Discovery of benzisoxazoles as potent inhibitors of chaperone heat shock protein 90

Article abstract of DOI:10.1021/jm701385c

Heat shock protein 90 (Hsp90) is a molecular chaperone that is responsible for activating many signaling proteins and is a promising target in tumor biology. We have identified small-molecule benzisoxazole derivatives as Hsp90 inhibitors. Crystallographic

Full text of DOI:10.1021/jm701385c

J. Med. Chem. 2008, 51, 373–375
373
Letters
Chart 1. Examples of Hsp90 Inhibitors
Discovery of Benzisoxazoles as Potent
Inhibitors of Chaperone Heat Shock Protein
90
Ariamala Gopalsamy,*,† Mengxiao Shi,† Jennifer Golas,#
Erik Vogan,‡ Jaison Jacob,‡ Mark Johnson,‡
Frederick Lee,†, Ramaswamy Nilakantan,†
Roseann Petersen,# Kristin Svenson,‡ Rajiv Chopra,‡,§
May S. Tam,‡ Yingxia Wen,‡ John Ellingboe,† Kim Arndt,#
and Frank Boschelli#
Chemical and Screening Sciences, Wyeth Research, 401 N.
Middletown Road, Pearl RiVer, New York 10965, Chemical and
Screening Sciences, Wyeth Research, 200 Cambridge Park DriVe,
Cambridge, Massachusetts 02139, and DiscoVery Oncology, Wyeth
Research, Pearl RiVer, New York 10965
natural products and synthetic small molecules have emerged
as Hsp90 inhibitors, and most bind to the Hsp90 N-terminal
domain.13 Natural products (Chart 1) such as the benzoquinone
ansamycin geldanamycin 1 and its 17-amino derivatives like
17-AAG 2 and 17-DMAG 3 are the most advanced. Purine
analogues like PU3 4, radicicol 5, and related resorcinol
derivatives are other classes of N-terminal binders of Hsp90.
Through a high-throughput screening effort followed by
database mining, we identified the benzisoxazole derivative 6
(Figure 1) as an Hsp90 inhibitor. Compound 6 exhibited an IC50
of 0.19 µM in the florescence polarization competition assay
using bodipy-geldanamycin.14 Binding of this low molecular
weight inhibitor was further evaluated by 1D NMR competition
STD experiments. The compound was found to bind to the ATP
binding domain as inferred from the presence of direct STD
from the compound and decrease in AMP-PNP STD, indicating
that it competes with AMP-PNP as well. A cocrystal structure
of 6 with N-terminal domain of Hsp90 was solved at 1.6 Å
resolution, clearly indicating that the compound binds in the
active site (the ATP binding site).15 However, this molecule
had suboptimal aqueous solubility (4 µg/mL at pH 7.4) and
probably the poor activity of this compound in the immuno-
fluorescence-based HER2 degradation assay in the SKBR3 cell
line (IC50 g 20 µM) and in the cellular proliferation assay (IC50
g 20 µM) in the HCT116 cell line can be attributed to low
aqueous solubility. Herein, we describe our optimization efforts
on this hit to obtain favorable biochemical and physical chemical
profiles using structure-based design.
ReceiVed NoVember 5, 2007
Abstract: Heat shock protein 90 (Hsp90) is a molecular chaperone
that is responsible for activating many signaling proteins and is a
promising target in tumor biology. We have identified small-molecule
benzisoxazole derivatives as Hsp90 inhibitors. Crystallographic studies
show that these compounds bind in the ATP binding pocket interacting
with the Asp93. Structure based optimization led to the identification
of potent analogues, such as 13, with good biochemical profiles.
Since multiple signal transduction pathways contribute to the
etiology of human cancer, considerable skepticism exists that
inhibition of a single molecular target will be broadly efficacious
in cancer treatment regimens. Clinical trials with signal trans-
duction agents evolve into multiple agent affairs of increasing
complexity, unpredictability, and expense. Kinase inhibitors with
serendipitously defined multiple activities provide one means
of target multitasking.1 However, a more defined path to global
inhibition of multiple activated pathways in tumor cells is
preferable and is possible via inhibition of molecular chaperone
complexes.2–4 Chaperone function is required for folding,
stabilization, and activation of many “clients” (protein kinases,
steroid hormone receptors, and other diverse signaling mol-
ecules).5–8 Loss of chaperone function leads to loss of client
activity and destabilization.
Several essential molecular chaperones and cochaperones
have been identified. Of these, Hsp90 has perhaps been the most
extensively studied.9–11 Its broad clientele includes proteins as
structurally and functionally different as telomerase, the actin
organizer N-WASP, nitric oxide synthase, a range of nuclear
hormone receptors, and an ever-growing selection of protein
kinases.12 The activity of the complex depends on the Hsp90
ATPase activity, which resides in its N-terminal domain, for
which 3D structures have been reported.10,11 A number of
The benzisoxazole hit 6 and additional derivatives like 14-16
and 19 were prepared following the route previously described
from our laboratories.16 Derivatives designed to introduce or
modify substituents to infer SAR were prepared using the
appropriately substituted benzophenone intermediates 9 as
shown in Scheme 1. Buchwald coupling using the iodo precursor
* To whom correspondence should be addressed. Phone: 845-602-2841.
Fax: 845-602-3045. E-mail: gopalsa@wyeth.com.
†
Chemical and Screening Sciences, Pearl River, NY.
Discovery Oncology, Pearl River, NY.
Chemical and Screening Sciences, Cambridge, MA.
#
‡
Current address: Kean University, Union, NJ 07083.
Current address: Novartis Institute of Biomedical Research, 250
§
Massachusetts Avenue, Cambridge, MA 02139.
Figure 1. Hsp90 hit structure.
10.1021/jm701385c CCC: $40.75
2008 American Chemical Society
Published on Web 01/16/2008

374 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Letters
Scheme 1a
Table 1. Hsp90 Inhibition Activity of Benzisoxazole Derivatives
17
FP IC50
compd R1 R2 R3
R4
R5
std dev
(µM)
6
H
H
H
H
H
H
H
H
Br
H
Me
Cl
H
H
H
H
H
OH
OH
OH
OH
0.19 0.089
14
19
15
16
17
18
12
13
15.2
(n ) 1)
a
0.06 0.03
Me Me Cl
H
H
Me Me Cl -NHCH2CH2-morpholino
H
OMe >20
H
H
Cl N-methylpiperizino-
Br -NHCH2CH2-morpholino
H
H
H
H
0.8
0.189
0.04 0.001
>20
0.03 0.012
0.02 0.009
H
Cl -NHCH2CH2-morpholino
Geldanamycin
a In the case of 19, the compound fluorescence was interfering under
the assay condition.
a Reagents: (a) ZnO, room temp, overnight, 50%; (b) (1) NH2OH,
pyridine, 115 °C, 16 h; (2) NaH, DMF, room temp, overnight, 90% for
two steps; (c) morpholinoethylamine, Pd2(dba)3, BINAP, t-BuONa, toluene,
80 °C, 35%; (d) BBr3, DCM, room temp, 8 h, 90%.
Table 2. Biochemical Profile of Analogue 13
assay
IC50 (µM) (n)
std dev
Hsp90 FP binding
HCT 116 cell proliferation
SKBR3 cell proliferation
LnCap cell proliferation
DU145 cell proliferation
0.03 (5)
0.28 (4)
0.37 (3)
0.17 (3)
0.29 (3)
0.23 (3)
0.33 (6)
0.33 (4)
0.05 (54)
0.012
0.06
0.06
0.006
0.08
0.02
0.1
H157 cell proliferation
SKBR3 Her-2 degradation
LnCAP androgen receptor degradation
HCT 116 cell proliferation (geldanamycin)
0.06
0.035
In the crystal structure, the 5′ bromo group is pointing toward
a small hydrophobic pocket interacting with Phe138 and Leu107.
Analogue 14, which lacks a hydrophobic group in this position,
leads to a significant loss in potency, indicating the importance
of this favorable interaction. Replacing the bromo group with
chloro group (analogue 15) did not dramatically change the
activity. From the cocrystal structure it was also evident that
the benzisoxazole scaffold by itself makes some key interactions
with the binding pocket. The N of the ring interacts with a
conserved water molecule, which in turn is in proximity to
Asp93 carboxylate to form a hydrogen bond. However, the
5-hydroxyl group on the benzisoxazole group was well exposed
to the solvent.
As mentioned earlier, although 6 showed favorable potency
in the FP binding assay, its lack of potency in the cellular assays
was of concern. On the basis of the structural information, it is
very clear that the solvent exposed C-5 and C-6 regions of the
benzisoxazole scaffold are very well suited for incorporating
water-solubilizing groups. Analogue 17 with the N-methyl-
piperizino group as the water solubilizing group in the C-5
position was found to be slightly less potent in the Hsp90
binding assay but showed modest potency in the proliferation
assay (IC50 ) 9.4 µM) in the HCT-116 cell line. Probably the
rigidity posed by the piperizine ring is not highly favorable for
the binding. To address this, analogue 13 with a flexible linker
to append the water-solubilizing group (aminoethylmorpholino
group) was prepared. This compound showed improved potency
(Table 2) in the proliferation assay and maintained or slightly
enhanced the potency in the binding assay. The corresponding
bromo analogue 18 was equipotent as well. Compound 13 was
cocrystallized with the amino terminal domain of Hsp90 (1.6
Å)15 (Figure 3) and was indeed found to bind in the same
Figure 2. Atomic interactions of 6 with human Hsp90R. Polar
interactions closer than 3.2 Å are shown as dashed lines.
10 introduced the water solubilizing groups on the aromatic ring.
Deprotection of the methoxy group afforded the final benzisox-
azole derivatives like 13, 17, and 18.
The cocrystal structure of 6 with N-terminal domain of human
Hsp90R (Figure 2) indicated that the bound conformation of
the protein was similar to the apo structure. It is also very clear
that the 2′ hydroxyl of the resorcinol moiety plays a crucial
role in the ATP binding pocket interacting with Asp 93 and
one of the conserved water molecules. The second hydroxyl
group (4′) is involved in maintaining the network of hydrogen
bonds established by the other two conserved water molecules
resulting in interaction with some of the backbone residues like
Leu48. This interaction is reflected in the observed SAR,
wherein analogues 16 and 12 with methoxy in the place of
hydroxyl groups were much less potent in the binding assay
(Table 1).

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 375
Melissa Lin for spectral data. We also thank Dr. Tarek Mansour
for support of this work.
Supporting Information Available: Experimental details and
spectral data for the compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
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PI3K-R, PDK-1, MK2, IKK-2, ActRIIB, and m-TOR (IC50
20 µM).
>
In summary, we have identified benzisoxazole derivatives as
potent and selective inhibitors of molecular chaperone Hsp90.
The hit to lead optimization was guided by structure-based
design facilitated by the cocrystallization efforts. Inhibitors with
improved physical properties resulting in enhanced potency in
the cellular systems are disclosed.
(17) The values represent an average of at least three independent
determinations unless otherwise mentioned.
Acknowledgment. We thank Wyeth Dicovery Analytical
Department for compound characterization, in particular Dr.
JM701385C