Novel synthesised flavone derivatives provide significant insight into the structural features required for enhanced anti-proliferative activity

Article abstract of DOI:10.1039/c6ra11041j

With many cancers showing resistance to current chemotherapies, the search for novel anti-cancer agents is attracting considerable attention. Natural flavonoids have been identified as useful leads in such programmes. However, since an in-depth understanding of the structural requirements for optimum activity is generally lacking, further research is required before the full potential of flavonoids as anti-proliferative agents can be realised. Herein a broad library of 76 methoxy and hydroxy flavones, and their 4-thio analogues, was constructed and their structure-activity relationships for anti-proliferative activity against the breast cancer cell lines MCF-7 (ER +ve), MCF-7/DX (ER +ve, anthracycline resistant) and MDA-MB-231 (ER -ve) were probed. Within this library, 42 compounds were novel, and all compounds were afforded in good yields and >95% purity. The most promising lead compounds, specifically the novel hydroxy 4-thioflavones 15f and 16f, were further evaluated for their anti-proliferative activities against a broader range of cancer cell lines by the National Cancer Institute (NCI), USA and displayed significant growth inhibition profiles (e.g. compound-15f: MCF-7 (GI₅₀ = 0.18 μM), T-47D (GI₅₀ = 0.03 μM) and MDA-MB-468 (GI₅₀ = 0.47 μM) and compound-16f: MCF-7 (GI₅₀ = 1.46 μM), T-47D (GI₅₀ = 1.27 μM) and MDA-MB-231 (GI₅₀ = 1.81 μM)). Overall, 15f and 16f exhibited 7-46 fold greater anti-proliferative potency than the natural flavone chrysin (2d). A systematic structure-activity relationship study against the breast cancer cell lines highlighted that free hydroxyl groups and the B-ring phenyl groups were essential for enhanced anti-proliferative activities. Substitution of the 4-CO functionality with a 4-CS functionality, and incorporation of electron withdrawing groups at C-4′ of the B-ring phenyl, also enhanced activity. Molecular docking and mechanistic studies suggest that the anti-proliferative effects of flavones 15f and 16f are mediated via ER-independent cleavage of PARP and downregulation of GSK-3β for MCF-7 and MCF-7/DX cell lines. For the MDA-MB-231 cell line, restoration of the wild-type p53 DNA binding activity of mutant p53 tumour suppressor gene was indicated.

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Novel synthesised flavone derivatives provide significant
insight into the structural features required for enhanced
anti-proliferative activity
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Divyashree Ravishankara, Kimberly A. Watsonb, Francesca Grecoa†, Helen M. I.
Osborna†
www.rsc.org/
With many cancers showing resistance to current chemotherapies, the search for novel anti-cancer agents is attracting
considerable attention. Natural flavonoids have been identified as useful leads in such programmes. However, since an
in-depth understanding of the structural requirements for optimum activity is generally lacking, further research is
required before the full potential of flavonoids as anti-proliferative agents can be realised. Herein a broad library of 76
methoxy and hydroxy flavones, and their 4-thio analogues, was constructed and their structure-activity relationships for
anti-proliferative activity against the breast cancer cell lines MCF-7 (ER+ve), MCF-7/DX (ER+ve, anthracycline resistant)
and MDA-MB-231 (ER-ve) were probed. Within this library, 42 compounds were novel, and all compounds were
afforded in good yields and > 95% purity. The most promising lead compounds, specifically the novel hydroxy 4-
thioflavones 15f and 16f, were further evaluated for their anti-proliferative activities against a broader range of cancer
cell lines by the National Cancer Institute (NCI), USA and displayed significant growth inhibition profiles (e.g Compound-
15f: MCF-7 (GI50 = 0.18 µM), T-47D (GI50 = 0.03 µM) and MDA-MB-468 (GI50 = 0.47 µM) and compound-16f: MCF-7 (GI50
=
1.46 µM), T-47D (GI50 = 1.27 µM) and MDA-MB-231 (GI50 = 1.81 µM) . Overall, 15f and 16f exhibited 7-46 fold greater
anti-proliferative potency than the natural flavone chrysin (2d). A systematic structure-activity relationship study
against the breast cancer cell lines highlighted that free hydroxyl groups and the B-ring phenyl groups were essential for
enhanced anti-proliferative activities. Substitution of the 4-C=O functionality with
a 4-C=S functionality, and
incorporation of electron withdrawing groups at C4’ of the B-ring phenyl, also enhanced activity. Molecular docking and
mechanistic studies suggest that the anti-proliferative effects of flavones 15f and 16f are mediated via ER-independent
cleavage of PARP and downregulation of GSK-3β for MCF-7 and MCF-7/DX cell lines. For the MDA-MB-231 cell line,
restoration of the wild-type p53 DNA binding activity of mutant p53 tumour suppressor gene was indicated.
areas1. For example, analysis of plant-based
compounds has highlighted many potential clinical
Introduction
Natural products and their derivatives provide a applications for flavonoids2. Within the area of
flavonoids as anticancer agents3–11, the success of such
significant number of new chemical leads for drug
approaches has been exemplified through the
development of flavone derivatives such as flavopiridol
(for chronic lymphocytic leukemia)12,13,14, silibinin (for
prostate cancer)15, and quercetin16 and its derivative
QC1217 (for ovarian cancer), which are at various stages
of clinical application. Although the natural flavonoids
demonstrate anti-proliferative activities and can
themselves act as useful leads, these activities have
often been reported at pharmacologically non-
achievable high micromolar concentrations (50-100
µM)18–21. Therefore, medicinal chemistry strategies are
essential to optimise the pharmacokinetic profiles of
discovery programmes within a wide range of clinical
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these compounds, in order to ensure that the full of i) dihydroxy flavones with hydroxyl substitution on
clinical applications for flavonoids are realised. In this ring-A at the C-7,8 or C-5,7 positions, ii) tetrahydroxy
study, we have proposed methylation of hydroxyls, and flavones with dihydroxyl substitution on ring-A (at the
conversion of 4-C=O to 4-C=S, as a promising strategy C-7,8 or C-5,7 positions) and on ring-B (at the C-3’and
to improve the activities of flavonoids, since these C-4’ positions), and iii) pentahydroxy flavones with
structural motifs could lead to flavonoids with desirable dihydroxyl substitution on ring-A (at the C-5,7 position
physiochemical properties such as increased only) and on ring-B (at the C-3’and C-4’ positions) as
lipophilicity and metabolic stability. Therefore, the aim well as a hydroxyl group at the C-3 position on ring-C
of this study was to study the effects of methylation of were designed and synthesised.
The analogous
free hydroxyls and 4-C=S substitution on the anti- methoxy and 4-thio derivatives were also prepared
proliferative properties of the flavonoids by (compounds-1c-f to 5c-f, Series-1, Figure-1).
undertaking the systematic design, synthesis and
The pentahydroxy flavone (quercetin) derivatives (5c-f)
biological analysis of a well-defined library of hydroxy
were obtained starting from pentahydroxy flavone
flavone analogues. In addition, synthesis of this library
(purchased) according to Scheme-327,28 in 36% overall
of compounds would also allow structure-activity
yield and with 99% purity by HPLC. This represents an
relationships (SARs) to be determined. This is of
improvement on the recently reported synthesis of 5f,
both in terms of overall yield and purity29 (22% overall
particular importance for flavonoids as in-depth
understanding of the SARs for anticancer properties of
yield and 95% purity). The panel of flavones studied
flavones is still limited22,23. Specifically, the significance
herein also contained the well-known natural flavones
of some of their core structural features for activity,
chrysin (2d), luteolin (4d) and quercetin (5d) to allow
such as hydroxyl substitutions at different positions on
comparisons to be made between the novel analogues
the flavonoid rings, and the effect of methylation of the
prepared in our laboratory and some standard
free hydroxyls on activity, are poorly defined. Also, the
flavonoids. The design of further series of flavones was
impact of modifying the 4-C=O substituent on the
guided by the anti-proliferative activities of Series-1
anticancer activities of flavones has not been
compounds. Therefore, compound-2f, a dihydroxy 4-
extensively studied.
thio flavone that was identified to possess promising
anti-proliferative activity in in vitro screening (discussed
Results and discussion
below), was considered as a candidate for further
Design and Synthesis
optimisation through the bioisostere approach and
Topliss scheme30. This resulted in the synthesis of a
To realise the aims of this study, structurally related
hydroxy and methoxy flavones, and their 4-thio
focused set of dihydroxy flavone analogues (bearing C-
derivatives, were synthesised. The synthesis of the
7,8 and C-5,7 hydroxyls on ring-A) with phenyl
methoxy and hydroxy flavone scaffolds was achieved
bioisosteres (Series-2, Figure-1), and phenyl groups
using
a synthetic strategy involving the Baker-
with electron-withdrawing substituents at the C-4’
position (based on the Topliss scheme for the
optimisation of phenyl ring substitution in drug
design30) (Series-3, Figure-1) as ring-B. The Series-2
bioisosteric analogues of 2f comprised of dihydroxy and
dimethoxy flavones, as well as their 4-thioflavone
derivatives, with a five-membered 2-thienyl and 2-
furanyl group, and a six-membered 3-pyridyl group
[compounds 6c-f to 11c-f]. Series-3 involved 7,8 and 5,7
dihydroxy, dimethoxy flavones, and their 4-thio flavone
Venkataraman rearrangement as a key step (Scheme-
1).24,25 The 4-thio analogues were obtained according to
Scheme-226 in 20-30% overall yield (see experimental
section in supporting information material for full
details).
First, the effects of methylation and 4-thio
modifications on flavones with different numbers of
hydroxyl groups and different positions of hydroxyls on
ring-A, were investigated. Thus, 20 flavones comprising
2 | J. Name., 2012, 00, 1-3
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analogues with electron-withdrawing groups (EWGs) Overall, multiple series of flavone derivatives
such as fluoro (-F), chloro (-Cl), bromo (-Br) and cyano (- comprising of hydroxy flavones (free -OH and 4-C=O),
CN) at the C-4’ position of the phenyl ring (ring-B) hydroxy 4-thioflavones (free -OH and 4-C=S), methoxy
[compounds 12c-f to 19c-f]. It is noteworthy that for flavones (-OMe and 4-C=O) and methoxy 4-thioflavones
the synthesis of novel flavones containing nitrogen (-OMe and 4-C=S) with different B-ring substitutions
atoms (eg the 3-pyridyl and 4-cyano phenyl groups), were successfully synthesised (summarised in Figure-
during the cyclisation step (step iii-a) in the presence of 1). Out of 76 compounds, 42 compounds (3e
concentrated sulfuric acid in glacial acetic acid, the 1,3- 7c-f 8c-f 9c 9e 9f 10e 10f 11e 11f 12e
diketone intermediates (compound 10b 11b and 19b 13f 14c-f 15e 15f 16c-f 17e 17f 18c 18e
,
3f
12f
18f
,
4f
,
6d-
f
,
,
,
,
,
,
,
,
,
,
,
,
13e
,
,
,
)
,
,
,
,
,
,
,
,
,
,
19c
were found to undergo hydrolysis instead of cyclisation 19e and 19f) were prepared and characterised for the
and dehydration, to yield the corresponding methoxy first time. All of the synthesised compounds were
1
flavones. Therefore, the cyclisation of such 1,3- characterised by H and 13C NMR and IR spectroscopy,
diketones was instead accomplished in high yields (70- and high-resolution mass spectrometry. The purity of
80%) by the addition of concentrated sulfuric acid into these compounds was established by reverse phase
the solution of the 1,3-diketone in CHCl3 at 0 °C 31 (step HPLC and the purities were found to be >95% in all
iii-b).
cases.
Scheme 1- Synthesis of methoxy flavones and hydroxy
flavones (1c-d to 19c-d)
Scheme 3- Synthesis of methoxy and 4-thiocarbonyl
analogues
of
pentahydroxy
flavone
(5c-f)
(i) Dimethyl sulfate, 15% KOH, room temperature to 90 °C,
overnight (ii) Dry toluene, Lawesson’s reagent, 110 °C, 4 h (iii) Dry
DCM, BBr3, 40 °C, overnight.
(i) Pyridine, 75 °C, 1 h; (ii) Pyridine, KOH, 50-75 °C, 2 h; (iii) a-Glacial
acetic acid, 1% H2SO4, 90-110 °C, 1 h, b- CHCl3, 0 °C to room
temperature, 30 min (iv) Dry DCM, BBr3, room temperature/40 °C, 4
h/overnight.
Scheme 2- Synthesis of 4-thiocarbonyl analogues of
the hydroxy and methoxy flavones (1e-f to 19e-f)
Figure-1. Library of the synthesised flavone derivatives. Novel
compounds are highlighted in bold.
Anti-proliferative evaluation
As flavonoids have been reported to show activity
against breast cancer cell lines32–34
, all of the
(i) Dry toluene, Lawesson’s reagent, 110 °C, 4 h (ii) Dry DCM, BBr3,
room temperature/40 °C, 4 h/overnight.
synthesised compounds (1c-f to 19c-f) were evaluated
for their anti-proliferative activity against the estrogen-
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responsive breast cancer cell lines MCF-7, and the derivatives, and in particular the hydroxy compounds,
clinically relevant anthracycline-resistant MCF-7/DX exhibited greater anti-proliferative activity than the
(which mimics the multi-drug resistant scenario in methoxy and hydroxy flavone analogues. However, no
patients), as well as the estrogen-independent, triple derivatives reached the IC50 at 10 µM (See supporting
negative breast cancer cell line MDA-MB-231, using the information, Figure-S1).
MTT assay. The initial series of compounds (Series 1,
As compound-2f was identified as a promising lead
i.e. 1c-f to 5c-f) was first evaluated against MCF-7 and
compound, further novel analogues of compound-2f
MCF-7/DX cell lines to investigate the effects of
were prepared to discover more active flavone
methylation and 4-thio substitution on flavones with
derivatives. Thus a series of bioisosteric analogues of
differing numbers of hydroxyl groups as well as with
the B-ring phenyl group of compound-2f (Series-2,
different positions on the A-ring. The IC50 values (half
compounds 6c-f to 11c-f), and a series of compound-2f
maximal inhibitory concentration) of these compounds
analogues with electron-withdrawing substituents at
were determined as a measure of their respective
the C4’position (Series-3, compounds 12c-f to 19c-f),
cytotoxicity and these values are tabulated in Table-1.
along with their methoxy (both 4-C=O and 4-C=S) and
For this series, compound-2f with the dihydroxy groups
their 7,8-
at the C-5,7 positions on ring-A, and 4-thio substitution,
showed the highest anti-proliferative potency with IC50
Table 1. IC50 values of the compounds from Series 1
values of 7.1 ± 0.51 µM and 34.93 ± 5.75 µM against
(
1c-f to 5c-f) against the MCF-7 and MCF-7/DX cell lines
MCF-7 and MCF-7/DX cell lines, respectively.
Compounds 1f 3e 3f 4d and 5d showed moderate
Chemical structure
Compound
Substitution
R1,2= OMe, X= O
R1,2= OH, X= O
R1,2= OMe, X= S
R1,2= OH, X= S
IC50 in MCFꢀ7
35.2 ± 4.4 µM
38.0 ± 0.6 µM
34.7 ± 1.7 µM
14.7 ± 1.0 µM
IC50 in MCFꢀ7/DX
77.0 ± 4.2 µM
86.7 ± 0.9 µM
90.3 ± 0.8 µM
47.2 ± 1.2 µM
,
,
,
1c
1d
1e
1f
cytotoxicity with IC50 values of 25.6 ± 1.26 µM, 23.5 ±
4.27 µM and 11.8 ± 1.79 µM, respectively however,
these compounds were weakly anti-proliferative
against the MCF-7/DX cell line with IC50 values of 176.3
± 1.61 µM, >250 µM and >250 µM, respectively.
2c
2d
2e
2f
R1,2= OMe, X= O
R1,2= OH, X= O
R1,2= OMe, X= S
R1,2= OH, X= S
35.2 ± 4.4 µM
25.6 ± 1.2 µM
23.5 ± 4.2 µM
7.9 ± 0.2 µM
>250 µM
176.3 ± 1.6 µM
>250 µM
34.9 ± 5.7 µM
3c
3d
3e
R1,2,3,4= OMe, X= O
R1,2,3,4= OH, X= O
R1,2,3,4= OMe, X= S
>250 µM
>250 µM
183.9 ± 9.0 µM
>250 µM
97.5 ± 6.7 µM
11.8 ± 1.7 µM
Compounds 1c, 1d, 1e, 9c, 3c, 3d, 4c, 4e, 5c, 5e and 5f
3f
R1,2,3,4= OH, X= S
16.9 ± 1.2 µM
66.8 ± 0.7 µM
4c
4d
4e
R1,2,3,4= OMe, X= O
R1,2,3,4= OH, X= O
R1,2,3,4= OMe, X= S
>250 µM
21.6 ± 0.8 µM
>250 µM
>250 µM
81.1 ± 1.7 µM
>250 µM
were found to be less active against both MCF-7 and
MCF-7/DX cell lines with IC50 values >30 µM and >85
µM, respectively. The IC50 values for the well-known
natural flavones in this series, 2d (chrysin), 4d (luteolin)
and 5d (quercetin) were found to be 25.6 ± 1.26 µM,
21.6 ± 0.81 µM and 13.7 ± 0.61 µM, respectively,
against the MCF-7 cell line and the obtained values are
in the range of their literature reported values35,36. In
general, all compounds exhibited a 3-10 fold lower
anti-proliferative activity against MCF-7/DX, which
could be because these compounds might act as
substrates of the efflux pump P-gp, which is generally
over expressed in the MCF-7/DX cell line37.
4f
R1,2,3,4= OH, X= S
27.3 ± 1.5 µM
75.9 ± 3.0 µM
5c
5d
5e
5f
R1,2,3,4,5= OMe, X= O
R1,2,3,4,5= OH, X= O
R1,2,3,4,5= OMe, X= S
R1,2,3,4,5= OH, X= S
140.0 ± 1.6 µM
13.7 ± 0.6 µM
>250 µM
245.1 ± 2.5 µM
80.4 ± 1.7 µM
>250 µM
102.6 ± 5.3 µM
139.3 ± 6.8 µM
Note: Most active compound of the series is highlighted in bold.
regioisomeric analogues (both 4-C=O and 4-C=S), were
synthesised and evaluated for their anti-proliferative
activities. Since compound-2f was identified to be
active below 10 µM and 50 µM against MCF-7 and
MCF-7/DX,
respectively,
these
subsequently
synthesised compounds were initially assessed for their
anti-proliferative activities at 10 µM concentration
against the MCF-7 and MDA-MB-231 cell lines, and at
50 µM against the MCF-7/DX cell line. Compounds that
reduced the cell viability below 50% in the initial
screening were arbitrarily classified as ‘active’ and they
were then evaluated at a range of concentrations
Next, Series 1 compounds (1c-f to 5c-f) were assessed
for their anti-proliferative activities at 10 µM
concentration against an ER-ve hormone-independent
breast cancer cell line, MDA-MB-231. The 4-thio
4 | J. Name., 2012, 00, 1-3
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concentration against the MCF-7/DX cell line. Data are expressed as
the mean ± standard error of the mean (SEM) (n = 3). Cells without
treatment serve as control. Statistical significance was estimated,
with respect to the control, by one-way ANOVA, followed by
Bonferroni’s post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001 and
****p < 0.0001). Dashed line corresponds to 50% cell viability and
those compounds showing < 50% cell viability were considered as
active. Colour coding: blue-hydroxy flavone (-OH, 4-C=O), red-
hydroxy 4-thioflavone (-OH, 4-C=S), purple-methoxy flavone (-OMe,
4-C=O) and green-methoxy 4-thioflavone (-OMe, 4-C=S).
(between 0.1 and 100 µM) against the respective cell
line to determine their IC50 values.
In the initial screening, Series-2 compounds with
bioisosteric
replacements
showed
a
decreased/complete loss of activity with cell viability >
50% (See supporting information, Figure-S2). Among
Series-3, the novel compounds 14f and 16f were found
to be active against all three cell lines, and novel
compounds 13f, 15f, 17f and 19f were found to be
Novel insights into structure-activity relationships
(SARs): Comparison of the anti-proliferative activities of
methoxy flavones (both 4-C=O and 4-C=S) with their
corresponding hydroxy flavones illustrates that the
methoxy flavones were less anti-proliferative than their
hydroxy analogues with the exception of compound-3e
active against both the MCF-7 and MCF-7/DX cell lines.
Novel compounds 12f and 18f were found to be active
only against the MCF-7 cell line (Figure-2). These active
compounds were further tested against the respective
cell lines at different concentrations ranging from 0.1
to 100 µM to determine their IC50 values. The
determined IC50 values are listed in Table-2. Among
these, novel compounds 15f (IC50= 1.0 ± 0.1 µM and 9.1
± 0.1 µM against MCF-7 and MCF-7/DX cell lines
respectively) and 16f (IC50= 4.9 ± 0.7 µM, 6.5 ± 0.4 µM
and 8.9 ± 0.9 µM against MCF-7, MCF-7/DX and MDA-
MB-231 cell lines respectively) were identified as active
compounds.
[
7,8,3’,4’-tetramethoxy 4-thioflavone, IC50 = 11.8 ± 1.79
µM against the MCF-7 cell line]. For example, hydroxy
flavone 4d possesses an IC50 value of 21.6 ± 0.8 µM,
whereas the IC50 value of its methoxy flavone
derivative-4c was found to be >250 µM. This highlights
that the free hydroxyls are important for anti-
proliferative activities (for the hydrogen bonding
interactions with the potential biological cellular
target).
Table 2. IC50 values of the compounds 12f to 19f
against the MCF-7, MCF-7/DX and MDA-MB-231 cell
lines and their correlation with theoretical Log P values.
Chemical
Compound
Log P
IC50 in MCFꢀ7 IC50 in MCFꢀ7/DX IC50 in MDAꢀMBꢀ231
structure
18f
2.87
25.9 ± 1.6 µM
8.0 ± 0.1 µM
7.9 ± 2.4 µM
4.9 ± 0.7 µM
8.9 ± 0.6 µM
5.9 ± 0.7 µM
1.0 ± 0.1 µM
2.5 ±0.1 µM
> 50 µM
> 50 µM
> 10 µM
> 10 µM
12f
14f
16f
19f
13f
15f
17f
3.00
3.40
3.67
2.87
3.00
3.40
3.67
34.0 ± 4.6 µM
6.50 ± 0.4 µM
27.3 ± 1.8 µM
41.3 ± 1.5 µM
9.1 ± 0.8 µM
15.4 ± 0.8 µM
10.9 ± 2.4 µM
8.9 ± 0.8 µM
> 10 µM
> 10 µM
> 10 µM
> 10 µM
Note: Most active compounds of the series are highlighted in bold. Data
represented is mean ± SEM, n=3. Log P values were determined using
ChemDraw ultra 12.1 software.
Figure-2. Anti-proliferative activities of compounds 12c-f to 19c-f
against (A) MCF-7 cell line (B) MCF-7/DX cell line (C) MDA-MB-231 The importance of free hydroxyls for anti-proliferative
cell line. Cell viability was determined using MTT assay in the
activity has previously been debated in the literature,
presence of compounds 12c-f to 19c-f at 10 µM concentration
with contradicting results. Thus, the results from our
against the MCF-7 cell line and MDA-MB-231 cell lines, and 50 µM
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38,39
programme support findings
that indicate that Effect of bioisosteric replacements: Replacement of
polymethoxylation decreases the anti-proliferative the B-ring phenyl group with a bioisostere (eg either a
activity, but contradict earlier findings that suggested five-membered 2-thienyl, a 2-furanyl group, or a six-
higher anti-proliferative activities for polymethoxylated membered 3-pyridyl group) resulted in a significant loss
flavones against MCF-720,40, human leukemic HL-6041 of anti-proliferative activity. This highlighted that the
and melanoma cell lines19.
Further, from the comparison of anti-proliferative anti-proliferative activity.
activities between the hydroxy flavones (free -OH and Influence of electron-withdrawing
presence of a phenyl group as ring-B is favourable for
groups:
4-C=O) and their 4-thio analogues (free -OH and 4-C=S), Interestingly, introduction of EWGs at the C4’ position
it was apparent that the substitution of 4-C=S for 4-C=O of the phenyl group, mainly with halogens such as F
enhances the anti-proliferative activity [compound-2f
(free -OH and 4-C=S), IC50= 7.9 ± 0.8 µM < compound- found to enhance anti-proliferative activities in
2d (free -OH and 4-C=O), IC50= 25.6 ± 1.2 µM]. comparison to the flavones with an unsubstituted
(12f and 13f), Cl (14f and 15f) and Br (16f and 17f) was
Influence of number and position of hydroxyls: By phenyl B-ring (compounds 1f and 2f). In particular, the
comparing the anti-proliferative activities of flavone introduction of Cl and Br groups increased the anti-
derivatives (free -OH and 4-C=O) with different proliferative potencies 2-7 fold (14f, 15f, 16f and 17f)
numbers of hydroxy groups, it was observed that the against MCF-7 and MCF-7/DX cell lines, and also their
anti-proliferative activities against the MCF-7 cell line introduction afforded 7,8-dihydroxy 4-thioflavones
were in the order pentahydroxy flavone (5d) [IC50 = 13.7 with an increased anti-proliferative activity towards
± 0.6 µM] > tetrahydroxy flavone (4d) [IC50 = 21.6 ± 0.8 MDA-MB-231 cells (compound-1f: cell viability at 10
µM] ≈ dihydroxy flavone (2d) [IC50 = 25.6 ± 0.6 µM]. µM was 79%, whereas, compound-14f (containing -Cl)
However, an opposite correlation was observed in the and compound-16f (containing -Br) showed 49% and
case of the hydroxy 4-thioflavone derivatives (free -OH 20% cell viability at 10 µM against MDA-MB-231). The
and 4-C=S), where the order of anti-proliferative pronounced activity observed for the -F, -Cl and -Br
activities was found to be dihydroxy 4-thioflavone (2f
[IC50 = 7.9 ± 0.3 µM] > tetrahydroxy 4-thioflavone (4f
[IC50 = 27.3 ± 1.5 µM] ≈ pentahydroxy 4-thioflavone (5f
)
)
)
substituted 4-thioflavones could be attributed to their
increased lipophilicity, as can be observed by the
decrease in IC50 values (more active) for increasing
[IC50 = 102.6 ± 5.4 µM]. This indicates that the number theoretical Log P values (Table-2). A summary of SARs
of hydroxyls is not a determining factor for the anti- for the anti-proliferative activities of flavones analysed
proliferative activities, in the case of hydroxy flavones herein is depicted in Figure-3.
(free -OH and 4-C=O). In complete contrast, the
number of hydroxyl groups in hydroxy 4-thioflavones
plays an important role in their anti-proliferative
activities and the dihydroxy 4-thioflavones were found
to be the most active flavones.
In terms of the position of the hydroxyls, flavones with
5,7-hydroxyls on the A-ring were more active than their
corresponding 7,8-hydroxy flavones. For example,
Figure-3. Summary of the SARs for the anti-proliferative activities of
5,7,3’,4’-tetrahydroxy flavone (4d) [IC50 = 21.6 ± 0.8
µM] > 7,8,3’,4’-tetrahydroxy flavone (3d) [IC50 = 97.5 ±
6.7 µM] and 5,7-dihydroxy 4-thioflavone (2f) [IC50 = 7.9
± 0.2 µM] > 7,8-dihydroxy 4-thioflavone (2e) [IC50 = 14.7
± 1.0 µM].
flavones analysed herein.
Molecular mechanism of action: Next, the probable
molecular mechanism of the anti-proliferative activities
of the flavonoids was explored.
Two plausible
molecular mechanisms were postulated. (a) As the
majority of flavones evaluated herein exhibited a
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strong and selective anti-proliferative activity against proliferative effects via antagonism of ERα, compound-
the estrogen-responsive breast cancer cell line that 15f (IC50 = 1 µM) compound-16f (IC50 = 4.9 µM) and
expresses high levels of estrogen receptor-α (ERα)42, it compound-3e (IC50 = 11.8 ± 1.79 µM), that were potent
was anticipated that these compounds may act as against MCF-7 cells, were assessed for their antagonist
estrogen-receptor antagonists by interacting with ERα. activity at four-point concentrations (10 µM - 0.1 µM)
(b) As these compounds have been reported to interact in the presence of a constant concentration of 17β-
with cell signalling proteins43,4,44 it was hypothesised estradiol (3.2 nM, EC75). The known antagonist
that these compounds could modulate the cell fulvestrant was used as a positive control. Compounds
signalling proteins that are involved in cell survival and 15f and 16f did not show any decrease in the luciferase
cell death.
activity (induced by 17β-estradiol), which was in
contrast to fulvestrant. However, compound-3e
showed a dose-dependent decrease in the luciferase
activity [see supporting information (Figure-S4) for
details]. These results support the findings from the in
silico study that the majority of these compounds
(except compound-3e) most likely act through an
estrogen-independent mechanism.
a) Interaction with ERα: The interaction of flavones
1c-19f with the estrogen receptor-α (ERα) was first
investigated using a molecular docking approach. For
this, compounds 1c-19f were docked sequentially into
the binding site of the prepared X-ray crystallographic
structure of human ERα complexed with an antagonist
2-phenyl-1-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-1,2,3,4-
tetrahydro-isoquinolin-6-ol [native ligand] (PDB code b) Interactions with cell signalling proteins: Next, the
1UOM, 2.28 Å resolution)45 using a validated docking signals mediated by compounds 15f and 16f in ER+ve
procedure (See experimental section). In general no MCF-7 and MCF-7/DX cells and ER-ve MDA-MB-231
correlation was observed between the order of anti- cells were examined using the PathScan® Intracellular
proliferative activity and their binding affinities with Signalling Array Kit, which allows simultaneous
ERα (as determined by their respective docking scores). detection of 18 significant and well-characterised
One notable exception was compound-3e which signalling molecules that undergo covalent post-
showed higher binding affinity towards the ERα translational modifications, such as phosphorylation
receptor [(docking score of 10.02, versus the native and proteolysis (cleavage). Protein samples obtained
ligand docking score of 13.0), see supporting from the treatment of MCF-7, MCF-7/DX and MDA-MB-
information (Figure-S3) for details]. This indicates that 231 cells with compounds 15f (IC50 < 10 µM against
the majority of the flavonoid derivatives mediate their MCF-7 and MCF-7/DX) and 16f (IC50 < 10 µM against
anti-proliferative effects via estrogen-independent MCF-7, MCF-7/DX and MDA-MB-231 cells) were
mechanisms. A similar observation has been reported analysed (Figure-4). The results showed that the anti-
for 5-aminoflavones, which showed remarkable anti- proliferative effects were mediated by significant
proliferative activity against the ER+ve MCF-7 cell line inhibition of the Akt associated downstream target
without ER competition, as determined by the ER GSK-3β, which is a key signalling molecule for cell
binding assay46.
survival and proliferation. In addition, compound-15f,
To confirm this finding, an ERα antagonism assay was which showed greater potency towards MCF-7 cells
carried out using the human estrogen receptor alpha (IC50 = 1 µM for 15f vs IC50 = 4.9 µM for 16f), was found
assay kit which consists of ERα reporter cells in which to trigger cell death through PARP cleavage, which is a
an ERα-responsive promoter is functionally linked to hallmark of the cell death pathway47. This explains the
the luciferase reporter gene, and allows quantification greater potency of compound-15f towards MCF-7 cells.
of changes in the ERα activity via measurement of the It is noteworthy that no caspase-3 cleavage was
changes in luciferase expression. In order to evaluate observed; this suggests that the observed PARP
whether the flavone derivatives mediate their anti- cleavage is caspase-3 independent and the activation of
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PARP cleavage in MCF-7 cells might have been be due to restoration of the wild-type p53 DNA binding
executed via other caspases such as caspases6/9.48 In activity of the mutant p53 tumour suppressor gene.
the case of MCF-7/DX cells, compound-16f, that has
greater activity towards MCF-7/DX cells than
compound-15f, mediated its anti-proliferative effects
through a significant reduction in the phosphorylation
of several cell survival proteins such as mTOR, GSK-3β
and Stat3, along with induction of cell death through
caspase-3 and PARP cleavage. However, compound-15f
was found to elicit its anti-proliferative effects through
a reduction in the phosphorylation of GSK-3β as well as
through induction of cell death via PARP cleavage.
Thus, the higher potency of compound-16f could be
related to its ability to downregulate multiple cell
survival proteins (mTOR, GSK-3β and Stat3) along with
its ability to induce cell death via caspase-3 and PARP
cleavage.
Interestingly, treatment of MDA-MB-231 cells with
compound-16f showed a 2-fold increase in the levels of
p53 compared to the control. MDA-MB-231 cells have a
mutant p53 gene49,50 that is more stable and oncogenic
unlike the wild type p53 in MCF-7 cells51. The presence
of mutant p53 drives the invasiveness of the
tumour52,53. However, the increase in the Serine-15
phosphorylated p53 (mutant) in compound-16f treated
MDA-MB-231 cells [than the control (untreated)] linked
with its profound activity suggest that compound-16f
induces Ser-15 phosphorylation of a mutant p53 in
MDA-MB-231 cells, which probably leads to the
restoration of wild-type p53 DNA binding activity and
induction of cell death. A similar observation has been
reported for resveratrol (a chalcone) wherein cell death
was induced by restoring the wild-type p53 DNA
binding activity via the phosphorylation of a mutant
p53 at Serine-15 in prostate cancer DU145 cells54–56
.
This might explain the higher activity of compound-16f
against MDA-MB-231 cells (IC50 = 8.9 µM for 16f vs IC50
> 10 µM for 15f).
Overall, these results correlate well with the in vitro
findings, and suggest that for the MCF-7 and MCF-7/DX
cell lines, the anti-proliferative effects are mediated via
ER-independent cleavage of PARP and downregulation
of GSK-3β. For the MDA-MB-231 cell line, activity could
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were analysed for phosphatidylserine externalization
(apoptosis marker)57,58 using an annexin V-
FITC/propidium iodide (PI) apoptosis detection assay
and flow cytometry. As shown in Figure-5, a significant
increase in the percentage of apoptotic cells in
comparison to the untreated control was observed in
the MCF-7 cells treated with either 15f or 16f, and in
the MDA-MB-231 cells treated with 16f. These results
clearly indicate that the flavones 15f and 16f
potentially induce apoptosis in the MCF-7 and MDA-
MB-231 cells, whilst only 16f potentially induces
apoptosis in MDA-MB-231 cells. These observations are
in line with the cytotoxicity data/protein array data.
Therefore, from the molecular mechanism of action
studies, and the apoptotic assay, for compounds 15f
and 16f it can be concluded that these compounds
induce apoptotic cell death via PARP cleavage in MCF-7
cells, and 16f triggers apoptosis by p53 induction in the
MDA-MB-231 cell line.
Figure-4. Chemiluminescent array images of the PathScan
Intracellular
Signalling
array
kit
revealing
various
phosphorylated/cleaved signalling nodes in (A) MCF-7 cells, (B)
MCF-7/DX cells and (C) MDA-MB-231 cells after the treatment with
compounds 15f and 16f at 10 µM for 24h. Cells without treatment
serve as control. Bar chart representing the fold change in the
integrated density of phosphorylated/cleaved signalling nodes in
the array image of (A1) MCF-7 cells, (B1) MCF-7/DX cells and (C1)
MDA-MB-231 cells after the treatment with and without
compounds 15f and 16f at 10 µM for 24 h. Data are expressed as
the mean ± standard error of the mean (SEM) (n = 4). Statistical
significance was estimated with respect to the control by one-way
ANOVA, followed by Bonferroni’s post hoc test (*p < 0.05, **p <
0.01 and ***p < 0.001). (D) Molecular mechanism of action of 15f
and 16f in MCF-7, MCF-7/DX and MDA-MB-231 cell lines.
Figure-5. Apoptosis detection in MCF-7 and MDA-MB-231 cells by
flow cytometry. MCF-7 cells were treated with compounds 15f and
16f at 1 µM and 5 µM respectively for 24 h and MDA-MB-231 cells
were treated with compounds 15f and 16f at 10 µM and 9 µM
respectively for 24 h. The quadrants were set based on the
population of healthy, unstained cells in untreated samples
(control) compared to cells treated with 5 μM camptothecin for 24
h. A- Representative figures showing population of unstained live
(annexin V- PI-, lower left), apoptotic (annexin V+ PI-, lower right),
dead (annexin V+ PI+, upper right) and necrotic (annexin V- PI+,
upper left) cells. B- Bar chart represent the quantification of the
apoptotic cells induced upon the treatment of MCF-7 and MDA-MB-
Apoptosis determination: As compounds 15f and 16f
were found to act via induction of the apoptotic
signalling pathway (PARP, caspase-3 and p53), these
compounds were further assessed for their ability to
induce apoptosis. For this, MCF-7 and MDA-MB-231
cells were treated (at their IC50 concentration for 24 h)
with compounds 15f (1
ꢀ
M and 10
ꢀ
M against MCF-7
M and 9 ꢀM
and MDA-MB-231, respectively) or 16f (5
ꢀ
against MCF-7 and MDA-MB-231, respectively) and
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231 cells with compounds 15f and 16f. Data are expressed as
mean± standard error of the mean (SEM), n = 3. Statistical
significance was estimated with respect to the control (untreated
sample) by one-way ANOVA, followed by Bonferroni’s post hoc test
(ns = nonsignificant; *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p
< 0.0001).
OVCAR-4 cell line, 43 fold and 24 fold more active than
chrysin against colon HCT-15 and 5 fold and 15 fold
more active than chrysin against the NSCLC NCI-H460
cell line, respectively (Figure-6).
NCI 60 cell line screening: Finally, to further probe the
promising and significant anti-proliferative activities of
the novel flavonoids 15f and 16f, they were screened
for in vitro cytotoxicity by the National Cancer Institute
Developmental Therapeutic Program59, USA (NCI/DTP,
USA). Initial evaluation was performed at a single dose
of 10 µM against 60 different human tumour cell lines
using the Sulforhodamine B cytotoxic assay.60 Both
compounds showed potential growth inhibition against
a wide range of cancer cell lines (See Supporting
information Information). As a result, they were
selected for further 5-dose assay screening against the
60 cell lines (0.01 µM - 100 µM). Three end points,
specifically the GI50 (concentration that causes 50%
growth inhibition), TGI (total growth inhibition) and
LC50 (the concentration of the drug at which the
original cell number is reduced by 50%) were
determined. The mean inhibitory doses (MID) against
60 cell lines for compound-15f and compound-16f were
2.81 µM and 2.39 µM respectively. The GI50 values
determined for these two compounds against 60 cell
lines are presented in Table-3. Interestingly, both
compounds 15f and 16f exhibited nanomolar range
activity various cell lines. Specifically, compound-15f
showed the highest cytotoxicity against the breast
Figure-6. Comparison of GI50 values of compounds 15f and 16f with
2d (chrysin, a well-known natural flavone). Data for 2d (chrysin)
were obtained from NCI/DTP screening: December 2010
(NSC407436).
Table 3. GI50 values for 15f and 16f against 60 cell lines
obtained from NCI/DTP screening
GI50 (µM)
Panel/Cell line
15f
16f
Leukaemia
CCRF-CEM
HL-60
4.13
2.99
3.39
3.93
2.95
3.97
2.53
1.66
2.47
2.16
1.32
2.54
K-562
MOLT-4
RPMI-8226
SR
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
9.20
17.3
1.95
1.76
14.6
1.95
2.23
3.09
2.57
3.26
17.9
1.50
1.69
7.22
1.52
1.89
0.97
1.16
HOP-62
HOP-92
NCI-H226
NCI-H23
cancer cell lines MCF-7 (GI50 = 0.18 µM), T-47D (GI50
0.03 µM) and MDA-MB-468 (GI50 = 0.47 µM), HCT-15
(colon cancer, GI50 = 0.48 µM), OVCAR-4 (ovarian, GI50
=
NCI-H322M
NCI-H460
NCI-H522
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
=
0.57 µM). Also, compound-16f, showed significant
cytotoxicity against NCI-H460 (NSCLC, GI50 = 0.97 µM)
and HCT-15 (colon cancer, GI50 = 0.85 µM). The
cytotoxicity profile of the well-known flavone chrysin
16.8
1.74
2.78
0.48
19.4
2.17
3.85
10.7
2.28
2.22
0.85
4.56
3.35
2.60
HT29
(2d) [NSC407436] was retrieved from the NCI database
KM12
(mean dose graph provided in supporting information)
for comparison. Overall, compounds-15f and 16f were
found to be 7-8 fold more potent than chrysin (MID-
20.10 µM, against 51 cell lines). In particular,
compounds-15f and 16f were found to be 46 fold and
13 fold more active than chrysin against an ovarian
SW-620
CNS Cancer
SF-268
SF-295
3.57
8.97
3.48
3.85
2.91
2.18
4.01
1.98
2.95
4.55
SF-539
SNB-19
SNB-75
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3.36
2.44
0.1 µM against MCF-7 and MCF-7/DX cell lines,
respectively) and 16f (IC50 = 4.9 ± 0.7 µM, 6.5 ± 0.4 µM
and 8.9 ± 0.8 µM against MCF-7, MCF-7/DX and MDA-
MB-231 cell lines, respectively)). A systematic SAR
study highlighted the presence of free hydroxyl groups,
and the B-ring phenyl groups, as essential for enhanced
anti-proliferative activities. Replacement of the 4-C=O
functional group with the 4-C=S functional group also
enhanced the anti-proliferative activities. Incorporation
of lipophilic electron withdrawing groups at C4’ of the
B-ring phenyl was found to be favourable, and the
increased lipophilicity correlates well with the greater
anti-proliferative activities. Molecular mechanistic
studies have suggested that the anti-proliferative
effects of flavones 15f and 16f are mediated via ER-
independent cleavage of PARP and downregulation of
GSK-3β in the case of MCF-7 and MCF-7/DX cell lines.
For the MDA-MB-231 cell line, a probable restoration
of the wild-type p53 DNA binding activity of mutant
p53 tumour suppressor gene was indicated. Taken
together, these results demonstrate that the novel
thioflavones 15f and 16f offer considerable potential as
anti-proliferative agents within medicinal chemistry
programmes focussed on cancer. In addition, the SARs
derived herein are likely to contribute significantly to
the design and synthesis of novel thioflavones for
further therapeutic applications.
Melanoma
LOX IMVI
MALME-3M
M14
3.17
1.81
3.25
3.46
2.69
4.67
2.05
2.93
3.27
1.18
1.87
2.01
2.98
2.27
2.94
1.79
2.88
2.09
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian Cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
1.14
1.96
0.57
12.1
3.34
2.82
3.61
2.56
1.23
2.03
5.83
2.28
1.43
2.60
Renal Cancer
786-0
7.35
1.22
2.53
3.12
4.22
2.83
4.60
1.28
2.89
2.15
2.35
2.65
2.51
2.61
4.16
1.02
A498
ACHN
CAKI-1
RXF393
SN12C
TK-10
UO-31
Prostate Cancer
PC-3
3.21
4.26
2.92
2.67
DU-145
Breast Cancer
MCF-7
0.18
2.20
5.26
3.40
0.03
0.47
1.46
1.81
6.69
2.19
1.27
2.08
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
MDA-MB-468
Experimental
Note: Sub-micromolar range GI50 values are highlighted in bold.
Materials and methods
Conclusions
Cell line and culture: MCF-7 (ER +ve breast cancer cell
line) was provided by Tenovus Centre for Cancer
Research (Cardiff, UK), MCF-7/DX (a doxorubicin
resistant phenotype of MCF-7) was obtained as a gift
from Professor Luigi Quintieri from the University of
Padova, Italy and MDA-MB-231 (ER –ve breast cancer
cell line) were purchased as a frozen stock from the
European Collection of Cell Cultures (ECACC). MCF-7
and MCF-7/DX cells were cultured in RPMI 1640
supplemented with 5% fetal bovine serum, MDA-MB-
231 cells were cultured in DMEM supplemented with
10% fetal bovine serum. All of the cell culture reagents
were obtained from Lonza, UK. The PathScan
A library of 76 compounds containing structurally
related methoxy and hydroxy flavones, and their 4-thio
analogues, has been designed, synthesised and
evaluated for anti-proliferative activity against the
breast cancer cell lines MCF-7 (ER+ve), MCF-7/DX
(ER+ve, anthracyline resistant) and MDA-MB-231 (ER-
ve) . Within this library, 42 compounds were prepared
and characterised for the first time within our
laboratory. The study provided significant insight into
the structural features required for enhancing the anti-
proliferative profiles of flavones, and identified two
novel hydroxy 4-thioflavones 15f and 16f, as lead anti-
proliferative agents (15f (IC50 = 1.0 ± 0.1 µM and 9. ±
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Intracellular Signalling array kit was purchased from after 48 h exposure to the test compound, C =
Cell Signaling (Catalog number-7323). ERα receptor Absorbance of control after 48 h and T0 = Absorbance
anatagonist kit was purchased from Indigo biosciences, at time zero.
USA. Annexin V/propidium iodide apoptosis detection
Molecular docking
kit I was procured from BD, Pharmingen™, UK. For the
Docking validation: To validate the accuracy of the
cell culture experiments, the stock solutions of the test
docking procedure to be used, the original ligands were
compounds (10 mg/mL) were prepared in sterile DMSO
extracted from the coordinate files (taken from the
and ethanol (1:1 v/v) and these stocks were then
Protein Data Bank PDB), and then docked again into
appropriately diluted with the complete culture
the corresponding crystal structure of the proteins,
medium and, the ethanol and DMSO levels were
using the automated docking procedure in the program
Surflex-Dock (SFXC)62,63, as provided by SYBYL-X-2.1.
IC50 values were calculated using MS excel 2010.
maintained below 1% in the test concentrations. The
The resulting ligand conformation from the docking
Molecular docking was performed using SYBYL-X-2.1
procedure was compared with the ligand conformation
software.
as found in the actual crystal structure of the complex.
Synthesis: See the supporting information.
Comparative structural orientation of the ligand was
calculated as the root mean square deviation (RSMD)
between the docked ligand and the ligand as found in
the crystal structure, using the programme LSQKAB, as
provided in the CCP464 suite. If the root mean square
deviation (RMSD) value between the real and the best-
scored conformations is equal to or less than 2.0 Å
(representing the grid spacing used for the docking
procedure), then the docking process was considered
successful65. In this case the docking procedure was
first validated by re-docking the extracted co-
crystallised ligand of human estrogen receptor-α (ERα)
(2-phenyl-1-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-
Anti-proliferative assay: Anti-proliferative activities of
the compounds were assessed using the MTT assay at
72 h of treatment. For this, MCF-7 and MCF-7/DX cells
were seeded at a density of 4 x 104 cells/mL and MDA-
MB-231 cells were seeded at a density of 2 x 104
cells/mL into 96 well plates and incubated for 24 h to
allow attachment. After 24 h, the cells were treated
with these synthesised derivatives at a range of
concentrations (0 to 250 µM) or at single doses (10 µM
against MCF-7 and MDA-MB-231 cells, and 50 µM
against MCF-7/DX cells) for 67 h. After 67 h, MTT
assay61 was carried out by the addition of 20 µL of MTT
(5mg/mL) solution in PBS into each well and the cells
were incubated for 5 h. The purple crystals formed
were dissolved in 100 µL of DMSO and the plates were
read at 570 nm using a SPECTRA max UV spectrometer
(Bio-Rad). The data represented are the mean of the
three individual experiments. The cell viability of the
control is considered to be 100%.
1,2,3,4-tetrahydro-isoquinolin-6-ol) into the prepared
target protein to be used for docking. The RMSD
between the docked conformation, as generated by the
docking algorithm and the native co-crystallised ligand
conformation was found to be 0.12 Å, which was well
within the 2 Å grid spacing used in the docking
procedure, indicating that the docking method to be
used was reliable and valid. Furthermore, the
interactions between the docked ligand and the
prepared target receptor mimicked those observed in
the crystal structure of the same.
IC50 values presented here correspond to the
concentration (T/C) x 100 = 50, where T = Absorbance
of the test well after 72 h exposure to the test
compound, C = Absorbance of control well after 72 h.
Docking procedure: Docking studies were performed
using the programme Surflex-Dock (SFXC)62 as provided
by Sybyl-X 2.1. The X-ray crystallographic structures of
human estrogen receptor-α (ERα) complexed with an
GI50 values presented here involve a correction for the
cell count at time zero. Therefore the GI50 value for a
test drug corresponds to the concentration [(T - T0)/(C -
T0)] x 100 = 50, where T = Absorbance of the test well
antagonist
2-phenyl-1-[4-(2-piperidin-1-yl-ethoxy)-
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phenyl]-1,2,3,4-tetrahydro-isoquinolin-6-ol (PDB code- greater than 3.3 Å, but less than 4.5 Å, was considered
1UOM, 2.28 Å resolution)45 was retrieved from the a van der Waal interaction.
Protein Data Bank. The protein structure was prepared
ERα antagonist assay: ERα reporter cells consisting of
for docking using the Biopolymer Structure Preparation
an ERα-responsive promoter gene functionally linked to
Tool with the implemented default settings provided in
the luciferase gene were defrosted and seeded into a
the SYBYL programme suite. Hydrogens were added to
96-well plate and these cells were immediately dosed
the protein structures in idealised geometries and an
with the test compounds at different concentrations
overall energy minimisation of each protein was
(10-0.1 µM) and with 17β estradiol (3.2 nM, EC75
performed using the MMFF94 force field, employing a
concentration) according to the manufacturer’s
conjugate gradient algorithm66 with a convergence
protocol. After 24 h incubation in the presence of the
criterion of 0.5 kcal/mol A−1and up to 5000 iterations.
test compound or solvent (DMSO), the cell viability of
Finally, before the docking run, all water molecules
these treated/untreated reporter cells was measured
were removed and the ligand 2-phenyl-1-[4-(2-
to eliminate false positives using the fluorescence-
piperidin-1-yl-ethoxy)-phenyl]-1,2,3,4-tetrahydro-
based live cell multiplex (LCM) assay. The fluorescence
isoquinolin-6-ol was extracted from the coordinate file
from the live cells was measured using the plate reader
of the ER α (PDB-1UOM). The protomol, representing
with the filter combination of [485nmEx | 535nmEm].
the ligand binding groove, was generated using a ligand
Following this, fold reduction in the luciferase intensity,
directed method, which allows the docking of ligands
which is the measure of the antagonist activity was
into predefined sites, as defined by occupancy of any
measured by using a luminometer (TECAN) according
co-crystallised ligand at the site of interest.
to the manufacturer’s protocol. The data are expressed
as the fold reduction as compared to the control. The
mean of three experiments and the standard error is
reported.
The Surflex-X docking algorithm docks a given ligand to
a receptor using a flexible ligand and a semi-flexible
receptor; in this case the peptides were allowed to be
fully flexible while the receptor was semi-flexible. This
approach allows for optimisation of potentially
favourable molecular interactions, such as those
defined by hydrogen bond and van der Waal forces. The
docking results yield a docking score, which takes into
consideration entropic, polar, hydrophobic, repulsive
and desolvation factors. Here, the docking scores were
expressed in –log10 (Kd) units to represent binding
affinities, where Kd is the dissociation constant. The free
energy of binding of the ligand to the protein was
extrapolated from equation (1).
PathScan sandwich immunoassay: The PathScan
Intracellular Signalling array kit was used for the
simultaneous detection of 18 significant and well-
characterised cellular proteins and signalling nodes that
were phosphorylated or cleaved at the specific
residues.
Preparation of cell lysate: MCF-7 and MCF-7/DX cells (8
x 104 cell/mL) and MDA-MB-231 cells (4 x 104 cells/mL)
were seeded into 24 well plates (2 mL/well) and
incubated for 24 h. After 24 h, the cells were treated
with the test compounds at 10 µM concentration for 24
h. Following this 24 h exposure, the cells were washed
with ice-cold 1X phosphate-buffered saline and lysed in
1X cell lysis buffer provided (phosphotase and protease
inhibitors added). These lysates were quantified using
the BCA protein assay.
Free energy of binding = RTlogeKd...... (1)
The docking results were visualised using the
programme PyMOL67,68 and the molecular interactions
of the docked ligands were analysed by the programme
CONTACTS, as provided in the CCP4 suite
of programmes64,69,70. Potential hydrogen bonds were
assigned if the distance between two electronegative
atoms was less than 3.3 Å, whereas any separation
Assay procedure: The array blocking buffer was added
to each well of the glass slide provided and incubated
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Journal Name
for 15 min at room temperature. Subsequently, the cell
lysate, diluted to 0.3 mg/mL in array diluents, was
added to each well and incubated for 2 h at room
temperature. Subsequent to washing, the detection
antibody cocktail was added to each well and incubated
for 1 h at room temperature. Horseradish peroxidase
(HRP)-linked streptavidin was added to each well and
incubated for 30 min at room temperature. The slide
was then covered with LumiGLO/Peroxide reagent (Cell
Signaling Technology) and exposed to film for 2–30 sec.
The image was captured by a digital imaging system,
ImageQuant LAS 4000 (GE Healthcare).
Acknowledgements
Financial support to DR from the Felix trust is gratefully
acknowledged. We thank Amit Kumar Rajora for his help with
the sample preparation for microarray experiments. We also
thank the University of Reading for the provision of the
Chemical Analysis Facility and Dr Philip Dash, University of
Reading, UK for providing MDA-MB-231 cells. We gratefully
acknowledge NCI, USA for accepting and screening our
compounds against 60 cell lines [NSC reference numbers-
783090 (15f) and 783091 (16f)].
Abbreviations
DMSO-Dimethylsulfoxide, DCM-Dichloromethane, EtOAc-
Apoptosis detection assay: Apoptosis in MCF-7 and
MDA-MB-231 cells was assayed by annexin V and
propidium iodide (PI) co-staining using an Annexin-V-
FITC staining kit according to the manufacturer’s
instruction. 1 x 106 MCF-7 and MDA-MB-231 cells were
plated into a 6 well plate (3.4 x 105 cells/ mL, 3
mL/well). After 24 h incubation, cells were treated
without and with test compounds (at their IC50
concentration) for 24 h. Cells were harvested using
Accutase™ Cell Detachment Solution (1 mL/well) for 5
min at 37 ºC. Accutase was inactivated by addition of
complete medium (2 mL). Cells were collected by
centrifugation at 100 x g and the pellet was washed
twice with cold PBS and then resuspended in 1 mL of
annexin-binding buffer. 100 ꢀL of the cell suspension
was transferred to 1.5 mL eppendorf tube and 5 μl of
FITC-conjugated annexin V and 5 μl of PI was added and
incubated in dark for 15 min at room temperature.
After 15 min incubation 400 ꢀL of annexin binding
buffer was added to the cells and the fluorescence was
measured using BD accuriTM instrument flow cytometry.
The instrument was set for FL1 (annexin V-FITC) vs FL3
(PI) bivariant analysis. Data from 10,000 cells/sample
was collected and dot plots of FL1 vs FL3 were
generated. The quadrants were set based on the
population of healthy, unstained cells in untreated
samples compared to cells treated with a known
apoptotic inducer camptothecin (5 μM) for 24 h. BD
CSamplerTM software was used to calculate the
percentage of the cells in the respective quadrants. The
experiment was performed in triplicate.
Ethylacetate,
MTT-3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide, NMR-Nuclear magnetic
resonance, IR-Infrared; FTMS-Fourier transform mass
spectroscopy, ESI-Electron spray ionisation, ERK1/2-
extracellular signal-regulated kinase, mTOR-mammalian
target of rapamycin, PARP-Poly (ADP-ribose) polymerase,
AMPKα-5' adenosine monophosphate-activated protein
kinase, p53-phosphoprotein53, GSK-3β- Glycogen synthase
kinase-3β, Bad-Bcl-2-associated death promoter, PRAS40-
proline-rich
Akt
substrate
of
40
kDa,
PI3K-
Phosphatidylinositol-4,5-bisphosphate-3-kinase, MMFF94-
Merck Molecular Force Field94.
Notes
The authors declare no competing financial interest.
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