Neo-tanshinlactone inspired synthesis, in vitro evaluation of novel substituted benzocoumarin derivatives as potent anti-breast cancer agents

Article abstract of DOI:10.1016/j.bmcl.2010.09.040

A small library of novel benzocoumarin derivatives based on naturally occurring neo-tanshinlactone scaffold was constructed and their antiproliferative activities against breast cancer cells MCF-7 and MDA-MB-231 were evaluated. A number of derivatives sho

Full text of DOI:10.1016/j.bmcl.2010.09.040

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7127–7131
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Neo-tanshinlactone inspired synthesis, in vitro evaluation of novel substituted
benzocoumarin derivatives as potent anti-breast cancer agents q
Koneni V. Sashidhara a, , Jammikuntla N. Rosaiah a, Manoj Kumar a, Rishi Kumar Gara b,
⇑
Lakshma Vadithe Nayak b, Kamini Srivastava b, Hemant Kumar Bid b, Rituraj Konwar b,
a Medicinal and Process Chemistry Division, Central Drug Research Institute, CDRI-CSIR, Lucknow 226 001, India
b Endocrinology Division, Central Drug Research Institute, CDRI-CSIR, Lucknow 226 001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2010
Revised 7 September 2010
Accepted 8 September 2010
Available online 21 September 2010
A small library of novel benzocoumarin derivatives based on naturally occurring neo-tanshinlactone scaf-
fold was constructed and their antiproliferative activities against breast cancer cells MCF-7 and MDA-
MB-231 were evaluated. A number of derivatives showed good anti-breast cancer activity, in some cases
higher to that of the reference compound tamoxifen. In particular, benzocoumarins Bc-5, Bc-8 and Bc-9
strongly inhibited the proliferation of MCF-7 cancer cell line with the IC50 values of 3.8, 7.9 and 6.5 lM,
respectively. The compounds were capable of inducing nuclear fragmentation, cell cycle arrest and cas-
pase dependent apoptosis in MCF-7 cell lines. In addition, these derivatives were devoid of cytotoxic
effect against normal osteoblast cells. These synthetic benzocoumarins hold promises for developing
safer alternative to the existing anti-breast cancer agents.
Keywords:
Synthesis
Neo-tanshinlactone
Benzocoumarins
Anti-breast cancer agents
Apoptosis
Ó 2010 Elsevier Ltd. All rights reserved.
Breast cancer, a leading pandemic, affects women of all ages.1 In
hormone dependent breast carcinoma, estrogens play a critical
role, stimulating cancer cell proliferation.2,3 Since high serum lev-
els of estrogens favor the progression of breast cancer, estrogen
receptor antagonist approaches have made a huge contribution
in the breast cancer chemotherapy.4 Tamoxifen, a selective estro-
gen receptor modulator (SERM) has been used thoroughly in breast
cancer patients for more than three decades.5 Although tamoxifen
is most widely used clinical agent, but overcoming drug resistance6
and increase risk of uterine cancer in postmenopausal patients
merits development of potent anti-breast cancer agents with novel
scaffold or new mode of action.7,8
Natural and synthetic coumarins have been found to exhibit a
variety of pharmacological activities like anti-HIV,9 anticoagu-
lant,10 antibacterial,11 antioxidant,12 and anti-inflammatory.13
Among the diverse biological activities of coumarin the most
intriguing bioactivity is the effect against breast cancer.14–16
Figure 1 shows the chemical structures of some potent anti-breast
cancer molecules that contain a coumarin in their molecular make-
up and in this regard it is worth mentioning that 667 COUMATE is
in phase1 clinical trials for the treatment of hormone dependent
breast cancer in postmenopausal women.17 Furthermore, Lee and
co-workers reported that Neo-tanshinlactone, a steroid-like tetra-
cyclic natural product (Fig. 1) showed significant inhibition against
two estrogen receptor positive human breast cancer cell lines and
was 10-fold more potent and 20-fold more selective as compared
to tamoxifen.18 It’s analog, 4-ethyl neo-tanshinlactone exhibited
selective and potent in vivo anti-breast cancer activity in three dif-
ferent mouse models.19 A lot of studies aimed at better under-
standing of SAR for tanshinlactone are continuing to emerge
from Lee and co-workers20 These encouraging studies and our long
standing interest in this class of compounds21 prompted us to de-
sign and synthesize novel benzocoumarin derivatives to explore
their anti-breast cancer potential.
In order to establish chemical library with various functional-
ities and structural diversity, we have synthesized two different
series of coumarins starting from dicarbaldehyde Bc-2. In the first
series, we began with the synthesis of benzocoumarins and their
Schiff bases derivatives, the detailed synthetic methodology of
which has been previously reported by us and the reaction condi-
tions are shown in Scheme 1.22 Thus, the Pechmann reaction on
naphthalene 1, 5-diol 1, furnished 7-hydroxy-4-methyl-
benzo(h)chromene-2-one Bc-1 which on Duff formylation gave
dicarbaldehyde adduct Bc-2. This dialdehyde was then condensed
with various primary amines to give regioselective Schiff bases
that existed in keto-enamine form. These derivatives (Bc-1-10)
were evaluated for their in vitro anti-breast cancer activity in
MCF-7 and MDA-MB-231 cell lines. Out of ten compounds
q
Part VI in the series, ‘Advances in drug design and discovery’.
⇑
Corresponding author. Tel.: +91 9919317940; fax: +91 522 2623405.
E-mail addresses: kv_sashidhara@cdri.res.in, sashidhar123@gmail.com (K.V.
Sashidhara), r_konwar@cdri.res.in (R. Konwar).
Biological activity.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.040

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K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7127–7131
Figure 1. Examples of some potent coumarins having anti-breast cancer activity.
O
O
O
2
1
O
H
O
H
O
3
OH
O
O
10
7
9
8
b
4
d
a
H
5
6
OH
Bc-1
OH
O
O
OH
NH
Bc-2
R
Bc-3-10
1
c
R1
O
R1
O
O
Bc-3 -butyl
t
O
R =
Bc-4
Bc-5
isopropyl
methyl
O
O
d
Bc-6 heptyl
Bc-7 ethyl
Bc-8 propyl
Bc-9 butyl
H
O
NH
OH
Bc-10 2-morpholino
O
R
ethyl
Bc-16-35
Bc-11-15
Bc-11
phenyl
Bc-16
Bc-17
Bc-18 R = t -butyl,
R = butyl,
R = ethyl
R1 = phenyl
R1 = phenyl
R1 = phenyl
R1
=
Bc-12
Bc-13
Bc-14
tolyl
p-methoxy phenyl
2,4-dimethyl phenyl
Bc-19 R = isopropyl, R1 = phenyl
Bc-15 4-hydroxy phenyl
Bc-20
Bc-21
Bc-22
R = butyl,
R = ethyl
R = t -butyl,
R1 = tolyl
R1 = tolyl
R
1 = tolyl
Bc-23 R = isopropyl, R1 = tolyl
Bc-24 R = butyl,
Bc-25
Bc-26
R =
Bc-27
Bc-28 R = butyl,
Bc-29 R = ethyl,
Bc-30
R =
Bc-31
Bc-32
R1 = p-methoxy phenyl
R1 = p-methoxy phenyl
R1 = p-methoxy phenyl
R = ethyl,
t -butyl,
R = isopropyl,
R
1 = p-methoxy phenyl
R1 = 2,4-dimethyl phenyl
R1 = 2,4-dimethyl phenyl
R1 = 2,4-dimethyl phenyl
t -butyl,
R = isopropyl,
R = butyl,
R
1 = 2,4-dimethyl phenyl
R1 = 4-hydroxy phenyl
R1 = 4-hydroxy phenyl
R1 = 4-hydroxy phenyl
Bc-33 R = ethyl,
Bc-34 R =
Bc-35
t -butyl,
R = isopropyl,
R
1 = 4-hydroxy phenyl
Scheme 1. Reagents and conditions: (a) EAA, PTSA, 75 °C, 8 h; (b) (i) HMTA, TFA, 120 °C, 4 h; (ii) aq H2SO4, 100 °C, 1 h; (c) R1–COOH3, concd HCl, dioxane, reflux, 5 h; (d) R–
NH2, C2H5OH, rt, 10 min.
evaluated, three compounds showed IC50 in between 3.8 and
7.9 M which was lower than tamoxifen (Table 1). It is an interest-
ing to reveal that though all compounds were screened against
both MCF-7 (estrogen receptor—positive) and MDA-MB-231
l

K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7127–7131
7129
Table 1
Benzocoumarins induce loss of cell viability in MCF-7. Three com-
pounds Bc-5, Bc-8 and Bc-9 induced loss of cell viability of MCF-7
cells in a concentration-dependent fashion and their activity was
comparable to tamoxifen (Fig. 2). A significantly higher activity
Anticancer activity (IC50, lM) of benzocoumarins in MCF-7 cells
Compounds
IC50
(
l
M)
Compounds
IC50 (lM)
Bc-1
Bc-2
Bc-3
Bc-4
Bc-5
Bc-6
Bc-7
Bc-8
>100
>100
20
Bc-19
Bc-20
Bc-21
Bc-22
Bc-23
Bc-24
Bc-25
Bc-26
Bc-27
Bc-28
Bc-29
Bc-30
Bc-31
Bc-32
Bc-33
Bc-34
Bc-35
Tamoxifen
>100
>100
>100
97
21
92
of Bc-5 at 10, 20 and 30
served as compared to tamoxifen. Particularly, Bc-5 induced signif-
icant loss of cell viability even at 10 M, while, Bc-8 induced
significant loss of cell viability at 20 M that was observed as po-
lM and for Bc-8 at 20 and 30 lM was ob-
130
l
3.8
>100
>100
7.9
6.5
>100
>100
>100
>100
>100
>100
100
l
tent as tamoxifen. The calculated IC50 of antiproliferative activity
in MCF-7 cells of the three compounds were found to be lower than
tamoxifen (Table 1). Out of the three active compounds, Bc-5 had
lowest IC50 (Table 1).
Benzocoumarins induce increase in sub-diploid population and G0/
G1 arrest. Flow cytometry of PI stained treated cell analysis for cell
cycle phase distribution showed that all three compounds Bc-5,
Bc-8 and Bc-9 induced significant increase in sub-diploid popula-
tion (Fig. 3). Thus, the molecular events involved in cellular re-
sponse to the effective compounds, were investigated and to this
end, the compound Bc-5 and Bc-8 differed from Bc-9 as they
caused significant blockade at G0/G1 phase of cell cycle, whereas
Bc-9 caused G2 arrest (Fig. 3).
30
>100
>100
>100
>100
>100
35
40
85
>100
32
11.8
Bc-9
Bc-10
Bc-11
Bc-12
Bc-13
Bc-14
Bc-15
Bc-16
Bc-17
Bc-18
87
92
(estrogen receptor—negative) breast cancer cell lines, these deriv-
atives exhibited selective and significant inhibition only in MCF-7
cell lines. Furthermore, these active compounds did not affect the
viability of osteoblasts.
Benzocoumarins induce apoptotic changes. Manual field quantita-
tion of percent apoptotic cells based on cytoplasmic condensation,
presence of apoptotic body, nuclear fragmentation and relative
In the second series of compounds dicarbaldehyde Bc-2 was
used as the molecular template and an active pharmacophore for
further diversification. As a free aldehyde group was present in
the active compounds, for structural diversity, we wanted to intro-
duce chalcones at this position, which are well known for their
anticancer potential (e.g., Licochalcone-A, isolated from the licorice
root)23 and these compounds were synthesized by an efficient and
novel method employing the reagent controlled regioselective syn-
thesis of chalcones in aromatic dialdehydes that was recently dis-
covered by us.24,25
Thus, the regioselective synthesis of chalcones from dicarbalde-
hyde Bc-2 was achieved by using acid catalyzed Claisen–Schmidt
reaction, by condensation with appropriate ketones (Bc-11-15),
which was transformed to (Bc-16-35) by reacting them with differ-
ent aliphatic amines (Scheme 1).26 The structures of all the novel
compounds were confirmed by 1H, 13C, 2D NMR spectroscopy
and mass spectrometry (see Supplementary data). In all the chal-
cones synthesized the trans double bond was obtained exclusively.
The idea of synthesizing these benzocoumarins was not without
reason, it was envisaged that since these molecules are fluorescent
(due to extended conjugation) they would enhance the anticancer
activity due to their photosensitization properties.27 However, on
evaluation these compounds (Bc-11-35) they were found to be less
active than parent structure. Only compounds Bc-23, Bc-25, Bc-31,
Bc-32 and Bc-35 showed moderate inhibition against MCF-7 cell
lines (Table 1).
Figure 2. Loss of viability of MCF-7 cells by benzocoumarin derivatives: 1 Â 105
MCF-7 cells of 70–80% confluence were incubated 16 h with increasing concentra-
tions of benzocoumarin derivatives. Percent inhibition of cell growth (Total/
Control) was determined with MTT assay. Tamoxifen was used as positive control
*
(n = 9, p <0.05 vs Tam).
Of the two different set of compounds synthesized, the first ser-
ies of compounds in which the benzocoumarins were derivatized
with keto-enamine Schiff bases (Bc-3-10) were more active than
the other (Bc-11-15) and (Bc-16-35). Overall, within this series,
Bc-5, Bc-8 and Bc-9 were found to be more effective in inhibiting
cell growth of breast cancer cells, indicating in terms of SAR, that
the functional group substituted at the position 10 may be size
dependent and substitution at this position with bulky group’s re-
sults in loss of activity. Because of high selective cytotoxic effect in
MCF-7 cells and with no effect in primary osteoblast cells, we took
Bc-5, Bc-8 and Bc-9 for subsequent biological experiments (see
Supplementary data).28 Furthermore, in an attempt to better char-
acterize the cellular basis of their anti proliferative effects, the abil-
ity of these selected compounds to induce apoptosis was
investigated and the results of various studies are discussed next.
Figure 3. Effect of benzocoumarin derivatives (10 lM each) on cell cycle progres-
sion of MCF-7 cells: 5 Â 105 cells/well in 6-well plates with 70–80% confluence was
serum starved before treatment. After 24 h treatment in phenol red free media, cells
were fixed in 70% ethanol, re-hydrated in PBS, and stained with PBS containing PI
(50 lg/ml) and ribonuclease A (100 lg/ml) as described in materials and methods.
Data are expressed as % of cell count in each phase of cell cycle induced by each
*
compound. ( p <0.05 vs control).

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K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7127–7131
fluorescence in cells treated with test compounds revealed that
compound Bc-5, Bc-8 and Bc-9 treatment in MCF-7 cells resulted
in significant increase in percentage of apoptotic cell (Fig. 4). Out
of the three compounds, Bc-5 induced significantly higher apopto-
tic changes in treated cells.
Benzocoumarins enhanced Annexin-V expression. MCF-7 cells
were exposed for 24 hours to 10 lM concentration of each com-
pounds and analyzed by flow cytometry using Annexin-V-FITC
and PI. Data showed that all three compounds Bc-5, Bc-8 and Bc-
9 significantly induced percentage of Annexin-V-FITC positive
and PI negative cells compared with untreated controls indicating
increase in the proportion of cells undergoing early stages of apop-
tosis (Fig. 5). Significantly higher Annexin-V positivity was ob-
served with Bc-5 treatment under similar condition.
Benzocoumarins induced apoptosis via caspase activation. Homo-
geneous caspase activity was increased in MCF-7 cells by all three
compounds Bc-5, Bc-8 and Bc-9. The caspase activity upon treat-
ment of compound was Bc-5, Bc-8 and Bc-9 significantly high ver-
sus untreated control indicating that all these compounds
activated of general caspases (Fig. 6).
Figure 5. Quantitative determination of apoptosis by flow cytometry: MCF-7 cells
(5 Â 105 cells) with 70–80% confluence were incubated in DMEM supplemented
with 10% FBS without (control) or with 10 lM of benzocoumarin derivatives. After
24 h, the cells were harvested, stained with Annexin-V-FITC and PI, and analyzed by
flow cytometry. Data are expressed as % of apoptotic (Annexin V-FITC-positive and
*
PI-negative) and necrotic (PI-positive) cells. ( p <0.05 vs control).
Benzocoumarins do not affect viability of osteoblasts. Effect of ben-
zocoumarins on the viability of osteoblasts was studied at 10 and
20 lM concentrations. None of these compounds showed loss of
cell viability of primary osteoblasts (Fig. 7). Instead, these three
compounds exhibited increase in osteoblast numbers at 10 and
20 lM concentration (Fig. 7). Our choice of osteoblast was not
without reason as the onset of metastatic breast cancer often coin-
cides with bone loss disorders. Our data showed that whereas all
three benzocoumarins had no effect on the viability of osteoblasts,
all could actually increase osteoblast number compared to un-
treated cells indicating the possibility in their involvement in
osteoblast proliferation. Recently, furanocoumarin, a class of cou-
marin derivative, was found to promote bone formation by stimu-
lating differentiation and mineralization of osteoblasts.29 It is
possible that these derivatives, particularly Bc-9, beside its cell
growth promoting action, could have similar osteogenic effect
which is beyond the scope of this study.
Figure 6. Benzocoumarin derivatives activate homogeneous caspase in MCF-7
cells: 1 Â 104 cells/well with 70–80% confluence were grown 16 h, treated with
10 lM each compounds for 16 h. Flurometric determination of general caspase was
In conclusion, in this study, among the synthesized
benzocoumarins, Bc-5, Bc-8 and Bc-9 were identified to be very
done as described in materials and methods with an excitation filter 465 nm and
*
emission filter 520 nm. ( p <0.05 vs negative control).
Figure 4. Benzocoumarin derivatives induce apoptosis of MCF-7 cells: (a) MCF-7 cells of 70–80% confluence were treated with 10 lM benzocoumarin derivatives for 18 h in
complete growth medium. Representative photomicrographs from Bc-5 treated cells after Hoechst 33342 staining were acquired with Â40 objective. Arrows indicate
cytoplasmic condensation, apoptotic body and nuclear fragmentation. Tamoxifen was used as a positive control for apoptosis in MCF-7. (b) Quantitation of data for % of
*
apoptotic nuclei based on the shape and intensity of Hoechst 33342 staining in cells normalized with control data. ( p <0.05 vs control).

K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7127–7131
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results.
Figure 7. Effect of benzocoumarin derivatives in the proliferation of osteoblasts:
Osteoblasts were cultured as described in the materials and methods. 3 Â 103 cells/
well were treated with 10 and 20
l
M of each compound. MTT assay was performed.
*
(n = 9, p >0.05 vs control).
promising candidates, their excellent antiproliferative potency, to-
gether with their remarkable apoptosis-inducing activity, make
them leads of great interest for further studies. Also, since these
derivatives are highly fluorescent, the utility of these compounds
to act as novel angiogenesis inhibitors with high selectivity over
tumor cells is under investigation as it is potentially possible to in-
hibit and detect tumor angiogenesis simultaneously.
Acknowledgments
The authors are grateful to the Director, CDRI, Lucknow, India
for constant encouragement in drug development program, S.P.
Singh for technical support, SAIF for NMR, IR, and Mass spectral
data. Rosaiah and Manoj thank UGC and CSIR, New Delhi, India
for financial support. This is CDRI communication number 7438.
26. Synthetic procedure for compounds: (a) Bc-3: To a mixture of Bc-2 (2 mmol)
and tert-butylamine (4 mmol), absolute ethanol (10 ml) was added at room
temperature. After completion of the reaction solvent was evaporated and the
residue was washed with hexane to afford Bc-3 in good yield. (b) Bc-11: A
solution of Bc-2 (1 g, 3.54 mmol) and acetophenone (0.390 g, 3.54 mmol) in
dioxane was treated with conc. HCl (5 mL) and refluxed for 5 h. Most of the
excess solvent was evaporated under reduced pressure and the residue was
neutralized with aq NaHCO3 solution. To this residue, water (50 mL) was added
and extracted 3 fold with 25 mL of CHCl3. The combined organic layer was
dried over anhydrous Na2SO4 and evaporated, which was chromatographed
over silica gel to afford compound Bc-11. (c) Bc-16: A solution of Bc-11 (0.2 g,
0.52 mmol) and butyl amine (0.057 g, 0.78 mmol) in ethanol (10 mL) was
stirred for 10 min at room temperature. After completion of the reaction
solvent was evaporated and the residue was washed with hexane to afford Bc-
16.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.09.040.
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