Synthesis, structure activity relationship and mode of action of 3-substitutedphenyl-1-(2,2,8,8-tetramethyl-3,4,9,10-tetrahydro-2H,8H-pyrano[2, 3-f]chromen-6-yl)-propenones as novel anticancer agents in human leukaemia HL-60 cells

Article abstract of DOI:10.1016/j.ejmech.2013.01.027

A novel class of 3-substitutedphenyl-1-(2,2,8,8-tetramethyl-3,4,9,10- tetrahydro-2H,8H-pyrano[2,3-f]chromen-6-yl)-propenones were designed, synthesized and evaluated for their antiproliferative activity against the human cancer cell lines of diverse origin. Structure activity relationship was elucidated with various substitutions on the benzene ring and these variations significantly affected the potency. Most of the twelve tested compounds inhibited the growth of aggressive cancer cell lines. Moreover, three compounds 4j, 4k and 4l displayed excellent cytotoxic profile by inhibiting >90% cell proliferation in HL-60 and Caco-2 cells at 50 μM concentration. Further studies to elucidate the mode of action revealed that these three compounds induced G0/G1 cell cycle arrest and apoptosis, which was accompanied by loss of mitochondrial membrane potential, DNA fragmentation and nuclear morphology in HL-60 cells.

Full text of DOI:10.1016/j.ejmech.2013.01.027

European Journal of Medicinal Chemistry 62 (2013) 545e555
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, structure activity relationship and mode of action of
3-substitutedphenyl-1-(2,2,8,8-tetramethyl-3,4,9,10-tetrahydro-2H,
8H-pyrano[2,3-f]chromen-6-yl)-propenones as novel anticancer
agents in human leukaemia HL-60 cells
,
a,
Y.L.N. Murthy a , K.P. Suhasini 1, A.S. Pathania b, S. Bhushan b, Y. Nagendra Sastry a
*
a Dept. of Organic Chemistry, Andhra University, Visakhapatnam 530003, India
b Cancer Pharmacology Division, Indian Institute of Integrative Medicine (C.S.I.R), Canal Road, Jammu 18001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A
novel class of 3-substitutedphenyl-1-(2,2,8,8-tetramethyl-3,4,9,10-tetrahydro-2H,8H-pyrano[2,3-f]
Received 18 September 2012
Received in revised form
15 January 2013
Accepted 22 January 2013
Available online 29 January 2013
chromen-6-yl)-propenones were designed, synthesized and evaluated for their antiproliferative activity
against the human cancer cell lines of diverse origin. Structure activity relationship was elucidated with
various substitutions on the benzene ring and these variations significantly affected the potency. Most of
the twelve tested compounds inhibited the growth of aggressive cancer cell lines. Moreover, three
compounds 4j, 4k and 4l displayed excellent cytotoxic profile by inhibiting >90% cell proliferation in HL-
60 and Caco-2 cells at 50 mM concentration. Further studies to elucidate the mode of action revealed that
Keywords:
these three compounds induced G0/G1 cell cycle arrest and apoptosis, which was accompanied by loss of
mitochondrial membrane potential, DNA fragmentation and nuclear morphology in HL-60 cells.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Chromanochalcones
Antiproliferative activity
Cytotoxicity
Cell cycle analysis
Apoptosis
Anticancer efficacy
1. Introduction
On the other hand, chroman, or 3,4-dihydro-2H-1-benzopyran,
is itself not found in nature, but the chroman unit is an important
Cancer has become the major cause of death in both developed
and developing countries with the changes in the living habitat of
people and environment [1]. Accordingly, much attention has been
triggered throughout the world research laboratories to develop
new classes of chemotherapeutic agents [2].
Chalcones or 1,3-diaryl-2-propen-1-ones are cancer-preventive
food components found in human diet rich in fruits and vegeta-
bles [3]. In general, natural or synthetic chalcones, many reports
have documented their biologically active properties such as anti-
cancer [4], antibacterial [5], antimalarial [6], antileishmanial [7],
antitumour [8], anti-inflammatory [9], immunomodulatory [10]
and antimitotic [11]. A number of reports focused on their syn-
thesis, bio-evaluation [12] and mechanism of action [13]. Their
simple structure and the ease of preparation make chalcones as an
attractive drug scaffold with important therapeutic potential [14].
core structure in many biologically active natural products [15].
Chroman derivatives are interesting due to their significant phar-
macological activities such as anticancer [16], antioxidant [17],
antiestrogen [18], anticonvulsion [19], antirhinovirus [20], neuro-
protection [21], etc. Due to the various biological functions asso-
ciated with this skeleton, it has been frequently employed as a key
scaffold in the drug research [22] and assorted pharmaceuticals [23].
The ultimate biological and pharmaceutical importance of both
chromans and chalcones, incited us to envisage a possibility of
pharmacomodulation, by incorporating the above two active
pharmacophores in a single molecular frame work to yield a set
of new chemical entities i.e., 3-substitutedphenyl-1-(2,2,8,8-
tetramethyl-3,4,9,10-tetrahydro-2H,8H-pyrano[2,3-f]chromen-6-
yl)-propenones or chromanochalcones (4ael) and to examine
their efficacy on a panel of human cancer cell lines.
To establish more advanced structure activity relationship
around chromanochalcones, chroman ring A was fixed, and more
diverse substituents (both electron-releasing and electron-
withdrawing groups) such as methoxy, methyl, halogen, nitro and
cyano groups were introduced on ring B of chalcone at different
* Corresponding author.
E-mail addresses: murthyyln@gmail.com, murthyyln@yahoo.com (Y.L.N. Murthy).
1
Part of Ph.D thesis.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.027

546
Y.L.N. Murthy et al. / European Journal of Medicinal Chemistry 62 (2013) 545e555
positions (Fig. 1) with a goal to explore whether the electron den-
sity on chalcone may influence the cytotoxicity. To assess this, the
compounds were evaluated for their antiproliferative potential in
various human cancer cell lines viz., prostate cancer PC-3, breast
cancer MCF-7, human leukaemia HL-60, pancreatic cancers MIA
PaCa-2 and AsPC, melonoma MDA-MB-431 and colon Caco-2
in vitro by means of MTT assay.
Most of the tested compounds being active cytotoxic agents,
three of them that exhibit efficient antiproliferative activity in the
preliminary screening were further evaluated against four repre-
hensive human cancer cell lines of HL-60, PC-3, MIA PaCa-2 and
Caco-2. Interestingly, all the three compounds displayed substantial
cytotoxicity towards the leukaemia cell line HL-60 with IC50 values
the different cancer cell lines. With compounds 4ael, the substi-
tution pattern significantly affected potency against different cell
lines, as evident from the percentage (%) of growth inhibition from
Table 1. Precursor compound 2 was also included in the screening
to allow for the direct comparison of cytotoxic effect of 4ael.
Among the precursor compound 2 and unsubstituted parent
compound 4a, it was noticeable that the introduction of the ‘enone’
moiety in 4a improved the activity against all the cell lines except
MCF-7 and MIA PaCa-2. Comparing the methyl derivative 4b with
the unsubstituted parent compound 4a, the latter was more active
in five (PC-3, HL-60, AsPC, MDA-MB-435 and Caco-2) of the seven
cell lines examined, with the greatest difference observed in the
Caco-2 cells. Turning specifically to 4cee, having electron releasing
groups, the cytotoxicity was increased with the number of methoxy
groups against MCF-7, HL-60 cancer cell lines and was dramatically
diminished against MDA-MB-435. In some extent, the in vitro
antiproliferative activity did not differ greatly with the number of
methoxy groups and not significant. Indeed, compounds with
electron withdrawing groups, 4fel showed highly variable po-
tencies. Among the halogen derivatives (4fei), the meta-chloro
derivative 4h was much more active than its ortho-congener 4g
except in MIA PaCa-2. meta-Chloro derivative 4h has comparable
antiproliferative activity with the para-bromo derivative 4i against
four (MCF-7, AsPC, MDA-MB-435 and Caco-2) of the seven cell lines
tested. From the results, the small substituent like fluorine, seemed
to have the moderate activity among the halogen derivatives. In
case of the nitro substituted compounds, para-position is less fav-
oured than meta-position. This is supported by comparing the ac-
tivity of meta substituted compound 4j and para substituted
compound 4k against all the cell lines screened. The anti-
proliferative activity of the meta-nitro derivative 4j was not greatly
different from that of the para-cyano derivative 4l towards all the
cell lines tested. However, 4l is more effective among all the com-
pounds tested (4ael), as the cyano group is more electron with-
drawing and also much more polar than the other groups. A closure
look into the structure activity relationship (SAR) studies, indicated
that the hydrophobic substituents such as halogens, nitro and
cyano groups are better substituents to inhibit the cell proliferation
and electron donating substituents are unfavourable.
in the range of 6e15 mM and the modality of cell death induced by
these compounds was investigated in detail with HL-60 cells in
terms of cell cycle analysis and mitochondrial potential assay by
flow cytometry, DNA fragmentation assay on agarose gel electro-
phoresis and nuclear morphology under fluorescent microscope.
2. Results and discussion
2.1. Chemistry
With the objective of developing effective new anticancer
agents, a series of twelve chromanochalcones (4ael) were syn-
thesised in quantitative yields from the commercially available 2,4-
dihydroxy acetophenone 1 in two steps. In the first step, 1-(2,2,8,8-
tetramethyl-3,4,9,10-tetrahydro-2H,8H-pyrano[2,3-f]chromen-6-
yl)-ethanone 2 was obtained by the reaction of 1 with 2-methyl-3-
buten-2-ol in the presence of polyphosphoric acid (PPA) in excel-
lent yield. Coupling of 2 with different substituted benzaldehydes
3ael was accomplished with borontrifluoride etherate in dioxane
afforded the target compounds 4ael via Claisen Schmidt con-
densation in subsequent step. The synthetic steps are depicted in
Scheme 1. The structures of compounds 4ael were confirmed by 1H
NMR, 13C NMR, EI-MS, IR and elemental analyses. The configuration
of compounds 4ael is of the trans form, as the coupling constants of
two protons on vinyl bond is above 15.0 Hz evidenced in 1H NMR.
Over all, it can be seen that three chalcones in particular 4j
(R ¼ 3-NO2), 4k (R ¼ 4-NO2) and 4l (R ¼ 4-CN) have shown
excellent cytotoxic profile (>90%) against HL-60, Caco-2 cell lines
and appreciable level of cell growth inhibition (>50%) in rest of
2.2. Cancer biology
2.2.1. In vitro antiproliferative activity and structure activity
relationship (SAR) of chromanochalcones (4ael)
The in vitro cytotoxicity of all the synthesised compounds 4ael
was evaluated by performing a comprehensive screen at a single
cells except pancreatic cell line AsPC (Table 1) at 50
tration level. Further, the compounds that exhibited >50% inhibi-
tion were repeated screening at lower concentration (30 M),
mM concen-
m
dose of 50 mM in various human cancer cell lines encompassing
which resulted in the identification of compounds 4j, 4k and 4l are
the most potent among all the compounds (4ael). These encour-
aging results prompted us to determine their concentration needed
for 50% of inhibition (IC50 value) in vitro against HL-60, PC-3, MIA
PaCa-2 and Caco-2 cells at different concentrations between 1 and
prostate cancer cell line PC-3, breast cancer MCF-7, promyelocytic
human leukaemia HL-60, pancreatic cancer cell lines MIA PaCa-2 and
AsPC, melanoma MDA-MB-435 and colon cancer cell line Caco-2 by
employing MTT assay with mitomycin (Miot-C)/adriamycin (Adria.)/
5-fluorouracil (5-FU) as positive controls. The results are summarised
in Table 1 and expressed as percentage (%) of growth inhibition.
The data shown in Table 1 demonstrate the effects of different
substituents on the phenyl ring B for activity and selectivity against
100 mM (Fig. 2). Interestingly, compounds 4j, 4k and 4l were more
pronounced against HL-60 cells and resistant to pancreatic cancer
cells MIA PaCa-2 in terms of their cytotoxic profile (Fig. 2).
A clear cytotoxic effect of 4j, 4k and 4l was observed on cell
proliferation of HL-60 cell line upon 24 h and 48 h exposure at
different concentrations (Fig. 3). The IC50 values of these com-
pounds for 24 h are in range of 16e25
mM which further decrease to
6e15 M after 48 h exposure of compounds. Further experiments
m
were aimed to examine the mechanistic details of compounds 4j,
4k and 4l principally in HL-60 cells.
2.2.2. Cell cycle analysis of chromanochalcones
With a view to investigate the mode of action underlying the
anti-proliferative nature of the chromanochalcones, the cell cycle
Fig. 1. Basic structure of chromanochalcones.

Y.L.N. Murthy et al. / European Journal of Medicinal Chemistry 62 (2013) 545e555
547
Scheme 1. Steps involved in the synthesis of 4ael. Reagents and conditions: (i) 2 methyl-3-buten-2-ol, methylene chloride, stirring, rt, 4 h. (ii) boron trifluoride etherate, dioxane,
stirring, rt.
distribution of HL-60 cells was analyzed by flow cytometry. The HL-
60 cells were exposed to 4j, 4k and 4l compounds at 5, 10, 15 and
20 mM concentrations for 24 h, stained with propidium iodide and
analyzed by Modfit software to determine the distribution of the
total population in different phases (G0/G1, S, and G2/M) of cell
cycle. Fig. 4 represents the percentage of HL-60 cells in each stage of
the cell cycle following incubation with 4j, 4k and 4l for a period of
24 h. Untreated cells used as negative control and camptothecin
treated cells used as positive control (Fig. 4, Panel A). Treated HL-60
cells resulted in the accumulation of cells in the G0/G1 phase of cell
cycle with a concomitant decrease in the number of cells in both
the S and G2/M phases in a dose dependent manner. The
Table 1
In vitro cytotoxic profile of 4ael at a single concentration (50 mM) against different panel of human cancer cell lines.
Compound
Conc. (
m
M)
% Growth inhibition ꢀ SDa
PC-3 prostate
MCF-7 breast
HL-60 leukaemia
AsPC pancreatic
MIA PaCa pancreatic
MDA-MB-43 melanoma
Caco-2 colon
2
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
50
50
50
50
50
50
50
50
50
50
50
50
50
10
10
02
19 ꢀ 0.9
51 ꢀ 0.8
48 ꢀ 1.8
63 ꢀ 1.1
36 ꢀ 1.2
62 ꢀ 1
18 ꢀ 2
14 ꢀ 3
15 ꢀ 1.4
7 ꢀ 0.7
15 ꢀ 1.5
26 ꢀ 1.2
17 ꢀ 1.1
9 ꢀ 0.5
16 ꢀ 1
16 ꢀ 1.3
89 ꢀ 1.3
52 ꢀ 2.8
78 ꢀ 1
N.Db
25 ꢀ 1
32 ꢀ 1.5
18 ꢀ 0.5
5 ꢀ 1
5 ꢀ 1.1
17 ꢀ 0.6
2 ꢀ 0.7
12 ꢀ 0.9
2 ꢀ 1.7
5 ꢀ 1.2
28 ꢀ 1.2
15 ꢀ 1.3
24 ꢀ 1
27 ꢀ 1.3
48 ꢀ 1.5
27 ꢀ 1.2
48 ꢀ 0.8
N.D
25 ꢀ 2.4
24 ꢀ 0.5
41 ꢀ 2
30 ꢀ 3
17 ꢀ 1.1
27 ꢀ 1.3
38 ꢀ 1
31 ꢀ 1.8
24 ꢀ 1.2
45 ꢀ 0.73
80 ꢀ 0.9
74 ꢀ 1.3
76 ꢀ 1.5
N.D
2 ꢀ 0.6
47 ꢀ 1.1
38 ꢀ 0.7
53 ꢀ 0.6
52 ꢀ 0.9
32 ꢀ 1.2
52 ꢀ 1.1
33 ꢀ 2.6
54 ꢀ 3.4
52 ꢀ 1.9
88 ꢀ 1.2
78 ꢀ 1.2
88 ꢀ 0.9
N.D
20 ꢀ 2
34 ꢀ 1
11 ꢀ 1.5
16 ꢀ 2
23 ꢀ 2
17 ꢀ 1.3
12 ꢀ 1.8
11 ꢀ 1.2
31 ꢀ 1.5
28 ꢀ 3
93 ꢀ 1.4
73 ꢀ 1.6
93 ꢀ 2
N.D
27 ꢀ 1.2
77 ꢀ 1.3
38 ꢀ 2.6
41 ꢀ 1.5
60 ꢀ 1.2
25 ꢀ 1
92 ꢀ 1.9
91 ꢀ 1.9
93 ꢀ 2
84 ꢀ 0.7
N.D
59 ꢀ 0.9
52 ꢀ 2
59 ꢀ 3
33 ꢀ 0.7
72 ꢀ 1.5
61 ꢀ 2
4l
76 ꢀ 1.3
72 ꢀ 0.33
68 ꢀ 0.88
N.D
5-FU
Mito-C
Adria.
N.D
90 ꢀ 3
N.D
N.D
N.D
N.D
N.D
N.D
N.D
N.D
N.D
a
Data are presented as the mean of three independent experiments ꢀ standard deviation.
b
N.D not determined.

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Y.L.N. Murthy et al. / European Journal of Medicinal Chemistry 62 (2013) 545e555
Fig. 2. Inhibition of cell proliferation by compounds 4j, 4k and 4l on HL-60, PC-3, MIA PaCa-2 and Caco-2 cancer cell lines. Mitochondrial competence of MTT reduction as an index
of cell viability determined. Optical density (OD) of untreated control was taken as 100% viability. IC50 values of the compounds are depicted in the figure. Data are presented as the
mean ꢀ SD (n ¼ 8 wells) of three independent experiments.
population changes were observed even at the lowest concentra-
tion (5 M) used, and doubling the concentration of chroma-
nochalcones also doubled the sub-G0/G1 population. In particular,
88% at 20 mM as compared to control that is 48% (Fig. 4, Panel D).
m
Thus the exposure of HL-60 cells to compounds 4j, 4k and 4l leads
to an accumulation of cells in G0/G1 phase with increase in hypo
diploid population (sub-G0/G1) in a concentration dependent
manner. It indicates that during the rise in concentration from 5 to
compound 4j increases G0/G1 population of HL-60 cells to 86% at
20
mM concentration which is nearly equal to positive control
camptothecin (88%). Sub-G0/G1 population (<2n DNA), an indica-
tive of apoptosis also increased in 4j in dose dependent manner up
to 58% as compared to control (<1%). (Fig. 4, Panel B). While 4k
showed a less pronounced G0/G1 arrest (59%) in comparison with
4j (86%) and 4l (88%) in the same cell line accompanied with
20 mM, an increase in the population of G0/G1 phase was associated
with the population increase in the sub-G0/G1 phase, indicating
that the arrested cells entered into apoptosis. However, further
studies were conducted to confirm the induction of apoptosis by
these novel compounds, as the presence of hypo diploid cells is not
conclusive proof of apoptotic death [24].
a significant reduction of cells in both S and G2/M phases at 20
concentration. The sub-G0/G1 population is 32% in 4k at 20
m
m
M
M
concentration as compared to control (<1%). (Fig. 4, Panel C).
Among the three tested compounds, 4l is most potent in inducing
apoptosis indicated by sub-G0/G1 fraction with 71% cells, which is
2.2.3. DNA fragmentation
The apoptotic potential of 4j, 4k, and 4l was further confirmed
through induction of DNA fragmentation in HL-60 cells (Fig. 5),
which is known as the biochemical hallmark of apoptosis [25]. HL-
60 cells (2 ꢁ 106/3 ml/well) were treated with above compounds for
more than positive control camptothecin (69%) at 20
mM concen-
tration as compared to control (<1%) and the G0/G1 population is
Fig. 3. Inhibition of cell proliferation by compounds 4j, 4k and 4l on human leukaemic HL-60 cell line. MTT assay was performed at various concentrations of compounds. OD of
untreated control was taken as 100% viability. Data are presented as the mean ꢀ SD (n ¼ 8 wells) of three independent experiments.

Y.L.N. Murthy et al. / European Journal of Medicinal Chemistry 62 (2013) 545e555
549
Fig. 4. Representative graphs of HL-60 cell cycle distribution after 24 h, Panel A: control and positive control camptothecin (4
15 M, 4j at 20 M. Panel C: 4k at 5 M, 4k at 10 M, 4k at 15 M, 4k at 20 M. Panel D: 4l at 5 M, 4l at 10 M, 4l at 15 M, 4l at 20
of diploid cells in G0/G1, S and G2/M phases of the cell cycle, hypo diploid cells appear as a sub-G0/G1 peak and indicated as apoptosis.
m
M, for 6 h). Panel B: 4j at 5
mM. The ModFit software calculates the number
m
M, 4j at 10
m
M, 4j at
m
m
m
m
m
m
m
m
m
24 h, then the genomic DNA was isolated and electrophoresis is
performed. Results obtained clearly showed that DNA from com-
pounds treated cells exhibited such fragments typical of apoptosis.
The DNA fragmentation analysis revealed that compounds 4j, 4k
and 4l induced a discrete ladder pattern in HL-60 cell line at 20 mM
after 24 h of incubation thereby showing significant fragmentation,
of several mitochondrial proteins into the cytoplasm, which consti-
tutes the key event of mitochondrial mediated apoptosis. Loss of
mitochondrial membrane potential leads to depolarization of mito-
chondrial membrane which results less uptake of rhodamine-123,
a fluorescent dye by mitochondria. Compounds 4j, 4k and 4l caused
concentration dependent mitochondrial damage and hence
decrease in mitochondrial membrane potential (Fig. 6). They showed
48%, 56% and 61% dissipation of mitochondrial membrane potential
while no fragmentation was observed in untreated cells (Fig. 5).
2.2.4. Mitochondrial membrane potential (DJm) loss
when used at concentration of 20 mM in HL-60 cells for 24 h (Fig. 6).
Apoptotic stimuli results the convergence of many signals at
mitochondria and most of these stimuli trigger a change of the
mitochondrial membrane permeability [26] resulting in the release
Hence, treated HL-60 cells in the presence of 4j, 4k and 4l exhibited
a marked shift in fluorescence compared with untreated control cells
(7%) and the positive control camptothecin (51%).

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Y.L.N. Murthy et al. / European Journal of Medicinal Chemistry 62 (2013) 545e555
Fig. 4. (continued).

Y.L.N. Murthy et al. / European Journal of Medicinal Chemistry 62 (2013) 545e555
551
compounds exhibited significant in vitro antiproliferative activity at
micromolar ( M) concentration. Three compounds, 4j, 4k and 4l
demonstrated the most marked effect in the human leukaemia HL-
60 cancer cell line with IC50 values of 6e15 M range. With regard
m
m
to the proposed mode of action, cell cycle analysis in the HL-60 cell
line has shown a significant increase in the G0/G1 and sub-G0/G1
population, which is suggestive of induction of cell cycle arrest
and apoptosis. Moreover, DNA fragmentation, mitochondria depo-
larization and nuclear morphology studies evidenced that these
compounds inhibit the cancer cell growth through apoptosis.
Therefore, it was confirmed that 4j, 4k and 4l are an excellent
scaffolds for further study in the field of cancer chemotherapy.
4. Experimental section
4.1. Chemistry
Fig. 5. DNA fragmentation of compounds 4j, 4k and 4l in HL-60 cancer cells. Lane-1:
control. Lane-2: 4j at 20 M. Lane-3: 4k at 20 M. Lane-4: 4l at 20 M.
m
m
m
All the chemicals and solvents used in this work were of ana-
lytical reagent grade (anhydrous) and purchased from Sigma
Aldrich. Organic solutions were dried over Na2SO4. Melting points
were determined on an electrothermal apparatus in an open
capillary tube and are uncorrected. The 1H NMR and 13C NMR were
recorded on Bruker Avance-II 300 MHz in CDCl3 using tetrame-
2.2.5. Morphological appearance of HL-60 cancer cells treated with
4j, 4k and 4l
To confirm the apoptosis-induction of 4j, 4k and 4l compounds,
morphological analysis was done by Hoechst 33258 staining that
selectively binds DNA and allows monitoring of nuclear morpho-
logical changes under fluorescence microscopy. In Fig. 7, control
cells showed an even distribution of staining of homogeneous
nuclei features. Compounds 4j, 4k and 4l caused nuclear con-
densation and blebbing in HL-60 cells after 24 h of treatment in
thylsilane (TMS) as internal reference. Chemical shifts (d) are given
in ppm (parts per million). Infrared spectra were recorded from KBr
discs on Thermo Nicolet Avatar 330 spectrometer. The main bands
are given in cmꢂ1. Electron induced (EI) mass spectra were recorded
on an Ionspec QFT FT-ICR mass spectrometer. Elemental analyses
were performed on an Elementar Vario EL elemental analyzer. All
tested compounds yielded data consistent with a purity of at least
98% as compared with the theoretical values. Reaction mixtures
were monitored by TLC on silica gel (precoated F254 Merck plates),
and compounds were visualized with aqueous KMnO4. Flash
chromatography was performed using 230e400 mesh Merck silica
gel and the indicated corresponding solvent system.
concentration dependent manner. At 20 mM concentration all the
three compounds showed apoptotic bodies in most of cells indi-
cated by arrows in Fig. 7, confirming these compounds trigger cell
demise by apoptosis.
3. Conclusion
In conclusion, a series of twelve novel 3-substitutedphenyl-1-
(2,2,8,8-tetramethyl-3,4,9,10-tetrahydro-2H,8H-pyrano[2,3-f]chro-
men-6-yl)-propenones (4ael) have been designed, synthesised and
evaluated for their cytotoxic potential against seven human cancer
cell lines (PC-3, MCF-7, HL-60, MIA PaCa-2, AsPC, MDA-MB-435 and
Caco-2). A comprehensive structure activity relationship (SAR)
study established the substitutional requirements that have a pos-
itive impact and those that have a negative impact on the biological
activity of these novel compounds. Most of the twelve tested
4.1.1. General procedure for synthesis of compound 2
To a solution of 2,4-dihydroxy acetophenone 1 (1.52 g, 10 mmol)
in methylenedichloride (20 ml) was added polyphosphoric acid
(0.2 equiv) followed by 2-methyl-3-buten-2-ol (1.72 g, 20 mmol)
and stirred for 4 h at room temperature. The solvent was evapo-
rated under reduced pressure and the compound was purified by
flash chromatography using benzene/ether (5:9.5) (v/v) as an
eluent.
Fig. 6. Compounds 4j, 4k and 4l induced loss of mitochondrial membrane potential in HL-60 cells at 15 mM and 20 mM concentrations. Figures are representative of one of three
similar experiments.

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Y.L.N. Murthy et al. / European Journal of Medicinal Chemistry 62 (2013) 545e555
Fig. 7. Nuclear morphological changes of compounds 4j, 4k and 4l at 15 mM and 20 mM concentrations towards HL-60 cells.
4.1.1.1. 1-(2,2,8,8-Tetramethyl-3,4,9,10-tetrahydro-2H,8H-pyrano[2,3-
f]chromen-6-yl)-ethanone (2). White sticky solid; yield 68%; mp
155.8, 190.8; IR (KBr) nmax: 817, 892, 999, 1117, 1139, 1287, 1328, 1450,
1577, 1610, 1650, 2929, 2973 cmꢂ1; MS: m/z 390.2 (Mþ), 334.1, 279.1,
217.0,178.2,117.0. Anal. Calcd for C26H30O3: C, 79.97; H, 7.74. Found: C,
79.95; H, 7.78.
76 ꢃC; 1H NMR (300 MHz, CDCl3):
d
1.32 (s, 6H, 2 ꢁ CH3), 1.35 (s, 6H,
2 ꢁ CH3),1.77 (t, 4H, J ¼ 6.9 Hz, 2 ꢁ CH2), 2.57 (s, 3H, CH3), 2.70 (t, 4H,
J ¼ 6.6 Hz, 2 ꢁ CH2), 7.48 (s,1H, Ar); 13C NMR (75 MHz, CDCl3):
d 17.0,
21.6, 26.7, 26.8, 27.0, 31.6, 32.6, 74.5, 75.1, 104.5, 109.2, 111.7, 119.8,
129.0, 153.6, 156.0, 198.5; IR (KBr) nmax: 881, 921, 1121, 1151, 1118,
1299, 1358, 1456, 1579, 1660, 2931, 2973 cmꢂ1; MS: m/z 288 (Mþ),
273, 245, 219, 203,177, 165, 149,123, 109. Anal. Calcd for C18H24O3: C,
74.97; H, 8.39. Found: C, 74.95; H, 8.41.
4.1.2.3. 3-(4-Methoxyphenyl)-1-(2,2,8,8-tetramethyl-3,4,9,10-
tetrahydro-2H,8H-pyrano[2,3-f]chromen-6-yl)-propenone (4c). Yellow
oil; yield 78%; 1H NMR (300 MHz, CDCl3):
d
1.33 (s, 6H, 2 ꢁ CH3), 1.35
(s, 6H, 2 ꢁ CH3), 1.70e1.81 (m, 4H, 2 ꢁ CH2), 2.62 (t, 2H, J ¼ 6.9 Hz,
CH2), 2.72 (t, 2H, J ¼ 6.9 Hz, CH2), 3.83 (s, 3H, OCH3), 6.89 (d, 2H,
J ¼ 8.1 Hz, Ar), 7.03 (s, 1H, Ar), 7.27 (d, 2H, J ¼ 8.1 Hz, Ar), 7.40 (d, 1H,
J ¼ 15.9 Hz, Ha), 7.57 (d, 1H, J ¼ 15.6 Hz, Hb); 13C NMR (75 MHz,
4.1.2. General procedure for the synthesis of compounds (4ael)
To a stirred solution of 1-(2,2,8,8-tetramethyl-3,4,9,10-tetrahydro-
2H,8H-pyrano[2,3-f]chromen-6-yl)-ethanone 2 (2.88 g,10 mmol) and
corresponding substituted benzaldehyde 3ael (10 mmol) was added
gradually BF3$Et2O (0.71g, 5 mmol) at room temperature in dioxane.
The reaction mixture was stirred at ambient temperature till the
disappearance of the starting material 2. After completion of reaction,
the reaction mixture was diluted with moist ether (50 ml), washed
with water to discharge the colour and the BF3$Et2O complex. The
extracted ethereal solution was dried over anhydrous Na2SO4, con-
centrated and purified by column chromatography over SiO2 using
hexane/ethylacetate (1:9) as an eluent toyield the final products 4ael.
CDCl3): d 16.3, 20.3, 21.2, 25.6, 24.3, 30.7, 32.5, 74.3, 74.8, 109.2, 119.9,
120.7, 127.3, 127.9, 129.8,132.1, 139.7, 140.0,152.7, 155.3, 188.8; IR (KBr)
nmax: 818, 892, 929, 1110, 1139, 1229, 1319, 1436, 1529, 1604, 1636,
2935, 2936 cmꢂ1; MS: m/z 406.2 (Mþ). Anal. Calcd for C26H30O4: C,
76.82; H, 7.44. Found: C, 75.95; H, 7.40.
4.1.2.4. 3-(2,5-Dimethoxyphenyl)-1-(2,2,8,8-tetramethyl-3,4,9,10-
tetrahydro-2H,8H-pyrano[2,3-f]chromen-6-yl)-propenone (4d). Light
brown oil; yield 75%; 1H NMR (300 MHz, CDCl3):
d 1.33 (s, 6H,
2 ꢁ CH3), 1.34 (s, 6H, 2 ꢁ CH3), 1.70e1.81 (m, 4H, 2 ꢁ CH2), 2.62 (t, 2H,
J ¼ 6.9 Hz, CH2), 2.75 (t, 2H, J ¼ 6.9 Hz, CH2), 3.84 (s, 3H, OCH3), 3.89 (s,
3H, OCH3), 6.83 (dd, 2H, J ¼ 8.2 and 2.3 Hz, Ar), 6.99 (s, 1H, Ar), 7.37
(m, 2H, Ar and Hb), 7.18 (d, 1H, J ¼ 15.9 Hz, Ha); 13C NMR (75 MHz,
4.1.2.1. 3-Phenyl-1-(2,2,8,8-tetramethyl-3,4,9,10-tetrahydro-2H,8H-
pyrano[2,3-f]chromen-6-yl)-propenone (4a). Yellow crystals; yield
CDCl3): d 16.8, 21.2, 26.9, 26.2, 29.7, 30.3, 32.2, 55.3, 54.3, 74.3, 75.2,
72%; mp 127 ꢃC; 1H NMR (300 MHz, CDCl3):
d
1.33 (s, 6H, 2 ꢁ CH3),
96.3, 108.2 110.1, 112.9, 116.5, 120.1, 124.8, 128.1, 135.5, 143.3, 150.2,
154.3, 155.3, 189.9; IR (KBr) nmax: 831, 929, 1026, 1113, 1227, 1223,
1348, 1436,1539, 1527, 1606, 1633,1732, 2822, 2934, 2948 cmꢂ1; MS:
m/z 436.2 (Mþ), 380.1, 325.1, 273.1, 217.0, 178.9, 163.0. Anal. Calcd for
C27H32O5: C, 74.29; H, 7.39. Found: C, 74.25; H, 7.34.
1.37 (s, 6H, 2 ꢁ CH3), 1.71e1.81 (m, 4H, 2 ꢁ CH2), 2.34 (s, 3H, CH3),
2.67 (t, 2H, J ¼ 6.9 Hz, CH2), 2.73 (t, 2H, J ¼ 6.9 Hz, CH2), 7.18 (d, 2H,
J ¼ 7.8 Hz, Ar), 7.45 (s,1H, Ar), 7.48 (m, 3H, Ar), 7.61 (d,1H, J ¼ 15.9 Hz,
Ha), 7.69 (d, 1H, J ¼ 15.6 Hz, Hb); 13C NMR (75 MHz, CDCl3):
d 17.1,
20.3, 21.6, 26.9, 27.5, 31.3, 32.6, 74.4, 75.2, 108.5, 112.9, 120.7, 127.39,
127.9, 129.2, 133.3, 139.9, 141.2, 152.0, 154.7, 190.5; IR (KBr) nmax: 819,
887, 987, 1125, 1135, 1293, 1337, 1493, 1529, 1608, 1654, 2935,
2965 cmꢂ1; MS: m/z 376.2 (Mþ), 320.1, 265.0, 217, 178, 103.0. Anal.
Calcd for C25H28O3: C, 79.75; H, 7.50. Found: C, 79.70; H, 7.48.
4.1.2.5. 3-(2,4,6-Trimethoxyphenyl)-1-(2,2,8,8-tetramethyl-3,4,9,10-
tetrahydro-2H,8H-pyrano
[2,3-f]chromen-6-yl)-propenone
(4e).
Deep brown oil; yield 72%; 1H NMR (300 MHz, CDCl3):
d
1.33 (s, 6H,
2 ꢁ CH3),1.35 (s, 6H, 2 ꢁ CH3),1.78e1.79 (m, 4H, 2 ꢁ CH2), 2.63 (t, 2H,
J ¼ 7 Hz, CH2), 2.73 (t, 2H, J ¼ 7Hz, CH2), 3.86 (s, 6H, 2 ꢁ OCH3), 3.92
(s, 3H, OCH3), 6.50 (s, 1H, Ar), 7.14 (s, 1H, Ar), 7.45 (s, 1H, Ar), 7.60 (d,
1H, J ¼ 15.9 Hz, Ha), 8.01 (d, 1H, J ¼ 15.9 Hz, Hb); 13C NMR (75 MHz,
4.1.2.2. 3-(4-Methylphenyl)-1-(2,2,8,8-tetramethyl-3,4,9,10-
tetrahydro-2H,8H-pyrano[2,3-f]chromen-6-yl)-propenone
(4b). Pale
yellow crystals; yield 73%; mp 122 ꢃC; 1H NMR (300 MHz, CDCl3):
CDCl3):
d 17.1, 21.6, 26.8, 27.0, 31.8, 32.7, 55.9, 56.1, 56.4, 74.3, 75.0,
d
1.34 (s, 6H, 2 ꢁ CH3), 1.37 (s, 6H, 2 ꢁ CH3), 1.71e1.82 (m, 4H,
109.2, 110.1, 112.0, 116.5, 121.1, 125.8, 129.6, 135.2, 143.1, 151.5, 152.7,
153.9, 155.5, 190.9; IR (KBr) nmax: 849, 926, 1033, 1117, 1209, 1296,
1316, 1454, 1513, 1564, 1603, 1640, 1735, 1773, 2851, 2926,
2972 cmꢂ1; MS: m/z 467.3 (Mþ), 411.2, 355.1, 273.1, 217.0, 178.9,
193.0, 160.0; Anal. Calcd. for C28H34O6: C, 72.08; H, 7.35. Found: C,
71.96; H, 7.37.
2 ꢁ CH2), 2.36 (s, 3H, CH3), 2.64 (t, 2H, J ¼ 6.9 Hz, CH2), 2.73 (t, 2H,
J ¼ 6.9 Hz, CH2), 7.18 (d, 2H, J ¼ 7.8 Hz, Ar), 7.46 (s, 1H, Ar), 7.48 (d, 2H,
J ¼ 7.8 Hz, Ar), 7.61 (d, 1H, J ¼ 15.9 Hz, Ha), 7.69 (d,1H, J ¼ 15.6 Hz, Hb);
13C NMR (75 MHz, CDCl3):
d 17.1, 21.3, 21.6, 26.8, 27.0, 31.7, 32.6, 74.5,
75.1, 109.3, 112.0, 120.7, 127.0, 127.9, 129.6, 133.1, 139.7, 140.2, 153.0,

Y.L.N. Murthy et al. / European Journal of Medicinal Chemistry 62 (2013) 545e555
553
4.1.2.6. 3-(4-Fluorophenyl)-1-(2,2,8,8-tetramethyl-3,4,9,10-
tetrahydro-2H,8H-pyrano[2,3-f]chromen-6-yl)-propenone (4f). Light
brown crystals; yield 65%; mp 122 ꢃC; 1H NMR (300 MHz, CDCl3):
26.8, 27.0, 31.7, 32.6, 74.9, 75.4, 109.5,112.4,119.9, 121.5, 123.5, 129.7,
129.9, 130.9, 134.0, 136.6, 137.8, 148.6, 153.5, 156.6, 189.5; IR (KBr)
nmax: 836, 980, 1116, 1139, 1277, 1330, 1452, 1527, 1574, 1609, 1650,
2936, 2977 cmꢂ1; MS: m/z 421.1 (Mþ), 365.1, 310.0, 217.0, 178.9,
148.0. Anal. Calcd for C25H27NO5: C, 71.24; H, 6.46; N, 3.32. Found:
C, 71.22; H, 6.49; N, 3.30.
d
1.34 (s, 6H, 2 ꢁ CH3), 1.36 (s, 6H, 2 ꢁ CH3), 1.76e1.82 (m, 4H,
2 ꢁ CH2), 2.63 (t, 3H, J ¼ 6.6 Hz, CH2), 2.73 (t, 3H, J ¼ 6.9 Hz, CH2),
7.34 (d, 2H, J ¼ 8.4 Hz, Ar), 7.50 (d, 2H, J ¼ 8.4 Hz, Ar), 7.48 (s, 1H, Ar),
7.57 (d, 1H, J ¼ 15.6 Hz, Ar), 7.70 (d, 1H, J ¼ 15.9 Hz, Ar); 13C NMR
(75 MHz, CDCl3):
d
17.1, 21.6, 26.8, 27.0, 31.7, 32.6, 74.6, 75.2, 109.4,
4.1.2.11. 3-(4-Nitrophenyl)-1-(2,2,8,8-tetramethyl-3,4,9,10-tetrahydro-
112.2, 120.4, 128.6, 128.9, 129.0, 129.7, 134.4, 135.1, 138.4, 153.2,
156.1, 190.2; IR (KBr) nmax: 834, 998, 1117, 1142, 1284, 1327, 1450,
1577, 1607, 1650, 2972, 2932 cmꢂ1; MS: m/z 394.1 (Mþ), 338.1,
283.0, 217.0, 178.9, 121.0. Anal. Calcd. for C25H27FO3: C, 76.12; H,
6.90. Found: C, 76.10; H, 6.91.
2H,8H-pyrano[2,3-f]chromen-6-yl)-propenone (4k). Yellow crystals;
yield 69%; mp 179 ꢃC; 1H NMR (300 MHz, CDCl3):
d 1.35 (s, 6H,
2 ꢁ CH3),1.37 (s, 6H, 2 ꢁ CH3), 1.62e1.82 (m, 4H, 2 ꢁ CH2), 2.64 (t, 2H,
J ¼ 6.9 Hz, CH2), 2.74 (t, 2H, J ¼ 6.6 Hz, CH2), 7.51 (s, 1H, Ar), 7.62 (d,
1H, J ¼ 15.9 Hz, Ha), 7.70 (d, 2H, J ¼ 8.7 Hz, Ar), 7.84 (d,1H, J ¼ 15.9 Hz,
Hb), 8.24 (d, 2H, J ¼ 8.7 Hz, Ar); 13C NMR (75 MHz, CDCl3):
d 17.0, 21.6,
4.1.2.7. 3-(2-Chlorophenyl)-1-(2,2,8,8-tetramethyl-3,4,9,10-
tetrahydro-2H,8H-pyrano[2,3-f]chromen-6-yl)-propenone (4g). Pale
yellow crystals; yield 68%; mp 132 ꢃC; 1H NMR (300 MHz, CDCl3):
26.8, 27.0, 31.7, 32.6, 74.8, 75.4, 109.5, 112.5, 119.9, 122.9, 124.0, 128.2,
128.6, 129.9, 132.0, 136.5, 142.3, 146.1, 153.4, 156.6, 189.5; IR (KBr)
nmax: 846, 989, 1119, 1140, 1283, 1340, 1453, 1516, 1577, 1601, 1650,
2930, 2974 cmꢂ1; MS: m/z 421.1 (Mþ), 365.1, 310.0, 217.0,178.9,148.0.
Anal. Calcd for C25H27NO5: C, 71.24, H, 6.46; N, 3.32. Found: C, 71.26;
H, 6.44; N, 3.30.
d
1.34 (s, 6H, 2 ꢁ CH3), 1.36 (s, 6H, 2 ꢁ CH3), 1.76e1.82 (m, 4H,
2 ꢁ CH2), 2.63 (t, 2H, J ¼ 6.6 Hz, CH2), 2.74 (t, 2H, J ¼ 6.9 Hz, CH2),
7.24e7.28 (m, 2H, Ar and Ha), 7.40 (dd, 1H, J ¼ 6.9 and 2.4 Hz, Ar),
7.48 (s, 1H, Ar), 7.68e7.73 (m, 2H, Ar), 8.03 (s, 1H, J ¼ 15.9 Hz, Hb);
13C NMR (75 MHz, CDCl3):
d
17.1, 21.6, 26.8, 27.0, 31.7, 32.6, 74.6,
4.1.2.12. 3-(4-Cyanophenyl)-1-(2,2,8,8-tetramethyl-3,4,9,10-
tetrahydro-2H,8H-pyrano[2,3-f] chromen-6-yl)-propenone (4l). Bright
yellow crystals; yield 62%; mp 209 ꢃC; 1H NMR (300 MHz, CDCl3):
75.2, 109.3, 112.2, 120.4, 126.7, 127.2, 129.8, 130.0, 130.3, 134.1, 135.1,
135.7, 153.1, 156.1, 190.3; IR (KBr) nmax: 866, 978, 1138, 1118, 1277,
1329, 1448, 1586, 1608, 1660, 2931, 2974 cmꢂ1; MS: m/z 411.2 (Mþ),
413.3 (Mþ2), 353.0, 299.0, 271.1, 233.0, 217.0, 178.9, 137.0. Anal.
Calcd. for C25H27ClO3: C, 73.07; H, 6.62. Found: C, 73.01; H, 6.66.
d
1.34 (s, 6H, 2 ꢁ CH3), 1.36 (s, 6H, 2 ꢁ CH3), 1.77e1.83 (m, 4H,
2 ꢁ CH2), 2.63 (t, 3H, J ¼ 6.9 Hz, CH2), 2.74 (t, 3H, J ¼ 6.6 Hz, CH2), 7.50
(s,1H, Ar), 7.57 (d,1H, J ¼ 15.9 Hz, Ha), 7.63 (d, 2H, J ¼ 8.4 Hz, Ar), 7.67
(d, 2H, J ¼ 8.4 Hz, Ar), 7.80 (d, 1H, J ¼ 15.9 Hz, Hb); 13C NMR (75 MHz,
4.1.2.8. 3-(3-Chlorophenyl)-1-(2,2,8,8-tetramethyl-3,4,9,10-
tetrahydro-2H,8H-pyrano[2,3-f]chromen-6-yl)-propenone
(4h). Yellow crystals; yield 68%; mp 125 ꢃC; 1H NMR (300 MHz,
CDCl3): d 17.0, 21.6, 26.8, 27.0, 31.7, 32.6, 74.8, 75.4, 109.4, 112.3,112.4,
118.5, 120.0, 128.1, 129.9, 131.3, 132.4, 137.1, 140.4, 153.4, 156.5, 189.7;
IR (KBr) nmax: 830, 994, 1119, 1140, 1287, 1329, 1452, 1575, 1609, 1651,
2224, 2926, 2974 cmꢂ1; MS: m/z 401.2 (Mþ), 345.1, 290.0, 217.0,
178.9, 163.0, 128.0. Anal. Calcd for C26H27NO3: C, 77.78; H, 6.78; N,
3.49. Found: C, 77.75; H, 6.75; N, 3.44.
CDCl3):
d
1.35 (s, 6H, 2 ꢁ CH3), 1.37 (s, 6H, 2 ꢁ CH3), 1.75e1.85 (m,
4H, 2 ꢁ CH2), 2.63 (t, 2H, J ¼ 6.9 Hz, CH2), 2.75 (t, 2H, J ¼ 6.6 Hz,
CH2), 7.32 (s, 1H, Ar), 7.39 (d, 1H, J ¼ 8.1 Hz, Ar), 7.51 (d, 1H,
J ¼ 15.6 Hz, Ha), 7.62 (d, 1H, J ¼ 7.5 Hz, Ar), 7.90 (d, 1H, J ¼ 15.9 Hz,
Hb), 8.17e8.20 (m, 1H, Ar), 8.49 (s, 1H, Ar); 13C NMR (75 MHz,
4.2. Cancer biology
CDCl3): d 17.1, 21.6, 26.8, 27.1, 31.7, 32.6, 74.8, 75.2,109.3,112.4,119.9,
121.5, 123.4, 129.6, 129.8, 130.5, 134.2, 135.36, 137.6, 147.3, 153.6,
156.3, 189.5; IR (KBr) nmax: 839, 984, 1126, 1268, 1343, 1450, 1575,
1611, 1650, 2986, 2977 cmꢂ1; MS: m/z 411.2 (Mþ), 413.3 (Mþ2),
353.0, 299.0, 271.1, 233.0, 217.0, 178.9, 137.0. Anal. Calcd. for
C25H27ClO3: C, 73.07; H, 6.62. Found: C, 73.01; H, 6.64.
4.2.1. Cancer cell lines
Human cancer cell lines for promyelocytic human leukaemia
HL-60, prostate PC-3, pancreatic MIA PaCa-2 and AsPC, colon Caco-
2 were procured from European Collection of Cell Cultures (ECACC).
Breast cancer cell line MCF-7 and melanoma cancer cell line MDA-
MB-435 were procured from National Cancer Institute, Frederick,
U.S.A. Cells were grown in RPMI-1640/DMEM/MEM medium con-
4.1.2.9. 3-(4-Bromophenyl)-1-(2,2,8,8-tetramethyl-3,4,9,10-
tetrahydro-2H,8H-pyrano[2,3-f]chromen-6-yl)-propenone (4i). Dark
yellow crystals; yield 66%; mp 138 ꢃC; 1H NMR (300 MHz, CDCl3):
taining 10% FCS, 100 units penicillin and 100 mg of streptomycin per
ml medium. Cells were grown in CO2 incubator (Thermocon Elec-
tron Corporation, USA) at 37 ꢃC with 95% humidity and 5% CO2 gas
environment. Cells were treated with tested compounds dissolved
in DMSO, while the untreated control cultures received only the
vehicle (DMSO, <0.2%).
d
1.34 (s, 6H, 2 ꢁ CH3), 1.36 (s, 6H, 2 ꢁ CH3), 1.76e1.82 (m, 4H,
2 ꢁ CH2), 2.63 (t, 2H, J ¼ 6.9 Hz, CH2), 2.73 (t, 2H, J ¼ 6.6 Hz, CH2),
7.43 (d, 2H, J ¼ 8.7 Hz, Ar), 7.50 (d, 2H, J ¼ 8.7 Hz, Ar), 7.48 (s, 1H, Ar),
7.55 (d, 1H, J ¼ 15.9 Hz, Ha), 7.71 (d, 1H, J ¼ 15.9 Hz, Hb); 13C NMR
(75 MHz, CDCl3):
d 17.1, 21.6, 26.8, 27.0, 31.7, 32.6, 74.6, 75.2, 109.4,
112.2, 120.4, 123.4, 128.6, 129.2, 129.7, 131.9, 134.8, 138.5, 153.2,
156.2, 190.3; IR (KBr) nmax: 817, 981, 1117, 1138, 1290, 1324, 1448,
1574, 1611, 1649, 2922, 2970 cmꢂ1; MS: m/z 454.1 (Mþ), 456.8
(Mþ2), 398.0, 342.9, 217.0, 180.9, 178.9. Anal. Calcd for C25H27BrO3:
C, 65.94, H, 5.98. Found: C, 65.95; H, 5.95.
4.2.2. Chemicals and reagents
RPMI-1640, minimal essential medium (MEM), Dulbecco’s modi-
fied eagle medium (DMEM), rhodamine-123 (Rh-123), PBS, protein-
ase K, agarose, propidium iodide (PI), DNase-free RNase, Hoechst-
33258, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT), 5-fluorouracil, mitomycin C, adriamycin and campto-
thecin were purchased from Sigma Chemical Company. Other
reagents used were of analytical grade and available locally.
4.1.2.10. 3-(3-Nitrophenyl)-1-(2,2,8,8-tetramethyl-3,4,9,10-
tetrahydro-2H,8H-pyrano[2,3-f]chromen-6-yl)-propenone (4j). Deep
yellow crystals; yield 63%; mp 161 ꢃC; 1H NMR (300 MHz, CDCl3):
d
1.34 (s, 6H, 2 ꢁ CH3), 1.41 (s, 6H, 2 ꢁ CH3), 1.77e1.85 (m, 4H,
4.3. MTT cell proliferation assay
2 ꢁ CH2), 2.64 (t, 2H, J ¼ 6.9 Hz, CH2), 2.74 (t, 2H, J ¼ 6.6 Hz, CH2),
7.53 (s, 1H, Ar), 7.57 (d, 1H, J ¼ 8.1 Hz, Ar), 7.64 (d, 1H, J ¼ 15.6 Hz,
Ha), 7.82 (d,1H, J ¼ 7.5 Hz, Ar), 7.90 (d,1H, J ¼ 15.9 Hz, Hb), 8.17e8.20
Cells from suspension and adherent cultures grown in 96-well
plates were exposed to indicate concentrations of compounds for
48 h. MTT solution (2.5 mg/ml in PBS) was added to each well 3 h
(m, 1H, Ar), 8.49 (s, 1H, Ar); 13C NMR (75 MHz, CDCl3):
d 17.0, 21.6,

554
Y.L.N. Murthy et al. / European Journal of Medicinal Chemistry 62 (2013) 545e555
before termination. The plates were centrifuged and the superna-
tant was discarded while the MTT-formazan crystals [27] were
dissolved in 100 ml DMSO. The OD measured at 570 nm with ref-
observed for any nuclear morphological alterations and apoptotic
bodies under inverted fluorescence microscope (Olympus 1X70,
magnification 10ꢁ) using UV excitation.
erence wavelength of 620 nm.
Appendix A. Supplementary data
4.4. Cell cycle analysis
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2013.01.027.
Cells (0.5 ꢁ 106/ml) were treated with different concentrations
of 4j, 4k and 4l for 24 h and collected at 300 g for 5 min. Cell pellets
were washed with PBS twice and fixed in 70% alcohol for overnight
References
at 4 ꢃC. Cells were again washed, suspended in 250
m
l of PBS and
g/ml) at 37 ꢃC for about 1 h and
stained with propidium iodide [28] (10 g/ml) for 30 min. Cells
incubated with RNase A (400
m
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4.5. DNA agarose gel electrophoresis
DNA fragmentation typical of apoptosis was analysed by elec-
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various treatments, HL-60 cells were centrifuged at 300 g for 5 min,
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