Synthesis and docking studies on styryl chromones exhibiting cytotoxicity in human breast cancer cell line

Article abstract of DOI:10.1016/j.bmcl.2010.05.108

The search for small molecules that preferentially target the functionally important surfaces of estrogen receptor and disrupt the transcriptional activity in the cell has emerged as a promising area towards rationale based drug design. Herein, we report substituted styryl chromones as a new class of compounds that exhibit selectivity for ERβ binding at the second binding site of HT and antiproliferative activity in human breast cancer cell line.

Full text of DOI:10.1016/j.bmcl.2010.05.108

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4945–4950
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and docking studies on styryl chromones exhibiting cytotoxicity
in human breast cancer cell line
b
Seema Bhatnagar a, , Shakti Sahi , Puneet Kackar c,d, Swati Kaushik a, Manan K. Dave a,
*
Akshara Shukla a, Ashita Goel a
a Chemical Synthesis Group, Amity Institute of Biotechnology, Amity University Sector 125, Noida, India
b School of Biotechnology, Gautam Buddha University, Greater Noida 201 308, India
c Bioinformatics Group, Amity Institute of Biotechnology, Amity University Sector 125, Noida, India
d Unit of Drug Discovery and Development Italian Institute of Technology Central Research Labs—Genoa Headquarter, Via Morego, 30, 16163 Genoa, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The search for small molecules that preferentially target the functionally important surfaces of estrogen
receptor and disrupt the transcriptional activity in the cell has emerged as a promising area towards
rationale based drug design. Herein, we report substituted styryl chromones as a new class of compounds
that exhibit selectivity for ERb binding at the second binding site of HT and antiproliferative activity in
human breast cancer cell line.
Received 9 April 2010
Revised 27 May 2010
Accepted 31 May 2010
Available online 10 June 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Estrogen receptor
Styryl chromones
Estrogen receptor (ER) is a member of the nuclear hormone
receptor superfamily and functions as ligand-activated transcrip-
tion factor to regulate genetic networks controlling cell growth
and differentiation, inflammatory responses, and metabolism.
The ligand binding domain (LBD) of ER is the primary target for nu-
clear receptor (NR) drug discovery. Traditional approaches com-
prise of type I and type II antiestrogens which were designed to
modulate NR activity based on competitive binding to the cognate
ligand-binding domain. The recent discovery of the second binding
site for tamoxifen in the ER1 has provided additional avenues
worth exploring towards better understanding of mode of action
of type I antiestrogens and targeted drug discovery. The ability of
small molecules to modulate nuclear receptor-dependent gene
expression has made NR super family a favoured target for drug
discovery. Targeting the functionally important surfaces of the
receptor which disrupt transcriptional activity in the cell is emerg-
ing as a promising area towards rationale based drug design. In this
context several heterocyclic ring systems such as triazenes, pyrim-
idines, trithianes, and cyclohexanes have recently been explored
which have been reported to act as coactivator binding inhibitors
and quercetin which also share the benzopyran scaffold have been
reported to possess anti cancer properties and behave in a biphasic
manner at different concentrations.4
The above facts prompted us to evaluate the estrogenicity of
styryl chromones that are a rare class of oxygen heterocycles that
have been reported to possess a wide spectrum of biological activ-
ity. The natural and synthetic analogs of styryl chromones have
been reported to exhibit antineoplastic, antiallergic, antihepato-
protective, and estrogenic activity.4 Herein, we report substituted
styryl chromones as a new class of compounds that exhibit selec-
tivity for ERb binding along with antiproliferative and cytotoxic
activity on human breast cancer cell line.
Chromones can be considered as substituted flavones that be-
have as masked 1,3-dicarbonyl systems.5 Although chromones
are known to be Michael acceptors towards nucleophiles; the nat-
ure of the nucleophile attacking the pyrone ring determines
whether attack will be at the carbonyl carbon or at the b carbon
of the enone system. Also, the susceptibility of Ca@Cb bond of
styryl chromones to the attack by electron donor6 and acceptor
systems6 posed a formidable challenge in our goal towards regio-
selective functionalization of C-3 and C-4 of the chromone ring.
The synthesis of selected derivatives of styryl chromones was done
according to Scheme 1.
(CBI) in ERa 2
. Among the heterocyclic ring systems studied; the
pyrimidine-based CBIs appear to be the first small molecule inhib-
itors of NR coactivator binding. Among oxygen heterocycles there
are several reports implicating the benzopyran scaffold as selective
estrogen receptor (ER) b agonists.3 Flavonoids such as genestien
E-3-Hydroxy-2-styryl chromone 1(a–c) on reaction with mal-
ononitrile led to a mixture of products (Scheme 1).
The possible explanation could be the susceptibility Ca@Cb
bond of styryl chromones to the attack of electron donor systems;
* Corresponding author. Tel.: +91 1204392145.
as well as the electrophilicity of the carbonyl carbon due to alpha
E-mail address: sbhatnagar@aib.amity.edu (S. Bhatnagar).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.108

4946
S. Bhatnagar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4945–4950
R
R
R
O
Ac2O,Pyridine
O
O
+
(Reflux)
OCOCH3
OCOCH3
OH
O
O
O
1(a-c)
2(a-c)
3(a-c)
(CH2(CN)2,
ET3N, EtOH
(CH2(CN)2,
ET3N, EtOH
R
R
O
C
O
Mixture of Products
+
OCOCH3
OCOCH3
CN
C
CN CN
CN
R= a) C6H5
b)2'OCH3(C6H4)
c) CH3
4(a-c)
5(a-c)
Scheme 1.
Table 1
Conformational energy of E,Z isomers of compounds
Compound
Conformation
Conformational energy
of the (Kcal/mol)
O
O
R2
R1
Ia
1b
Ic
E
E
E
Z
Z
Z
E
E
E
Z
Z
Z
E
E
E
Z
E
20.161
22.931
12.439
22.4210
23.688
17.90
22.959
25.820
15.302
31.3308
34.322
29.741
32.357
36.078
25.859
17.205
19.850
O
I
O
II
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
7a
R1=OAc
R2=Br
Figure 1.
of compounds 2(a–c). Compound 2b was chosen as the representa-
tive compound for this purpose. Compound 2b can exist in two
possible conformations I and II depicted in Figure 1.
It was observed that H-8 in compound 2b appeared up field at d
6.60. Previous reports where compounds of similar stereochemis-
try were studied suggested the presence of shielding environment
in the vicinity of H-8 proton by the phenyl of styryl group that led
to the up field shifting of H-8 proton.7 A comparison of the chem-
ical shifts obtained for H-8 in case of compound 6a where Z isomer
existed in conformation II (Fig. 1) further validated our results as in
this case the H-8 proton appeared downfield at d 7.85 (in press).
Therefore, the proposed stereochemistry of the compounds
2(a–c) was proposed as I. The coupling constant value obtained
hydroxyl group which might be leading to multiple products.
Therefore, as an alternative strategy compounds 1(a–c) were first
reacted with acetic anhydride and pyridine. A racemic mixture of
E, Z 3-acetoxy-2-styryl chromones was obtained in which the Z iso-
mers 2(a–c) were formed in minor amounts while E isomers 3(a–c)
predominated. Detailed spectroscopic and conformational energy
studies (Table 1) were undertaken to assign the stereochemistry
for H
a and Hb protons in compounds 2(a–c) was 12 Hz which indi-
cated Z vinylic protons. COSY experiments depicted cross peaks
CH2Cl2,NBS
O
R
R
Br
R
O
6a
O
OH
CH3OH,NBS
O
O
O
1a
Br
7a
R= a) C6H5
Scheme 2.

S. Bhatnagar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4945–4950
4947
Table 2
Table 2 (continued)
Binding energy of the complexes
Compound
Structure
Docking score
Glide energy
Compound
Structure
Docking score
Glide energy
of the model
(Kcal/mol)
of the model
(Kcal/mol)
CH3
O
5c
6a
ꢀ3.307
ꢀ3.768
ꢀ18.610
ꢀ26.325
O
Ia
ꢀ4.434
ꢀ28.239
OCOCH3
C
OH
CN
CN
O
O
CH3O
Br
Br
O
1b
ꢀ4.431
ꢀ4.264
ꢀ3.881
ꢀ31.924
ꢀ24.449
ꢀ30.842
O
OH
O
7a
ꢀ4.523
ꢀ39.391
O
O
CH3
O
Ic
OH
O
between Ha and Hb protons at d 7.43 and 7.45 ppm; 7.26 (H-7) and
6.60 (H-8); 7.40 (Hb) and 7.20 (H-60); 6.89 (H-6) and 7.26 (H-7).
Another interesting observation made was that in case of com-
pounds 3a and 3b the Hb proton was remarkably shielded and ap-
peared upfield at d 5.83 ppm probably due to the shielding effect of
the phenyl ring of the styryl group. Such an effect was absent in
case of compound 3c and the Hb proton appeared downfield at
6.93 ppm. Compounds 2 and 3(a–c) on reaction with malononitrile
stereoselectivly furnished 4 and 5(a–c) which were again a mix-
ture of E, Z isomers and the E isomer predominated as the product.
The Z isomer was identified on the basis of the upfield shift in the
position of the H-8 proton as well as COSY experiments. The signif-
icant correlations obtained in case of compound 4b were between
3a
O
O
OCOCH3
CH3O
3b
3c
5a
ꢀ4.124
ꢀ4.801
ꢀ3.686
ꢀ36.469
ꢀ30.663
ꢀ33.427
O
OCOCH3
O
O
O
CH3
OCOCH3
O
C
OCOCH3
CN
CN
CH3O
5b
O
C
ꢀ4.172
ꢀ33.956
OCOCH3
CN
Figure 2. Three dimensional structure of ERb docked with compound 5a. Also
shown here is HT complexed with the receptor. The compound 5a does not bind to
the cognate binding site but preferentially binds in the coactivator binding groove.
CN

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S. Bhatnagar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4945–4950
Figure 3. Interaction of compound 5a with receptor using Ligplot.
d 7.70 (H
a
) and 7.15 (Hb); 7.15 (Hb) and 7.38 (H-60); 7.00 (H-7)
is a complex of ER
defining the grid as well as blind docking of HT with ER
the same results as reported earlier. Docking of compounds was
also done with ER and the results not only validated our docking
a
with HT, was also taken. Docking studies by
and 6.50 (H-80). In case of the E isomer 5b a significant downfield
shift in the position of H-8, H-b were observed. H-8 appeared
downfield at d 7.90. COSY experiments on compound 5a reveal cor-
a
gave
a
relation peaks between 7.72 (H
a
) and 7.63(Hb); 7.90 (H-8) and
protocol but also established the following findings.
7.58 (H-7); 7.90(H-8) and 6.98 (H-6). Compounds 6a and 7a were
prepared by reaction of 1a with NBS (Scheme 2). The stereochem-
istry of compounds 6a and 7a were assigned on the basis spectro-
scopic techniques and energy calculations (in press).
The docking studies showed that the compounds 1–5(a–c) were
binding to the second binding site of HT in the co activator groove.
The docking scores and energies of the models have been shown in
(Table 2). Amongst the analogs synthesized significant interactions
were seen in few cases. Foremost amongst them was compound
5a. The comparison of compound 5a docked with ERb shows that
it has a binding site similar to HT in the coactivator groove
(Fig. 2). Majority of the poses share the following common resi-
dues: Trp335, Leu331, Glu332, and Leu306 as compared with HT.
Whereas Trp335 of ERb was involved in the interaction with the
phenyl ring and the ethyl group in case of HT; in our case we see
interactions with the carbonyl oxygen of the acetate side chain in
compound 5a.
Preliminary evaluation of the estrogenicity of the synthesized
compounds was carried out by docking studies. The compounds
were modeled using BUILD application of Maestro 8.0. The molec-
ular modeling studies were performed on Schrodinger Maestro8
and the binding affinities of the synthesized compounds with the
receptor were compared using ligplot. The pdb entries chosen for
the above studies were 2QTU3 and 2FSZ.1 The selection of 2QTU en-
try was based on structural similarity of the synthesized ligands
with benzopyranones. Due to the absence of native ERb pdb entry:
2FSZ was selected which is a complex of hydroxytamoxifen with
ERb. This pdb entry was selected as a dataset in order to compare
the binding of synthesized molecules with that of hydroxytamox-
ifen (HT) which is a well established ER agonist /antagonist. Fur-
ther, to validate our docking protocol the pdb entry 3ERT9 which
Also in case of compound 5a the Van der Waals interactions
were seen with Leu331, Leu306, and Ile 310 were with the phenyl
ring of the styryl group as compared to Van der Waals interaction
of Glu332 with phenolic ring of HT. Few other poses were also ob-
tained but they did not have any significant interactions.

S. Bhatnagar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4945–4950
4949
Figure 4. Interaction of compound 3b with receptor using Ligplot.
Compound 5a also depicted specific non-bonded interactions
with the following residues; Val307, Leu306, Ile310, Leu331,
Gln327, and Val328 (Fig. 3). The glide energy for the docked com-
plex was ꢀ33.427 K cal/mol. In case of compound 5b specific inter-
actions were seen with the following common residues as
compared to HT Val328, Ile310, and Leu331. On the other hand
compound 5c did not show any specific interaction either with
the LBD or the coactivator groove. There were few conformations
which showed interactions with Lys401, Tyr397, and His279. Pre-
vious studies based on structural prerequisites required for the
binding in ERb indicate that presence of phenyl group facilitated
binding in the receptor which may probably be the reason why
compounds 5c and 1c did not exhibit any significant interactions.
The other analog exhibiting specific interaction in the receptor
was 3a which had specific interactions with Trp335, Leu331,
Glu332, and Leu306. Few conformations were also seen to have
interactions with His 279. Similarly 3b had specific interactions
with Trp335, Leu331, and Glu332 (Fig. 4). Compound 3c had spe-
cific interaction with Trp335.
Compound 1a had specific interactions with Trp335 and com-
pound 1b showed specific interaction with Trp335, Ile310, and
Leu306. However, Compound 1c did not show any specific interac-
tion with the residues although it occupies the second coactivator
groove. In case of compound 6a the only interactions seen were
with Tyr397. Compound 7a showed interactions with Trp345. This
Figure 5. Three dimensional structure of ERb docked with compound 5a using
Maestro. Beta sheet is shown in blue color and the compound is shown as ball and
stick model.
Figure 6. Comparative study between control and compound 5a at different
concentrations using MTT assay.

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S. Bhatnagar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4945–4950
Figure 7. %Cytotoxity of compound 5a at different concentrations.
could be probably because of a bulky substituent at C-3 due to
which compounds 6a and 7a did not exhibit binding in the coacti-
vator groove. Based on the above results it was hypothesized that
phenyl ring at position C-2 was essential for binding to the active
site. Substitution/removal of the phenyl ring by alkyl group in
the side chain resulted in weaker binding affinity and no significant
interactions with the receptor were observed. Although the inter-
action energy of compound 7a was lowest (ꢀ39.39 Kcal/mol) but
it neither exhibited binding to the second binding site of HT nor
the ligand binding site. Substitution of hydroxy group at C-3 in
compounds 1(a–c) by a bulky substituent like a bromo group in-
stead of an acetate group hindered the ligand from binding to
the second binding site. Interaction energies and interactions be-
tween the ligand and the receptor residues clearly indicated that
none of the compounds studied had affinity for the ligand binding
domain of ERb (Fig. 5), however, the above data suggests binding
affinity to the second binding site of HT.
Recent literature reports indicate that the interaction of HT at
second binding site is of considerably low affinity and ligand bind-
ing at this site is responsible for antagonist activity.10 Accordingly,
if styryl chromones exhibited binding at this site they may be ex-
pected to exhibit antagonistic activity. Cytotoxicity studies were
therefore carried out using MTT assay protocol on candidate com-
pound 5a. The MTT assay revealed that compound 5a was cyto-
toxic and antiproliferative on MCF-7 cell line (Fig. 6) in a dose
dependent manner (Fig. 7), which was in accordance with antago-
nistic role of second binding site of HT.
study. The authors thank Research Associate S.K., and students
M.K.D., A.S., and A.G. who have equally contributed in the synthesis
and purification of compounds. The invaluable contribution of Dr.
V. Pooja and Ms. Sonali Kumari for the cytotoxicity studies is grate-
fully acknowledged. The assistance provided by Indian Institute of
Technology, New Delhi; Central Drug Research Institute, Luck now
for analytical analysis of the samples are gratefully acknowledged.
All authors gratefully acknowledge infrastructure facilities pro-
vided by Amity Institute of Biotechnology, Amity University, Noida.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.05.108.
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Acknowledgements
The author S.B. gratefully acknowledges the financial assistance
provided by the Department of Science and Technology for the