7-Substituted umbelliferone derivatives as androgen receptor antagonists for the potential treatment of prostate and breast cancer

Article abstract of DOI:10.1016/j.bmcl.2016.02.088

The clinically used androgen receptor (AR) antagonists (bicalutamide, flutamide and nilutamide) bind with low affinity to AR and can induce escape mechanisms. Furthermore, under AR gene amplification or mutation conditions they demonstrate agonist activity and fail to inhibit AR, causing relapse into castration resistant prostate cancer (CRPC). Discovery of new scaffolds distinct from the 4-cyano/nitro-3-(trifluoromethyl)phenyl group common to currently used antiandrogens is urgently needed to avoid cross-resistance with these compounds. In this study, a series of twenty-nine 7-substituted umbelliferone derivatives was prepared and their antiproliferative activities were evaluated. The most active compound 7a demonstrated submicromolar inhibitory activity in the human prostate cancer cell line (22Rv1); IC₅₀ = 0.93 μM which represents a 50 fold improvement over the clinical antiandrogen bicalutamide (IC₅₀ = 46 μM) and a more than 30 fold improvement over enzalutamide (IC₅₀ = 32 μM). Interestingly, this compound showed even better activity against the human breast cancer cell line (MCF-7); IC₅₀ = 0.47 μM. Molecular modelling studies provided a plausible theoretical explanation for our findings.

Full text of DOI:10.1016/j.bmcl.2016.02.088

Bioorganic & Medicinal Chemistry Letters 26 (2016) 2000–2004
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
7-Substituted umbelliferone derivatives as androgen receptor
antagonists for the potential treatment of prostate and breast cancer
⇑
Sahar Kandil , Andrew D. Westwell, Christopher McGuigan
School of Pharmacy & Pharmaceutical Sciences, Cardiff University, King Edward VII Avenue, Cardiff CF10 3NB, Wales, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
The clinically used androgen receptor (AR) antagonists (bicalutamide, flutamide and nilutamide) bind
with low affinity to AR and can induce escape mechanisms. Furthermore, under AR gene amplification
or mutation conditions they demonstrate agonist activity and fail to inhibit AR, causing relapse into cas-
tration resistant prostate cancer (CRPC). Discovery of new scaffolds distinct from the 4-cyano/nitro-3-
Received 6 December 2015
Revised 28 February 2016
Accepted 29 February 2016
Available online 2 March 2016
(trifluoromethyl)phenyl group common to currently used antiandrogens is urgently needed to avoid
cross-resistance with these compounds. In this study, a series of twenty-nine 7-substituted umbellifer-
one derivatives was prepared and their antiproliferative activities were evaluated. The most active com-
pound 7a demonstrated submicromolar inhibitory activity in the human prostate cancer cell line
Keywords:
Androgen receptor (AR)
Prostate cancer (PC)
Breast cancer (BC)
Steroids
Triple negative breast cancer (TNBC)
Coumarin
(
22Rv1); IC50 = 0.93
tamide (IC50 = 46
Interestingly, this compound showed even better activity against the human breast cancer cell line
l
M which represents a 50 fold improvement over the clinical antiandrogen bicalu-
l
M) and a more than 30 fold improvement over enzalutamide (IC50 = 32 M).
l
(MCF-7); IC50 = 0.47
our findings.
lM. Molecular modelling studies provided a plausible theoretical explanation for
Umbelliferone
Ó 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license
http://creativecommons.org/licenses/by/4.0/).
(
The androgen receptor (AR) is a member of the steroid nuclear
receptor superfamily, which consists of estrogen, progesterone,
glucocorticoids, mineralocorticoids and androgen receptors.
However, the mainstay of advanced stage PC treatment is blockade
of the AR signaling pathway by the use of AR antagonists. Initially
this androgen ablation treatment is effective, but eventually this
treatment strategy fails with the development of a more aggressive
form of PC, namely castration resistant prostate cancer (CRPC).7
Currently, the major antiandrogens in clinical use worldwide are
bicalutamide, flutamide and nilutamide, Figure 1. However, these
compounds bind with low affinity and can induce escape mecha-
1
Androgenic steroids have reproductive and anabolic actions in
2
–5
both men and women.
The androgen receptor (AR) is expressed
in many cell types and AR signaling has been found to have impor-
tant roles in modulating tumourigenesis and metastasis in several
6
cancers including prostate, bladder, kidney, lung, breast and liver.
7
,8
While supplementing androgens is necessary in the treatment of
hypogonadism and may be beneficial in aging individuals to main-
tain muscle mass, over the last 60–70 years there has been consid-
erable research interest in reducing circulating levels of
testosterone and/or blocking the androgen receptor for the clinical
management of some diseases such as prostate cancer and poly-
cystic ovarian syndrome. However, more recently, there is also a
growing appreciation of the need for selective androgen modula-
tors that would demonstrate tissue-selective agonist or antagonist
activity.6
nisms. Furthermore, under AR gene amplification or mutation
conditions these compounds demonstrate partial agonist activity
6
and fail to inhibit AR. These observations indicate that there is
an urgent need to discover a broader chemotype spectrum of AR
antagonists distinct from the 4-cyano/nitro-3-(trifluoromethyl)
phenyl group in flutamide, nilutamide, bicalutamide and enzalu-
tamide, Figure 1, to avoid the cross-resistance with these com-
pounds. A new structural motif without partial agonist activity
would be particularly useful.
,7
Prostate cancer and breast cancer share similarities as hormone
related cancers with a wide heterogeneity. The AR is involved in
both benign and malignant settings in both sexes. Targeting the
AR pathway is central to PC therapy. Despite AR expression in
breast cancer (BC) was known almost 50 years ago, unlike PC the
AR signalling has an important role in initiation and progression
of many hormone-related cancers. Prostate cancer (PC) is one of
the leading causes of cancer related death in men. The early stages
of PC are treated by surgical excision and radiation therapy.
6
,9
antiandrogen narrative for BC is still in its infancy.
Recently,
there has been increased interest in the role of the AR in BC devel-
opment and growth, with results indicating AR expression across
⇑
Corresponding author.
E-mail address: Kandils1@cardiff.ac.uk (S. Kandil).
http://dx.doi.org/10.1016/j.bmcl.2016.02.088
960-894X/Ó 2016 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
0

S. Kandil et al. / Bioorg. Med. Chem. Lett. 26 (2016) 2000–2004
2001
Figure 1. Chemical structure of the clinically used nonsteroidal antiandrogens; flutamide, nilutamide, bicalutamide and enzalutamide.
all intrinsic subtypes of BC. Targeting the AR axis is an evolving
field with novel therapies in development, which may ultimately
be applicable to both PC and BC tumour types. Insights into the
novel drugs in development for targeting this signaling pathway
varying the terminal aromatic group of the ketone linkage and
exploring the impact of replacing the 4-CH group of the coumarin
ring with the more lipophilic 4-CF since the former is assumed to
interact with a hydrophobic subpocket within the AR.
over, a closely related AR selective modulator (LGD2226) features
a CF group in the 4 position of the quinolinone ring that interacts
with the same hydrophobic subpocket as confirmed by the X-ray
3
3
14,15
More-
9
has been thoroughly reviewed recently by Proverbs-Singh et al.
X-ray crystallographic studies of AR have elucidated the struc-
tures of the AR ligand binding domain (LBD) bound to agonists
and of mutant AR bound to antagonists in an agonist like confor-
3
1
9,20
crystal structure (PDB: 2HVC).
Generally, introduction of
mation. However, there are no crystal structures of the AR-LBD
fluorinated substituents into drug candidates can impart a unique
variety of properties owing to the special combination of elec-
tronegativity, size and lipophilicity features of fluorinated groups.
These factors can have a substantial impact on the molecular con-
formation, which in turn affects the binding affinity to the target
in an antagonist conformation reported so far.1
0,11
Several studies
have used the available structural information, computer modeling
and biochemical observations in order to identify new structurally
distinct nonsteroidal small molecule competitive AR antago-
nists.1
2–18
In two independent and recently published studies, a
protein.
21–25
computer aided drug discovery platform was used to identify alter-
native chemical architectures of AR antagonists. In the first study,
the in silico pharmacophore based virtual screening of small mole-
cule libraries led to the identification of six distinct chemotypes, all
of which proved to function in vitro as pure antagonists with IC50
In addition to the substantial role of antiandrogens in the treat-
ment of PC, there has been growing evidence that the androgen
signaling pathway can play a critical role in malignant breast tis-
sue. The AR is the most prevalent sex steroid receptor in in situ,
invasive, and metastatic breast cancers, occurring in up to 90% of
primary tumours and 75% of metastases. AR expression has been
9
14
values of approximately 5 lM. Of particular interest to us was
chemotype A, represented by compound 1, Figure 2, that did not
show cross reactivity with the related steroid receptors; glucocor-
ticoid receptor (GR) and progesterone receptor (PR).14 In the sec-
ond study a different strategy was utilised to elucidate a
pharmacophore model used in the virtual screening of different
commercially available small molecule libraries.15 Interestingly,
one of the three chemotypes identified in this study i.e. chemotype
reported in over 70% of estrogen receptor ER positive breast cancer
and in 45–50% of patients with ER negative breast cancers.2
6–28
The
tumour AR expression level was shown to be inversely associated
2
6
with the survival of BC patients. Ablation of the AR in the human
BC epithelial cell line, MCF-7, has been shown to suppress cell pro-
liferation, suggesting that AR might have a positive role in promot-
2
7
ing BC progression. Therefore, AR is a highly prevalent target in
BC, with potential significance for therapeutic management of both
B, represented by compounds 2 (IC50 = 3.4
lM) and 3 (IC50 = 5.1 -
9
lM), Figure 2, was structurally similar to chemotype A of the first
primary and advanced disease. For example, triple negative breast
study. Both of these two chemotypes feature a 4-methyl coumarin
core fragment and b-keto ether substitution at the 7-position of the
coumarin ring. More importantly, neither of these chemotypes
cancers (TNBC) are characterized by aggressive tumour biology
resulting in a poor prognosis. The androgen receptor (AR) is
reported to be a newly emerging biomarker in TNBC.2
8,29
Enzalu-
show any significant agonistic activity against AR and its mutants
(
Noting the value of the findings of these two independent stud-
ies, we sought to improve upon these starting point structures by
tamide, the second generation AR antagonist, was FDA approved
in 2012 for use in prostate cancer. However, recent studies support
the initiation of clinical evaluation of enzalutamide for treatment
of AR positive breast tumours regardless of ER status, since it
W741C and T877A) and act as pure antagonists.1
4,15
1
2
3
IC50 = 5 µM
IC50 = 3.76 µM
IC50 = 5.09 µM
Figure 2. Structures of chemotype A (represented by compound 1) and chemotype B (represented by compounds 2 and 3).

2
002
S. Kandil et al. / Bioorg. Med. Chem. Lett. 26 (2016) 2000–2004
blocks both androgen- and estrogen-mediated tumour growth.28–33
For the above mentioned reasons we decided to screen our com-
pounds against the MCF-7 (human breast cancer) cell line along-
side 22Rv1 (human prostate cancer) and interestingly
comparable results in both cell lines were observed.
The b-ketoethers (7a–n) and (8a–o) were prepared in good to
excellent yields (70–93%)34 by triethylamine-promoted alkylation
of 7-hydroxy-2H-1-benzopyran-2-ones (4 and 5) with the appro-
priate bromoketones (6a–o) as outlined in Scheme 1.
The homology model for the AR-LBD in the antagonist confor-
mation was constructed using Molecular Operating Environment
(MOE) (Chemical Computing Group, Montreal, Canada). It was con-
structed using the sequence of the hAR in the agonist conformation
from the crystal structure of hAR-LBD with dihydrotestosterone
3
9
(DHT) [2AMA, PDB]. To model the antagonistic conformation of
hAR-LBD, the progesterone crystal structure [2OVH, PDB] was used
as a template since it has sequence identity 54% with the human
4
0
AR. Applying the default parameters of MOE homology modeling
module and using the AMBER99 force field, a total of 10 homology
models were generated. The quality of each model was assessed
within MOE, and the best model was chosen for the docking stud-
ies. The chemical structures of our compounds were constructed,
rendered and minimized with the MMFF94x force field in MOE.
Docking simulations were performed using Glide SP in Maestro
(Glide, version 9.5, Schrödinger, LLC, New York, NY. http://www.
schrodinger.com). The putative docking mode of the most active
compounds 7a and 7b is shown in Figure 4A and B, respectively.
Key interactions include two H-bonds between the lactone car-
bonyl group and both the guanidine group of Arg 752 of helix 5
and the amino group of Gln 711 of helix 3 residues. Another H-
bond between the terminal carbonyl group and the side chain
OH group of residue Thr 877, and the H-bond between the methy-
lene group and the side chain carbonyl group of Asn 705 residue of
helix 3 were also observed. In addition, hydrophobic interactions
were noticed between the 4-methyl coumarin moiety and the sur-
rounding hydrophobic pocket formed of residues; Trp 741, Met
745, Leu 712 and Met 787. The significantly higher activity of com-
pound 7a appears to be a result of the extra bulk conferred by the
Compounds (7a–n) and (8a–o) were screened for antiprolifera-
35
tive activity using Oncotest’s monolayer assay against the pros-
tate cancer cell line 22Rv1 and the breast cancer cell line MCF-7.
Bicalutamide and enzalutamide were used as positive controls.
Anticancer activity was assessed after four days of treatment with
3
5
the compounds using a propidium iodide based monolayer assay.
Potency is expressed as absolute IC50 values, calculated by non lin-
ear regression analysis. The twenty-nine compounds were tested
at 10 concentrations in half log increments up to 100 lM in tripli-
cate. The results summarised in Table 1 indicated that four
compounds (7a–d) have better antiproliferative activity
(
(
IC50 = 0.93–20.37
lM) than the positive controls; bicalutamide
IC50 = 46.25 M) and enzalutamide (IC50 = 31.76
l
l
M). The 3,5-
bis-trifluoromethyl analogue 7a proved to be the most potent com-
pound with submicromolar inhibitory activity in prostate cancer
22Rv1; IC50 = 0.93 lM), which represents an approximate 50 fold
improvement over bicalutamide and more than 30 fold improve-
ment over enzalutamide. Interestingly, this compound showed
even better activity against breast cancer (MCF-7; IC50 = 0.47 lM).
The second most active compound 7b, with m-methoxy substitu-
(
tion of the terminal phenyl group, displayed a low micromolar
3
bis-CF group on the terminal aromatic ring. This steric interaction
activity against prostate cancer (22Rv1; IC50 = 8.41
l
M) and breast
ensures that helix 12 is pushed away from the binding pocket and
is thus more likely to retain activity in the resistant W741L mutant
variant of AR for the same reason. It is worth mentioning that none
of the inactive compounds was able to demonstrate H-bond inter-
actions with the Arg 752 residue in our model, which may indicate
the importance of this key interaction for the AR inhibitory activity.
Generally, our SAR analysis for this umbelliferone based family of
cancer (MCF-7; IC50 = 2.21 M). Both 7c (p-tert-butyl) and 7d (o-
l
trifluoromethyl) analogues displayed about two fold improvement
over bicalutamide in 22Rv1. Compound 7e interestingly showed
moderate activity (IC50 = 38.38
lM) only against MCF-7 but not
2
2Rv1. The dose response curves of compounds (7a–d) are repre-
sented in Figure 3. On the other hand, there was no or only
marginal antagonist activity observed for compounds (7e–n) and
3
compounds indicates that replacement of the 4-CH group of the
(
8a–o). Interestingly, all the analogues with antiproliferative activ-
ity have the 4-methyl group in the coumarin ring rather than the
-trifluoromethyl analogues. Considering the surprising drop of
activity upon small chemical changes (e.g. 4-CH in 7a–d vs. 4-
CF in 8a–d), we suspect that an ‘activity ridge’ has been identified.
coumarin core ring with 4-CF seems to be detrimental to the
3
antiproliferative activity in both cell lines; compounds (7a–d) ver-
sus (8a–d). Docking studies suggest that this detrimental effect can
be attributed to the steric intolerance of the binding sub-pocket of
4
3
3
the coumarin ring to the slight increase in size from CH
to CF in (8a–d) counterparts.
3
in (7a–d)
This phenomenon is also known as SAR discontinuity and it reveals
structural modifications that are of critical importance for biologi-
cal activity. Generally, activity ridges provide significant opportu-
nities for further SAR study in medicinal chemistry.3
3
In summary, 7-substituted umbelliferone derivatives have been
shown to be potentially useful AR antagonists. Through optimiza-
tion of the scaffold with regard to the substitution of the terminal
6–38
3
Scheme 1. Synthesis of b ketoethers (7a–n) and (8a–o). Reagents and condition: (a) THF, Et N, rt, 24 h.

S. Kandil et al. / Bioorg. Med. Chem. Lett. 26 (2016) 2000–2004
2003
M)
Table 1
Mean in vitro antiproliferative activity (IC50
l
M) of compounds (7a–n) across two human cancer cell lines (MCF-7 and 22Rv1)
ID
R1
R2
22Rv1 IC50
(
l
M)
MCF-7 IC50
(
lM)
ID
R1
R2
22Rv1 IC50
>100
(lM)
MCF-7 IC50
>100
(l
F C
3
7
a
CH
3
0.93
0.47
7h
CH
3
H3CO
F
CF
3
H3CO
7
7
b
c
CH
CH
3
3
8.41
2.21
7i
7j
CH
CH
3
3
>100
>100
>100
>100
22.27
20.72
NC
O
CF
3
7
7
7
7
d
e
f
CH
CH
CH
CH
3
3
3
20.37
>100
>100
43.21
38.38
>100
7k
7l
CH
CH
CH
CH
3
3
3
>100
>100
>100
>100
>100
>100
O
F
C
3
7m
7n
F3CO
g
3
>100
>100
—
3
>100
>100
—
F
C
3
Bicalutamide
46.25
Enzalutamide
31.76
Figure 3. Dose response curves of compounds 7a (A), 7b (B), 7c (C) and 7d (D) in the Oncotest monolayer assay of MCF-7 (red) and 22Rv1 (green) cell lines.
A
B
Figure 4. Putative binding modes of compounds 7a (A) and 7b (B) inside the antagonistic hAR-LBD showing hydrogen bond interactions with key amino acids; Arg752,
Gln711, Thr877 and Asn705.

2
004
S. Kandil et al. / Bioorg. Med. Chem. Lett. 26 (2016) 2000–2004
aryl and the 4-position of the coumarin core ring, significantly
potent analogues were identified such as compound 7a, which
demonstrated sub-micromolar inhibitory activity in prostate can-
cer (22Rv1); IC50 = 0.93 lM (around 50 fold improvement over
bicalutamide activity and more than 30 fold improvement over
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These findings provide a basis for further development of umbellif-
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2
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Supplementary data
3
1. Cochrane, D. R.; Bernales, S.; Jacobsen, B. M.; Cittelly, D. M.; Howe, E. N.;
D’Amato, N. C.; Spoelstra, N. S.; Edgerton, S. M.; Jean, A.; Guerrero, J.; Gómez, F.;
Medicherla, S.; Alfaro, I. E.; McCullagh, E.; Jedlicka, P.; Torkko, K. C.; Thor, A. D.;
Elias, A. D.; Protter, A. A.; Richer, J. K. Breast Cancer Res. 2014, 16, R7.
2. Ando‘, S.; De Amicis, F.; Rago, V.; Carpino, A.; Maggiolini, M.; Panno, M. L.;
Lanzino, M. Mol. Cell. Endocrinol. 2002, 193, 121.
Supplementary data (experimental procedures and spectro-
scopic characterizations data of the compounds 7a–n and 8a–o)
associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmcl.2016.02.088.
3
3
3
3. Hickey, T. E.; Robinson, J. L. L.; Carroll, J. S.; Tilley, W. D. Mol. Endocrinol. 2012,
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6, 1252.
4. General procedure for preparation of umbelliferone b keto ether derivatives (7a–n)
and (8a–o): The appropriate bromoketone (6a–o) (1.7 mmol) and
triethylamine (1.6 mmol) were added to a solution of either 7-hydroxy-4-
methyl-2H-chromen-2-one 4 (1.4 mmol) or 7-hydroxy-4-(trifluoromethyl)-
2H-chromen-2-one 5 (1.4 mmol) in THF (20 mL). The mixture was stirred at
room temperature for 24 h, filtered and the solvent was evaporated under
reduced pressure. The solid residue was purified by column chromatography
eluting with DCM/MeOH 9:1 to afford (7a–n) and (8a–o).
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