3-arylcoumarin derivatives manifest anti-proliferative activity through Hsp90 inhibition

Article abstract of DOI:10.1021/ml300018e

The potential therapeutic benefits associated with Hsp90 modulation for the treatment of cancer and neurodegenerative diseases highlight the importance of identifying novel Hsp90 scaffolds. KU-398, a novobiocin analogue, and silybin were recently identified as new Hsp90 inhibitors. Consequently, a library of 3-arylcoumarin derivatives that incorporated the structural features of KU-398 and silybin was designed, synthesized, and evaluated against two breast cancer cell lines. Western blot analysis confirmed that the resulting 3-arylcoumarin hybrids target the Hsp90 protein folding machinery.

Full text of DOI:10.1021/ml300018e

Letter
pubs.acs.org/acsmedchemlett
3-Arylcoumarin Derivatives Manifest Anti-proliferative Activity
through Hsp90 Inhibition
Huiping Zhao, Bin Yan, Laura B. Peterson, and Brian S. J. Blagg*
Department of Medicinal Chemistry, 1251 Wescoe Hall Drive, Malott 4070, The University of Kansas, Lawrence, Kansas 66045-7563,
United States
S
* Supporting Information
ABSTRACT: The potential therapeutic benefits associated with Hsp90 modulation
for the treatment of cancer and neurodegenerative diseases highlight the importance
of identifying novel Hsp90 scaffolds. KU-398, a novobiocin analogue, and silybin
were recently identified as new Hsp90 inhibitors. Consequently, a library of 3-
arylcoumarin derivatives that incorporated the structural features of KU-398 and
silybin was designed, synthesized, and evaluated against two breast cancer cell lines.
Western blot analysis confirmed that the resulting 3-arylcoumarin hybrids target the
Hsp90 protein folding machinery.
KEYWORDS: heat shock protein 90, Hsp90 inhibitors, novobiocin, silybin, 3-arylcoumarin, breast cancer
of cancer or, conversely, induce the pro-survival pathways to aid
cells exposed to toxic insults without causing client protein
degradation, such as those found in neurodegenerative diseases,
including Alzheimer's, Parkinson's, Prion, and Hodgkin's dis-
eases.10−12 The existing and emerging therapeutic benefits
associated with Hsp90 modulation highlight the importance of
identifying novel Hsp90 inhibitors that can differentiate these
attributes.
Hsp90 inhibitors for the treatment of cancers are under
extensive investigation, and recently, a lead molecule named
KU-398 was shown to exhibit promising anticancer activity.13−19
Alongside KU-398, the natural product, silybin, was also recently
identified as an Hsp90 inhibitor through an Hsp90-dependent
firefly luciferase rematuration and Hsp90-dependent heme-
regulated eIF2α kinase (HRI) activation assay.20 These studies
suggested that similar to KU-398, silybin also binds to the
Hsp90 C-terminus. Structural alignment of KU-398 and silybin
suggests that the coumarin and flavonone ring systems may
be interchangeable (Figure 1) and that the corresponding
3-arylcoumarin derivatives could target the Hsp90 protein fold-
ing machinery. Interestingly, 3-aryl-containing coumarins are
commonly produced by plants and often exhibit antioxidant,
anti-inflammatory, and antimicrobial activities, but more
he 90 kDa heat shock protein (Hsp90) is an ATP-
Tdependent chaperone that is responsible for the folding,
activation, and stabilization of more than 200 client proteins, of
which approximately 50 are kinases, hormone receptors, and/or
transcription factors directly associated with signaling pathways
that regulate cell growth.1,2 Hsp90 exists as a homodimer in
normal cells and acts as a house-keeping chaperone to maintain
protein homeostasis.3 However, in malignant cells, Hsp90 forms
a superchaperone complex that binds to cochaperones and
immunophilins to modulate the function of proteins associated
with all six hallmarks of cancer. These oncogenic Hsp90-
dependent clients include Raf-1, Akt, CDK4, Src, hTERT, and c-
Met, all of which have developed an addiction to Hsp90 for their
activity in transformed cells.4,5 Not surprisingly, inhibition of the
Hsp90 protein folding machinery results in simultaneous
disruption of these signaling cascades and induces client protein
degradation through the ubiquitin-proteasome pathway, which
can eventually lead to cell death.6,7 More than 16 small molecule
inhibitors that target the Hsp90 N-terminus are under clinical
evaluation in an effort to validate Hsp90 as a therapeutic target
for the treatment of cancer.8,9 During Hsp90 N-terminal inhi-
bition, induction of the pro-survival heat shock response also
occurs at the same concentration needed to promote client
protein degradation, which can often lead to cytostatic activity in
lieu of cytotoxicity. Ideally, it would be beneficial to segregate
these two activities so that one can induce client protein degra-
dation, without inducing a pro-survival pathway for the treatment
Received: January 18, 2012
Accepted: February 26, 2012
Published: February 26, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
Figure 1. Rationale for the design of 3-arylcoumarin 1. Left: Structural alignment of KU-398 and silybin. Right: Overlay of energy minimized KU-
398 (blue) with silybin (green).
recently, they were shown to also manifest anticancer activ-
ity.21−24 As a consequence of these observations, compound 1
was designed to maintain the coumarin ring present in KU-398
while incorporating the 3-aryl appendage of silybin to provide a
new class of Hsp90 C-terminal inhibitors.
Western blot analyses of MCF-7 cell lysates confirmed selective
degradation of Hsp90-dependent clients at concentrations that
parallel the observed anti-proliferative activity (Figure 2), suggesting
that 3-arylcoumarin derivatives target the Hsp90 protein folding
machinery.
Synthesis of compound 1 commenced with commercially
available 2-methyl resorcinol (2), which was formylated under
Vilsmeier−Haack conditions using phosphorus oxychloride
(POCl3) and N,N-dimethylformamide (DMF), followed by hy-
drolysis to give formyl-resorcinol 3 (Scheme 1).25 Condensation
Scheme 1. Synthesis of 3-Arylcoumarin 1
Figure 2. Western blot analyses of Hsp90-dependent client proteins
from MCF-7 breast cancer cell lysates upon treatment with 3-
arylcoumarin 1 for 24 h. Concentrations (in μM) were indicated above
each lane, and geldanamycin (G, 0.5 μM) and dimethylsulfoxide (D)
were employed as positive and negative controls.
To generate structure−activity relationships for the phenyl
appendage, commercially available coumarin 8, which lacks the
8-methyl substituent, was used. Bromination of 8 proceeded
poorly following literature procedures.26−28 Therefore, com-
pound 8 was first methylated with iodomethane in the presence
of sodium hydride in DMF to afford compound 9, which
underwent bromination with N-bromosuccinimide in the pres-
ence of catalytic sodium acetate. Subsequent demethylation of
10 with tribromoborane, followed by Mitsunobu coupling with
N-methyl-4-hydroxylpiperidine, generated intermediate 12. The
final products were then produced by Suzuki coupling of 12
with a series of phenylboronic acids (13a−p) or fused hetero-
arylbronic acids (15a,b) to give a library of 3-arylcoumarin
derivatives, 14a−p and 16a,b (Scheme 2).
Upon construction of this library, the corresponding 3-aryl-
coumarins were evaluated for anti-proliferative activity against
SKBr3 and MCF-7 breast cancer cell lines. As shown in Table 1,
3-arycoumarin derivatives are more efficacious against MCF-7
than SKBr3 cells. Any substitution on the phenyl ring was
determined beneficial; however, electron-deficient groups are
more effective than electron-rich substitutions (14d/14h vs 14f).
Substitution at the para position resulted in a slightly
of 3 with 3,4-methylenedioxyphenylacetic acid in the presence of
acetic anhydride and pyridine produced the acylated 3-arylcoumarin,
5, which upon hydrolysis of the ester yielded compound 6. Finally,
a Mitsunobu etherification proceeded upon treatment of 6 with
N-methyl-4-hydroxylpiperidine to afford 3-arylcoumarin, 1.17
Biological evaluation of 3-arylcoumarin 1 for anti-proliferative
activity against SKBr3 (estrogen receptor negative, Her2 over-
expressing breast cancer cells) and MCF-7 (estrogen receptor
positive breast cancer cells) cell lines was performed.18 Because
Her2 and the estrogen receptor are two Hsp90-dependent client
proteins and their stability and function are highly dependent
upon Hsp90 folding machinery, studies with these two different
cell lines were used. Compound 1 was shown to manifest anti-
proliferative activity with an IC50 value of 5.21
1.14 μM
against SKBr3 cells and 3.71 0.11 μM against MCF-7 cells.
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ACS Medicinal Chemistry Letters
Letter
detrimental (14b vs 14d and 14o vs 14p). It appears that bulky
substitutions (14n) or a phenoxyl group (14m) at the para
position can also lead to compounds that exhibit increased anti-
proliferative activity. The decreased activity observed for 14l,
when compared to compound 1, clearly demonstrates that a
methyl group at the 8-position of the coumarin ring increases
anti-proliferative activity. More interestingly, benzo[b]-
thiophene derivative 16a exhibited activity against both cell
lines at concentrations below 1 μM and could serve as a lead
compound for further diversification.
Scheme 2. Synthesis of 3-Arylcoumarins
To further confirm that 8-methyl substituents are beneficial
for anti-proliferative activity, a small set of commercial available
phenylacetic acids 17a−e were similarly condensed with 3 in
the presence of acetic anhydride, followed by hydrolysis to give
19a−e, which underwent etherication with N-methyl-4-
hydroxylpiperidine to afford 8-methyl-3-arylcoumarin deriva-
tives, 20a−e (Scheme 3).
As suggested by related compounds, the 8-methyl-3-
arylcoumarin derivatives manifested improved anti-proliferative
activity, when compared to the 8-desmethyl analogues against
the MCF-7 breast cancer cell line, suggesting that substitution
at the 8-position of the coumarin ring is important (Table 2).
Table 1. Anti-proliferative Activity for 3-Arylcoumarin
Derivatives
Table 2. Anti-proliferative Activity for 8-Methyl-3-
arylcoumarins
R
SKBr3 (μM)
MCF-7 (μM)
a
14a
14b
14c
14d
14e
14f
H
38.41 2.31
25.38 9.24
9.46 0.84
8.24 1.59
11.23 1.11
29.93 15.44
9.21 1.67
5.78 0.59
10.60 1.27
43.85 0.32
24.86 9.72
14.80 0.01
4.43 0.14
4.42 1.75
15.63 4.35
3.33 0.69
0.98 0.01
5.84 0.06
28.98 14.96
10.58 1.50
5.79 0.53
3.89 0.61
8.60 0.25
16.92 2.34
4.64 0.69
4.12 0.37
8.42 0.12
10.50 0.57
7.67 1.46
8.79 1.32
1.45 0.11
2.36 0.30
15.62 0.50
3.08 0.09
0.81 0.02
4.93 0.06
o-Cl
m-Cl
p-Cl
R
SKBr3 (μM)
MCF-7 (μM)
p-CH3
p-OCH3
m-CF3
p-OCF3
m-CN
p-F
a
20a
20b
20c
20d
20e
H
14.27 0.54
5.52 1.44
4.94 0.03
7.38 0.23
4.51 0.42
7.72 1.64
1.97 0.11
1.24 0.06
3.83 0.00
1.65 0.16
14g
14h
14i
p-OCH3
p-Cl
p-F
14j
p-OCF3
a
14k
14l
m-F
Values represent means
standard deviations for at least two
3,4-methylenedioxyl
p-phenoxy
p-t-butyl
separate experiments performed in triplicate.
14m
14n
14o
14p
16a
16b
Although compound 14f, which contains an electron-rich methoxy
group at the para position, exhibited decreased activity when
compared to 8-desmethyl compounds against both SKBr3 and
MCF-7 cell lines (see Table 1), its 8-methyl counterpart (20b)
manifested comparable activity to the other 8-methyl deriv-
atives (20c−e).
o-phenyl
p-phenyl
a
Values represent means
standard deviations for at least two
separate experiments performed in triplicate.
The 3-benzo[b]thiophenecoumarin 16a exhibited the most
potent anti-proliferative activity against two breast cancer cell
lines evaluated. The anti-proliferative activities exhibited by 16a
result from Hsp90 inhibition as demonstrated by Western blot
increased activity as compared to substitutions at the meta
position (14d vs 14c), while substitution at the ortho position is
Scheme 3. Synthesis of 8-Methyl-3-arylcoumarins
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ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures for the synthesis and characterization
of new compounds (1H and 13C NMR, HRMS). This material
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 785-864-2288. Fax: 785-864-5326. E-mail: bblagg@
ku.edu.
Funding
We gratefully acknowledge support of this project by the NIH/
NCI (CA120458).
Notes
The authors declare no competing financial interest.
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