Synthesis and evaluation of Hsp90 inhibitors that contain the 1,4-naphthoquinone scaffold

Article abstract of DOI:10.1016/j.bmc.2008.11.064

High-throughput screening of a library of diverse molecules has identified the 1,4-naphthoquinone scaffold as a new class of Hsp90 inhibitors. The synthesis and evaluation of a rationally-designed series of analogues containing the naphthoquinone core sca

Full text of DOI:10.1016/j.bmc.2008.11.064

Bioorganic & Medicinal Chemistry 17 (2009) 634–640
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of Hsp90 inhibitors that contain the
1,4-naphthoquinone scaffold
M. Kyle Hadden a, Stephanie A. Hill a, Jason Davenport b, Robert L. Matts b, Brian S. J. Blagg a,
*
a Department of Medicinal Chemistry, The University of Kansas, 1251 Wescoe Hall Dr., Malott 4070, Lawrence, KS 66045-7563, United States
b Department of Biochemistry and Molecular Biology, NRC 246, Oklahoma State University, Stillwater, OK 74078, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
High-throughput screening of a library of diverse molecules has identified the 1,4-naphthoquinone scaf-
fold as a new class of Hsp90 inhibitors. The synthesis and evaluation of a rationally-designed series of
analogues containing the naphthoquinone core scaffold has provided key structure–activity relationships
for these compounds. The most active inhibitors exhibited potent in vitro activity with low micromolar
IC50 values in anti-proliferation and Her2 degradation assays. In addition, 3g, 12, and 13a induced the
degradation of oncogenic Hsp90 client proteins, a hallmark of Hsp90 inhibition. The identification of
these naphthoquinones as Hsp90 inhibitors provides a new scaffold upon which improved Hsp90 inhib-
itors can be developed.
Received 8 October 2008
Revised 20 November 2008
Accepted 24 November 2008
Available online 3 December 2008
Keywords:
Hsp90
Cancer
Naphthoquinone
MCF-7
Ó 2008 Elsevier Ltd. All rights reserved.
Anti-proliferation
High-throughput screening
1. Introduction
relationship (SAR) studies for these compounds have provided in-
sight into key structural features necessary for optimal Hsp90
The 90 kDa family of heat shock proteins (Hsp90) has become a
validated target for the treatment of cancer because of their role as
molecular chaperones responsible for the folding of nascent poly-
peptides into conformationally active three-dimensional struc-
tures.1 Client proteins of Hsp90 have been implicated in all six
hallmarks of cancer and inhibition of Hsp90 can provide a multi-
faceted attack on cancer and malignant cell growth. Studies have
revealed two nucleotide binding pockets in the Hsp90 protein,
one at the N-terminus and the other in the C-terminal domain.2,3
Only the N-terminal ATP-binding pocket manifests ATPase activity
and facilitates client protein maturation, while the C-terminus ap-
pears to exhibit allosteric properties.4 In addition to its potential as
an anti-cancer target, regulation of Hsp90 also exhibits promising
activity that may be utilized for the treatment of numerous neuro-
degenerative disorders, including Alzheimer’s and Parkinson’s Dis-
ease, in which protein aggregation is a common etiology.5,6
Natural product Hsp90 inhibitors currently being evaluated in
anti-cancer models include geldanamycin (GDA) and radicicol
(RDC) (Fig. 1). Both of which bind to the N-terminal ATP-binding
site and exhibit potent in vitro activity.7 However, various side ef-
fects have slowed their clinical development. In addition, the nat-
ural products novobiocin and coumermycin A1 have been
identified as C-terminal inhibitors of Hsp90 and structure–activity
inhibitory activity.8–10 The promise that Hsp90 holds as a thera-
peutic target is illustrated by the number of research groups in
both academia and industry that developed high-throughput
screens in an effort to identify new inhibitory scaffolds that are
amenable to rapid syntheses.11–15
Utilizing a high-throughput screen recently developed with
rabbit reticulocyte lysate,
a library of small molecules was
screened for their ability to inhibit Hsp90 via the renaturation of
thermally denatured firefly luciferase, which is dependent upon
Hsp90.16 In the presence of an Hsp90 inhibitor, the natural biolu-
minescence of luciferin is significantly reduced as a consequence
of the inability to renature luciferase. Both GDA and novobiocin
significantly reduce the renaturation of luciferase in this assay,
indicating that it is a suitable assay for identification of both N-
and C-terminal inhibitors of Hsp90. As an example of the utility
of this assay, the natural product derrubone was recently identified
and characterized as an Hsp90 inhibitor (Fig. 1).17
During this screen, several compounds that contain a naphtho-
quinone core were identified as ‘hits’ based on their ability to inhi-
bit the renaturation of luciferase at 20 lM (HTS1-3, Table 1). The
similar core structures present in these inhibitory scaffolds sug-
gested these compounds bind to the same region of Hsp90 and
provided preliminary SAR directly from the high throughput
screen. Similar anti-proliferative and Her2 degradation activity in
in vitro assays further supported optimization of the naphthoqui-
none scaffold. Interestingly, HTS1 demonstrated fourfold greater
* Corresponding author. Tel.: +1 785 864 2288; fax: +1 785 864 5326.
E-mail address: bblagg@ku.edu (B.S.J. Blagg).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.064

M. Kyle Hadden et al. / Bioorg. Med. Chem. 17 (2009) 634–640
635
OH
OMe
HO
OH
O
O
OH
R
H
N
O
R
O
O
O
O
NH2
O
O
O
O
MeO
OH
NH
O
MeO
O
O
O
OH
Novobiocin
Radicicol (RDC)
OH
H2N
O
R = OMe; Geldanamycin (GDA)
R = NHallyl; 17-AAG
OH
O
HN
H
H
N
O
O
N
OH
O
O
H
O
O
O
N
O
O
O
O
O
O
HO
O
OMe
MeO
O
O
OH
OH
Coumermycin A1
Derrubone
O
NH
Figure 1. Natural product inhibitors of Hsp90.
were combined to develop a library of analogues to further probe
SAR for Hsp90 inhibition. Library synthesis began with commer-
cially available 2-amino-3-chloro-1,4-naphthoquinone, 1. The
original synthetic route for these compounds included Boc protec-
tion of the amine, conversion of the chloride to the corresponding
iodide and subsequent Suzuki coupling with various aryl boronic
acids. Numerous attempts to protect the amine were unsuccessful,
highlighting its reduced nucleophilicity as a consequence of its
participation in the electron deficient naphthoquinone ring sys-
tem. This reduced activity combined with previous reports of suc-
cessful Suzuki couplings of vinyl chlorides conjugated to electron-
withdrawing groups18,19 altered the original route and led to direct
coupling of the starting naphthoquinone to a series of arylboronic
acids instead (Fig. 2). Reflux of 1 in the presence of 4 mol % tetra-
kis(triphenylphosphine)palladium(0), an arylboronic acid, and so-
dium carbonate resulted in the phenylnaphthalene diones, 2a–i,
in moderate yield (25–46%) (Scheme 1).
Table 1
In Vitro results of HTS hits with the naphthoquinone scaffold
O
O
O
O
NH
N
N
N
N
O
O
N
O
HTS1
O
HTS2
O
O
HTS3
Luciferase Assaya
Her2 ELISA IC50
Anti-proliferation IC50
c
c
Compound
MCF-7
SKBr3
HTS1
HTS2
HTS3
0.25b
0.38
0.02
3.3
1.2 0.2
1.2 0.3
1
0.21 0.02
0.82 0.02
2.9 0.5
0.87 0.23
19
4
0.83 0.13
a
All values are reported in lM.
Values represent the IC50 of a representative luciferase refolding assay.
Values are mean SEM of three separate experiments performed in triplicate.
b
c
To probe the size of the binding region for the amide side chain
of HTS2, we prepared both acetamide and benzamide derivatives.
The unreactive nature of the amine prevented use of standard pep-
tide coupling conditions and thus, more reactive conditions were
employed to provide the requisite acetamides (3a–i) and benzam-
ides (4a–i) (Scheme 2). Reflux of the arylated amines, 2a–i, in acet-
yl chloride with catalytic sulfuric acid afforded the acetamides in
quantitative yield.20 In contrast, treatment of the amine with so-
dium hydride and benzoyl chloride produced the benzamide com-
pounds, 4a–i, in moderate yield (35–62%). In addition, 1 was
acetylated (5) or benzylated (6) to provide compounds that contain
chlorine in the 3-position.
anti-proliferative activity against MCF-7 cells (ER
a+ human breast
cancer cell line) compared to SKBr3 cells (ER -, Her2 over-express-
a
ing human breast cancer cell line), indicating this scaffold may pro-
vide a useful probe to study estrogen-dependent cancers. This
manuscript details the synthesis, optimization, and in vitro activity
of the naphthoquinone scaffold as a new class of Hsp90 inhibitors.
2. Results and discussion
2.1. Chemistry
Structural similarity shared between the naphthoquinone core
scaffold, the central coumarin ring of novobiocin, and the isoflav-
one of derrubone have led to our current hypothesis that these
Optimization of the naphthoquinone scaffold was based upon
the structural features present in compounds HTS1–HTS3 that
OMe
O
R
O
O
H
OH
O
O
O
N
OMe
NH
O
O
O
O
HO
Ph
O
MeO
O
HO
Novobiocin analogue (NA)
Derrubone
Naphthoquinones
OH
Figure 2. Structural similarity of novobiocin analogue (NA), derrubone, and naphthoquinone scaffolds.

636
M. Kyle Hadden et al. / Bioorg. Med. Chem. 17 (2009) 634–640
(Fig. 2).10 Based on the improved inhibitory activity of the biaryl
O
O
moiety when incorporated into novobiocin analogues and in an ef-
fort to identify synergistic structural modifications between these
three classes of inhibitors, the biaryl acid 9 was prepared as previ-
ously described,10 converted to the corresponding acid chloride
and coupled with the phenylnaphthalene dione 2d to give the
tri-aryl naphthoquinones, 11 (Scheme 3). The para-methoxy dione
was chosen as the core scaffold for benzamide SAR for two reasons:
(1) its structural similarity to the benzamide of NA and (2) previ-
ously elucidated SAR for derrubone21 identified this functionality
as important for Hsp90 inhibition. The starting dione 1 was also
coupled directly to the biaryl acid to give analogue 12 for direct
comparison of the necessity of the p-methoxy aryl ring in the pres-
ence of the biaryl functionality. In addition, 2d was coupled with a
variety of benzoyl chlorides that contain substitutions at the
ortho-, meta-, and para-positions to determine optimal substitu-
tions at these positions of the benzamide ring (Scheme 4).
NH2
NH2
Cl
K2CO3, Pd(PPh3)4
boronic acids, Δ
O
O
1
R
2a-i
OH
B
OH
OH
B
B
HO
HO
HO
MeO
OMe
a
b
c
OH
OH
OH
B
B
B
HO
HO
HO
OMe
Cl
2.2. Biological activity
Cl
d
e
f
OH
B
OH
B
OH
B
The individual boronic acids (Scheme 1) were chosen to (1)
probe the hydrogen bond donor and acceptor properties for this re-
gion of the molecules as well as (2) to determine the potential for
HO
HO
HO
Cl
OPh
O
hydrophobic and
p-stacking attributes of the aromatic ring. Each
g
h
compound was tested for its anti-proliferative activity against
two distinct human breast cancer cell lines, an estrogen receptor
positive cell line, MCF-7, and an estrogen receptor negative, Her2
over-expressing cell line, SKBr3. In addition, the ability to down-
regulate the Hsp90 client protein, Her2, was measured via ELISA
assay in the SKBr3 cell line.22 There were two general trends ob-
served for the anti-proliferative activity manifested by both the
acetamide 3a–i, 5 (Table 2) and benzamide 4a–i, 6 (Table 3) series
of compounds: (1) the analogues were more active against the
MCF-7 cell line, particularly for the benzamide analogues and (2)
the analogues containing biaryl ethers (3h-i and 4h–i) were signif-
icantly less active than the naphthoquinones incorporating a single
phenyl group. In addition, both series of compounds demonstrated
reduced anti-proliferative activity as compared to the most active
HTS hits (HTS1 and HTS3), however, their comparable activity in
the Her2 ELISA assay (low micromolar IC50 values) suggests they
are equipotent at Hsp90 inhibition. Nonspecific protein binding
in vitro or increased redox activity of the HTS hits may account
for the observed anti-proliferation discrepancies. Both the acetam-
ide and the benzamide appear to be well tolerated, suggesting that
the binding region for this portion of the scaffold may be amenable
to additional functionalities of varying size. In addition, the
i
Scheme 1. Suzuki coupling of naphthoquinone analogues.
O
NHCOCH3
CH3COCl
H2SO4, Δ
O
O
3a-i
NH2
R
O
O
R
NHCOPh
2a-i
NaH, THF
C6H5COCl
O
R
4a-i
O
NHCOCH3
Cl
anti-proliferative activity of
5 against the SKBr3 cell line
CH3COCl
H2SO4, Δ
(0.6 0.003 M) was significantly increased compared to MCF-7
l
cells and Her2 degradation, suggesting the potential for an addi-
tional target for this compound within the SKBr3 cell line.
O
5
O
NH2
Cl
Naphthoquinone analogues containing the 3-position p-meth-
oxy aryl functionality and varying benzamides were prepared
and evaluated for two specific reasons: (1) to probe the structural
requirements for improving activity at each position of the benz-
amide ring and (2) to apply SAR developed for other Hsp90 inhib-
itory scaffolds to the naphthoquinone series. The biaryl benzamide
that exhibits increased anti-proliferative activity when incorpo-
rated in novobiocin analogues10 was completely inactive when
O
O
1
NHCOPh
Cl
NaH, THF
C6H5COCl
O
6
combined with the p-methoxy aryl ring (11, IC50 > 100 lM, see Ta-
Scheme 2. Preparation of acetamide and benzamide naphthoquinone analogues.
ble 4). The increased steric bulk of the tri-aryl ring system appears
too large to be accommodated in the proposed binding site and is
thus unable to maintain favorable binding interactions. The im-
proved anti-proliferative activity of compound 12 (IC50 = 1.7–
3.0 lM) compared to 11 may suggest that the flexibility of the biar-
yl benzamide moiety allows it to adopt an orientation in which the
compounds may bind Hsp90 in a similar region. Previous struc-
ture–activity relationship studies for the benzamide moiety of
novobiocin from our laboratory identified the biaryl functionality
present in novobiocin analogue (NA) as exhibiting improved anti-
proliferative activity over the novobiocin prenylated benzamide
m-methoxy phenyl ring of the benzamide can occupy the binding

M. Kyle Hadden et al. / Bioorg. Med. Chem. 17 (2009) 634–640
637
OH
B
OMe
I
Pd(dppf)Cl2
2 M K2CO3,
OMe
HO
MeO2C
MeO2C
7
8
OMe
1. LiOH
OMe
OMe
2. (COCl)2, DMF, 1 or 2d
9
OMe
OMe
O
O
O
O
OMe
NH
NH
Cl
O
O
OMe
12
11
Scheme 3. Preparation of biaryl naphthoquinone analogues.
Table 2
R
In vitro activity of acetamide naphthoquinones
O
O
a
O
NH2
Compound
Her2 ELISA IC50
Anti-proliferation IC50
MCF-7 SKBr3
O
O
NaH, RPhOCl
NH
HTS1
3a
3b
3c
3d
3e
3f
3g
3h
3i
3.3
1
0.21 0.02
1.6 0.5
2.2 0.7
2.7 0.7
2.2 0.3
3.0 0.3
1.3 0.5
1.4 0.2
2.4 0.3
2.5 0.2
1.7 0.1
0.82 0.02
1.5 0.4
1.7 0.3
3.9 0.7
2.7 0.3
3.6 0.2
2.5 0.1
1.8 0.4
6.8 0.5
12.1 0.4
0.6 0.003
3.2 2.0
2.5 1.4
4.8 1.4
2.2 0.1
2.3 0.2
2.0 0.2
5.3 2.0
13.3 2.6
24.6 1.5
6.6 0.9
OMe
2d
OMe
13a-i
Cl
ClOC
ClOC
Cl
ClOC
Cl
5
a
b
e
c
f
a
Values are mean SEM of three separate experiments performed in triplicate
OMe
and reported in lM.
ClOC
ClOC
ClOC
OMe ClOC
OMe
Table 3
d
In vitro activity of benzamide naphthoquinones
a
a
Compound
Her2 ELISA IC50
Anti-proliferation IC50
MCF-7
SKBr3
ClOC
HTS1
4a
4b
4c
4d
4e
4f
4g
4h
4i
3.3
1
0.21 0.02
2.1 0.4
2.2 0.1
1.4 0.1
1.4 0.02
1.8 0.1
4.8 2.0
2.6 0.1
6.0 0.1
23 1.9
0.82 0.02
6.8 0.1
1.9 0.7
2.6 0.02
4.0 1.8
4.0 0.4
12.7 0.6
8.5 0.6
>100
COCl
3.8 0.6
3.0 1.4
2.7 0.7
2.8 0.9
1.8 0.4
6.3 1.5
1.8 0.5
>100
g
h
i
Scheme 4. Preparation of benzamide analogues.
site normally occupied by the 3-position aryl functionalities. In
general, these analogues 13a–i also exhibit increased anti-prolifer-
ative activity against MCF-7 cells over SKBr3 cells. Hydrogen bond
acceptors were well tolerated at both the ortho- and para-posi-
tions, however, meta substituents were detrimental to Her2 degra-
>100
30.1 1.1
>100
6
1.8 0.3
2.1 0.1
a
Values are mean SEM of three separate experiments performed in triplicate
M.
and reported in
l
dative activity.
A 6- and 15-fold decrease in activity was
demonstrated for the meta chloro (13b) or methoxy (13e) substit-
uents, respectively. The threefold decrease in Her2 activity exhib-
ited by 13g compared to the corresponding benzamide (4d)
suggests that the increased steric bulk of the naphthalene ring sys-
tem prevents optimal binding site interactions. In addition, orien-
tation of the naphthalene ring is important as evidenced by
complete loss of Her2 degradative activity for the 2-napthyl ana-
logue (13h). Finally, 13i exhibits Hsp90 inhibitory activity compa-
rable to the corresponding benzamide (4d) suggesting the aryl ring
is not essential for inhibitory activity.
Compounds representing each series of naphthoquinone ana-
logues were tested for their ability to inhibit Hsp90-dependent
refolding of firefly luciferase in the HTS screening assay (Table 5).

638
M. Kyle Hadden et al. / Bioorg. Med. Chem. 17 (2009) 634–640
Table 4
In vitro activity of p-OMe/benzamide derivatives
a
Compound
Her2 ELISA IC50
Anti-proliferation IC50
MCF-7
SKBr3
HTS1
11
3.3
>100
1
0.21 .02
>100
0.82 .02
>100
12
1.7 0.04
2.0 0.06
12.9 0.4
3.0 0.2
5.8 0.8
44.2 4.0
2.4 0.08
9.9 0.9
>100
3.0 0.4
2.2 0.1
3.4 1.5
3.3 1.2
2.1 0.1
1.8 0.1
1.5 0.3
2.2 0.3
6.8 0.6
1.9 0.6
1.7 0.5
5.5 0.9
9.5 2.3
3.0 1.7
7.9 0.8
6.4 0.2
6.4 0.1
5.8 1.1
>100
13a
13b
13c
13d
13e
13f
13g
13h
13i
2.5 1.2
4.8 1.3
a
Values are mean SEM of three separate experiments performed in triplicate
and reported in lM.
Table 5
Firefly luciferase renaturation
a
Compound
IC50
HTS1
3b
3g
4b
4g
5
11
12
0.25b
1.6 0.5
0.2 0.03
39 16
1.4 0.3
5.0 1.5
86 22
1.6 0.4
2.5 1.6
24.0 15
1.4 0.3
13e
13f
13i
a
Values are mean SEM of three separate experiments performed in triplicate
M.
Value represents the IC50 value from a luciferase refolding assay.
and reported in
l
b
Figure 3. Induced degradation of Hsp90 client proteins via 3g (top), 12 (middle), or
13a (bottom) inhibition of Hsp90. Compound, at varying concentrations, ( M) was
l
evaluated for its ability to downregulate several client proteins as described in
Section 4. GA (500 nM) and DMSO were used as positive and negative controls,
respectively.
As a general trend, Hsp90 inhibition with these compounds
compared favorably with their anti-proliferative and Her2 degra-
dation activities. The acetamide derivatives evaluated were more
active than the corresponding benzamide derivatives, suggesting
that the smaller functionality provides improved inhibitory
activity. The inhibition of luciferase renaturation elicited by the
3. Conclusion
Identification of the 1,4-naphthoquinone scaffold as a new class
of Hsp90 inhibitors led to the synthesis and evaluation of a ratio-
nally-designed series of analogues that contain the naphthoqui-
none core. Structure–activity relationship studies for these
compounds have provided insight into the functionalities that pro-
duce optimal in vitro activity. While both the benzamide and acet-
amide derivatives were active in cell-based assays, the acetamides
were more potent in the luciferase refolding assay, a direct mea-
sure of Hsp90 inhibition. Compounds that contained three aryl
rings branched from the central naphthoquinone core were less ac-
tive, suggesting that increased steric bulk prevents these analogues
from binding similarly to the proposed site. Based on these ana-
logues, the rational design and evaluation of naphthoquinone
mimics lacking the redox-active quinone moiety are currently
underway in an effort to further develop this class of inhibitors.
most active compound, 3g (IC50 = 0.2 0.03 lM), compared
favorably to that observed from the original high throughput
screen. As expected, 11 was unable to inhibit luciferase refolding,
while 12 exhibited low micromolar activity, further supporting
the hypothesis that the presence of three bulky aryl moieties
prevents optimal binding/inhibition of Hsp90. In addition, the
low micromolar activity exhibited by 13i corresponds well to its
in vitro activity suggesting that the aryl ring may not be essential
for activity.
The naphthoquinones 3g, 12, and 13a were further evaluated for
their ability to downregulate numerous oncogenic Hsp90 client
proteins, including Her2, Raf-1, and Akt in MCF-7 cells via western
blot analysis as shown inFigure 3. All three compounds induced the
degradation of these client proteins in a concentration-dependent
manner while producing no similar effects on actin, a non Hsp90-
dependent client protein used as a positive control. Compound
13a induced Hsp90 client protein degradation at concentrations
4. Experimental
comparable to its anti-proliferative activity (IC50s ꢀ5–10
contrast, both 3g and 12 were slightly less active than 13a at client
M). The concentrations at
which 3g decreased levels of Hsp90-dependent client proteins
correlates directly with its activity in cell-based assays rather than
its ability to inhibit luciferase refolding.
lM). By
4.1. 2-Amino-3-phenylnaphthalene-1,4-dione (2a)
protein degradation (IC50s ꢀ10–20
l
A
solution of 2-amino-3-chloro-naphthoquinone (300 mg,
1.4 mmol) and tetrakis(triphenylphosphine)palladium(0) (4 mol %,
64.7 mg, 56 mol) in 11 mL THF:2 M K2CO3 (10:1) was stirred at rt
for 30 min. Phenyl boronic acid (341 mg, 2.8 mmol) in THF (3 mL)
l

M. Kyle Hadden et al. / Bioorg. Med. Chem. 17 (2009) 634–640
639
was added and the solution stirred for 30 min at rt. The solution was
heated to reflux and stirred for 12 h. The solution was cooled, filtered
through Celite, and diluted with EtOAc (50 mL). The EtOAc was
washed with H2O (50 mL) and saturated aqueous sodium chloride
(50 mL), dried (Na2SO4), filtered, and concentrated. Chromatogra-
phy (SiO2, Hex:EtOAc, 3:1) afforded 2a as a red solid (34%).1H NMR
(CD2Cl2, 400 MHz) d 8.13 (t, J = 7.3 Hz, 2H), 7.80 (t, J = 7.3 Hz, 1H),
7.61 (t, J = 7.3 Hz, 1H), 7.54 (t, J = 7.9 Hz, 2H), 7.41 (m, 3H) 5.25 (br,
NH2). 13C NMR (CD2Cl2, 500 MHz) d 182.2, 181.9, 145.5, 135.0,
133.6, 133.3, 132.6, 131.0, 130.5 (2C), 129.3 (2C), 128.4, 126.7,
126.1, 117.13. mmax 3138, 3022, 1674, 1622, 1572, 1555, 1354,
1230, 1047, 733 cmꢁ1. ESI-HRMS m/z calcd for C16H12NO2 [M+H]+
250.0868, found 250.0860.
and the mixture stirred at rt for 1 h. The THF was removed and the
mixture redissolved in EtOAc (5 mL) and H2O (5 mL). The aqueous
layer was removed, the organic layer washed with saturated aque-
ous sodium chloride (5 mL), dried (Na2SO4), and concentrated. Pre-
parative TLC (Hex:EtOAc 2:1) afforded 11 as a red oil (46%). 1H
NMR (CD2Cl2, 400 MHz) d 8.39 (s, 1H), 8.16 (t, J = 7.4 Hz, 2H),
7.80 (m, 3H), 7.73 (s, 1H), 7.40 (d, J = 8.8 Hz, 1H), 7.35 (t,
J = 8.8 Hz, 1H), 7.07 (t, J = 8.3 Hz, 3H), 6.92 (d, J = 8.1 Hz, 4H), 3.89
(s, 3H), 3.85 (s, 3H), 3.81 (s, 3H). 13C NMR (CD2Cl2, 500 MHz) d
184.1, 182.8, 163.6, 160.4, 160.1, 159.8, 139.2, 138.3, 134.9,
134.1, 133.8, 132.9, 131.2 (2C), 131.1, 131.0, 130.7, 129.4, 129.4,
127.2, 126.5, 126.3, 126.2, 122.3, 115.7, 113.7 (2C), 113.2, 111.4,
56.2, 55.7, 55.5. mmax 2956, 2916, 2846, 1663, 1608, 1574, 1520,
1478, 1292, 1285, 1186, 667 cmꢁ1
. ESI-HRMS m/z calcd for
4.2. N-(1,4-Dioxo-3-phenyl-1,4-dihydronaphthalen-2-
C32H26NO [M+H]+ 520.1760, found 520.1761.
yl)acetamide (3a)
4.5. Anti-proliferative effects of naphthoquinones
To a solution of 2a (10 mg, 0.04 mmol) in 5 mL acetyl chloride at
rt was added a catalytic amount of concentrated sulfuric acid. The
solution was refluxed for 15 min (or until the solution changed
from deep red to yellow). The acetyl chloride was removed and
the mixture redissolved in EtOAc (5 mL) and H2O (5 mL). The aque-
ous layer was removed, the organic layer washed with saturated
aqueous sodium chloride (5 mL), dried (Na2SO4), and concentrated.
Preparative TLC (Hex:EtOAc, 2:1) afforded 3a in quantitative yield
as a yellow oil. 1H NMR (CD2Cl2, 400 MHz) d 8.17 (d, J = 6.8 Hz, 2H),
7.83 (m, 2H), 7.75 (s, 1H), 7.46 (m, 2H), 7.38 (m, 3H), 2.01 (s, 3H).
13C NMR (CD2Cl2, 500 MHz) d 183.9, 182.7, 166.7, 138.0, 135.0,
134.9, 134.0, 133.9, 132.7, 131.0, 129.7 (2C), 128.7, 128.2 (2C),
127.2, 126.5, 24.2. mmax 3109, 3058, 2962, 1664, 1612, 1595,
1491, 1477, 1326, 1294, 1157, 1016, 731 cmꢁ1. ESI-HRMS m/z calcd
for C18H14NO3 [M+H]+ 292.0974, found 292.0973.
MCF-7 and SKBr3 cells were maintained in a 1:1 mixture of Dul-
becco’s Modified Eagle’s Medium:Ham’s F-12 (Gibco) supple-
mented with non-essential amino acids,
streptomycin (500 g/mL), penicillin (100 units/mL) and 10% fetal
bovine serum. Cells were grown to confluence in a humidified
atmosphere (37 °C, 5% CO2). Cells (2000/well, 100 L) were seeded
L-glutamine (2 mM),
l
l
in 96-well plates and allowed to attach overnight (37 °C, 5% CO2).
Compounds or GA at varying concentrations in DMSO (vehicle)
were added (1% DMSO final concentration) and cells returned to
the incubator (37 °C, 5% CO2) for 72 h. At 72 h, the number of viable
cells was determined using an MTS/PMS cell proliferation kit (Pro-
mega) per the manufacturer’s instructions. Cells incubated in 1%
DMSO were used as 100% proliferation and values were adjusted
accordingly.
4.3. N-(1,4-Dioxo-3-phenyl-1,4-dihydronaphthalen-2-
yl)benzamide (4a)
4.6. Her2. ELISA of naphthoquinones
SKBr3 cells were grown as described above and seeded (3000
cells/well) in 96-well plates and allowed to attach overnight
(37 °C, 5% CO2). Compounds, at varying concentrations, were
added and the plates returned to the incubator for 24 h. Media
was removed and the cells washed three times with ice-cold buf-
fer (PBS with 1% Tween). Methanol (ꢁ20 °C) was added and the
plates placed at 4 °C for 10 min to permeabilize and fix the cells.
The plates were washed again with ice-cold buffer and incubated
in blocking buffer (5% BSA in PBST) for 1 h at rt. The plates were
incubated with a Her2 specific antibody (rabbit IgG; 1:500 dilu-
tion in blocking buffer) at 4 °C overnight. The plates were
washed again and incubated at room temperature for 2 h in
the presence of an HRP-conjugated anti-rabbit IgG (1:1000 in
blocking buffer). Plates were rinsed, chemiluminescent reagent
was added and the plates immediately read on a luminometer
(Molecular Devices).
To a solution of 2a (7.5 mg, 0.03 mmol) in anhydrous THF was
added sodium hydride (4.3 mg of a 56% dispersion, 0.1 mmol)
and the mixture stirred at rt for 30 min. Benzoyl chloride
(5.6 mg, 4.6 lL, 0.04 mmol) was added and the mixture stirred at
rt for 1 h. The THF was removed and the mixture redissolved in
EtOAc (5 mL) and H2O (5 mL). The aqueous layer was removed,
the organic layer washed with saturated aqueous sodium chloride
(5 mL), dried (Na2SO4), and concentrated. Preparative TLC (benzene
or benzene/EtOAc, TLC plate developed 8–10 times) afforded 4a as
a red oil (56%). 1H NMR (CD2Cl2, 400 MHz) d 8.47 (s, 1H), 8.21 (t,
J = 6.7 Hz, 2H), 7.86 (m, 2H), 7.77 (d, J = 7.8 Hz, 2H), 7.60 (t,
J = 6.7 Hz, 2H), 7.40–7.52 (m, 5H), 7.34 (m, 1H). 13C NMR (CD2Cl2,
500 MHz) d 183.8, 182.8, 163.9, 138.4, 135.1, 134.4, 134.1, 134.0,
133.9, 133.0, 132.9, 131.0, 129.6 (2C), 129.2 (2C), 128.7, 128.2
(2C), 128.0 (2C), 127.3, 126.6. mmax 3059, 3034, 2869, 1663, 1502,
1474, 1294, 1273, 1070, 1026, 914, 717 cmꢁ1. ESI-HRMS m/z calcd
for C23H16NO3 [M+H]+ 354.1130, found 354.1155.
4.7. Luciferase renaturation assay
4.4. 30,6-Dimethoxy-N-(3-(4-methoxyphenyl)-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)biphenyl-3-carboxamide (11)
The ability of the naphthoquinones to inhibit the Hsp90-depen-
dent renaturation of firefly luciferase was determined as previ-
ously described.16
Acid 1010 (43 mg, 0.17 mmol) was dissolved in anhydrous
CH2Cl2 (5 mL) under argon at rt. Oxalyl chloride (42.6 mg,
28.8 lL, 0.34 mmol) and catalytic DMF were added and the mix-
4.8. Western blot analyses of naphthoquinones
ture stirred at rt for 12 h. The CH2Cl2 was removed and the result-
ing yellow oil was dryed under high vacuum for 3 h and used
MCF-7 cells were seeded (1 ꢂ 106/plate) in culture dishes and
allowed to attach overnight. Compounds 3g, 12, and 13a were
added at varying concentration and incubated (37 °C, 5% CO2) for
24 h. Cells were harvested and analyzed for Hsp90 client protein
degradation as described previously.17
without further purification. To
a solution of 2d (13.5 mg,
0.05 mmol) in anhydrous THF (5 mL) was added sodium hydride
(12.9 mg of a 56% dispersion, 0.15 mmol) and the mixture stirred
at rt for 30 min. Biaryl acid chloride (17 mg, 0.06 mmol) was added

640
M. Kyle Hadden et al. / Bioorg. Med. Chem. 17 (2009) 634–640
7. Hadden, M. K.; Lubbers, D. J.; Blagg, B. S. J. Curr. Top. Med. Chem. 2006, 6, 1173.
Supporting Information Available
8. Burlison, J. A.; Blagg, B. S. J. Org. Lett. 2006, 8, 4855.
9. Le Bras, G.; Radanyi, C.; Peyrat, J.-F.; Brion, J.-D.; Alami, M.; Marsaud, V.; Stella,
B.; Renoir, J.-M. J. Med. Chem. 2007, 50, 6189.
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J. J. Org. Chem. 2008, 73, 2130.
General synthetic procedures, full characterization of key inter-
mediates and all final analogues. This material is free of charge via
the Internet.
11. Zhou, V.; Han, S.; Brinker, A.; Klock, H.; Caldwell, J.; Gu, X.-j. Anal. Biochem.
2004, 331, 349.
12. Cheung, K.-M. J.; Matthews, T. P.; James, K.; Rowlands, M. G.; Boxall, K. J.;
Sharp, S. Y.; Maloney, A.; Roe, S. M.; Prodromou, C.; Pearl, L. H.; Aherne, G. W.;
McDonald, E.; Workman, P. Bioorg. Med. Chem. Lett. 2005, 15, 3338.
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Acknowledgments
This work was supported by the National Institutes of Health
(NIH-1R01CA125392 and), OAES project 1975, and the Kansas Can-
cer Center (Postdoctoral fellowship, M.K.H.). SAH was the recipient
of a KINBRE Scholarship.
16. Galam, L.; Hadden, M. K.; Ma, Z.; Ye, Q.-Z.; Yun, B.-G.; Blagg, B. S. J.; Matts, R. L.
Bioorg. Med. Chem. 2007, 15, 1939.
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