Novel, potent small-molecule inhibitors of the molecular chaperone Hsp90 discovered through structure-based design

Article abstract of DOI:10.1021/jm050355z

The crystal structure of a previously reported screening hit 1 (CCT018159) bound to the N terminal domain of molecular chaperone Hsp90 has been used to design 5-amide analogues. These exhibit enhanced potency against the target in binding and functional a

Full text of DOI:10.1021/jm050355z

4212
J. Med. Chem. 2005, 48, 4212-4215
Novel, Potent Small-Molecule Inhibitors
of the Molecular Chaperone Hsp90
Discovered through Structure-Based
Design
Brian W. Dymock,† Xavier Barril,† Paul A. Brough,†
Julie E. Cansfield,† Andrew Massey,†
Edward McDonald,‡ Roderick E. Hubbard,†
Allan Surgenor,† Stephen D. Roughley,† Paul Webb,†
Paul Workman,‡ Lisa Wright,† and
Martin J. Drysdale*,†
Vernalis Ltd., Granta Park, Great Abington, Cambridge
CB1 6GB, U.K., and Cancer Research U.K. Centre for
Cancer Therapeutics, The Institute of Cancer Research,
15 Cotswold Road, Sutton, Surrey SM2 5NG, U.K.
Figure 1. Structures of known Hsp90 inhibitors.
Received April 15, 2005
enzyme ATPase activity assay. The structure of this
compound bound to Hsp90 guided the design of a
fluorescence polarization (FP) assay,16 allowing the
measurement of binding competition with a fluorescent
probe. Importantly, 1 inhibited the proliferation of
HCT116 human colon tumor cells with the expected
pharmacodynamic (PD) “signature” of up-regulation of
Hsp70 and down-regulation of one of Hsp90’s important
client protiens, in this case Raf-1.2,17
Abstract: The crystal structure of a previously reported
screening hit 1 (CCT018159) bound to the N terminal domain
of molecular chaperone Hsp90 has been used to design 5-amide
analogues. These exhibit enhanced potency against the target
in binding and functional assays with accompanying appropri-
ate cellular pharmacodynamic changes. Compound 11 (VER-
49009) compares favorably with the clinically evaluated 17-
AAG.
Molecular chaperones are protein machines that are
responsible for the correct folding, stabilization, and
function of other proteins in the cell.1 Exposure of cells
to environmental stress, including heat shock, alcohols,
heavy metals, and oxidative stress, results in the
cellular accumulation of these chaperones, commonly
known as heat shock proteins (Hsp’s). Hsp90 has
emerged over the past few years as being of particular
interest because of its role in the evolution, develop-
ment, and disease pathology of cancer.1-3 Hsp90 is an
ATP-dependent chaperone protein essential for the
maturation and activity of a varied group of proteins
involved in signal transduction, cell cycle regulation,
and apoptosis.4,5 The ATPase activity can be inhibited
with some selectivity by natural product antibiotics such
as geldanamycin and its derivatives 17-AAG and 17-
DMAG or radicicol and its oxime derivatives (Figure 1).6
In vivo and clinical data with 17-AAG supports the
hypothesis that the Hsp90 family may be an appropriate
target for anticancer drug development.7-11
Structures of the N-terminal domain of human Hsp90R
have been published as unliganded protein and in
complex with a variety of inhibitors.6,12 A seven-
stranded â sheet forms the backbone of the protein, and
four R helices are arranged such that they form a
compact cavity in which resides the ATP binding site.
There has been significant recent interest in the
identification of small-molecule Hsp90 inhibitors,13 and
this includes our report of the discovery of a novel series
based on a pyrazole template with pendant aryl groups.
Discovery of this series was made via a high-throughout
screen using an ATPase assay14,15 and identified
CCT018159 (1, Table 1), which exhibited activity in the
Even though the potency of 1 was better than 17-AAG
in the FP binding assay and ATPase functional assay,
this did not translate to potent inhibition of HCT116
cell proliferation (Table 1). The GI50 of 0.16 µM for 17-
AAG is surprisingly low and has been the subject of
much discussion in the literature, and one report
suggests that this better than expected potency is due
to accumulation within the cell.19
Herein we report potent derivatives of 1 discovered
through a structure-driven design paradigm. One of
these new compounds inhibits the proliferation of tumor
cells with potency close to that of 17-AAG and repre-
sents one of the first small-molecule inhibitors of Hsp90
to be described that demonstrates the required potency
and potential to become a clinical candidate.
Information gained from the crystal structure of 1
bound to the N-terminal domain of human Hsp9014,17,20
was used to drive the lead development program (Figure
2). The structure is essentially identical to the previ-
ously reported structure bound to the N-terminal of
yeast Hsp90R.17 The resorcinol groups are clearly
central to the binding mode of the compound, particu-
larly the 2′-hydroxyl, which makes a hydrogen bond to
the residue Asp93. A key water molecule at the base of
the pocket interacts with Asp93 and the pyrazole N2 of
the inhibitor. This water is part of a network with Asp93
and two other waters that are seen in all reported
crystal structures of Hsp90. The ethyl group on the
resorcinol ring of 1 fills a small lipophilic pocket that,
upon binding of the weak inhibitor PU3,21-23 can
undergo a major conformational rearrangement to ac-
commodate much bigger lipophilic groups. This binding
mode is entirely consistent with the work of Kreusch et
al. who have published the crystal structures of two
dihydroxyphenylpyrazoles bound to human N-terminal
Hsp90R.24 Further analysis of the crystal structure of
* To whom correspondence should be addressed. Phone: +44 1223
895 313. Fax: +44 1223 895 556. E-mail: m.drysdale@vernalis.com.
† Vernalis Ltd.
‡ The Institute of Cancer Research.
10.1021/jm050355z CCC: $30.25 © 2005 American Chemical Society
Published on Web 06/02/2005

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4213
Scheme 1a
Table 1. Biological Data for Hsp90 Inhibitors
FP IC50
ATPase
GI50
compd R1
R2
R3
R4
(µM)b IC50 (µM)a,b (µM)b
17-AAG
radicicol
1.27
0.15
0.28
4.73
0.21
4.40
0.062
0.150
0.51
0.053
13.4
0.20
5.70
23.0
0.65
36.9
1.90
1.7
0.16
0.13
5.8
34.8
14.9
33.3
6.1
6.7
>80
1.1
0.68
0.938
0.26
1.9
1
Et -O(CH2)2O- Me
-O(CH2)2O- Me
Cl -O(CH2)2O- Me
2
H
3
4
H
OMe
H
H
H
H
H
H
H
H
H
Me
Me
Me
5
Br OMe
Cl OMe
Cl OMe
Cl OMe
Cl OMe
Br OMe
Cl OMe
Cl OMe
6
a Reagents and conditions: (a) 4-methoxyphenylacetic acid,
BF3‚OEt2, 90 °C, 90 min; (b) Ac2O, K2CO3, DMF, 115 °C, 90 min;
(c) hydrazine hydrate, EtOH, 78 °C, 5 h; (d) BnNMe3+Br3-, DCM,
room temp, 90 min.
7
COOH
2.3
8
CONH2
CONHMe 0.039
CONHEt
CONHEt
0.42
0.11
0.390
0.14
1.5
9
10
11
12
0.070
0.025
CONHiPr 0.12
the aliphatic chain of Lys58. This could make favorable
hydrophobic interactions with apolar groups provided
by a ligand. Finally, there are several charged side
chains between 6 and 12 Å away from the 5-methyl
carbon (in order of increasing distance: Lys58, Asp102,
His154, Glu62, and Lys69) that could form polar inter-
actions with relatively large substituents. However,
owing to the solvation effect and the flexible nature of
the side chains of these residues, we believe that it is
highly unlikely that this would yield increased potency.
On the basis of this analysis of the structure, we gave
priority to appending a hydrogen bond donor from the
pyrazole 5-position, which we anticipated would result
in a new hydrogen bond to the backbone of the protein.
The synthesis of 1-3 in Table 1 has been described
previously17,18 (1 and 2 are also commercially available;
see Supporting Information), and the same synthetic
route was utilized (Scheme 1) for the synthesis of 4-6.
Freidel-Crafts acylation of phenols 13 and 14 afforded
the acetophenone derivatives 15 and 16, which were
converted to the chromen-4-ones 17 and 18. Reaction
with hydrazine hydrate afforded the 2,4-dihydroxyphen-
ylpyrazole compounds described. The data for this initial
set of compounds (Table 1) demonstrate the importance
of the substituent R1 being lipophilic in nature, with R1
being chloro and ethyl appearing to afford more potent
binding than the corresponding unsubsituted compound
(1 and 3 vs 2). Replacement of the ethylenedioxyaryl
group of 1-3 with the 4-methoxyaryl group of 4-6 does
not have significant effect on the binding as previously
predicted on the basis of the available structural
information.
a ATPase activity determined with full length yeast Hsp90
protein. b GI50, FP, and ATPase IC50 values are averages of at least
n ) 2.
Figure 2. X-ray structure of 1 bound to the ATP binding site
of human Hsp90R.
1 revealed that the 4-aryl group was pointing toward
the solvent in a quite open part of the binding site,
providing little potential for potency gains.17
Although the 5-methyl group is located in another
solvent channel, it is closer to the protein and there are
a number of potential interaction sites that could be
accessed by substituents at this point.
The most attractive group to interact with is the
carbonyl oxygen of Gly97, located less than 4 Å away
from the 5-methyl carbon. This hydrogen bond acceptor
is 2.7 Å from the pyrazole’s N1, but the interaction is
out of the plane of the peptide bond, leaving the lone
pairs on the oxygen free to interact with other hydrogen
bond donors. Amides (-CONHR), methylamines (-CH2-
NHR) and methyl alcohol (-CH2OH) are examples of
C5 derivatives theoretically able to form hydrogen bonds
with this atom of the backbone of the protein with the
correct geometry. The second site of interest in the
vicinity of the 5-methyl is a small solvent-exposed
hydrophobic patch formed by the side chain of Ile96 and
We selected an amide as the moiety to incorporate
the requisite hydrogen bond donor functionality at the
5-position of the pyrazole. The synthesis of amide
compounds 8, 9, 11, and 12 was achieved from carbox-
ylic acid 7 via a standard coupling protocol (Scheme 2).
The carboxylic acid 7 was itself prepared from chromene
19 by the route outlined in Scheme 1 using chlo-
rooxoacetic acid methyl ester in place of acetic anhydride
in step b. Bromo analogue 10 was prepared in an
analogous method to compound 5 from the correspond-
ing R1 ) H series.
Data presented in Table 1 show that the new com-
pounds 8-12 display high binding affinity, as evident

4214 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13
Letters
Scheme 2a
Figure 4. Raf-1 down-regulation following inhibition of Hsp90
by 8, 9, 11, and 12. HCT116 cells were exposed to 1 × GI50 or
2 × GI50 8, 9, 11, and 12 for 48 h, and Raf-1 levels were
determined by Western blotting. GAPDH was used to confirm
equal loading.
potency seen with the 5-amides is due to the interaction
with the key residue Gly97, and this is further cor-
roborated by structural data from other amides from
this series that do not make an interaction with Lys58.
To add support to the premise that these new com-
pounds exert their antiproliferate effects via inhibition
of Hsp90, we studied the effect of compounds 8, 9, 11,
and 12 on the PD marker Raf-1 (a known client of
Hsp90 and down-regulated upon Hsp90 inhibition) and
the cochaperone Hsp70 (which is up-regulated upon
Hsp90 inhibition).2
a Reagents and conditions: (a) NaHCO3(aq), MeOH, 65 °C, 5
h; (b) hydrazine hydrate, EtOH, 78 °C, 5 h; (c) RNH2, HOBt, EDCI,
DCM, NMM, room temp, 18 h; (d) BnNMe3+Br3-, MeCN, DMF.
As shown in Figure 4, exposure of HCT116 cells to
amides 8, 9, 11, and 12 caused significant reduction in
cellular levels of Raf-1 at concentrations equal to the
concentration that caused cell growth arrest. Up-regula-
tion of Hsp70 (measured using an Hsp70 specific
ELISA) was also observed at similar concentrations
(data not shown), and 11 caused a decrease in cellular
levels of the client protein CDK4 (data not shown).
These results are consistent with the observed effects
on cellular growth arrest being through inhibition of
cellular Hsp90.
In conclusion, we have discovered a new series of
small-molecule Hsp90 inhibitors that display inhibition
of cell proliferation similar to clinically evaluated 17-
AAG. We have determined a crystal structure of the
most potent new compound (11) bound to the target
enzyme, rationally explaining the observed improve-
ments. Further reports will describe structure-activity
relationships in the wider series.
Figure 3. X-ray structure of 11 bound to the ATP binding
site of human Hsp90R.25
from the FP IC50 values. More importantly, however,
the amides 8-12 now exhibit significantly improved
potency in the inhibition of growth of HCT116 cells, with
11 (VER-49009) displaying a value close to that of 17-
AAG. Interestingly the bromo analogue 10 is 3-fold less
active in all assays compared to 11.
Acknowledgment. The authors are grateful to
Adam Hold for compound purification and analysis,
Heather Simmonite for NMR results, and Joanne
Wayne and Kate Grant for assay determinations.
These changes in binding potency can be rationalized
by analysis of the crystal structure of 11 bound to Hsp90
(Figure 3). Key aspects of the binding mode such as the
resorcinol hydrogen bonding with the Asp93 and waters
remain identical to 1. As predicted from modeling, the
new amide group has made a hydrogen bond with
Gly97. This new interaction satisfactorily explains the
tighter binding of the amide series. It is interesting to
note that the flexible Lys58 residue makes a secondary
H-bond interaction with the carbonyl moiety of the
amide in 11. It is reported that the carboxyl groups of
5-carboxy-2′,4′-dihydroxyphenylpyrazoles make a salt
bridge with Lys58; however, this interaction did not
appear to have a significant effect on binding potency.24
This is in agreement with the binding affinity for 7 and
is not surprising because the side chain of Lys58 is
completely solvent-exposed and unconstrained. Thus,
any interaction made by its amino group will be
counteracted by major solvation and entropic penalties.
It is highly probable therefore that the increased
Supporting Information Available: Synthetic proce-
dures with supporting spectral and HPLC data for 4-12 and
key parameters relating to X-ray crystallography and methods
of protein production. This material is available free of charge
via the Internet at http://pubs.acs.org.
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