ¼ 6.4 Hz, 3Hminor), 1.25
(d, J ¼ 7.2 Hz, 3Hmajor), 1.18 (d, J ¼ 7.2 Hz, 3Hminor); 13C NMR
d
192.1, 163.9, 161.2, 159.7, 157.8, 156.4, 140.8, 132.4, 129.8, 128.8,
127.9, 127.2, 115.8, 115.2, 112.5, 110.0, 80.9, 80.2, 46.5, 28.4, 28.1, 21.4,
19.7, 10.1 ppm; HRMS (ESI): [MþH]þ Calcd for C26H26N3O4S,
476.1644; found 476.1653.
(100 MHz, CDCl3): d 193.6, 193.5, 192.1, 192.0, 161.4, 161.2, 159.8,
159.5,159.1,156.5,156.4,150.7,137.7,132.5,132.4,128.8,120.9,115.7,
115.3, 114.8, 112.2, 112.1, 112.1, 110.1, 110.0, 81.1, 80.2, 77.9, 77.2, 46.5,
44.9, 29.6, 28.4, 28.3, 28.2, 19.7, 16.1, 10.1, 9.2 ppm; HRMS (ESI):
[MþH]þ Calcd for C24H23N4O4S, 463.1440; found 463.1466.
4.1.14. 2,3,8,8-Tetramethyl-4-oxo-N-(5-(3,4,5-trimethoxyphenyl)-
1,3,4-thiadiazol-2-yl)-2,3,4,8-tetrahydropyrano[2,3-f]chromene-6-
carboxamide (15c)
The reaction was carried out similar to 15a using acid 11
(400 mg, 1.32 mmol), DIPEA (375 mg, 2.90 mmol), HOBt (196 mg,
1.45 mmol), EDCI$HCl (278 mg, 1.45 mmol) and 5-(3,4,5-
4.1.17. 2,3,8,8-Tetramethyl-4-oxo-N-(5-(thiophen-2-yl)-1,3,4-
thiadiazol-2-yl)-2,3,4,8-tetrahydropyrano[2,3-f]chromene-6-
carboxamide (15f)
The reaction was carried out similar to 15a using acid 11
(400 mg, 1.32 mmol), DIPEA (375 mg, 2.90 mmol), HOBt (196 mg,
1.45 mmol), EDCI$HCl (278 mg, 1.45 mmol) and 5-(thiophen-2-yl)-
1,3,4-thiadiazol-2-amine (14f, 264 mg, 1.45 mmol) in DMF (5 mL)
for 8 h. The crude solid was purified through a silica gel column
chromatography (chloroform-methanol ¼ 9:1) to give title com-
pound 15f (501 mg, 81%, dr: trans/cis ¼ 93:7) as a white solid. M.p.
trimethoxyphenyl)-1,3,4-thiadiazol-2-amine
1.45 mmol) in DMF (5 mL) for 6 h. The crude solid was purified
through silica gel column chromatography (chloroform-
(14c,
387
mg,
a
methanol ¼ 9:1) to give title compound 15c (642 mg, 88%, dr: trans/
cis ¼ 85:15) as a white solid. M.p. 232e236 ꢁC; 1H NMR (400 MHz,
CDCl3):
d
11.26 (s, 1H), 8.74 (s, 1H), 7.22 (s, 2H), 6.72 (d, J ¼ 10.4 Hz,
1H), 5.77 (d, J ¼ 10.4 Hz, 1H), 4.77e4.74 (m, 1Hminor), 4.38e4.30 (m,
1Hmajor), 3.95 (s, 6H), 3.91 (s, 3H), 2.77e2.71 (m,1Hminor), 2.63e2.55
(m, 1Hmajor), 1.71e1.67 (m, 6H), 1.58 (d, J ¼ 6.0 Hz, 3Hmajor), 1.45 (d,
J ¼ 6.4 Hz, 3Hminor), 1.24 (d, J ¼ 6.8 Hz, 3Hmajor), 1.18 (d, J ¼ 7.2 Hz,
238e242 ꢁC; 1H NMR (400 MHz, CDCl3) for tce15f:
d 11.24 (s, 1H),
8.74 (s, 1H), 7.53 (d, J ¼ 3.2 Hz, 1H), 7.45 (d, J ¼ 5.2 Hz, 1H), 7.13 (dd,
J ¼ 4.8, 3.6 Hz, 1H), 6.72 (d, J ¼ 10.0 Hz, 1H), 5.77 (d, J ¼ 10.4 Hz, 1H),
4.77e4.74 (m, 1Hminor), 4.38e4.30 (m, 1Hmajor), 2.75e2.72 (m,
1Hminor), 2.63e2.55 (m, 1Hmajor), 1.70e1.66 (m, 6H), 1.58 (d,
J ¼ 6.0 Hz, 3Hmajor), 1.44 (d, J ¼ 6.4 Hz, 3Hminor), 1.24 (d, J ¼ 6.8 Hz,
3Hmajor), 1.18 (d, J ¼ 7.2 Hz, 3Hminor); 13C NMR (100 MHz, CDCl3) for
3Hminor); 13C NMR (100 MHz, CDCl3):
d 193.6, 192.1, 163.6, 161.3,
159.7, 159.4, 157.9, 156.5, 156.4, 153.7, 140.2, 132.5, 132.4, 128.8,
125.9, 115.8, 115.2, 114.8, 112.5, 112.4, 110.1, 110.0, 104.4, 81.0, 80.2,
77.9, 77.2, 60.9, 56.3, 46.5, 44.9, 28.4, 28.2, 19.7, 16.1, 10.1, 9.2 ppm;
HRMS (ESI): [MþH]þ Calcd for C28H30N3O7S, 552.1804; found
552.1773.
transe15f isomer:
d 192.1, 161.3, 159.7, 158.0, 157.5, 156.4, 133.0,
132.4, 128.9, 128.3, 128.2, 127.8, 115.8, 115.2, 112.4, 110.0, 81.0, 80.2,
77.2, 46.5, 28.4, 28.2, 19.7, 10.1 ppm; HRMS (ESI): [MþH]þ Calcd for
C23H22N3O4S2, 468.1052; found 468.1046.
4.1.15. 2,3,8,8-Tetramethyl-N-(5-(4-nitrophenyl)-1,3,4-thiadiazol-
2-yl)-4-oxo-2,3,4,8-tetrahydropyrano[2,3-f]chromene-6-
4.1.18. 2,3,8,8-Tetramethyl-4-oxo-N-(pyridin-3-yl)-2,3,4,8-
carboxamide (15d)
tetrahydropyrano[2,3-f]chromene-6-carboxamide (20a)
The reaction was carried out similar to 15a using acid 11
(400 mg, 1.32 mmol), DIPEA (375 mg, 2.90 mmol), HOBt (196 mg,
1.45 mmol), EDCI$HCl (278 mg, 1.45 mmol) and 5-(4-nitrophenyl)-
1,3,4-thiadiazol-2-amine (14d, 322 mg, 1.45 mmol) in DMF (5 mL)
for 12 h. The crude solid was purified through a silica gel column
chromatography (chloroform-methanol ¼ 9:1) to give title com-
pound 15d (502 mg, 75%, dr: trans/cis ¼ 90:10) as a yellow solid.
The reaction was carried out similar to 15a using acid 11
(400 mg, 1.32 mmol), DIPEA (375 mg, 2.90 mmol), and HOBt
(196 mg, 1.45 mmol), EDCI$HCl (278 mg, 1.45 mmol) and 3-
aminopyridine (16, 136 mg, 1.45 mmol) in DMF (5 mL) for 8 h.
The crude solid was purified through a silica gel column chroma-
tography (hexane-EtOAc ¼ 6:4) to give title compound 20a
(300 mg, 60%, dr: trans/cis ¼ 75:25) as a white solid. M.p.
M.p. 258e262 ꢁC; 1H NMR (400 MHz, CDCl3): for tc-15d:
d
11.36 (s,
189e193 ꢁC; 1H NMR (400 MHz, CDCl3):
d 9.92 (s, 1H), 8.72 (s, 1H),

P. Sakthivel et al. / European Journal of Medicinal Chemistry 122 (2016) 302e318
315
8.55 (d, J ¼ 2.4 Hz, 1H), 8.43 (dd, J ¼ 8.4, 1.2 Hz, 1H), 8.35 (dd, J ¼ 4.4,
1.2 Hz, 1H), 7.33e7.29 (m, 1H), 6.73 (d, J ¼ 10 Hz, 1H), 5.74 (d,
J ¼ 10 Hz, 1H), 4.75e4.70 (m, 1Hminor), 4.35e4.28 (m, 1Hmajor),
2.73e2.67 (m, 1Hminor), 2.60e2.52 (m, 1Hmajor), 1.66e1.62 (m, 6H),
1.57 (d, J ¼ 6.0 Hz, 3Hmajor), 1.43 (d, J ¼ 6.4 Hz, 3Hminor), 1.23 (d,
J ¼ 6.8 Hz, 3Hmajor), 1.16 (d, J ¼ 7.2 Hz, 3Hminor); 13C NMR (100 MHz,
7.56e7.51 (m, 3H), 6.77 (d, J ¼ 10.0 Hz, 1H), 5.75 (d, J ¼ 10.0 Hz, 1H),
4.37e4.29 (m, 1H), 2.62e2.54 (m, 1H), 1.69 (s, 3H), 1.65 (s, 3H), 1.58
(d, J ¼ 6.4 Hz, 3H), 1.24 (d, J ¼ 7.2 Hz, 3H); 13C NMR (100 MHz,
CDCl3):
d 192.4, 162.4, 158.8, 155.9, 134.1, 133.1, 132.5, 128.9, 128.4,
126.7, 126.1, 125.9,125.8, 124.9, 120.7, 120.1, 116.4, 115.9, 114.9, 109.8,
80.2, 80.0, 46.5, 28.4, 28.1, 19.7, 10.2 ppm; HRMS (ESI): [MþH]þ
Calcd for C27H26NO4, 428.1862; found 428.1839.
CDCl3):
d 193.8, 192.3, 162.4, 159.0, 155.6, 145.0, 141.0, 135.3, 132.0,
128.3, 128.2, 127.1, 123.8, 116.3, 116.2, 115.0, 114.9, 114.4, 109.8, 109.7,
80.1, 80.0, 77.7, 46.5, 44.9, 28.4, 28.4, 28.3, 28.1, 19.7, 16.1, 10.1,
9.2 ppm; HRMS (ESI): [MþH]þ Calcd for C22H23N2O4, 379.1658;
found 379.1656.
4.2. Biological methods
4.2.1. Cell lines
MCF-7, A549 and HeLa and cells were cultured in Dulbecco’s
Modified Eagle’s Medium: Nutrient Mixture F-12 (Ham) (D-MEM/
F-12) with 15 mM HEPES buffer, Lglutamine, and pyridoxine hy-
drochloride, supplemented with 1.2 g of sodium bicarbonate
(Invitrogen Corporation, CA), antibiotics (10,000 U Lꢀ1 penicillin
and 10 mg L1 streptomycin) (Himedia, Mumbai, Maharashtra India)
and 10% fetal bovine serum (FBS) (Invitrogen, Grand Island, NY,
USA). The cells were incubated at 37 ꢁC in a 5% CO2 and 95% hu-
midified incubator.
4.1.19. 2,3,8,8-Tetramethyl-4-oxo-N-(pyridin-4-yl)-2,3,4,8-
tetrahydropyrano[2,3-f]chromene-6-carboxamide (20b)
The reaction was carried out similar to 15a using acid 11
(400 mg, 1.32 mmol), DIPEA (375 mg, 2.90 mmol), HOBt (196 mg,
1.45 mmol), EDCI$HCl (278 mg, 1.45 mmol) and 4-aminopyridine
(17,136 mg, 1.45 mmol) in DMF (5 mL) for 12 h. The crude solid
was purified through a silica gel column chromatography (hexane-
EtOAc ¼ 6:4) to give title compound 20b (200 mg, 40%, dr: trans/
cis ¼ 90:10) as a white solid. M.p. 202e204 ꢁC; 1H NMR (400 MHz,
CDCl3):
d
9.99 (s, 1H), 8.71 (s, 1H), 8.53 (d, J ¼ 5.6 Hz, 2H), 7.58 (d,
J ¼ 6.0 Hz, 2H), 6.73 (d, J ¼ 10.0 Hz, 1H), 5.75 (d, J ¼ 10.0 Hz, 1H),
4.74e4.72 (m, 1Hminor), 4.34e4.30 (m, 1Hmajor), 2.72e2.70 (m,
1Hminor), 2.61e2.53 (m, 1Hmajor), 1.67e1.63 (m, 6H), 1.57 (d,
J ¼ 6.4 Hz, 3Hmajor), 1.43 (d, J ¼ 6.8 Hz, 3Hminor), 1.23 (d, J ¼ 7.2 Hz,
3Hmajor), 1.17 (d, J ¼ 7.2 Hz, 3Hminor); 13C NMR (100 MHz, CDCl3):
4.2.2. Cell viability (MTT assay)
The anticancer activity of synthesized hybrids were determined
by MTT assay (3-(4, 5-dimethylthiazol-2- yl)-2, 5-diphenyl tetra-
zolium bromide) assay [65]. Briefly, the cells in their exponential
growth phase were trypsinized and seeded in 96-well at-bottom
culture plates at a density of 1 ꢂ 104 cells per well in 100 mL of
complete Dulbecco’s modified eagle’s medium (DMEM). Then, the
cells were allowed to adhere and grow for 24 h at 37 ꢁC in the
presence of 5% CO2. After 24 h, the medium was replaced with
100 mL of fresh incomplete DMEM medium containing different
d
193.8, 192.3, 162.6, 159.2, 158.9, 155.7, 155.6, 150.3, 150.2, 145.6,
132.4, 132.3, 128.3, 116.3, 115.1, 114.8, 114.6, 114.2, 113.9, 109.9, 109.8,
80.2, 80.1, 77.8, 77.2, 46.5, 44.9, 28.5, 28.3, 28.2, 21.1, 19.7, 16.1, 10.1,
9.2 ppm; HRMS (ESI): [MþH]þ Calcd for C22H23N2O4, 379.1658;
found 379.1656.
concentrations of synthesized hybrids (10e100
mM) and further
4.1.20. 2,3,8,8-Tetramethyl-4-oxo-N-(thiazol-2-yl)-2,3,4,8-
tetrahydropyrano[2,3-f]chromene-6-carboxamide (21)
incubated for 24 h. At the end of the incubation, fresh media con-
taining 0.40 mg mL1 MTT were added to the 95-well plates and
they were incubated for another 4 h at 37 ꢁC in 5% CO2. The for-
mazan crystals formed after 4 h were solubilized in 100 mL of
DMSO after aspirating the medium. The absorbance was monitored
at 595 nm using a 96-well plate reader (Bio-Rad, Hercules, CA,
USA). The growth inhibition was determined using:
The reaction was carried out similar to 15a using acid 11
(400 mg, 1.32 mmol), DIPEA (375 mg, 2.90 mmol), HOBt (196 mg,
1.45 mmol), EDCI$HCl (278 mg, 1.45 mmol) and 2-aminothiazole
(18, 145 mg, 1.45 mmol) in DMF (5 mL) for 5 h. The crude solid
was purified through a silica gel column chromatography (hexane-
EtOAc ¼ 6:4) to give title compound 21 (458 mg, 90%, dr: trans/
cis ¼ 70:30) as a white solid. M.p. 236e239 ꢁC; 1H NMR (400 MHz,
Growth inhibition % ¼ (control O.D. - sample O.D.)/control O.D.
CDCl3):
d
11.05 (s, 1H), 8.76 (s, 1H), 7.50 (d, J ¼ 3.6 Hz, 1H), 7.00 (d,
J ¼ 3.6 Hz, 1H), 6.71 (d, J ¼ 10.0 Hz, 1H), 5.74 (d, J ¼ 10.0 Hz, 1H),
4.76e4.71 (m, 1Hminor), 4.36e4.29 (m, 1Hmajor), 2.75e2.68 (m,
1Hminor), 2.61e2.53 (m, 1Hmajor), 1.69e1.64 (m, 6H), 1.57 (d,
J ¼ 6.4 Hz, 3Hmajor), 1.43 (d, J ¼ 6.8 Hz, 3Hminor), 1.23 (d, J ¼ 7.2 Hz,
3Hmajor), 1.17 (d, J ¼ 7.2 Hz, 3Hminor); 13C NMR (100 MHz, CDCl3):
IC50 value was determined as the concentration of synthesized
hybrids that produced a 50% reduction in cell viability.
4.2.3. AO/EB staining
d
193.7, 192.2, 161.3, 161.2, 159.4, 159.0, 158.0, 156.3, 156.1, 137.9,
Acridine orange/Ethidium bromide (AO/EB) staining was carried
out to detect the morphological evidence of apoptosis induced by
synthesized hybrids (15b, 15d, 20a and 20b) in treated MCF-7 cells
[66]. For that, hybrids treated cells were harvested and suspended
in phosphate buffered saline solution (PBS), then the cells were
132.3, 132.2, 128.7, 115.9, 115.1, 114.6, 113.8, 113.2, 113.1, 109.9, 109.8,
80.5, 80.1, 77.8, 46.5, 44.9, 28.4, 28.3, 28.2, 28.1, 19.7, 16.1, 10.1,
9.2 ppm; HRMS (ESI): [MþH]þ Calcd for C20H21N2O4S, 385.1222;
found 385.1219.
stained with 5 mL of a cocktail mix containing AO (100 mg mL¡1
)
4.1.21. 2,3,8,8-Tetramethyl-N-(naphthalen-1-yl)-4-oxo-2,3,4,8-
tetrahydropyrano[2,3-f]chromene-6-carboxamide (22)
and EB (100 mg mL¡1) in PBS. Further, cell suspension of 10 mL was
applied to a glass slide, covered with a cover slip and cells were
viewed and photographed under a fluorescent microscope (Carl
Zeiss, Jena, Germany) fitted with a 377e355 nm filter, at ꢂ 400
magnification,. Viable cells were identified by a bright green nu-
cleus with intact structure. Early apoptotic cells show a bright green
nucleus with condensed chromatin, while nuclei of apoptotic cells
in the late phase were stained orange and showed a condensed
chromatin structure. Whereas necrotic cells displayed an orange
nucleus without condensed chromatin. Tests were done in tripli-
cate, counting a minimum of 100 cells in total for each.
The reaction was carried out similar to 15a using acid 11
(400 mg, 1.32 mmol), DIPEA (375 mg, 2.90 mmol), HOBt (196 mg,
1.45 mmol), EDCI$HCl (278 mg, 1.45 mmol) and 1-naphthylamine
(19, 208 mg, 1.45 mmol) in DMF (5 mL) for 8 h. The crude solid
was purified through a silica gel column chromatography (hexane-
EtOAc ¼ 7:3) to give title compound 22 (396 mg, 70%) as a white
solid. M.p. 209e212 ꢁC; For trans-22 isomer: 1H NMR (400 MHz,
CDCl3):
d
10.09 (s, 1H), 8.84 (s, 1H), 8.31 (d, J ¼ 7.6 Hz, 1H),
8.06e8.03 (m, 1H), 7.91e7.88 (m, 1H), 7.69 (d, J ¼ 8.4 Hz, 1H),

316
P. Sakthivel et al. / European Journal of Medicinal Chemistry 122 (2016) 302e318
4.2.4. Hoechst 33342 staining
analysis Bcl-2: forward 50-CTGCACCTGACGCCCTTCAC-30, reverse 50-
GCAGAGTCTTCAGAGACAGC-30; The thermal cycling conditions
included an initial denaturation step at 95 ꢁC for 3 min and 40
cycles at 95 ꢁC for 10 s, 62 ꢁC for 15 s and 72 ꢁC for 20 s. For
quantification, the cycle threshold number (Ct) that exhibits the
maximum curve growth rates was determined using the CFX
Manager Software. The relative gene expression of each sample,
normalized to that of glyceraldehyde-3-phosphate dehydrogenase
The nuclear changes and apoptotic bodies induced by the hy-
brids were detected using Hoechst 33258 staining. Briefly, MCF-
7 cells in 24-well plates were trypsinized, washed twice with PBS
buffer (pH 7.4) and 25 mL of the cell suspension was stained with
1 m
L of Hoechst 33342 (1 mg mLꢀ1) for 10 min at 37 ꢁC in the dark
condition. The condensed or fragmented nuclei of apoptotic cells
were observed using fluorescence microscopy fluorescent micro-
scope (Carl Zeiss, Jena, Germany) fitted with a 377e355 nm filter
and observed at ꢂ 400 magnification.
(GAPDH), was calculated by the formula 2Ct(GAPDH)_Ct(gene)
.
4.2.9. Western blot analysis
4.2.5. Mitochondrial membrane potential assay
MCF-7 cells were cultured in 6-well plates and then treated with
or without NPs for 24 h. The cells were then lysed in a buffer
containing 50mMTriseHCl (pH 8.0), 150 mM sodium chloride,
0.02% sodium azide, phenylmethane sulfonyl fluoride, aprotinin,
and 1% Triton X 100, and centrifuged at 12,000 rpm for 30 min at
Mitochondrial membrane potential, Djm is an important
parameter of mitochondrial function used as an indicator of cell
health. Briefly, MCF-7 cells were treated overnight with synthe-
sized hybrids (15b and 20b) at their respective IC50 (14.3 and
26.2
incubated for 1 h with 2
dium. The adherent cell layer was then washed three times with
PBS and dislodged with 250 L of trypsin-EDTA. Cells were
m
M) in 6-well plates. After that the cells were further and
4
ꢁC. Subsequently, the quantified proteins were electrophoresed
m
g mLꢀ1 of JC-1 containing culture me-
using sodium dodecyl sulfatepolyacrylamide gel electrophoresis
(SDS-PAGE). The separated proteins were transferred to nitrocel-
lulose membranes. Membranes were washed with tris-buffered
saline Tween-20 (TBST), blocked with 5% skimmed milk for 1 h at
37 ꢁC, incubated overnight at 4 ꢁC with goat anti-rabbit Bclꢀ2, Bax,
and GAPDH (loading control) antibodies (Sigma, Madison, WI)
primary antibodies in appropriate dilutions. Membranes were
washed four times with TBS/0.05% Tweenꢀ20 and incubated with
secondary antibodies conjugated with peroxidase (Bangalore
Genei, Bangalore, India) or alkaline phosphatase for 1 h. After
extensive washing, the reaction was developed with dia-
minobenzidine/1ꢀ4CN (DAB) chromogenic detection method and
then scanned.
m
collected in PBS/2% bovine serum albumin (BSA), washed twice by
centrifugation, resuspended in 0.3 mL of PBS/2% BSA, mixed gently,
and examined in the fluorescent microscope (Carl Zeiss, Jena,
Germany).
4.2.6. Analysis of mitochondrial membrane potential (Djm
MCF-7 cells were treated with the IC50 concentrations of hybrids
15b and 15d for 48 h and collected by centrifugation. Subsequently,
)
the cells were resuspended in pre-warmed JC-1 (5 mg/mL in PBS)
and incubated for 30 min at 37 ꢁC. After incubation, the cells were
washed twice with PBS, and analyzed immediately using flow cy-
tometer FACS Aria-II.
4.2.10. Molecular docking studies
The geometry of the molecules was fully optimized at the
Arguslab program. We performed docking by the Autodock Vina
(PyRx) program [58] in order to identify the molecules binding
sites. This approach permits a scoring function evaluation during
the docking process so that as many conformations as possible can
be obtained. The crystal structure of Bcl-2 promoter quadruplex
(PDB Code: 2F8U) has been retrieved from the protein data bank.
All possible poses have been considered as starting points and the
docking analysis was performed. Furthermore, both the total en-
ergy and the RMSD of each system were examined in order to
ascertain whether the molecule becomes equilibrated during mo-
lecular dynamics simulation. Visualization of the docked systems
has been further analyzed with PyMOL software package.
4.2.7. DNA fragmentation assay
DNA fragmentation assay was also adopted to examine the
development internucleosomal DNA fragments due to activation of
apoptosis (programmed cell death). The MCF-7 cells were seeded in
6 well plates and treated with compounds such as 140b, 140d, 154a
and 154b for 24 h. Further, the DNA fragments were isolated ac-
cording to the standard protocol for DNA isolation [67]. Briefly, after
the treatment for 24 h, cells were harvested, counted and washed
with 1X PBS at 4 ꢁC. The cells were pelleted by centrifugation at
200 ꢂ g at 4 ꢁC. The pellet was suspended in DNA lysis buffer
[20 mM Tris-HCl (pH ~7.5), 0.15 M NaCl, 1 mM EDTA, 0.5% SDS] and
incubated at 4 ꢁC for 30 min. After centrifugation (3000 rpm,
10 min, rt), 25 mL of the proteinase K (stock 20 mg/mL) was added to
the supernatant and incubated for 2 h at 37 ꢁC. It was centrifuged
(12,000 rpm, 4 ꢁC for 30 min), chilled absolute ethanol, and 0.1 M
NaCl were successively added to the supernatant for DNA precipi-
tation. Precipitated DNA was thoroughly washed with 70% ethanol,
dried, and dissolved in autoclaved distilled water. DNA fragmen-
tation was visualized by electrophoresis in 1.8% agarose gel con-
4.2.11. Hemocompatibility assay
Hemocompatibility assay was performed as per the earlier
report [68] with slight modification. Fresh blood was collected from
healthy volunteers in sterile lithium heparin vacutainers. Further,
Red blood cells (RBCs) were separated by centrifugation (1500 rpm
for 10 min at 4 ꢁC) and a ficoll density gradient. After discarding the
supernatant containing plasma and platelets, the RBCs were
washed thrice with sterile phosphate buffered saline (PBS). Then,
the pellets (1 mL) were resuspended in 3 ml of PBS. Then, 0.1 mL of
the diluted RBC suspension was added to synthesized hybrids 15b,
15d, 20a and 20b mixed in 0.5 mL PBS suspension at their
taining ethidium bromide and photographed in
documentation system (G: BOX, SYNGENE).
a
Gel
4.2.8. Real time RT-PCR analysis
Total RNA from MCF-7 cells was isolated using RNeasy Mini Kit
(Qiagen, MD) as per manufacturer instructions. 200 ng of the total
RNA was used in reverse transcription (RT) by the RevertAid First
Strand cDNA Synthesis Kit (Thermoscientific, USA). Real-time PCR
was then performed using iQ SYBR® Green Supermix (BioRad, USA)
according to the manufacturer’s protocol. The amplification process
was performed in CFX96 Touch™ Real-Time PCR Detection System
(Biorad, USA). Bcl-2 gene specific primers were designed and syn-
thesised. The following primers were used for Real time PCR
respective IC-50 concentration (14.3, 31.2, 28.7 and 26.2 mM) and
incubated at 37 ꢁC for 4 h. After incubation, all the samples were
centrifuged at 12,000 rpm at 4 ꢁC and supernatants were trans-
ferred to a 96-well plate. The hemolytic activity was determined by
measuring the absorbance at 570 nm (Biorad microplate reader
model 550, Japan). Control samples of 0% lysis (PBS buffer) and
100% lysis (in 1% Triton X-100) were employed in the experiment.
The percent of hemolysis was calculated as follows:

P. Sakthivel et al. / European Journal of Medicinal Chemistry 122 (2016) 302e318
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This study was approved by the Institutional Ethics Committee
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